NEW APPARATUS AND METHODS FOR DISEASE DETECTION

[01] This application claims priority to US Application No. 62/470,181, filed on March 10, 2017, US Application No. 62/627,481, filed on February 7, 2018, and US Application No. 62/635,866, filed February 27, 2018, the contents of which are incorporated by reference in their entirety.

Background of the Invention

[02] Many diseases are difficult to be detected by a single approach or methodology. In particular, many serious diseases with high morbidity and mortality, including cancer and heart diseases, are difficult to diagnose at an early stage with high sensitively, specificity and efficiency, by using one detection equipment. Current disease diagnosis devices typically detect and rely on a single type of macroscopic data and information such as body temperature, blood pressure, or scanned images of the body. For example, to detect serious diseases such as cancer, each of the diagnosis apparatus commonly used today is based on one imaging technology, such as x-ray, CT scan, or nuclear magnetic resonance (NMR). While used in combination, these diagnosis apparatus provide various degrees of usefulness in disease diagnosis. However, each of them alone cannot provide accurate, conclusive, efficient, and cost-effective diagnosis of such serious diseases as cancer at an early stage. Further, many of the existing diagnosis apparatus have a large size and are invasive with large footprint, such as x-ray, CT scan, or nuclear magnetic resonance (NMR).

[03] Even the newly emerged technologies such as those deployed in DNA tests usually rely on a single diagnosis technology and cannot provide a comprehensive, reliable, accurate, conclusive, and cost-effective detection for a serious disease. In recent years, there have been some efforts in using nano technologies for various biological applications, with most of the work focused on one type of gene mapping and moderate developments in the field of disease detection. For instance, Pantel et al. discussed the use of a MicroEelectroMechanical Systems (MEMS) sensor for detecting cancer cells in blood and bone marrow in vitro (see, e.g., Klaus Pantel et al, Nature Reviews, 2008, 8, 329); Kubena et al. disclose in U.S. Patent Number 6,922,118 the deployment of MEMS for detecting biological agents; and Weissman et al. disclose in U.S. Patent Number 6,330,885 utilizing MEMS sensor for detecting accretion of biological matter.

[04] In sum, to date, most of above described technologies have been limited to isolated diagnosis technology for sensing, using systems of relatively simple constructions and large dimensions but often with limited functions, and lack sensitivities and specificities. Further, the existing technologies require multiple times detection by multiple apparatus. This will increase costs and affect achieved degree of sensitivity and specificity as well.

[05] These drawbacks call for novel solutions that provide reliable and flexible diagnosis apparatus using multiple diverse technologies and bring improved accuracy, sensitivity, specificity, efficiency, non-invasiveness, practicality, conclusive, and speed in early-stage disease detection at reduced costs.

Summary of the Invention

[06] The present invention in general relates to a novel technology for detecting disease, in which a number of different classifications of biological information are collected in a device and processes or analyzed.

[07] In traditional technology, typically only one level of biological information is collected (one dimensional), while in this novel technology, at least two levels (classifications) of information can be collected (seven dimensional, or seven factor interactions). Compared with traditional technology which typically focuses on one parameter or one level (for example, bio-marker at protein level), signal and information collected in this novel technology can be collected in a number of forms, and non-linearly amplified. There are additional 2-factor and three-factor interactions which can be collected and analyzed, which maybe missing in other technologies, since they typically only measure one type of biological information.

[08] This novel technology can be used for cancer screening, assisting in diagnosis, prognosis, and follow-up tests with improved sensitivity and specificity, ability to detect cancer early, ability to detect over 10 types of cancer, cost effective, and no side effects.

[09] The novel technology offers several advantages that cannot be achieved by the traditional technology: (1) Ability to detect over 10 cancer types in one test, including some cancer types which cannot be detected by other in vitro tests (e.g., esophageal cancer, cerebral cancer), covering over 80% of all cancer incidences; (2) capable of early stage cancer detection; (3) high sensitivity and specificity (75%~ 90% on over 10 types of cancer); (4) no side effects; (5) high speed, fully automated operations without human intervention; (6) statistical difference between cancer group and non-cancer disease group including inflammation - significantly lower false positives (higher specificity) ; (7) easy process, no difference between fasting blood testing and non-fasting blood testing, and (8) highly cost effective. [010] Accordingly, one aspect of this invention provides an apparatus for detecting presence or monitoring progression of a disease in a biological subject. The biological subject can be a blood sample, a urine sample, or a sweat sample of a mammal. The apparatus comprises a chamber in which the biological subject passes through, and at least one detection transducer placed partially or completely in the chamber; wherein at least two types of information about the biological subject selected from the group consisting of chemical composition, cellular classification, molecular classification, and any combination thereof, are detected by the detection transducer and collected for analysis to determine whether the disease is likely to be present with the biological subject or to determine the status of the disease, therefore providing the ability to continuously determine or monitor progression of the disease.

[Oil] In some embodiments, the detection transducer detects at least one selected from the group consisting of a chemical composition, a cellular classification, a molecular

classification, and any combination thereof; and the detected information is collected for analysis to as to whether the disease is likely to be present with the biological subject.

[012] An example of the chemical composition includes protein (such as a sugar based protein, an embryonic protein, a protein-based antigen, and a carbohydrate antigen).

Examples of the molecular classification include DNA, RNA, or a biomarker.

[013] As used herein, the term "biomarker" means a measurable indicator of the severity or presence of some disease state, but more generally a biomarker is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism. A biomarker can be a substance that is introduced into an organism as a means to examine organ function or other aspects of health. For example, rubidium chloride is used in isotopic labeling to evaluate perfusion of heart muscle. It can also be a substance whose detection indicates a particular disease state, for example, the presence of an antibody may indicate an infection. More specifically, a biomarker indicates a change in expression or state of a protein that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. Biomarkers can be specific cells, molecules, or genes, gene products, enzymes, or hormones.

[014] Examples of the cellular classification include circulating tumor cells, cell surface properties, cell signaling properties, and cell geometrical properties.

[015] In some embodiments, the chemical composition, cellular classification, or molecular classification includes a property of the biological subject at microscope level selected from the group consisting of a thermal, optical, acoustical, biological, chemical, physical-chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, bio-physical, bio- chemical, bio-mechanical, bio-electrical, bio-physical-chemical, bio-electro-physical, bio- electro-mechanical, bio-electro-chemical, bio-chemical-mechanical, bio-electro-physical- chemical, bio-electro-physical-mechanical, bio-electro-chemical-mechanical, physical, an electric, magnetic, electro-magnetic, and mechanical property. The thermal property can be temperature or vibrational frequency; the optical property is optical absorption, optical transmission, optical reflection, optical-electrical property, brightness, or fluorescent emission; the radiation property is radiation emission, signal triggered by radioactive material, or information probed by radioactive material; the chemical property is pH value, chemical reaction, bio-chemical reaction, bio-electro-chemical reaction, reaction speed, reaction energy, speed of reaction, oxygen concentration, oxygen consumption rate, ionic strength, catalytic behavior, chemical additives to trigger enhanced signal response, bio-chemical additives to trigger enhanced signal response, biological additives to trigger enhanced signal response, chemicals to enhance detection sensitivity, bio-chemicals to enhance detection sensitivity, biological additives to enhance detection sensitivity, or bonding strength; the physical property is density, shape, volume, or surface area; the electrical property is surface charge, surface potential, resting potential, electrical current, electrical field distribution, surface charge distribution, cell electronic properties, cell surface electronic properties, dynamic changes in electronic properties, dynamic changes in cell electronic properties, dynamic changes in cell surface electronic properties, dynamic changes in surface electronic properties, electronic properties of cell membranes, dynamic changes in electronic properties of membrane surface, dynamic changes in electronic properties of cell membranes, electrical dipole, electrical quadruple, oscillation in electrical signal, electrical current, capacitance, three-dimensional electrical or charge cloud distribution, electrical properties at telomere of DNA and chromosome, capacitance, or impedance; the biological property is surface shape, surface area, surface charge, surface biological property, surface chemical property, pH, electrolyte, ionic strength, resistivity, cell concentration, or biological, electrical, physical or chemical property of solution; the acoustic property is frequency, speed of acoustic waves, acoustic frequency and intensity spectrum distribution, acoustic intensity, acoustical absorption, or acoustical resonance; the mechanical property is internal pressure, hardness, flow rate, viscosity, fluid mechanical properties, shear strength, elongation strength, fracture stress, adhesion, mechanical resonance frequency, elasticity, plasticity, or compressibility.

[016] The disease that can be detected or monitor for progress can be a cancer, an inflammatory disease, diabetes, a lung disease, a heart disease, a liver disease, a gastric disease, a biliary disease, or a cardiovascular disease. Examples of cancer comprise breast cancer, lung cancer, esophageal cancer, intestine cancer, cancer related to blood, liver cancer, stomach cancer, cervical cancer, ovarian cancer, rectum cancer, colon cancer, nasopharyngeal cancer, cardiac carcinoma, uterine cancer, oophoroma, pancreatic cancer, prostate cancer, brain tumor, and circulating tumor cells; examples of the inflammatory disease include acne vulgaris, asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, chronic prostatitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, and tasculitis; examples of the lung disease include asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, acute bronchitis, cystic fibrosis, pneumonia, tuberculosis, pulmonary edema, acute respiratory distress syndrome, pneumoconiosis, interstitial lung disease, pulmonary embolism, and pulmonary hypertension; examples of the diabetes include Type 1 diabetes, Type 2 diabetes, and gestational diabetes; examples of the heart disease include coronary artery disease, enlarged heart (cardiomegaly), heart attack, irregular heart rhythm, atrial fibrillation, heart rhythm disorders, heart valve disease, sudden cardiac death, congenital heart disease, heart muscle disease (cardiomyopathy), dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, pericarditis, pericardial effusion, marfan syndrome, and heart murmurs; examples of the liver disease include fascioliasis, hepatitis, alcoholic liver disease, fatty liver disease (hepatic steatosis), hereditary diseases, Gilbert's syndrome, cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and Budd-Chiari syndrome; examples of the gastric disease include gastritis, gastric polyp, gastric ulcer, benign tumor of stomach, acute gastric mucosa lesion, antral gastritis, and gastric stromal tumors; examples of the biliary disease include calculus of bile duct, cholecystolithiasis, cholecystitis, cholangiectasis, cholangitis, and gallbladder polyps; the cardiovascular disease comprises coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, congenital heart disease, rheumatic heart disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, and renal artery stenosis.

[017] In some other embodiments, the apparatus can further include a sensor positioned to be partially inside the chamber and capable of detecting a property of the biological subject at the microscopic level. [018] In some other embodiments, the apparatus can further include a read-out circuitry which is connected to at least one sensor and transfers data from the sensor to a recording device.

[019] The connection between the read-out circuit and the sensor can be digital, analog, optical, thermal, piezo-electrical, piezo-photronic, piezo-electrical photronic, opto-electrical, electro-thermal, opto-thermal, electric, electromagnetic, electromechanical, or mechanical.

[020] The sensor can be positioned on the interior surface of the chamber.

[021] In some other embodiments, each sensor is independently a thermal sensor, optical sensor, acoustical sensor, biological sensor, chemical sensor, electro-mechanical sensor, electro-chemical sensor, electro-optical sensor, electro-thermal sensor, electro-chemical- mechanical sensor, bio-chemical sensor, bio-mechanical sensor, bio-optical sensor, electro- optical sensor, bio-electro-optical sensor, bio-thermal optical sensor, electro-chemical optical sensor, bio-thermal sensor, bio-physical sensor, bio-electro-mechanical sensor, bio-electrochemical sensor, bio-electro-optical sensor, bio-electro-thermal sensor, bio-mechanical- optical sensor, bio-mechanical thermal sensor, bio-thermal-optical sensor, bio-electrochemical-optical sensor, bio-electro-mechanical optical sensor, bio-electro-thermal-optical sensor, bio-electro-chemical-mechanical sensor, physical sensor, mechanical sensor, piezo- electrical sensor, piezo-electro photronic sensor, piezo-photronic sensor, piezo-electro optical sensor, bio-electrical sensor, bio-marker sensor, electrical sensor, magnetic sensor, electromagnetic sensor, image sensor, or radiation sensor. For example, the thermal sensor comprises a resistive temperature micro-sensor, a micro-thermocouple, a thermo-diode and thermo-transistor, and a surface acoustic wave (SAW) temperature sensor; the image sensor comprises a charge coupled device (CCD) or a CMOS image sensor (CIS); the radiation sensor comprises a photoconductive device, a photovoltaic device, a pyro-electrical device, or a micro-antenna; the mechanical sensor comprises a pressure micro-sensor, micro- accelerometer, flow meter, viscosity measurement tool, micro-gyrometer, or micro flow- sensor; the magnetic sensor comprises a magneto-galvanic micro-sensor, a magneto-resistive sensor, a magneto diode, or magneto-transistor; the biochemical sensor comprises a conductimetric device, a bio-marker, a bio-marker attached to a probe structure, or a potentiometric device.

[022] In some other embodiments, at least one sensor is a probing sensor and applies a probing or disturbing signal to the biological subject. [023] In some other embodiments, at least another sensor, different from the probing sensor, is a detection sensor and detects a response from the biological subject upon which the probing or disturbing signal is applied.

[024] The chamber can have a length ranging from 1 micron to 50,000 microns, from 1 micron to 15,000 micron, from 1 micron to 10,000 microns, from 1.5 microns to 5,000 microns, or from 3 microns to 1,000 microns. On the other hand, the chamber can have a width or height ranging from 0.1 micron to 100 microns; from 0.1 micron to 25 microns, from 1 micron to 15 microns, or from 1.2 microns to 10 microns.

[025] In some other embodiments, the apparatus comprises at least four sensors which are located on one side, two opposite sides, or four sides of the interior surface of the chamber. For example, the two sensors in the micro-cylinder can be apart by a distance ranging from 0.1 micron to 500 microns, from 0.1 micron to 50 microns, form 1 micron to 100 microns, from 2.5 microns to 100 microns, or from 5 microns to 250 microns; at least one of the panels comprises at least two sensors that are arranged in at least two arrays each separated by at least a micro sensor in a cylinder; or at least one array of the sensors in the panel comprises two or more sensors.

[026] In some other embodiments, the sorting unit or the detection unit further comprises an application specific integrated circuit chip which is internally bonded to or integrated into one of the panels or a micro-cylinder.

[027] In still some other embodiments, the sorting unit or the detection unit further comprises a memory unit, a logic processing unit, an optical device, imaging device, camera, viewing station, acoustic detector, piezo-electrical detector, piezo-photronic detector, piezo- electro photronic detector, electro-optical detector, electro-thermal detector, bio-electrical detector, bio-marker detector, bio-chemical detector, chemical sensor, thermal detector, ion emission detector, photo-detector, x-ray detector, radiation material detector, electrical detector , or thermal recorder, each of which is integrated into the a panel or a micro cylinder.

[028] In some other embodiments, one signal contains information related to the disease's location or where the disease is present in the source of the biological subject.

[029] In still some other embodiments, one signal contains information related to the occurrence or type of the disease.

[030] The apparatus of this invention is able to detect the presence of at least two different diseases at the same time or to determine the status or progression of a disease.

[031] In another aspect, the present invention provides a method for detecting the presence or progression of a disease in a biological subject, comprising detecting at least two types of information selected from the group consisting of chemical composition, cellular classification, molecular classification and any combination thereof of the biological subject, and analyzing the collected information to determine if the likely presence or progression of the status of the disease with the biological subject. Examples of the disease include cancer, an inflammatory disease, diabetes, lung diseases, liver diseases, gastric diseases, biliary diseases, or a cardiovascular disease. Specifically, the cancer can be breast cancer, lung cancer, esophageal cancer, intestine cancer, cancer related to blood, liver cancer, stomach cancer, cervical cancer, ovarian cancer, rectum cancer, colon cancer, nasopharyngeal cancer, cardiac carcinoma, uterine cancer, oophoroma, pancreatic cancer, prostate cancer, brain tumor, or circulating tumor cells; the inflammatory disease can be acne vulgaris, asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, chronic prostatitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, or tasculitis; the cardiovascular disease can be coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, congenital heart disease, rheumatic heart disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, or renal artery stenosis.

[032] The biological subject can be cells, a sample of an organ or tissue, DNA, RNA, virus, or protein. For example, the cells are circulating tumor cells or cancer cells, e.g., breast cancer, lung cancer, esophageal cancer, cervical cancer, ovarian cancer, rectum cancer, colon cancer, nasopharyngeal cancer, cardiac carcinoma, uterine cancer, oophoroma, pancreatic cancer, prostate cancer, brain tumor, intestine cancer, cancer related to blood, liver cancer, stomach cancer, or circulating tumor cells. In some other embodiments, the biological subject is contained in a media and transported into the first intra-unit channel.

[033] In yet another aspect, the invention provides a method for detecting presence or progression of a disease in a biological subject, which includes testing at least two types of information in the biological subject, with one of the at least two types of information indicating the disease's presence or progression in status and the other type of information indicating the disease's location.

[034] In some embodiments, the two levels of information each comprise protein level information, molecular level information, cellular level information, genetic-level information, or any combination thereof.

[035] In yet another aspect, the invention provides a method for detecting presence or progression of a disease in a biological subject, which comprising measuring at least one parameter correlated to a property at the protein, cellular, molecular, or genetic level.

[036] For instance, the property is a thermal, optical, acoustical, biological, chemical, physical-chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, biophysical, bio-chemical, bio-mechanical, bio-electrical, bio-physical-chemical, bio-electro- physical, bio-electro-mechanical, bio-electro-chemical, bio-chemical-mechanical, bio-electro- physical-chemical, bio-electro-physical-mechanical, bio-electro-chemical-mechanical, physical, an electric, magnetic, electro-magnetic, or mechanical property of the biologic subject. Specifically, for example, the thermal property is temperature or vibrational frequency; the optical property is optical absorption, optical transmission, optical reflection, optical-electrical property, brightness, or fluorescent emission; the radiation property is radiation emission, signal triggered by radioactive material, or information probed by radioactive material; the chemical property is pH value, chemical reaction, bio-chemical reaction, bio-electro-chemical reaction, reaction speed, reaction energy, speed of reaction, oxygen concentration, oxygen consumption rate, ionic strength, catalytic behavior, chemical additives to trigger enhanced signal response, bio-chemical additives to trigger enhanced signal response, biological additives to trigger enhanced signal response, chemicals to enhance detection sensitivity, bio-chemicals to enhance detection sensitivity, biological additives to enhance detection sensitivity, or bonding strength; the physical property is density, shape, volume, or surface area; the electrical property is surface charge, surface potential, resting potential, electrical current, electrical field distribution, surface charge distribution, cell electronic properties, cell surface electronic properties, dynamic changes in electronic properties, dynamic changes in cell electronic properties, dynamic changes in cell surface electronic properties, dynamic changes in surface electronic properties, electronic properties of cell membranes, dynamic changes in electronic properties of membrane surface, dynamic changes in electronic properties of cell membranes, electrical dipole, electrical quadruple, oscillation in electrical signal, electrical current, capacitance, three-dimensional electrical or charge cloud distribution, electrical properties at telomere of DNA and chromosome, capacitance, or impedance; the biological property is surface shape, surface area, surface charge, surface biological property, surface chemical property, pH, electrolyte, ionic strength, resistivity, cell concentration, or biological, electrical, physical or chemical property of solution; the acoustic property is frequency, speed of acoustic waves, acoustic frequency and intensity spectrum distribution, acoustic intensity, acoustical absorption, or acoustical resonance; the mechanical property is internal pressure, hardness, flow rate, viscosity, fluid mechanical properties, shear strength, elongation strength, fracture stress, adhesion, mechanical resonance frequency, elasticity, plasticity, or compressibility.

[037] In some other embodiments, the parameter can be simultaneously correlated to at least two levels of information each independently selected from the group consisting of chemical composition, cellular classification, molecular classification, genetic classification, and any combination thereof.

[038] For example, the parameter is a function of at least two levels of information each independently selected from the group consisting of chemical composition, cellular classification, molecular classification, genetic classification, and any combination thereof.

[039] In some other embodiments, the at least two levels of information interact with each other to amplify the measured parameter of the biological subject.

[040] For instance, the measured parameter can include a property at the protein level, cellular level, molecular level, or genetic level.

[041] In yet still another aspect of this invention is a method for detecting presence or monitoring progression of a disease in a biological subject, comprising testing at least two parameters of the biological subject for at least two different levels of information, processing the at least two different levels of information to result in a new parameter that has a stronger signal intensity than the sum of the signal intensities of the at least two levels of information.

[042] In some embodiments, the at least two levels parameters comprise information selected from the group consisting of chemical composition, cellular classification, molecular classification, and any combination thereof of the biological subject. For example, one testing parameter contains two biological levels of information, and its signal intensity is greater than the sum of the two signal intensities of the testing parameters with each containing one of the two biological levels. For another example, one signal has information related to the disease's location or where the disease is present in the biological subject. For still another example, one signal contains information related to the presence or type of the disease.

[043] The invention also provide a method for detecting presence or monitoring progression of a disease in a biological subject, which comprises tested one parameter containing at least two levels of signal, wherein the tested parameter's signal intensity is greater than the sum of the intensity of the at least two levels of signal.

[044] In some embodiments, the at least two levels of signal comprise information selected from the group consisting of chemical composition, cellular classification, molecular classification, and any combination thereof of the biological subject.

[045] As used herein, the term "or" is meant to include both "and" and "or". It may be interchanged with "and/or."

[046] As used herein, a singular noun is meant to include its plural meaning. For instance, a micro device can mean either a single micro device or multiple micro-devices.

[047] As used herein, the term "patterning" means shaping a material into a certain physical form or pattern, including a plane (in which case "patterning" would also mean

"planarization").

[048] As used herein, the term "a biocompatible material" refers to a material that is intended to interface with a living organism or a living tissue and can function in intimate contact therewith. When used as a coating, it reduces the adverse reaction a living organism or a living tissue has against the material to be coated, e.g., reducing the severity or even eliminating the rejection reaction by the living organism or living tissue. As used herein, it encompasses both synthetic materials and naturally occurring materials. Synthetic materials usually include biocompatible polymers, made either from synthetic or natural starting materials, whereas naturally occurring biocompatible materials include, e.g., proteins or tissues.

[049] As used herein, the term "a biological subject" or "a biological sample" for analysis or test or diagnosis refers to the subject to be analyzed by a disease detection apparatus. It can be a single cell, a single biological molecular (e.g., DNA, RNA, or protein), a single biological subject (e.g., a single cell or virus), any other sufficiently small unit or

fundamental biological composition, or a sample of a subject's organ or tissue that may having a disease or disorder.

[050] As used herein, the term "disease" is interchangeable with the term "disorder" and generally refers to any abnormal microscopic property or condition (e.g., a physical condition) of a biological subject (e.g., a mammal or biological species).

[51] As used herein, the term "subject" generally refers to a mammal, e.g., a human person.

[52] As used herein, the term "microscopic level" refers to the subject being analyzed by the disease detection apparatus of this invention is of a microscopic nature and can be a single cell, a single biological molecular (e.g., DNA, RNA, or protein), a single biological subject (e.g., a single cell or virus), and other sufficiently small unit or fundamental biological composition. [53] As used herein, an "apparatus" or a "micro-device" or "micro device" can be any of a wide range of materials, properties, shapes, and degree of complexity and integration. The term has a general meaning for an application from a single material to a very complex device comprising multiple materials with multiple sub units and multiple functions. The complexity contemplated in the present invention ranges from a very small, single particle with a set of desired properties to a fairly complicated, integrated unit with various functional units contained therein. For example, a simple micro-device could be a single spherical article of manufacture of a diameter as small as 100 angstroms with a desired hardness, a desired surface charge, or a desired organic chemistry absorbed on its surface. A more complex micro device could be a 1 millimeter device with a sensor, a simple calculator, a memory unit, a logic unit, and a cutter all integrated onto it. In the former case, the particle can be formed via a fumed or colloidal precipitation process, while the device with various components integrated onto it can be fabricated using various integrated circuit

manufacturing processes. In some places, a micro-device or micro device represents a sub- equipment unit.

[54] As used herein, the term "parameter" refers to a particular detection target (e.g., a property of microscopic level, physical property such as hardness , viscosity, current, or voltage, or chemical property such as pH value) of the biological subject to be detected , and can include micro-level property.

[55] As used herein, the term "level" refers to chemical composition (including biochemical composition such as protein, genetic materials, e.g., DNA and RNA), cellular classification, or molecular classification of the biological subject to be detected.

[56] As used herein, the term "component" refers a lower division or building block of a level described above. For instance, a protein level can include such components as alpha- feto protein or sugar protein; and the level of a cellular classification can include such components as surface voltage and membrane composition.

[57] As used herein, if not specifically defined, a "channel" or "chamber" can be either an inter-unit channel or an intra-unit channel.

[58] Biological subjects that can be detected by the apparatus include, e.g., blood, urine, saliva, tear, and sweat. The detection results can indicate the possible occurrence or presence of a disease (e.g., one in its early stage) in the biological subject.

[59] As used herein, the term "absorption" typically means a physical bonding between the surface and the material attached to it (absorbed onto it, in this case). On the other hand, the word "adsorption" generally means a stronger, chemical bonding between the two. These properties are very important for the present invention as they can be effectively used for targeted attachment by desired micro devices for measurement at the microscopic level.

[60] As used herein, the term "contact" (as in "the first micro-device contacts a biologic entity") is meant to include both "direct" (or physical) contact and "non-direct" (or indirect or non-physical) contact. When two subjects are in "direct" contact, there is generally no measurable space or distance between the contact points of these two subjects; whereas when they are in "indirect" contact, there is a measurable space or distance between the contact points of these two subjects.

[61] As used herein, the term "probe" or "probing," in addition to its dictionary meaning, could mean applying a signal (e.g., an acoustic, optical, magnetic, chemical, electrical, electro-magnetic, bio-chemical, bio-physical, or thermal signal) to a subject and thereby stimulating the subject and causing it to have some kind of intrinsic response.

[62] As used herein, the term "thermal property" refers to temperature, freezing point, melting point, evaporation temperature, glass transition temperature, or thermal conductivity.

[63] As used herein, the term "optical property" refers to reflection, optical absorption, optical scattering, wave length dependent properties, color, luster, brilliance, scintillation, or dispersion.

[64] As used herein, the term "electrical property" refers to surface charge, surface potential, electrical field, charge distribution, electrical field distribution, resting potential, action potential, or impedance of a biological subject to be analyzed.

[65] As used herein, the term "magnetic property" refers to diamagnetic, paramagnetic, or ferromagnetic.

[66] As used herein, the term "electromagnetic property" refers to property that has both electrical and magnetic dimensions.

[67] As used herein, the term "acoustical property" refers to the characteristics found within a structure that determine the quality of sound in its relevance to hearing. It can generally be measured by the acoustic absorption coefficient. See, e.g., United States Patent No. 3,915,016, for means and methods for determining an acoustical property of a material; T.J. Cox et al, Acoustic Absorbers and Diffusers, 2004, Spon Press.

[68] As used herein, the term "biological property" is meant to generally include chemical and physical properties of a biological subject.

[69] As used herein, the term "chemical property" refers to pH value, ionic strength, or bonding strength within the biological sample. [70] As used herein, the term "physical property" refers to any measurable property the value of which describes a physical system's state at any given moment in time. The physical properties of a biological sample may include, but are not limited to absorption, albedo, area, brittleness, boiling point, capacitance, color, concentration, density, dielectrical, electrical charge, electrical conductivity, electrical impedance, electrical field, electrical potential, emission, flow rate, fluidity, frequency, inductance, intrinsic impedance, intensity, irradiance, luminance, luster, malleability, magnetic field, magnetic flux, mass, melting point, momentum, permeability, permittivity, pressure, radiance, solubility, specific heat, strength, temperature, tension, thermal conductivity, flow rate, velocity, viscosity, volume, surface area, shape, and wave impedance.

[71] As used herein, the term "mechanical property" refers to strength, hardness, flow rate, viscosity, toughness, elasticity, plasticity, brittleness, ductility, shear strength, elongation strength, fracture stress, or adhesion of the biological sample.

[72] As used herein, the term "disturbing signal" has the same meaning as "probing signal" and "stimulating signal."

[73] As used herein, the term "disturbing unit" has the same meaning as "probing unit" and "stimulating unit."

[74] As used herein, the term "conductive material" (or its equivalent "electrical conductor") is a material which contains movable electrical charges. A conductive material can be a metal (e.g., copper, silver, or gold) or non-metallic (e.g., graphite, solutions of salts, plasmas, or conductive polymers). In metallic conductors, such as copper or aluminum, the movable charged particles are electrons (see electrical conduction). Positive charges may also be mobile in the form of atoms in a lattice that are missing electrons (known as holes), or in the form of ions, such as in the electrolyte of a battery.

[75] As used herein, the term "electrically insulating material" (also known as "insulator" or "dielectric") refers to a material that resists the flow of electrical current. An insulating material has atoms with tightly bonded valence electrons. Examples of electrically insulating materials include glass or organic polymers (e.g., rubber, plastics, or Teflon).

[76] As used herein, the term "semiconductor" (also known as "semiconducting material") refers to a material with electrical conductivity due to electron flow (as opposed to ionic conductivity) intermediate in magnitude between that of a conductor and an insulator.

Examples of inorganic semiconductors include silicon, silicon-based materials, and germanium. Examples of organic semiconductors include such aromatic hydrocarbons as the poly cyclic aromatic compounds pentacene, anthracene, and rubrene; and polymeric organic semiconductors such as poly(3-hexylthiophene), poly(p-phenylene vinylene), polyacetylene and its derivatives. Semiconducting materials can be crystalline solids (e.g., silicon), amorphous (e.g., hydrogenated amorphous silicon and mixtures of arsenic, selenium and tellurium in a variety of proportions), or even liquid.

[77] As used herein, the term "biological material" has the same meaning of "biomaterial" as understood by a person skilled in the art. Without limiting its meaning, biological materials or biomaterials can generally be produced either in nature or synthesized in the laboratory using a variety of chemical approaches utilizing organic compounds (e.g., small organic molecules or polymers) or inorganic compounds (e.g., metallic components or ceramics). They generally can be used or adapted for a medical application, and thus comprise whole or part of a living structure or biomedical device which performs, augments, or replaces a natural function. Such functions may be benign, like being used for a heart valve, or may be bioactive with a more interactive functionality such as hydroxyl-apatite coated hip implants. Biomaterials can also be used every day in dental applications, surgery, and drug delivery. For instance, a construct with impregnated pharmaceutical products can be placed into the body, which permits the prolonged release of a drug over an extended period of time. A biomaterial may also be an autograft, allograft, or xenograft which can be used as a transplant material. All these materials that have found applications in other medical or biomedical fields can also be used in the present invention.

[78] As used herein, the term "microelectronic technology or process" generally encompasses the technologies or processes used for fabricating micro-electronic and optical- electronic components. Examples include lithography, etching (e.g., wet etching, dry etching, or vapor etching), oxidation, diffusion, implantation, annealing, film deposition, cleaning, direct-writing, polishing, planarization (e.g., by chemical mechanical polishing), epitaxial growth, metallization, process integration, simulation, or any combinations thereof.

Additional descriptions on microelectronic technologies or processes can be found in, e.g., Jaeger, Introduction to Microelectronic Fabrication, 2 ^nd Ed., Prentice Hall, 2002; Ralph E. Williams, Modem GaAs Processing Methods, 2 ^nd Ed., Artech House, 1990; Robert F. Pierret, Advanced Semiconductor Fundamentals, 2 ^nd Ed., Prentice Hall, 2002; S. Campbell, The Science and Engineering of Microelectronic Fabrication, 2 ^nd Ed., Oxford University Press, 2001, the contents of all of which are incorporated herein by reference in their entireties.

[79] As used herein, the term "selective" as included in, e.g., "patterning material B using a microelectronics process selective to material A", means that the microelectronics process is effective on material B but not on material A, or is substantially more effective on material B than on material B (e.g., resulting in a much higher removal rate on material B than on material A and thus removing much more material B than material A).

[80] As used herein, the term "carbon nano-tube" generally refers to as allotropes of carbon with a cylindrical nanostructure. See, e.g., Carbon Nanotube Science, by P.J.F. Harris, Cambridge University Press, 2009, for more details about carbon nano-tubes.

[81] Through the use of a single micro-device or a combination of micro-devices integrated into a disease detection apparatus, the disease detection capabilities can be significantly improved in terms of sensitivity, specificity, speed, cost, apparatus size, functionality, and ease of use, along with reduced invasiveness and side-effects. A large number of micro-device types capable of measuring a wide range of microscopic properties of biological sample for disease detection can be integrated and fabricated into a single detection apparatus using micro-fabrication technologies and novel process flows disclosed herein. While for the purposes of demonstration and illustration, a few novel, detailed examples have been shown herein on how microelectronics or nano-fabrication techniques and associated process flows can be utilized to fabricate highly sensitive, multi-functional, and miniaturized detection devices, the principle and general approaches of employing microelectronics and nano-fabrication technologies in the design and fabrication of high performance detection devices have been contemplated and taught, which can and should be expanded to various combination of fabrication processes including but not limited to thin film deposition, patterning (lithography and etch), planarization (including chemical mechanical polishing), ion implantation, diffusion, cleaning, various materials, and various process sequences and flows and combinations thereof.

Brief Descriptions of the Figures

[82] Fig. 1 (a) illustrates a set of traditional detection apparatus each of which detects and relies on a single detection technology. Fig. 1 (b) and Fig. (c) are illustration of a detection apparatus of this invention where multiple sub-equipment units are integrated.

[83] Fig. 2 is a schematic illustration of a detection apparatus of this invention which comprises multiple sub-equipment units, a delivery system, and a central control system.

[84] Fig. 3 is a perspective illustration of a detection apparatus of this invention in which a biological sample placed in it or moving through it can be tested.

[85] Fig. 4 illustrates an apparatus of the present invention which comprises two slabs each of which is fabricated with one or more detection or probing units. [86] Fig. 5 is a perspective, cross-sectional illustration of a detection apparatus of this invention with multiple micro-devices placed at a desired distance for time of flight measurements with enhanced sensitivity, specificity, and speed, including time dependent or dynamic information.

[87] Fig. 6 is a perspective illustration of a novel set of microscopic probes, included in a detection apparatus of this invention, for detecting various electronic or magnetic states, configurations, or other properties of a biological sample (e.g., a cell).

[88] Fig. 7 is a perspective illustration of a novel four-point probe, included in a detection apparatus of this invention, for detecting weak electronic signal in a biological sample (e.g., a cell).

[89] Fig. 8 illustrates a fluid delivery system, which is a pretreatment part for the detection apparatus, and it delivers a sample or auxiliary material at a desired pressure and speed into a device.

[90] Fig. 9 illustrates how a micro-device in a disease detection apparatus of this invention can communicate, probe, detect, and optionally treat and modify biological subjects at a microscopic level.

[91] Fig. 10 illustrates another micro-device or sub-equipment that can detect the optical properties of the biological subject with a set of optical sensors.

[92] Fig. 11 illustrates another micro-device or sub-equipment that can separate biological subjects of different geometric size and detect their properties respectively.

[93] Fig. 12 illustrates a micro-device or sub-equipment that can measure the acoustic property of a biological subject.

[94] Fig. 13 illustrates a micro-device or sub-equipment that can measure the internal pressure of a biological subject.

[95] Fig. 14 illustrates a micro-device or sub-equipment that has concaves between the probe couples, in the bottom or ceiling of the channel.

[96] Fig. 15 illustrates another micro-device or sub-equipment that has concaves of a different shape from those illustrated in Fig. 14.

[97] Fig. 16 illustrates a micro-device or sub-equipment that has a stepped channel.

[98] Fig. 17 illustrates a micro-device or sub-equipment that has a set of thermal meters.

[99] Fig. 18 illustrates a micro-device or sub-equipment that includes a carbon nano-tube as the channel with DNA contained therein.

[100] Fig. 19 illustrates a micro-device or sub-equipment that includes a detecting device and an optical sensor. [101] Fig. 20 illustrates an integrated apparatus of this invention that includes a detecting device and a logic circuitry.

[102] Fig. 21 illustrated a micro-device or sub-equipment that includes a detecting device and a filter.

[103] Fig. 22 illustrates how apparatus of this invention can be used to measure a DNA' geometric factors.

[104] Fig. 23 illustrates an apparatus of this invention with a cover atop the trench to form a channel.

[105] Fig. 24 is a diagram of sub-equipment unit for detecting a disease in a biological subject.

[106] Fig. 25 shows an example of a sample filtration unit.

[107] Fig. 26 shows another example of a sample filtration unit.

[108] Fig. 27 is a diagram of a pre-processing unit of an apparatus of this invention.

[109] Fig. 28 is a diagram of an information processing unit of an apparatus of this invention.

[110] Fig. 29 shows the integration of multiple signals which results in cancellation of noise and enhancement of signal to noise ratio.

[Ill] Fig. 30 shows a novel disease detection method in which at least one probe object is launched at a desired speed and direction toward a biological subject, resulting in a collision.

[112] Fig. 31 shows a process of this invention for detecting a biological subject using disease detection apparatus.

[113] Fig. 32 shows another embodiment of disease detection process wherein diseased and healthy biological subjects are separated and the diseased biological subjects are delivered to further test.

[114] Fig. 33 shows an arrayed biological detecting device wherein a series of detecting devices fabricated into an apparatus.

[115] Fig. 34 shows another embodiment of a disease detection device of the current invention including inlet and outlet of the device, the channel where the biological subject passes through, and detection devices aligned along the walls of the channel.

[116] Fig. 35 shows an example of the apparatus of this invention packaged and ready for use.

[117] Fig. 36 shows another example of the apparatus of this invention that is packaged and ready for use. [118] Fig. 37 shows yet another example of the apparatus of this invention that is packaged and ready for use.

[119] Fig. 38 shows an apparatus of this invention that has a channel (trench) and an array of micro sensors.

[120] Fig. 39 shows another apparatus of this invention comprising several "sub-devices."

[121] Fig. 40 shows an example of the apparatus of this invention which includes an application specific integrated circuit (ASIC) chip with I/O pads.

[122] Fig. 41 is a diagram of the underlying principal of the apparatus of this invention which functions by combining various pre-screening and detection methods in unobvious ways.

[123] Fig. 42 shows cross-sectional and outside views of a channel into which a biological subject can flow.

[124] Fig. 43 shows a biological subject to be detected passing through a channel aligned with detectors along its passage in an apparatus of this invention.

[125] Fig. 44 is a view of the apparatus of this invention showing one or two sorting units therein.

[126] Fig. 45 shows an apparatus of this invention with a high number of desired structures fabricated simultaneously on the same chip.

[127] Fig. 46 shows another novel device layout for sorting, screening, separating, probing and detecting diseased biological entities, in which a desired component or multiple components through the middle channel into the middle chamber can play a wide range of roles.

[128] Fig. 47 shows that, compared with multiple stand-alone detection apparatuses, an apparatus of this invention with multiple sub-units of different functions and technologies assembled or integrated has a significantly reduced apparatus volume or size, therefore reduced costs since many common hardware (e.g., a sample handling unit, a sample measurement unit, a data analysis unit, a display, a printer, etc.) can be shared in an integrated apparatus.

[129] Fig. 48 shows that when multiple sub-units with different functions and technologies are assembled into one apparatus, a more diverse functionality, improved detection functionality, sensitivity, detection versatility, and reduced volume and cost can be achieved, where a number of common utilities including, e.g., input hardware, output hardware, sample handling unit, sample measurement unit, data analysis unit and data display unit can be shared. [130] Fig. 49 shows a number of different classifications of biological information are collected in a device and processed in the novel technology.

[131] Fig. 50 shows measured information in this novel technology includes protein, cellular and molecular level information, or combination of them.

[132] Fig. 51 shows signals from different biological classifications may interact, combine, and/or amplify to enhance signal in this novel technology.

[133] Fig. 52 shows detected signal in this novel technology as a function of cancer cell concentration. Signal increases with increasing amount of cancer cells.

[134] Fig. 53shows detected signal in this novel technology as a function of a bio-marker level. Signal increases with increasing level of bio-marker.

[135] Fig. 54 shows Advantage of this novel technology compared with traditional bio- marker (AFP) for liver cancer. Using 58 confirmed liver cancer samples, sensitivity of this novel technology is 79.3%, while that of AFP is 55.9%.

[136] Fig. 55 shows the results of detected signal CDA before and after adding molecular level reaction triggering agent.

[137] Fig. 56 shows the numbers of actual samples tested by this invention and the unexpected results achieved or shown by these tests.

[138] Fig. 57 shows the results of a multi-level detection system of this invention.

[139] Fig. 58 shows the CDA values of the control group, non-cancer disease group and cancer group.

Detailed Description of the Invention

[0140] One aspect of the present invention relates to apparatus for detecting a disease in a biological subject in vivo or in vitro (e.g., human being, an organ, a tissue, or cells in a culture). Each apparatus comprises a delivery system, at least two sub-equipment units, and optionally a central control system. Each sub-equipment is capable of measuring at least a microscopic property of a biological sample. Accordingly, the apparatus of this invention can detect different parameters of the biological subject and provide accuracy, sensitivity, specificity, efficiency, non-invasiveness, practicality, conclusive, and speed in early-stage disease detection at reduced costs. In addition, the apparatus of this invention have some major advantages, such as reducing effective foot print (e.g., defined as function per unit space), reducing space for the medical devices, reducing overall cost, and providing conclusive and effective diagnosis by one device. [0141] The delivery system can be a fluid delivery system. By the constant pressure fluid delivery system, microscopic biological subjects can be delivered onto or into one or more desired sub-equipment units of the apparatus.

[0142] As a key component of the apparatus, the micro-device should include means to perform at least the function of addressing, controlling, forcing, receiving, amplifying, or storing information from each probing address. As an example, the apparatus can further include a central control system for controlling the biological subject matter to be transported to one or more desired sub-equipment units and reading and analyzing a detected data from each sub-equipment unit. The central control system includes a controlling circuitry, an addressing unit, an amplifier circuitry, a logic processing circuitry, a memory unit, an application specific chip, a signal transmitter, a signal receiver, or a sensor.

[0143] In some embodiments, the fluid delivering system comprises a pressure generator, a pressure regulator, a throttle valve, a pressure gauge, and distributing kits. As examples of these embodiments, the pressure generator can include a motor piston system and a bin containing compressed gas; the pressure regulator (which can consist of multiple regulators) can down-regulate or up-regulate the pressure to a desired value; the pressure gauge feeds back the measured value to the throttle valve which then regulates the pressure to approach the target value.

[0144] The biological fluid to be delivered can be a sample of a biological entity to be detected for disease or something not necessarily to be detected for disease. In some embodiments, the fluid to be delivered is liquid (e.g., a blood sample or a lymph sample). The pressure regulator can be a single pressure regulator or multiple pressure regulators which are placed in succession to either down-regulate or up-regulate the pressure to a desired level, particularly when the initial pressure is either too high or too low for a single regulator to adjust to the desired level or a level that is acceptable for an end device or target.

[0145] Optionally, the apparatus includes additional features and structures to deliver a second liquid solution containing at least an enzyme, protein, oxidant, reducing agent, catalyst, radio-active component, optical emitting component, or ionic component. This second liquid solution can be added to the sample to be measured before or during sorting of the biological subject sample to be measured, or before or during the measurement (i.e., detection) of the biological subject sample, for the purposes of further enhancing the apparatus' measurement sensitivity.

[0146] In some other embodiments, the system controller includes a pre-amplifier, a lock-in amplifier, an electrical meter, a thermal meter, a switching matrix, a system bus, a nonvolatile storage device, a random access memory, a processor, or a user interface. The interface can include a sensor which can be a thermal sensor, a flow meter, an optical sensor, an acoustic detector, a current meter, an electrical sensor, a magnetic sensor, an electro-magnetic sensor, a pH meter, a hardness measurement sensor, an imaging device, a camera, a piezo-electrical sensor, a piezo-photronic sensor, a piezo-electro photronic sensor, an electro-optical sensor, an electro-thermal sensor, a bio-electrical sensor, a bio-marker sensor, a bio-chemical sensor, a chemical sensor, an ion emission sensor, a photo-detector, an x-ray sensor, a radiation material sensor, an electrical sensor, a voltage meter, a thermal sensor, a flow meter, or a piezo- meter..

[0147] In still some other embodiments, apparatus of this invention further include a biological interface, a system controller, a system for reclaiming or treatment medical waste. The reclaiming and treatment of medical waste can be performed by the same system or two different systems.

[0148] Another aspect of this invention provides apparatus for interacting with a cell, which include a device for sending a signal to the cell and optionally receiving a response to the signal from the cell.

[0149] In some embodiments, the interaction with the cell can be probing, detecting, sorting, communicating with, treating, or modifying with a coded signal that can be a thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-optical, bio- electro-optical, bio-thermal optical, electro-chemical optical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electrochemical-mechanical, electric, magnetic, electro-magnetic, physical, or mechanical signal, or a combination thereof.

[0150] In some other embodiments, the device or the sub-equipment unit contained in the apparatus can include multiple surfaces coated with one or more elements or combinations of elements, and a control system for releasing the elements. In some instances, the control system can cause release of the elements from the device surface via an energy including but not limited to thermal energy, optical energy, acoustic energy, electrical energy, electromagnetic energy, magnetic energy, radiation energy, or mechanical energy in a controlled manner. The energy can be in the pulsed form at desired frequencies.

[0151] In some other embodiments, the device or the sub-equipment unit contained in the apparatus includes a first component for storing or releasing one element or a combination of elements onto the surface of the cell or into the cell; and a second component for controlling the release of the elements (e.g., a circuitry for controlling the release of the elements). The elements can be a biological component, a chemical compound, ions, catalysts, Ca, C, CI, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, Zn, or a combination thereof. The signal, pulsed or constant, can be in the form of a released element or combination of elements, and it can be carried in a liquid solution, gas, or a combination thereof. In some instances, the signal can be at a frequency ranging from about lxlO ^"4 Hz to about 100 MHz or ranging from about lxlO ^"4 Hz to about 10 Hz, or at an oscillation concentration ranging from about 1.0 nmol/L to about 10.0 mmol/L. Also, the signal comprises the oscillation of a biological component, a chemical compound, Ca, C, CI, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, Zn, or a combination thereof, e.g., at desired oscillating frequencies.

[0152] In some embodiments, the signal to be sent to the cell can be in the form of oscillating element, compound, or an oscillating density of a biological component, and a response to the signal from the cell is in the form of oscillating element, compound, or an oscillating density of a biological component.

[0153] In some embodiments, the device or the sub-equipment unit can be coated with a biological film, e.g., to enhance compatibility between the device and the cell.

[0154] In some other embodiments, the device or the sub-equipment unit can include components for generating a signal to be sent to the cell, receiving a response to the signal from the cell, analyzing the response, processing the response, and interfacing between the device and the cell.

[0155] Still another aspect of this invention provides devices or sub-equipment units each including a micro-filter, a shutter, a cell counter, a selector, a micro-surgical kit, a timer, and a data processing circuitry. The micro-filter can discriminate abnormal cells by a physical property (e.g., dimension, shape, or velocity), mechanical property, electric property, magnetic property, electro-magnetic, thermal property (e.g., temperature), optical property, acoustical property, biological property, chemical property, electro-chemical property, biochemical property, bio-electro-chemical property, bio-electro-mechanical property, or electro-mechanical property. The devices each can also include one or more micro-filters. Each of these micro-filters can be integrated with two cell counters, one of which is installed at the entrance of each filter well, while the other is installed at the exit of each filter well. The shape of the micro-filter's well is rectangle, ellipse, circle, or polygon; and the micro- filter's dimension ranges from about 0.1 μιτι to about 500 μιτι or from about 5 um to about 200 um. As used herein, the term "dimension" means the physical or feature size of the filter opening, e.g., diameter, length, width, or height. The filter can be coated with a biological or bio-compatible film, e.g., to enhance compatibility between the device and the cell. [0156] In addition to separation of biological entity by its size and other physical features, the filter can also contain additional features and functions to perform biological entity separation via other properties, which comprise of mechanical property, electric property, magnetic property, electro-magnetic, thermal property (e.g., temperature), optical property, acoustical property, biological property, chemical property, electro-chemical property, bio-chemical property, bio-electro-chemical property, bio-electro-mechanical property, and electromechanical property.

[0157] In some embodiments of these devices, the shutter sandwiched by two filter membranes can be controlled by a timer (thus time shutter). The timer can be triggered by the cell counter. For instance, when a cell passes through the cell counter of the filter entrance, the clock is triggered to reset the shutter to default position, and moves at a preset speed towards the cell pathway, and the timer records the time as the cell passes through the cell counter at the exit.

[0158] Still a further aspect of this invention provides methods for fabricating a micro-device with micro-trench and probe embedded in the micro-trench's sidewalls. A micro-trench is an unclosed tunnel (see, e.g., Fig. 2(i), 2030), which can be coupled with another upended symmetric trench (see, e.g., Fig. 2(k), 2031) to form a closed channel (see, e.g., Fig. 2(1), 2020). The method may include chemical vapor deposition, physical vapor deposition, or atomic layer deposition to deposit various materials on a substrate; patterning the deposited layer(s) utilizing methods comprising of lithography, etch, and chemical mechanical polishing to form desired features (such as a trench); chemical mechanical planarization for surface planarization; chemical cleaning for particle removal; diffusion or ion implantation for doping elements into specific layers; or thermal anneal to reduce the crystal defects and activate diffused ions. An example of such method includes: depositing a first material onto a substrate; depositing a second material onto the first material and patterning the second material by a microelectronic process (e.g., lithography, etch) to form a detecting tip;

depositing a third material on the second material and then planarize the third material by a polishing process; depositing a fourth material on the third material and patterning the fourth material first by a microelectronic process (e.g., lithography, etch) and then by a

microelectronic process (e.g., another etch) to remove a portion of the third material and optionally a portion of the first material while this etch is typically selective to the second material (lower etch rate for the second material), in which the fourth material serves as a hardmask. A hardmask generally refers to a material (e.g., inorganic dielectric or metallic compound) used in semiconductor processing as an etch mask in lieu of polymer or other organic "soft" materials.

[0159] In some embodiments, the method further includes coupling two devices or sub- equipment units that are thus fabricated and symmetric (i.e., a flipped mirror) to form a detecting device with channels. The entrance of each channel can be optionally bell-mouthed, e.g., such that the size of channel's opening end (the entrance) is larger than the channel's body, thereby making it easier for a cell to enter the channel. The shape of each channel's cross-section can be rectangle, ellipse, circle, or polygon. The micro-trenches of the coupled two micro-devices can be aligned by the module of alignment marks designed on the layout of the micro-device. The dimension of the micro-trench can range from about 0.1 um to about 500 um.

[0160] Alternatively, the method can also include covering the micro-trench of the micro- device with a flat panel. Such a panel can comprise or be made with silicon, SiGe, S1O2, AI2O ₃ , quartz, low optical loss glasses, or other optical materials. Examples of other potentially suitable optical materials include acrylate polymer, AglnSbTe, synthetic alexandrite, arsenic triselenide, arsenic trisulfide, barium fluoride, CR-39, cadmium selenide, caesium cadmium chloride, calcite, calcium fluoride, chalcogenide glass, gallium phosphide, GeSbTe, germanium, germanium dioxide, glass code, hydrogen silsesquioxane, Iceland spar, liquid crystal, lithium fluoride, lumicera, METATOY, magnesium fluoride, agnesium oxide, negative index metamaterials, neutron supermirror, phosphor, picarin, poly(methyl methacrylate), polycarbonate, potassium bromide, sapphire, scotophor, spectralon, speculum metal, split-ring resonator, strontium fluoride, yttrium aluminum garnet, yttrium lithium fluoride, yttrium orthovanadate, ZBLAN, zinc selenide, and zinc sulfide.

[0161] In other embodiments, the method can further include integrating three or more sub- equipment units or devices thus fabricated to yield an enhanced device with an array of the channels.

[0162] Another aspect of this invention relates to a set of novel process flows for fabricating micro-devices (including micro-probes and micro-indentation probes) for their applications in disease detection by measuring microscopic properties of a biological sample. The micro- devices can be integrated into detection apparatus of this invention as sub-equipment units to measure one or more properties at microscopic levels. For example, a cancerous cell may have a different hardness (harder), density (denser), and elasticity than a normal cell.

[0163] Another aspect of this invention is to involve in cellular communications and regulate cellular decision or response (such as differentiation, dedifferentiation, cell division and cell death) with fabricated signals generated by the micro-devices disclosed herein. This could be further employed to detect and treat diseases.

[0164] Another aspect of the current application is that the inventive method or measured parameter in the method is a function of at least two levels F (level 1 , level 2), where level 1 can be a biological entity such as protein and level 2 can be another biological entity such as genetics, where the measured signal strength of F (level 1, level 2) is greater than the sum of the signal containing only level 1 information f (level 1) and the signal containing only level 2 information f (level 2):

Signal strength of F (level 1, level 2) >

signal strength of f (level 1) + signal strength of f (level 2)

[0165] The above novel feature and property can be extended to a measured parameter which is a function containing many levels F (level 1 , level 2, level 3 level n). One novel and unobvious feature of this innovation is that the measured signal in a parameter containing multiple biological levels is synergistically enhanced over the measured signals with each signal containing a single biological level only. With this approach, the typically weak detection signal in disease detection such as cancer detection (especially in early stage cancer detection) can be effectively enhanced or magnified, making early disease detection possible and more effective.

[0166] To further enhance measurement capabilities, multiple micro-devices can be implemented into a piece of detection apparatus as sub-equipment units employing the time of flight technique, in which at least one probing micro-device and one sensing micro-device placed at a preset, known distance. The probing micro-device can apply a signal (e.g., a voltage, a charge, an electrical field, a laser beam, a thermal pulse, a train of ions, or an acoustic wave) to the biological sample to be measured, and the detection (sensing) micro- device can measure response from or of the biological sample after the sample has traveled a known distance and a desired period of time. For instance, a probing micro-device can apply an electrical charge to a cell first, and then a detection (sensing) micro-device subsequently measures the surface charge after a desired period of time (T) has lapsed and the cell has traveled a certain distance (L).

[0167] The micro-devices or the sub-equipment units contained in the apparatus of this invention can have a wide range of designs, structures, functionalities, flexibilities, and applications due to their diverse properties, high degree of flexibilities, and ability of integration, miniaturization, and manufacturing scalability. They include, e.g., a voltage comparator, a four point probe, a calculator, a logic circuitry, a memory unit, a micro cutter, a micro hammer, a micro shield, a micro dye, a micro pin, a micro knife, a micro needle, a micro thread holder, micro tweezers, a micro laser, a micro optical absorber, a micro mirror, a micro wheeler, a micro filter, a micro chopper, a micro shredder, micro pumps, a micro absorber, a micro signal detector, a micro driller, a micro sucker, a micro tester, a micro container, a signal transmitter, a signal generator, a friction sensor, an electrical charge sensor, a temperature sensor, a hardness detector, an acoustic wave generator, an optical wave generator, a heat generator, a micro refrigerator and a charge generator.

[0168] Further, it should be noted that advancements in manufacturing technologies have now made fabrications of a wide range of micro-devices and integration of various functions onto the same device highly feasible and cost effective. The typical human cell size is about 10 microns. Using state-of-the-art integrated circuit fabrication techniques, the minimum feature size defined on a micro-device can be as small as 0.1 micron or below. Thus, it is ideal to utilize the disclosed micro-devices for biological applications.

[0169] In terms of materials for the micro-devices in the apparatus of this invention, the general principle or consideration is the material's compatibility with a biological entity. Since the time in which a micro-device is in contact with a biological sample (e.g., a cell) may vary, depending on its intended application, a different material or a different combination of materials may be used to make the micro-device. In some special cases, the materials may dissolve in a given pH in a controlled manner and thus may be selected as an appropriate material. Other considerations include cost, simplicity, ease of use and practicality. With the significant advancements in micro fabrication technologies such as integrated circuit manufacturing technology, highly integrated devices with minimum feature size as small as 0.1 micron can now be made cost-effectively and commercially. One good example is the design and fabrication of micro electro mechanical devices (MEMS), which now are being used in a wide variety of applications in the electronics industry and beyond.

[0170] Good disease (cancer and non-cancer) detection results in terms of measurement sensitivity and specificity have been obtained on multiple types of cancer tested,

demonstrating validity of the apparatus of this invention for improved ability to detect diseases (e.g., cancers), particularly in their early stages. The experimental results have also shown that multiple cancer types can be detected using the disclosed apparatus, which itself is an improvement over many existing detection apparatus. [0171] Specifically, studies utilizing the apparatus of this invention have been carried out on multiple types of cancer and non-cancer diseases (including an inflammatory disease, diabetes, a lung disease, a heart disease, a liver disease, a gastric disease, a biliary disease, or a cardiovascular disease). In these studies, whole blood samples were used within 5 days after being obtained and/or properly transported/stored in a 0.5-20 °C refrigerated environment. The samples of the control group were obtained from healthy people confirmed by physical examinations with normal AFP and CEA values (in normal ranges).

Table 1. Data from the Test for Lung Diseases

Table 2. Data from Tests for Diabetes

types

Table 3. Data from Tests for Heart Diseases

Table 4. Data from Tests for Liver Diseases

Hepatitis 56 63 24 - 76 54.27 52.50 43.32 43.84 7.74

Table 5. Data from Tests for Gastric Diseases

Table 6. Summary of Descriptive Statistics Biliary

28 57 21 - 85 60.11 60.50 45.75 46.57 11.82

Disease

Table 7. Results of ROC Curve Analysis

[0172] CDA value is obtained from an algorithm using calculation based on tested values from the studies. CDA value increases with risks of diseases. In other words, the higher the CDA values, the higher the risks of diseases.

[0173] As the above tables show, the CDA values are higher for various diseases (mid 40s) than those of control (healthy) group (around 36). Statistical analysis of CDA values for those two groups shows that there was a statistically significant difference in CDA values between those two groups. Accordingly, the studies above show that the apparatus and methods of this invention were able to distinguish some major diseases from control group, with sensitivity and specificity likely higher than existing technologies.

[0174] Set forth below are several illustrations or examples of apparatus of this invention containing a class of innovative micro-devices that are integrated as sub-equipment units.

[0175] Fig. 1 (a) illustrates a set of traditional detection apparatus each of which relies on a single detection technology. As shown in Fig. 1 (a), current diagnosis devices detect a disease on a narrow focus and typically by one single technology (e.g., x-ray machine or NMR machine).

[0176] Fig. 1 (b) and Fig. (c) are an illustration of a detection apparatus of this invention where multiple sub-equipment units are integrated into one piece of apparatus. As a result, the novel apparatus has a smaller size comparing to traditional devices.

[0177] Fig. 2 is a schematic illustration of a detection apparatus of this invention which comprises multiple sub-equipment units, a delivery system, and a central control system. The central control system comprises multiple processing units each of which can be a computer, data analysis unit, or display unit. The central control system is interacted with and used by multiple sub-equipment units. This resource sharing process can effectively reduce cost and size of the apparatus. The biological subject (e.g. a fluid sample) can flows to each sub- equipment units via the delivery system. The delivery system can also transport the biological subject to one or more desired sub-equipments for specific diagnosis purposes.

[0178] To enhance detection speed and sensitivity, a large number of micro-devices can be integrated into a single apparatus of this invention. Each micro-device can be a independent sub-equipment unit in the apparatus. To achieve the above requirements, the detection apparatus should be optimized with its surface area maximized to contact the biological sample and with large number of micro-devices integrated on the maximized surface.

[0179] Instead of measuring a single property of a biological subject for disease diagnosis, various micro-devices can be integrated into a detection apparatus to detect multiple properties. Various micro-devices can constitute different sub-equipment units. Fig. 3 is a perspective, cross-sectional illustration of a disease detection apparatus of this invention 133 with multiple micro-devices 311, 312, 313, 314, and 315, of different detection probes in which a sample 211 such as a blood sample placed in it or moving through it can be tested for multiple properties including but not limited to mechanical properties (e.g., density, hardness and adhesion), thermal properties (e.g., temperature), biological properties, chemical properties (e.g., pH), physical properties, acoustical properties, electrical properties (e.g., surface charge, surface potential, and impedance), magnetic properties, electromagnetic properties, and optical properties.

[0180] As illustrated herein, it is desirable to optimize the detection apparatus design to maximize measurement surface area, since the greater the surface area, the greater number of micro-devices that can be placed on the detection apparatus to simultaneously measure the sample, thereby increasing detection speed and also minimizing the amount of sample needed for the test. [0181] Fig. 4 is a perspective illustration of an apparatus or a sub-equipment unit of this invention. It includes two slabs separated by a narrow spacing with a sample such as a blood sample to be measured placed between the slabs, with multiple micro-devices placed at the inner surfaces of the slabs to measure one or more properties of the sample at microscopic levels.

[0182] Yet another aspect of this invention relates to a set of novel fabrication process flows for making micro-devices or sub-equipment units for disease detection purposes. Thus, a micro-device with two probes capable of measuring a range of properties (including mechanical and electrical properties) of biological samples is fabricated, using the above novel fabrication process flow.

[0183] Detection apparatus integrated with micro-devices disclosed in this application is fully capable of detecting pre-chosen properties on a single cell, a single DNA, a single RNA, or an individual, small sized biological matter level. In another further aspect, the invention provides the design, integration, and fabrication process flow of micro-devices capable of making highly sensitive and advanced measurements on very weak signals in biological systems for disease detection under complicated environment with very weak signal and relatively high noise background. Those novel capabilities using the class of micro-devices disclosed in this invention for disease detection include but not limited to making dynamic measurements, real time measurements (such as time of flight measurements, and combination of using probe signal and detecting response signal), phase lock-in technique to reduce background noise, and 4-point probe techniques to measure very weak signals, and unique and novel probes to measure various electronic, electromagnetic and magnetic properties of biological samples at the single cell (e.g., a telomere of DNA or chromosome), single molecule (e.g., DNA, RNA, or protein), single biological subject (e.g., virus) level.

[0184] For example, in a time of flight approach to obtain dynamic information on the biological sample (e.g., a cell, a substructure of a cell, a DNA, a RNA, or a virus), a first micro-device is first used to send a signal to perturb the biological subject to be diagnosed, and then a second micro-device is employed to accurately measure the response from the biological subject. In one embodiment, the first micro-device and the second micro-device are positioned with a desired or pre-determined distance L apart, with a biological subject to be measured flowing from the first micro-device towards the second micro-device. When the biological subject passes the first micro-device, the first micro-device sends a signal to the passing biological subject, and then the second micro-device detects the response or retention of the perturbation signal on the biological subject. From the distance between the two micro-devices, time interval, the nature of perturbation by the first micro-device, and measured changes on the biological subject during the time of flight, microscopic and dynamic properties of the biological subject can be obtained. In another embodiment, a first micro-device is used to probe the biological subject by applying a signal (e.g., an electronic charge) and the response from the biological subject is detected by a second micro-device as a function of time.

[0185] To further increase detection sensitivity, a novel detection process for disease detection is used, in which time of flight technique is employed. Fig. 5 is a perspective, cross-sectional illustration of detection apparatus 155 with multiple micro-devices 321 and 331 placed at a desired distance 700 for time of flight measurements to attain dynamic information on biological sample 211 (e.g., a cell) with enhanced measurement sensitivity, specificity, and speed. In this time of flight measurement, one or more properties of the biological sample 211 are first measured when the sample 211 passes the first micro-device 321. The same properties are then measured again when the sample 211 passes the second micro-device 331 after it has travelled the distance 700. The change in properties of sample 211 from at micro-device 321 to at micro-device 331 indicates how it reacts with its surrounding environment (e.g., a particular biological environment) during that period. It may also reveal information and provide insight on how its properties evolve with time. Alternatively, in the arrangement shown in Fig. 5, micro-device 321 could be used first as a probe to apply a probe signal (e.g., an electrical charge) to sample 211 as the sample passes the micro-device 321. Subsequently, the response of the sample to the probe signal can be detected by micro-device 331 as the sample passes it (e.g., change in the electrical charge on the sample during the flight). Measurements on biological sample 211 can be done via contact or non-contact measurements. In one embodiment, an array of micro-devices can be deployed at a desired spacing to measure properties of the biological subject over time.

[0186] The utilization of micro-devices (e.g., made by using the fabrication process flows of this invention) as discussed above and illustrated in Fig. 5 can be helpful for detecting a set of new, microscopic properties of a biological sample (e.g., a cell, a cell substructure, or a biological molecule such as DNA or RNA or protein) that have not been considered in existing detection technologies. Such microscopic properties can be thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-chemical- mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio- electro-chemical-mechanical, electrical, magnetic, electromagnetic, physical, or mechanical properties, or a combination thereof, of a biological sample that is a single biological subject (such as a cell, a cell substructure, a biological molecule - e.g., DNA, RNA, or protein - or a sample of a tissue or organ). It is known that biological matters includes from basic bonding such as OH, CO, and CH bonding, to complex, three dimensional structures such as DNA and RNA. Some of them have a unique signature in terms of its electronic configuration. Some of them may have unique thermal, optical, acoustical, biological, chemical, electromechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, electrical, magnetic, electromagnetic, physical, or mechanical properties and configurations, or a combination thereof. Normal biological subj ect and diseased biological subject may carry different signatures with respective to the above said properties. However, none of the above stated parameters or properties have been routinely used as a disease detection property.

Using a disease detection apparatus including one or more apparatus of this invention, those properties can be detected, measured, and utilized as useful signals for disease detection, particularly for early stage detection of serious diseases such as cancer.

[0187] Fig. 6 is a perspective illustration of a novel set of microscopic probes 341, 342, 343,

344, 345, 346, and 347 designed and configured to detect various electronic, magnetic, or electromagnetic states, configurations, or other properties at microscopic level on biological samples 212, 213, 214, and 215, which can be a single cell, DNA, RNA, and tissue or sample. As an example, in terms of measuring electronic properties, the shapes of biological samples 212, 213, 214, and 215 in Fig. 10 may represent electronic monopole (sample 212), dipole (samples 213 and 214), and quadruple (sample 215). The micro-devices 341, 342, 343, 344,

345, 346, and 347 are optimized to maximize measurement sensitivity of those said parameters including but not limited to electronic states, electronic charge, electronic cloud distribution, electrical field, and magnetic and electromagnetic properties, and the micro- devices can be designed and arranged in three dimensional configurations. For some diseases such as cancer, it is likely that electronic states and corresponding electronic properties differ between normal and cancerous cells, DNA, RNA, and tissue. Therefore, by measuring electronic, magnetic and electromagnetic properties at microscopic levels including at cell, DNA, and RNA levels, disease detection sensitivity and specificity can be improved.

[0188] In addition to the above examples in measuring electrical properties (e.g., charge, electronic states, electronic charge, electronic cloud distribution, electrical field, current, and electrical potential, and impedance), mechanical properties (e.g., hardness, density, shear strength, and fracture strength) and chemical properties (e.g., pH) in a single cell, and in Fig. 6 for measuring electrical, magnetic or electromagnetic states or configurations of biological samples at cell and biological molecular (e.g., DNA, RNA, and protein) levels, other micro- devices are disclosed in this application for sensitive electrical measurements.

[0189] Fig. 7 is a perspective illustration of a four-point probe for detecting weak electronic signal in a biological sample such as a cell, where a four point probe 348 is designed to measure electrical properties (impedance and weak electrical current) of a biological sample 216

[0190] One of the key aspects of this invention is the design and fabrication process flows of micro-devices and methods of use the micro-devices for catching and/or measuring biological subjects (e.g., cells, cell substructures, DNA, and RNA) at microscopic levels and in three dimensional space, in which the micro-devices have micro-probes arranged in three dimensional manner with feature sizes as small as a cell, DNA, or RNA, and capable of trapping, sorting, probing, measuring, and modifying biological subjects. Such micro- devices can be fabricated using state-of-the-art microelectronics processing techniques such as those used in fabricating integrated circuits. Using thin film deposition technologies such as molecular epitaxy beam (MEB) and atomic layer deposition (ALD), film thickness as thin as a few monolayers can be achieved (e.g., 4 A to 10 A). Further, using electron beam or x- ray lithography, device feature size on the order of nanometers can be obtained, making micro-device capable of trapping, probing, measuring, and modifying a biological subject (e.g., a single cell, a single DNA or RNA molecule) possible.

[0191] Another aspect of this invention relates to micro-indentation probes and micro-probes for measuring a range of physical properties (such as mechanical properties) of biological subjects. Examples of the mechanical properties include hardness, shear strength, elongation strength, fracture stress, and other properties related to cell membrane which is believed to be a critical component in disease diagnosis.

[0192] Another novel approach provided by this invention is the use of phase lock-in measurement for disease detection, which reduces background noise and effectively enhances signal to noise ratio. Generally, in this measurement approach, a periodic signal is used to probe the biological sample and response coherent to the frequency of this periodic probe signal is detected and amplified, while other signals not coherent to the frequency of the probe signal is filtered out, which thereby effectively reduces background noise. In one of the embodiments in this invention, a probing micro-device can send a periodic probe signal (e.g., a pulsed laser team, a pulsed thermal wave, or an alternating electrical field) to a biological subject, response to the probe signal by the biological subject can be detected by a detecting micro-device. The phase lock-in technique can be used to filter out unwanted noise and enhance the response signal which is synchronized to the frequency of the probe signal. The following two examples illustrate the novel features of time of flight detection arrangement in combination with phase lock-in detection technique to enhance weak signal and therefore detection sensitivity in disease detection measurements.

[0193] Fig. 8 illustrates a fluid delivery system that includes a pressure generator, a pressure regulator, a flow meter, a flow regulator, a throttle valve, a pressure gauge, and distributing kits. The pressure generator 805 sustains fluid with desired pressure, and the pressure is further regulated by the regulator 801 and then accurately manipulated by the throttle valve 802. Meanwhile, the pressure is monitored at real time and fed back to the throttle valve 802 by the pressure gauge 803. The regulated fluid is then in parallel conducted into the multiple devices where a constant pressure is needed to drive the fluid sample.

[0194] Fig. 9 illustrates how a micro-device in a disease detection apparatus of this invention can communicate, probe, detect, and optionally treat and modify biological subjects at a microscopic level. Fig. 9(a) illustrates the sequence of cellular events from signal recognition to cell fates determination. First, as the signals 901 are detected by receptors 902 on the cell surface, the cell will integrate and encode the signals into a biologically comprehensible message, such as calcium oscillation 903. Consequently, corresponding proteins 904 in the cell will interact with the message, then be modified and transform into ion-interacted proteins 905 accordingly. Through the translocation, these modified proteins 905 will pass the carried message to the nuclear proteins, and the controlled modification on nuclear proteins will modulate the expression of gene 907 which includes transcription, translation, epigenetic processes, and chromatin modifications. Through messenger RNA 909, the message is in turn passed to specific proteins 910, thereby changing their concentration - which then determines or regulates a cell's decision or activities, such as differentiation, division, or even death.

[0195] Fig. 9(b) illustrates a micro-device or sub-equipment of this invention which is capable of detecting, communicating with, treating, modifying, or probing a single cell, by a contact or non-contact means. The apparatus is equipped with micro-probes and micro- injectors which are addressed and modulated by the controlling circuitry 920. Each individual micro-injector is supplied with a separate micro-cartridge, which carries designed chemicals or compounds.

[0196] To illustrate how a micro-device can be used to simulate an intracellular signal, calcium oscillation is taken as an example mechanism. First, a Ca ²⁺ -release-activated channel (CRAC) has to be opened to its maximal extent, which could be achieved by various approaches. In an example of the applicable approaches, a biochemical material (e.g., thapsigargin) stored in the cartridge 924 is released by an injector 925 to the cell, and the CRAC will open at the stimulus of the biological subject. In another example of the applicable approaches, the injector 924 forces a specific voltage on cell membrane, which causes the CRAC to open as well.

[0197] The Ca ²⁺ concentration of a solution in the injector 928 can be regulated as it is a desirable combination of a Ca ²⁺ -containing solution 926, and a Ca ²⁺ free solution 927. While the injector 930 contains a Ca ²⁺ free solution, then injectors 928 and 930 are alternately switched on and off at a desired frequency. As such, the Ca ²⁺ oscillation is achieved and the content inside the cell membrane are then exposed to a Ca ²⁺ oscillation. Consequently, the cell's activities or fate is being manipulated by the regulated signal generated by the apparatus.

[0198] Meanwhile, the cell's response (e.g., in the form of a thermal, optical, acoustical, mechanical, electrical, magnetic, electromagnetic property, or a combination thereof) can be monitored and recorded by the probes integrated in this apparatus.

[0199] Fig. 9(c) illustrates another design of a micro-device or sub-equipment which is able to setup communication with a single cell. The apparatus is equipped with micro-probes which are coated with biologically compatible compounds or elements, e.g., Ca, C, CI, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, or Zn. These probes can generate oscillating chemical signals with such an element or compound to interact with the cell, and results into a response that affects the cell's activities or eventual fate as describe above. Likewise, this apparatus can probe and record the cell's response (e.g., in the form of an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electromechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical, mechanical property, or a combination thereof) as well.

[0200] As surface charge will affect the shape of a biological subject, by using novel and multiple plates, information on the shape and charge distribution of biological subjects can be obtained. The general principle and design of the micro-device can be extended to a broader scope, thereby making it possible to obtain other information on the biological subject via separation by applying other parameters such as ion gradient, thermal gradient, optical beam, or another form of energy.

[0201] Fig. 10 illustrates another micro-device or sub-equipment of this invention for detecting or measuring microscopic properties of a biological subject 1010 by utilizing a micro-device that includes a channel, a set of probes 1020, and a set of optical sensors 1032 (see, Fig. 10(a)). The detected signals by probes 1020 can be correlated to information including images collected by the optical sensors 1032 to enhance detection sensitivity and specificity. The optical sensors can be, e.g., a CCD camera, a florescence light detector, a CMOS imaging sensor, or any combination.

[0202] Alternatively, a probe 1020 can be designed to trigger optical emission such as florescence light emission 1043 in the targeted biological subject such as diseased cells, which can then be detected by an optical probe 1032 as illustrated in Fig. 10(c). Specifically, biological subjects can be first treated with a tag solution which can selectively react to diseased cells. Subsequently, upon reacting (contact or non-contact) with probe 1020, optical emissions from diseased cells occur and can be detected by optical sensors 1032. This novel process using the apparatus of this invention is more sensitive than such conventional methods as traditional florescence spectroscopy as the emission trigger point is directly next to the optical probe and the triggered signal 1043 can be recorded in real time and on-site, with minimum loss of signal.

[0203] Fig. 11 illustrates another embodiment of the apparatus of this invention, which can be used to separate biological subjects of different geometric size and detect their properties respectively. It includes at least an entrance channel 1110, a disturbing fluid channel 1120, an accelerating chamber 1130, and two selecting channels 1140 and 1150. The angle between 1120 and 1110 is between 0° and 180°. The biological subject 1101 flows in the x- direction from 1110 to 1130. The biocompatible distribution fluid 1102 flows from 1120 to 1130. Then the fluid 1102 will accelerate 1101 in y-direction. However, the acceleration correlates with the radius of the biological subjects and the larger ones are less accelerated than the small ones. Thus, the larger and smaller subjects are separated into different channels. Meanwhile, probes can be optionally assembled aside the sidewall of 1110, 1120, 1130, 1140, and 1150. They could detect electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, mechanical properties, or combinations thereof at the microscopic level. In the mean time, if desired, a cleaning fluid can also be injected into the system for dissolving and/or cleaning biological residues and deposits (e.g., dried blood and protein) in the narrow and small spaces in the apparatus, and ensuring smooth passage of a biological subject to be tested through the apparatus.

[0204] The channel included in the apparatus of this invention can have a width of, e.g., from 1 nm to 1 mm. The apparatus should have at least one inlet channel and at least two outlet channels. [0205] Fig. 12 shows another micro-device or sub-equipment of this invention with an acoustic detector 1220 for measuring the acoustic property of a biological subject 1201. This device includes a channel 1210, and at least an ultrasonic emitter and an ultrasonic receiver installed along the sidewall of the channel. When the biological subject 1201 passes through the channel 1210, the ultrasonic signal emitted from 1220 will be received after carrying information on 1201 by the receiver 1230. The frequency of the ultrasonic signal can be, e.g., from 2 MHz to 10 GHz, and the trench width of the channel can be, e.g., from 1 nm to 1 mm. The acoustic transducer (i.e., the ultrasonic emitter) can be fabricated using a piezo-electrical material (e.g., quartz, berlinite, gallium, orthophosphate, GaPC>4, tourmalines, ceramics, barium, titanate, BatiCb, lead zirconate, titanate PZT, zinc oxide, aluminum nitride, and polyvinylidene fluorides).

[0206] Fig. 13 shows another apparatus of this invention that includes a pressure detector for biological subject 1301. It includes at least one channel 1310 and whereon at least one piezo- electrical detector 1320. When the biologic subject 1301 passes through the channel, the piezo-electrical detector 1320 will detect the pressure of 1301, transform the information into an electrical signal, and send it out to a signal reader. Likewise, the trench width in the apparatus can be, e.g., from 1 nm to 1 mm, and the piezo-electrical material can be, e.g., quartz, berlinite, gallium, orthophosphate, GaPC>4, tourmalines, ceramics, barium, titanate, BatiC , lead zirconate, titanate PZT, zinc oxide, aluminum nitride, or polyvinylidene fluorides.

[0207] Fig. 14 shows another apparatus of this invention that include a concave groove 1430 between a probe couple, in the bottom or ceiling of the channel. When a biological subject 1410 passes through, the concave 1430 can selectively trap the biological subject with particular geometric characteristics and makes the probing more efficiently. The shape of concave's projection can be rectangle, polygon, ellipse, or circle. The probe could detect electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, mechanical properties, or combinations thereof. Similarly, the trench width can be, e.g., from 1 nm to 1 mm. Fig. 14(a) is an up-down view of this apparatus, Fig. 14(b) is a side view, whereas Fig. 14(c) is a perspective view.

[0208] Fig. 15 is another apparatus of this invention that also includes concave grooves 1530 (of a different shape from those shown in Fig. 14) on the bottom or ceiling of the channel. When a biological subject 1510 passes through, the concave grooves 1530 will generate a turbulent fluidic flow, which can selectively trap the micro-biological subjects with particular geometric characteristics. The probe could detect, e.g., electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, mechanical properties, or a combination thereof. The depth of the concave groove can be, e.g., from 10 nm to 1 mm, and the channel width can be, e.g., from 1 nm to 1 mm.

[0209] Fig. 16 illustrated a micro-device with a stepped channel 1610. When a biological subject 1601 passes through the channel 1610, probe couples of different distances can be used to measure different microscopic properties, or even the same microscopic at different sensitivity at various steps (1620, 1630, 1640) with probe aside each step. This mechanism can be used in the phase lock-in application so that signal for the same microscopic property can be accumulated. The probes can detect or measure microscopic electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, mechanical properties, or combinations thereof.

[0210] Fig. 17 illustrates another apparatus of this invention with thermal meters 1730. It includes a channel, a set of probes 1720, and a set of thermal meters 1730. The thermal meters 1730 can be an infrared sensor, a transistor sub-threshold leakage current tester, or thermister.

[0211] Fig. 18 illustrates a specific apparatus of this invention which includes carbon a nano- tube 1820 with a channel 1810 inside, probes 1840 which can detect at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, or mechanical property, or a combination thereof. The carbon nano-tube 1820 as shown contains a double-helix DNA molecule 1830. The carbon nano-tube can force and sense electrical signals by the probes 1840 aside. The diameter of the carbon nano tube diameter can be, e.g., from 0.5 nm to 50 nm, and its length can range from, e.g., 5 nm to 10 mm.

[0212] Fig. 19 shows an integrated apparatus of this invention that includes a detecting device (shown in Fig. 19(a)) and an optical sensor (shown in Fig. 19(b)) which can be, e.g., a CMOS image sensor (CIS), a Charge-Coupled Device (CCD), a florescence light detector, or another image sensor. The detecting device comprises at least a probe and a channel, and the image device comprises at least 1 pixel. Fig. 19(c-l) and Fig. 19(c-2) illustrate the device with the detecting device and optical sensor integrated. As illustrated in Fig. 19(d), when biological subjects 1901, 1902, 1903 pass through, the probe 1910 in the channel 1920, its electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, mechanical property or a combination thereof could be detected by the probe 1910 (see Fig. 19(e)), meanwhile its image could be synchronously recorded by the optical sensor (Fig. 19(f)). Both the probed signal and image are combined together to provide a diagnosis and enhanced detection sensitivity and specificity. Such a detecting device and an optical sensing device can be designed in a system-on-chip or be packaged into one chip.

[0213] Fig. 20 shows a micro-device or sub-equipment with a detecting micro-device (Fig. 20(a)) and a logic circuitry (Fig 20(b)). The detecting device comprises at least a probe and a channel, and the logic circuitry comprises an addressor, an amplifier, and a RAM. When a biological subject 2001 passes through the channel, its property could be detected by the probe 2030, and the signal can be addressed, analyzed, stored, processed, and plotted in real time. Fig. 20(c-l) and Fig. 20(c-2) illustrate the device with detecting device and Circuitry integrated. Similarly, the detecting device and the integrated circuit can be designed in a System-on-Chip or be packaged into one chip.

[0214] Fig. 21 shows a micro-device or sub-equipment of this invention that comprises a detecting device (Fig. 21(a)) and a filter (Fig. 21(b)). When a biological subject 2101 passes through the device, a filtration is performed in the filter, and irrelevant objects can be removed. The remaining subjects' property can then be detected by the probe device (Fig. 20(a)). The filtration before probing will enhance the precision of the device. The width of the channel can also range, e.g., from 1 nm to 1 mm.

[0215] Fig. 22 shows the geometric factors of DNA 2230 such as spacing in DNA's minor groove (2210) have an impact on spatial distribution of electrostatic properties in the region, which in turn may impact local biochemical or chemical reactions in the segment of this DNA. By probing, measuring, and modifying spatial properties of DNA (such as the spacing of minor groove) using the disclosed detector and probe 2220, one may detect properties such as defect of DNA, predict reaction/process at the segment of the DNA, and repair or manipulate geometric properties and therefore spatial distribution of electrostatic field/charge, impacting biochemical or chemical reaction at the segment of the DNA. For example, tip 2220 can be used to physically increase spacing of minor groove 2210.

[0216] Fig. 23 shows the fabrication process for an apparatus of this invention that has a flat cover atop of trench to form a channel. This will eliminate the need for coupling two trenches to form a channel, which can be tedious for requiring perfect alignment. The cover can be transparent and allow observation with a microscope. It can comprise or be made of silicon, SiGe, S1O2, various types of glass, or AI2O ₃ .

[0217] Fig. 24 is a diagram of an apparatus of this invention for detecting a disease in a biological subject. This apparatus includes a pre-processing unit, a probing and detecting unit, a signal processing, and a disposal processing unit.

[0218] Fig. 25 shows an example of a sample filtration sub-unit in the pre-processing unit, which can separate the cells with different dimensions or sizes. This device comprises at least one entrance channel 2510, one disturbing fluid channel 2520, one accelerating chamber 2530, and two selecting channels (2540 and 2550). The angle 2560 between 2520 and 2510 ranges from 0° to 180°.

[0219] The biological subject 2501 flows in the x direction from the entrance channel 2510 to the accelerating chamber 2530. A bio-compatible fluid 2502 flows from disturbing fluid channel 2520 to the accelerating chamber 2530, it then accelerates the biological subject 2501 in the y-direction. The acceleration correlates with the radius of the biological subject and the larger ones are less accelerated than the smaller ones. Then, the larger and smaller subjects are separated into different selecting channels. Meanwhile, probes can be optionally assembled on the sidewalls of the channels 2510, 2520, 2530, 2540, and 2550. The probes could detect, at the microscopic level, electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, biochemical, electro-mechanical, electro-chemical, electrochemical-mechanical, physical, mechanical properties, or combinations thereof.

[0220] Fig. 26 is a diagram of another example of a sample filtration unit in the apparatus of this invention. 2601 represents small cells, while 2602 represents large cells. When a valve 2604 is open and another valve 2603 is closed, biological subjects (2601 and 2602) flow towards exit A. Large cells that have larger size than the filtration hole are blocked against exit A, while small cells are flushed out through exit A. The entrance valve 2604 and exit A valve 2607 are then closed, and a bio-compatible fluid is injected through the fluid entrance valve 2606. The fluid carries big cells are flushed out from exit B. The larger cells are then analyzed and detected in the detection part of the invention.

[0221] Fig. 27 is a diagram of a pre-processing unit of an apparatus of this invention. This unit includes a sample filtration unit, a recharging unit or system for recharging nutrient or gas into the biological subject, a constant pressure delivery unit, and a sample pre-probing disturbing unit.

[0222] Fig. 28 is a diagram of an information or signal processing unit of an apparatus of this invention. This unit includes an amplifier (such as a lock-in amplifier) for amplifying the signal, an A/D converter, and a micro-computer (e.g., a device containing a computer chip or information processing sub-device), a manipulator, a display, and network connections.

[0223] Fig. 29 shows the integration of multiple signals which results in cancellation of noise and enhancement of signal/noise ratio. In this Figure, a biological 2901 is tested by Probe 1 during Δ t between tl and t2, and by Probe 2 during Δ t between t3 and t4. 2902 is 2901's tested signal from Probe 1, and 2903 is from Probe 2. Signal 2904 is the integration result from signal 2902 and 2903. The noise cancels out each other in certain extent and results in an improved signal strength or signal/noise ratio. The same principle can be applied to data collected from more than more than 2 micro-devices or probing units.

[0224] Fig. 30 shows a novel disease detection method of this invention in which at least one probe object is launched at a desired speed and direction toward a biological subject, resulting in a collision. The response(s) by the biological subject during and/or after the collision is detected and recorded, which can provide detailed and microscopic information on the biological subject such as weight, density, elasticity, rigidity, structure, bonding (between different components in the biological subject), electrical properties such as electrical charge, magnetic properties, structural information, and surface properties. For example, for a same type of cell, it is expected that a cancerous cell will experience a smaller traveling distance after the collision than that of a normal cell due to its denser, greater weight, and possibly larger volume. As shown in Fig. 30(a), a probe object 3011 is launched towards a biological subject 3022. After the collision with the probe object 3011, the biological subject 3022 may be pushed (scattered) out a distance depending on its properties as shown Fig. 30(b).

[0225] Fig. 30(c) shows a schematic of a novel disease detection device with a probe object launch chamber 3044, an array of detectors 3033, a probe object 3022 and a biological subject to be tested 3011. In general, a test obj ect can be an inorganic particle, an organic particle, a composite particle, or a biological subject itself. The launch chamber comprises a piston to launch the object, a control system interfaced to an electronic circuit or a computer for instructions, and a channel to direct the object.

[0226] Fig. 31 illustrates a method for detecting a disease in a biological subject. A biological subject 3101 passes through the channel 3131 at a speed v, and probe 3111 is a probe which can grossly detect the properties of the biological subject at high speed.

[0227] Probe 3112 is a fine probing device which is coated by a piezo-electrical material. There is a distance AL between probe 3111 and probe 3112.

[0228] When the biological subjects are tested when getting through 3111, if the entity is identified to be a suspected abnormal one, the system would trigger the piezo-electrical probe 3112 to stretch into the channel and probe particular properties after a time delay of At. And probe 3112 retracts after the suspected entity passed through.

[0229] The probing device is capable of measuring at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electro- mechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical or mechanical property, or a combination thereof, of the biological subject.

[0230] The width of the micro-channel can range from about 1 nm to about 1 mm.

[0231] Fig. 32 shows a process of detecting a disease in a biological subject. A biological subject 3201 passes through the channel 3231 at a speed v. Probe 3211 is a probe which can grossly detect the properties of the biological subject at high speed. 3221 and 3222 are piezo- electrical valves to control the micro-channel 3231 and 3232. 3212 is a fine probing device which can probe biological properties more particularly. 3231 is flush channel to rush out normal biological subjects. 3232 is detection channel where the suspected entities are fine detected in this channel.

[0232] When a biological subject is tested while getting through 3211, if it is normal, the valve 3221 of the flush channel is open, while the detection channel valve 3222 is closed, the biological subject is flushed out without a time-consuming fine detection.

[0233] When the biological subject is tested while getting through 3211, if it is suspected to be abnormal or diseased, the valve 3221 of the flush channel is closed, while the detection channel valve 3222 is open, the biological subject is conducted to the detection channel for a more particular probing.

[0234] The width of the micro-channel can range from about 1 nm to about 1 mm.

[0235] The probing device is capable of measuring at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electromechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical or mechanical property, or a combination thereof, of the biological subject.

[0236] Fig. 33 illustrates an arrayed biological detecting device. As shown in Fig. 33(a),

3301 are arrayed micro-channels which can get through the fluidics and biological subjects.

3302 are probing devices embedded aside the channels. The sensors are wired by bit-lines 3321 and word-lines 3322. The signals are applied and collected by the decoder RArow-select 3342 and decoder column select 3341. As illustrated in Fig. 33(b), the micro-channel arrayed biological detecting device 3300 can be embedded in a macro-channel 3301. The micro- channel's dimension ranges from about 1 um to about 1 mm. The shape of the micro-channel can be rectangle, ellipse, circle, or polygon.

[0237] The probing device is capable of measuring at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electro- mechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical or mechanical property, or a combination thereof, of the biological subject.

[0238] Fig. 34 illustrates a device of the current invention for disease detection. 3401 is inlet of the detecting device, and 3402 is the outlet of the device. 3420 is the channel where the biological subjects pass through. 3411 is the optical component of the detecting device.

[0239] As illustrated in Fig. 34(b), the optical component 3411 consists of an optical emitter 3412 and an optical receiver 3413. The optical emitter emits an optical pulse (e.g. laser beam pulse), when the biological subject 3401 passing through the optical component, and the optical sensor detects the diffraction of the optical pulse, then identify the morphology of the entity.

[0240] Fig. 35 shows an example of the apparatus of this invention packaged and ready for integration with a sample delivery system and data recording device. As illustrated in Fig. 35(a), the device 3501 is fabricated by micro-electronics processes described herein and has at least a micro-trench 3511, a probe 3522, and a bonding pad 3521. The surface of the device's top layer can include SixOyNz, Si, SixOy, SixNy, or a compound containing the elements of Si, O, and N. Component 3502 is a flat glass panel. In Fig. 35(b), the flat panel 3502 is shown to be bonded with micro-device 3501 on the side of micro-trench. The bonding can be achieved by a chemical, thermal, physical, optical, acoustical, or electrical means, or any combination thereof. Fig. 35(c) shows a conductive wire being bonded with the bonding pad from the side of the pads. As illustrated in Fig. 35(d), the device 3501 is then packaged in a plastic cube with only conducting wires exposed. In Fig. 35(e), a conical channel 3520 is carved through packaging material and connecting the internal channel of the device. As illustrated in Fig. 35(f), the larger opening mouth of the conical channel makes it operational and convenient to mount a sample delivery injector with the device, thereby better enabling the delivery of sample from an injector with relatively large size of injector needle into device with relatively small channels.

[0241] Fig. 36 shows another example of the apparatus of this invention packaged and ready for integration with a sample delivery system and data recording device. As shown in Fig. 36(a), a micro-device 3600 is fabricated by one or more micro-electronics processes as described in International Application No. PCT/US2011/042637, entitled "Apparatus for Disease Detection." The micro-device 3600 has at least a micro-trench 3604, a probe 3603, a connecting port 3602, and a bonding pad 3605. On the top of the micro-device 3600, the surface layer comprises SixOyNz, Si, SixOy, SixNy, or a compound consisting of Si, O, and N. The surface layer can be covered, and thus the micro-device 3600 is mounted, with a flat glass panel 3601. See Fig. 36(b). The mounting can be by a chemical, thermal, physical, optical, acoustical, or electrical means. As shown in Fig. 36(c), the conductive wire is bonded with bonding pad from the side of the pads. Fig. 36(d) illustrates that the micro- device 3600 can then be packaged in a cube with only conducting wires exposed. The packaging cube can comprise a packaging material such as plastic, ceramic, metal, glass, or quartz. As shown in Fig. 36(e), a tunnel 3641 is then drilled into the cube until the tunnel reaches the connecting port 3602. Further, as shown in Fig. 36(f), the tunnel 3641 is then being connected to other pipes which can delivery a sample to be tested into the micro-device 3600, and flush out the sample after the sample is tested.

[0242] Fig. 37 shows yet another example of the apparatus of this invention packaged and ready for integration with a sample delivery system and data recording device. As illustrated in Fig. 37(a), device 3700 is a micro-fluidic device which has at least one micro-channel 3701. 3703 is a pipe that conducts a fluidic sample. The micro-channel 3701 and the conducting pipe 3703 are aligned and submerged in a liquid, for example, water. Fig. 37(b) illustrates that, when the temperature of the liquid in which the micro-device and conducting pipe are submerged, is decreased to its freezing point or lower, the liquid solidifies into a solid 3704. As illustrated in Fig. 37(c), while the temperature of the liquid is maintained below the freezing point, the combination (including the solid 3704, the conducting pipe 3703, and the device 3700) is enclosed into a packaging material 3705 whose melting temperature is higher than that of the solid 3704, with only the conducting pipe exposed. Fig. 37(d) shows that, after the temperature is increased above the melting point of the solid 3704, the solid material 3704 melts and becomes a liquid and is then exhausted from the conducting pipe 3703. The space 3706 wherein the solid material 3704 once filled is now available or empty, and the channel 3701 and the conducting pipe 3703 are now connected through and sealed in the space 3706.

[0243] Fig. 38 shows an apparatus of this invention that has a channel (trench) and an array of micro sensors. In Fig. 38(a), 3810 is a device fabricated by microelectronics techniques; 3810 comprises micro-sensor array 3801 and addressing and read-out circuitry 3802. The micro-sensor array can include thermal sensors, piezo-electrical sensors, piezo-photronic sensors, piezo-optical electronic sensors, image sensors, optical sensors, radiation sensors, mechanical sensors, magnetic sensors, bio-sensors, chemical sensors, bio-chemical sensors, acoustic sensors, or a combination of them. Examples of thermal sensors include resistive temperature micro-sensors, micro-thermocouples, thermo-diodes and thermo-transistors, and SAW (surface acoustic wave) temperature sensor. Examples of image sensors include CCD (Charge Coupled Device) and CIS (CMOS image sensor). Examples of radiation sensors include photoconductive devices, photovoltaic devices, pyro-electrical devices, and micro- antennas. Examples of mechanical sensors include pressure micro-sensors, micro- accelerometers, micro-gyrometers, and micro flow-sensors. Examples of magnetic sensors include magneto-galvanic micro-sensors, magneto-resistive sensors, magneto diodes and magneto-transistors. Examples of biochemical sensors comprise conductimetric devices and potentiometric devices. Fig. 38(b) shows a micro-device 3820 that includes a micro-trench 3821. As illustrated in Fig. 38(c), 3810 and 3820 are bonded together to form the new micro- device 3830 which include a trench or channel 3831. The micro-sensor array 3801 is exposed in the channel 3831.

[0244] Fig. 39 shows another apparatus of this invention comprising several "sub-devices." Particularly, as illustrated in Fig. 39(a), the device 3910 composes "sub-devices" 3911, 3912, 3913, and 3914, among which 3911 and 3913 are devices which can apply disturbing signals, and 3912 and 3914 are micro-sensor arrays. Fig. 39(b) illustrates the functioning diagram of the device 3910, when biological samples 3921 under the test are passing through the channel 3910, they are disturbed by signal A applied by 3911, then being tested and recorded by detecting sensor array 1 of 3912. These biological samples are then disturbed by disturb probe 3913 of array 2, and being tested by detecting sensor 3914 of array 2. Disturbing probe 3911 of array 1 and disturbing probe 3913 of array 2 can apply the same or different signals. Likewise, detecting sensor 3912 of array 1 and detecting sensor 3914 of array 2 can sense or detect the same or different properties.

[0245] Fig. 40 shows an example of the apparatus of this invention which includes an application specific integrated circuit (ASIC) chip with I/O pads. Specifically, as illustrated in Fig. 40, 4010 is a micro-device with a micro-fluidic channel 4012 and I/O pads 4011. 4020 is an Application Specific Integrated Circuit (ASIC) chip with I/O pads 4021. 4020 and 4010 can be wired together through the bonding of I/O pads. As such, with an ASIC circuitry 4020, the micro-fluidic detecting device 4010 can perform more complicated computing and analytical functions.

[0246] Fig. 41 is a diagram of the underlying principal of the apparatus of this invention which functions by combining various pre-screening and detection methods in unobvious ways. In Fig. 41(a), a biological subject is first pre-screened for diseased biological entities, and then the diseased biological entities are separated from the normal (healthy or non- diseased) biological entities. The biological subject containing the diseased biological entities separated from the normal biological entities is detected using a desired disease detection method. In Fig. 41(b), a biological sample has gone through multiple, successive cell separation steps to concentrate diseased cells (or biological entities). In Fig. 41(c), after pre-screening to concentrate diseased biological entities, bio-marker is used to detect diseased biological entities. In Fig. 41 (d), bio-marker is first used to separate out diseased biological entities and then the sorted out, diseased biological entities are further detected by various detection methods. In short, this process includes initial screening, initial separation, further screening, further separation, probing with one or more disturbing signals or disturbing parameters (e.g., physical, mechanical, chemical, biological, bio-chemical, bio-physical, optical, thermal, acoustical, electrical, electro-mechanical, piezo-electrical, micro-electromechanical, or a combination thereof), and finally detection. This sequence can repeat one or more times. The effect of this process is concentrating the diseased entities for improved detection sensitivity and specificity, particularly for a biological subject with a very low concentration of diseased entities, such as circulating tumor cell (CTC).

[0247] In Fig. 41 (e) through Fig. 41(g), a set of novel processes include (a) pre-screening, pre-separation and initial separation for diseased biological entities, (b) further separation of diseased biological entities, (c) optionally carry out initial detection, and (d) detection using various processes and detection methods. In the pre-separation process, one of the embodiments utilizes nano-particles or nano- magnetic particles attached with bio-markers to sort out diseased biological entities. During pre-separation process, the diseased biological entities are concentrated for higher concentration, which will make further separation and/or following detection easier. The biological sample following pre-separation process can go through further separation process to further enhance the concentration of diseased biological entities. Finally, the biological sample gone through the pre-separation and follow-up separation steps will go through detection step(s), in which various detection techniques and processes can be used to determine diseased biological entities and their types. In some embodiments, multiple detection steps can be utilized to detect diseased biological entities.

[0248] Fig. 42(a) shows a cross-sectional view of a channel (4211) into which a biological subject can flow. Fig. 42(b) shows an outside view of the channel, along which an array of detectors (4222) are installed along the path of the flow of the biological subject.

Alternatively, both probes and detectors can be installed to both disturb the biological subject to be detected and detect response signals from such disturb signals. Fig. 42(c) shows a cross-section of the wall of the channel, where detectors (4222) are mounted through to contact the biological subject to be detected and also are making contact with the outside world (e.g., to connect to a detection circuitry).

[0249] Fig. 43(a) shows a biological subject (4333) to be detected passing through a channel (4311) aligned with detectors (4322) along its passage. The detectors can be the same type of detectors, or a combination of various detectors. Further, probes capable of sending out probing or disturbing signals to the biological subject to be detected can also be implemented along the channels, along with detectors which can detect response from the biological subject which has been probed or disturbed by the probe. The detected signals can be acoustical, electrical, optical (e.g., imaging), biological, bio-chemical, bio-physical, mechanical, bio-mechanical, electro-magnetic, electro-mechanical, electro-chemical- mechanical, electro-chemical-physical, thermal, and thermal-mechanical property related signals, or a combination of them. Fig. 43(b) shows an example of a set of detected signals (e.g., images, pressures, or electrical voltages) (4344) along the path of the biological subject, which recorded its behavior and properties as it passes through the channel. For example, for an optical detector, the size of the circle shown in the Fig. 43(b) could mean the optical emission from the biological subject (such as an optical emission from a florescence component attached to the biological subject), the strength of a strain (pressure) acting on the side wall of the channel detected by a piezo-electric detector or a piezo-photronic detector, or thermal emission from the biological subject detected by a thermal detector or an IR sensor. Such detected signals can be solely from the biological subject as it passes through the channel, or responses from the biological subject to a disturbing or probing signal by the probe.

[0250] Like Fig. 43(b), Fig. 43(c) through Fig. 43(e) show additional examples of various detected signal patterns (4344) as the biological subject passes through the channel and is detected by the novel detectors and processes disclosed in the application.

[0251] To effectively sorting, separating, screening, probing, or detecting of diseased biological entities, a chamber (or chambers) integrated with various channels can be deployed as shown Fig. 44(a), where incoming sample flowing into a chamber (4411) first. In the chamber, various techniques such as bio-markers and nano-technology (magnetic beads or nano-particles with bio-markers attached to them) based processes can be used to sort out, screen, and separate out the diseased biological entities. For example, a biological sample flowing from the left into the chamber can have its diseased entities separated out in the chamber, and passed downward through the bottom channel, while its normal entities can continue to flow from the chamber in the right hand direction, through the channel in the right side of the chamber. Depending upon the design, the diseased entities, having entered into the chamber on the left, can also be separated out in the chamber, and continue on towards right and flow into the channel on the right side of the chamber, while normal entities will continue to flow down toward and through the channel at the bottom of the chamber. Fig. 44(b) shows multiple chambers integrated with channels in which biological entities can be sorted, screened, separated, probed or detected. In the application of screening and separation, the multiple chambers can carry out multiple screening and separation steps. As shown in Fig. 44(b), for a biological sample flowing from the left toward the right direction, it will enter into the first chamber on the left (4433) and under go a first screening and separation. The biological sample can continue to flow towards the right, enter into the second chamber, the chamber on the right (4444), and undergo a second screening and further separation. In this way, through a multi-staged screening and separation process, the concentration of a diseased entity can be successively enhanced which can be helpful for a sensitive final or late stage detection. This type of device design and process could be very useful for defection of a biological sample with an initially very low concentration of diseased entity population, such as for the detection of circulating tumor cell (CTC) which is typically in the concentration of one part in one billion cells or 10 billion cells.

[0252] To significantly speed up the sorting, screening, probing and detection operations using the disclosed device and process, a high number of desired structures such as those discussed in Fig. 45 can be fabricated simultaneously on the same chip as shown in Fig. 45.

[0253] Fig. 46 shows another novel device layout for sorting, screening, separating, probing and detecting diseased biological entities, in which a desired component or multiple components through the middle channel into the middle chamber 4611 can play a wide range of roles. For example, the component flowing into the middle chamber could be a bio- marker which can be freshly added into the top chamber 4622 and bottom chamber 4633 when its (bio-marker) concentration needs to be adjusted. The timing, flow rate, and amount of component in the middle chamber 4611 need to be added into the top and bottom chambers (4622 and 4633) can be pre-programmed or controlled via a computer or software in real time. The component into the middle chamber 4612 could also be nano-particles or magnetic beads attached to bio-markers. In another novel embodiment, the component into the middle chamber 4611 could be a disturbing agent which will disturb the biological subject or samples to be detected in the top and bottom chambers.

[0254] Fig. 47 shows that, compared with multiple stand alone detection apparatuses (see Fig. 47(a), 4711, 4722, 4733, and 4744), an apparatus (4755) with multiple sub-units of different functions and technologies (4766) assembled or integrated has a significantly reduced apparatus volume or size (see Fig. 47(b)), therefore reduced costs since many common hardware (e.g., a sample handling unit, a sample measurement unit, a data analysis unit, a display, a printer, etc.) can be shared in an integrated apparatus. For example, such a multifunctional, integrated apparatus can include a bio-marker detector, an imaging based detector, a photo-detector, an x-ray detector, a nuclear magnetic resonance imaging detector, an electrical detector, and an acoustic detector all of which are assembled and integrated into the single apparatus, so that the apparatus can have improved detection functionality, sensitivity, detection versatility, and reduced volume and cost.

[0255] Fig. 48 shows that when multiple sub-units with different functions and technologies (2055) are assembled into one apparatus, a more diverse functionality, improved detection functionality, sensitivity, detection versatility, and reduced volume and cost can be achieved, where a number of common utilities including, e.g., input hardware, output hardware, sample handling unit, sample measurement unit, data analysis unit and data display unit (4811, 4833, and 4844) can be shared. For example, when a range of detection units utilizing various detection technologies are assembled into one apparatus, many functions and hardware such as sample handling unit, sample measurement unit, data transmission unit, data analysis unit, computer, and display unit can be shared, thereby significantly reducing the apparatus' equipment volume or size, costs, and complexity while improving measurement functionality and sensitivity.

[0256] Tests were carried out in the laboratory with the apparatus of this invention on certain cancerous tissue samples (with multiple samples for each type of cancer) although the apparatus of this invention can be used for detection of other types of cancer or other types of treatment. In the tests, healthy control samples were obtained from animals with no known cancer disease at the time of collection and no history of malignant disease. Both cancerous samples and healthy control samples were collected and cultured in the same type of culture solution. The cultured samples were then mixed with a dilution buffer and diluted to the same concentration. The diluted samples were maintained at the room temperature for different time intervals and processed within a maximum of 6 hours after being recovered. The diluted samples were tested at the room temperature (20-23 °C) and in the humidity of 30%~40%. The samples were tested with an apparatus of this invention under the same conditions and stimulated by the same pulse signal.

[0257] The tests show that, in general, the control groups' tested (measured) values (i.e., measured values in relative units for the testing parameter) were lower than the cancerous or diseased groups. Under the same stimulation (in terms of stimulation type and level) with a stimulating or probing signal applied by a probing unit of the tested apparatus of this invention, the difference shown in the measured values between the control groups and the cancerous groups became much more significant, e.g., ranging from 1.5 times to almost 8 times in terms of level of increase in such difference, compared with that without simulation. In other words, the cancerous groups' response to the stimulating signal was much higher than that of the control groups. Thus, the apparatus of this invention have been proven to be able to significantly enhance the relative sensitivity and specificity in the detection and measurement of diseased cells, in comparison to the control or healthy cells.

[0258] Further, the test results show that in terms of the novel parameter utilized by the apparatus of this invention, the cancerous group and the control group showed significantly different response. Such difference is significantly greater than the measurement noise. There was a large window to separate the control groups from the cancerous groups, showing a high degree of sensitivity of the novel measurement method and apparatus.

[0259] Compared with the traditional technology, signal and information collected by the apparatus and methods of this invention is linearly and can even be non-linearly amplified; and additional two-factor and three-factor (or higher order) interactions between various levels (cellular, protein, molecular or other levels) and components/parameters (exemplified in the following table) are not just novel, unique, but also exhibited unexpected reliable and sensitive results when compared to the traditional technology.

Traditional technology This Invention

P - protein based ffoio-raarker, AFP, CEA, PSA, etc.) P

C - cellular based (CFC, tiDNA) C

IWl - Molecular (genomics, DMA, R iA) M

+

=> one-diraensionai mforroatioii P-C

P M

+

M-C

=> seven dimensional Info -C-P

Other ievei/pararneter {Of + -C-P-O

= more dimensional info

[0260] Studies were also undertaken to examine the effect of adding molecular level reaction triggering agent on the efficacy of the apparatus and methods for detecting disease of this invention. The results provided in Fig. 55 show that the difference in signal between the control (healthy) group and cancer group was increased, indicating the detection system did detect molecular level information.

[0261] The apparatus and methods of this invention has been used in test of more than 20 different types of cancer in all stages of development and showed expectedly high sensitivity and specificity. Fig. 56 shows that to validate the usefulness and sensitivity of this invention, over 60,000 samples were collected, with 30,000 samples in retrospective investigation, and 30,000 samples in general screening, and remarkable sensitivity and selectivity of this invention was demonstrated from testing those samples.

[0262] Fig. 57 shows in a multi-level detection system of this invention, one biological level (for example, protein) can interact with another biological level(s) (such as genetic level), resulting in synergistic reactions and resultant amplification in signal.

[0263] Fig. 58 shows the CDA values of the control group, non-cancer disease group and cancer group. As detected by the apparatus and methods of this invention, the cancer group always has a higher CDA value than that of a non-cancer disease group, and this difference in CDA value between the cancer group and non-cancer disease group is statistically significant particularly for monitoring the progression of a disease state, e.g., from an inflammatory disease to a pre-cancer condition to a malignant cancer or tumor and then to a late stage cancer. In other words, CDA values can be used in a disease and cancer-differentiating analysis with the help of the apparatus and methods of this invention.

[0264] While for the purposes of demonstration and illustration, the above cited novel, detailed examples show how microelectronics and/or nano-fabrication techniques and associated process flows can be utilized to fabricate highly sensitive, multi-functional, powerful, and miniaturized detection devices, the principle and general approaches of employing microelectronics and nano-fabrication technologies in the design and fabrication of high performance detection devices have been contemplated and taught, which can and should be expanded to various combination of fabrication processes including but not limited to thin film deposition, patterning (lithography and etch), planarization (including chemical mechanical polishing), ion implantation, diffusion, cleaning, various materials, combination of processes and steps, and various process sequences and flows. For example, in alternative detection device design and fabrication process flows, the number of materials involved can be fewer than or exceed four materials (which have been utilized in the above example), and the number of process steps can be fewer or more than those demonstrated process sequences, depending on specific needs and performance targets. For example, in some disease detection applications, a fifth material such as a biomaterial-based thin film can be used to coat a metal detection tip to enhance contact between the detection tip and a biological subject being measured, thereby improving measurement sensitivity.

[0265] Applications for the detection apparatus and methods of this invention include detection of diseases (e.g., in their early stage), particularly for serious diseases like cancer. Since cancer cell and normal cell differ in a number of ways including differences in possible microscopic properties such as electrical potential, surface charge, density, adhesion, and pH, novel micro-devices disclosed herein are capable of detecting these differences and therefore applicable for enhanced capability to detect diseases (e.g., for cancer), particularly in their early stage. In addition micro-devices for measuring electrical potential and electrical charge parameters, micro-devices capable of carrying out mechanical property measurements (e.g., density) can also be fabricated and used as disclosed herein. In mechanical property measurement for early stage disease detection, the focus will be on the mechanical properties that likely differentiate disease or cancerous cells from normal cell. As an example, one can differentiate cancerous cells from normal cells by using a detection apparatus of this invention that is integrated with micro-devices capable of carrying out micro-indentation measurements.

[0266] Although specific embodiments of this invention have been illustrated herein, it will be appreciated by those skilled in the art that any modifications and variations can be made without departing from the spirit of the invention. The examples and illustrations above are not intended to limit the scope of this invention. Any combination of detection apparatus, micro-devices, fabrication processes, and applications of this invention, along with any obvious their extension or analogs, are within the scope of this invention. Further, it is intended that this invention encompass any arrangement, which is calculated to achieve that same purpose, and all such variations and modifications as fall within the scope of the appended claims.

[0267] All publications or patent applications referred to above are incorporated herein by reference in their entireties. All the features disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example of a generic series of equivalent or similar features.