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Title:
METHODOLOGY AND APPARATUS FOR THE DETECTION OF BIOLOGICAL SUBSTANCES
Document Type and Number:
WIPO Patent Application WO/2008/133719
Kind Code:
A3
Abstract:
A methodology and an apparatus for the detection of biological substances employing the integration of multiple functions and units designed into and implemented in a sensor unit and system. The deployment of a set of sensor units as a group results in a distributed detecting, discriminating, and alerting system and network. Distribution of the sensor units facilitates real-time, in the field detection of different biological substances such as viruses, bacteria, spores, allergens, and other toxins that can be suspended in multiple media such as air, liquid, and blood. Besides detection/sensing, the system and individual sensor units perform: data acquisition, data development, data storage, statistical analysis, and data transmission.

Inventors:
OLEYNIK VLADISLAV A (US)
Application Number:
PCT/US2007/083825
Publication Date:
January 15, 2009
Filing Date:
November 06, 2007
Export Citation:
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Assignee:
BIOWARN LLC (US)
OLEYNIK VLADISLAV A (US)
International Classes:
G01N25/18; G01N27/00
Foreign References:
US20050231855A12005-10-20
US20030134433A12003-07-17
US20060088960A12006-04-27
Attorney, Agent or Firm:
YIM, Peter, J. et al. (425 Market StreetSan Francisco, CA, US)
Download PDF:
Claims:
What xs claimed is:

1. A system for determining the presence of a biological target, comprising: a. at least one sensor element, each sensor element further comprising: l. a plurality of ligands of at least one ligand type applied to a coating on the sensor element; li. a voltage change generated by the sensor element from an interaction when one of the plurality of ligands of the at least one ligand type binds to at least one biological target type; in. a sensing surface, positioned in proximity to the at least one ligand for detecting the voltage change; lv. a detection means for detecting the voltage change; b. a controller to select and input samples of the voltage change from each sensor element; and c. means for indicating that a biological target has been detected when the voltage change of a sensor element exceeds a threshold voltage.

2. A system for determining the presence of a biological target m accordance with claim 1 wherein: a. the binding of the at least one ligand with the at least one biological target; b. the detecting of the voltage change; c. the controlling of the selecting of the input sample of the voltage change for each sensor element; and d. said binding, detecting and controlling, occurs xn real-time .

3. A system for determining the presence of a biological target in accordance with claim 1 further comprising generating a notification alert by the means for indicating that a biological target has been detected when the voltage change of a sensor element exceeds a threshold voltage.

4. A system for determining the presence of a biological target m accordance with claim 1 wherein the detection means is a field effect transistor (FET) provided with a source region, a gate region and a dram region.

5. The sensor unit in accordance with claim 4, wherein said

FET is an electron sensitive field effect transistor (ESFET) .

6. The sensor unit in accordance with claim 1, further comprising a biological amplification unit connected to at least one ligand type selected from the group consisting of a first ligand type and a second ligand type.

7. The sensor unit in accordance with claim 4, wherein each of the plurality of ligands comprises a ligand sensing surface and a ligand non-sensing surface opposite the ligand sensing surface, the non-sensing surface of at least a portion of the plurality of ligands is provided m proximity to the gate region of the FET.

8. The sensor unit in accordance with claim 4, further comprising a gel enveloping at least a portion of the plurality of ligands of the at least one ligand type in proximity to the gate region of the FET.

9. The sensor unit in accordance with claim 4, further comprising an electric current source connected between a

silicon base of the FET and a first electrode positioned opposite the gate region.

10. The sensor unit in accordance with claim 9, further comprising a dissolvable second electrode in proximity to the gate region, said second electrode connected to the electric current source.

11. The sensor unit in accordance with claim 10, further comprising a plurality of nanotubes provided between the first and second electrodes, wherein the non-sensing surfaces of the ligands attach to one of the nanotubes.

12. The sensor unit in accordance with claim 7, further comprising a catalyst provided on the gate region.

13. The sensor unit in accordance with claim 11, further comprising conductive or semi-conductive materials coating the surface of the plurality of nanotubes .

14. A system for determining the presence of a biological target, comprising: a. a sensor array comprising: l. at least one sensor element, each sensor element further comprising:

1. a plurality of ligands of at least one ligand type applied to a coating on the sensor element;

2. a voltage change generated by the sensor element from an interaction when one of the plurality of ligands of the at least one ligand type binds to at least one biological target type;

3. a sensing surface, positioned in proximity to the at least one ligand for detecting the electrostatic signal; and

4. a detection means for detecting the voltage change; b. a controller for controlling a sequencing of the selection of sensor elements within the array to sequentially select a column of the sensor array, to sequentially select each of the rows of the array and then to sequentially select and input a sample of the voltage change of each sensor element; and c. means for indicating that a biological target has been detected when the voltage change of a sensor element exceeds a threshold voltage.

15. A system for determining the presence of a biological target in accordance with claim 14 wherein: a. the binding of the at least one ligand with the at least one biological target; b. the detecting of the voltage change; c. the controlling of the sequencing of the selection of sensor elements within the array and selecting of the voltage change of each sensor element; and d. said binding, detecting and controlling, occurs in real-time .

16. A system for determining the presence of a biological target in accordance with claim 14 further comprising generating a notification alert by the means for indicating that a biological target has been detected when the voltage change of a sensor element exceeds a threshold voltage.

17. A system for determining the presence of a biological target m accordance with claim 14 wherein the detection means is a field effect transistor (FET) provided with a source region, a gate region and a drain region .

18. The sensor unit in accordance with claim 17, wherein said FET is an electron sensitive field effect transistor (ESFET) .

19. The sensor unit in accordance with claim 14, further comprising a biological amplification unit connected to at least one ligand type selected from the group consisting of a first ligand type and a second ligand type.

20. The sensor unit in accordance with claim 17, wherein each of the plurality of ligands comprises a ligand sensing surface and a ligand non-sensing surface opposite the ligand sensing surface, the non-sensing surface of at least a portion of the plurality of ligands is provided in proximity to the gate region of the FET.

21. The sensor unit in accordance with claim 17, further comprising a gel enveloping at least a portion of the plurality of ligands of at least the first ligand type in proximity to the gate region of the FET.

22. The sensor unit in accordance with claim 17, further comprising an electric current source connected between a silicon base of the FET and a first electrode positioned opposite the gate region.

23. The sensor unit in accordance with claim 22, further comprising a dissolvable second electrode in proximity to the gate region, said second electrode connected to the electric current source.

24. The sensor unit in accordance with claim 23, further comprising a plurality of nanotubes provided between the first and second electrodes, wherein the non-sensing surfaces of the ligands attach to one of the nanotubes.

25. The sensor unit in accordance with claim 17, further comprising a catalyst provided on the gate region.

26. The sensor unit in accordance with claim 24, further comprising conductive or semi-conductive materials coating the surface of the plurality of nanotubes .

Description:

METHODOLOGY AND APPARATUS FOR THE DETECTION OF BIOLOGICAL SUBSTANCES

FIELD OF THE INVENTION

The present invention is directed to a miniaturized sensor and group of sensors sensitive to various biological substances such as viruses, bacteria, spores, allergens and other toxins as well as a system for analyzing the outputs of the sensors .

BACKGROUND OF THE INVENTION

Due to the increased level of terrorism in the world as well as increased globalization, there is a need to be able to reliably and quickly detect, analyze and report, in real-time the existence of biological substances such as viruses, bacteria, spores, allergens and other toxins within the environment and within the human and animal population.

In addition to detecting toxins that may be introduced to the environment and to the human and animal population as a result of biological warfare and terrorist attacks, there also exists a need for the detection of naturally occurring biological organisms that may also be harmful to * rorld populations For example, avian influenza is a viral infection of the respiratory and pulmonary system. It is classed as an influenza Type A, and like other Type A influenzas, it is subject to gradual mutations and sudden changes in its surface proteins. This class of influenza may cause major pandemics. Type A viruses that cause avian influenza are identified by differences in two surface proteins called hemagglutinin (H) and neuraminidase (N) . There are 16 different hemagglutinin subtypes and 9 different neuraminidase subtypes. Hemagglutinin is a glycoprotein that binds the virus to a cell being infected and neuraminidase is an enzyme that helps the virus breach cell walls. They are both antigens that stimulate an immune response that results in the

production of antibodies. Detection may be accomplished indirectly by detection of antibodies produced and directly by detection of the antigens. The avian influenza virus is identified by the designation H5N1 because it includes a specific combination of hemagglutinin and neuraminidase subtypes. Other Type A viruses include but are not limited to HlNl, H2N2, H3N2, H3N8, H5N2 and H7N7.

Another example of requirements for detection of harmful biological substances is the detection of the presence of viral antigens in the blood. This approach may be used to diagnose HIV-I Human immunodeficiency virus (HIV-I)) infection. One of the more prevalent antigens for this purpose is the capsid antigen, P24, a viral structural protein that makes up most of the HIV virus core particle. Because high titers of P24 antigen are present in the serum of acutely infected individuals during the short period between infection and seroconversion, P24 antigen assays are useful in the diagnosis of HIV-I infection. The advantage of the P24 testing is that it can detect HIV infection days earlier, before antibodies develop and that it is a quantitative test that shows the intensity of HIV expression in the body which is a measure of how fast the disease is progressing. After seroconversion, the antigen is bound by P24- specific antibodies and becomes undetectable in the majority of infected individuals. For this reason, P24 antigen assays are not useful for diagnosing HIV-I infection in otherwise healthy individuals who are thought to have established infection. However, later in the course of the disease, the serum P24 antigen again becomes detectable m 30-79o of patients. The presence of detectable P24 antigens is associated with an increased risk of clinical progression of the HIV-I virus. Quantitative P24 assays are used to assess the antiviral activity of new drugs that are being tested to counteract the HIV-I virus.

SUMMARY OF THE INVENTION

The problems associated with preventing the widespread use of biological warfare and m reliably and quickly detecting, analyzing and reporting, in real-time, the existence of biological substances such as viruses, bacteria, spores, allergens and other toxins within the environment and within the human and animal population are addressed by the present invention which utilizes a self-contained, regular-scale as well as millimeter-sized miniaturized -scale sensing and communication platform for a massively distributed sensor network with flexible network hierarchy and secure data flow. Individual sensor units in the form of chips are designed and manufactured, and can be miniaturized to be as small as the size of a grain of sand, and contain sensors, a processor unit, a memory, bi-directional wireless communications, and an internal power supply. Each sensor unit is controlled by a self-contained microcontroller in the form of a digital signal processor (DSP) . This DSP controls both tasks performed by the sensor chip and, to conserve energy, power management between and for the various components of the system. Periodically, the DSP receives a reading from the sensor unit provided with one or more sensors contained on the chip, processes the data received from the sensors, and stores results in its memory. It also pseudo randomly activates the optical, acoustical and/or radio frequency (RF) transceiver provided on each sensor unit to monitor for incoming communication attempts. This communication may include new programs, data or messages from/to other sensor units or from/to a base station router (s) which controls the operation of a plurality of sensor units. In response to a message or upon initiation of a message, the DSP will use the RF transceiver, room re-transmitter (field operation station), or laser to transmit sensor data or a message to the router, another sensor unit or a centralized station. The router would also direct communication to or from the centralized station. To address the detection of different kinds of

biological substances such as viruses, bacteria, allergens, molds, proteins, and toxins (collectively, "targets") , the invention incorporates two classes of sensors with totally different manners of sensing and acquiring information.

The first of these sensors is acoustically based and may be used repeatedly without degradation. This sensor is functionally dependent on acoustical wave technologies. The sensor portion of the sensor unit is constructed as a microminiature mesh (net) on a silicon base, and has its own resonant frequency. For more accurate resonance readings other elements such as sapphire, quartz, or a germanium silica oxide (GSO) crystal, or a beryllium silica oxide (BSO) crystal may be used. The surface of the sensor unit is relatively small, approximately 1mm' of working surface. To achieve greater sensor sensitivity and selectivity to the targets, both sides of the sensor unit base are charged by static electricity. The acoustically based sensor unit operates m three primary modes - collecting data, measuring data, and cleaning the sensor unit. During the collecting mode, targets come in proximity to the sensor. The static electricity applied to each sensor unit surface will draw the targets toward the surface of the sensor and will stick to the sensor unit surface due to molecular adhesion forces. After a time increment determined by a timer provided in the DSP, the sensor unit will be switched to the measurement mode. At this juncture, static electricity will be switched off and the sensor surface will begin to resonate with high frequency oscillation conditions. If there are no targets adhered to the sensor unit surface, the surface will resonate at a first frequency. The sensor surface will resonate at a second frequency, unequal to the first frequency m the presence of particular targets. The power and frequency of that oscillation will be a function of the physical properties of the target particles. The oscillation would result in the target particle leaving the surface of the sensor, resulting m the generation of a pulse. The acoustical nature of the pulse will be analyzed by the DSP and compared to

data contained in a data base provided in the memory of the DSP. If any matching properties are found, this information will be relayed to the centralized station which could issue an alert. During the cleaning mode, the surface of the sensor will be cleaned by the simultaneous application of static electricity depolarization and high power pulses, at a third frequency. After cleaning, all modes may be repeated as required.

Sensor units will be calibrated to known target signatures. If the air has a preponderance of targets exhibiting the same or similar signature (mass, adhesion factor, form factor, etc.), an alert will be triggered providing the microbiological identity of the particles. This alert would be produced based upon communication between sensor units themselves, between communication with the routers and the sensing units and communication between the sensor units, the routers and the centralized system.

Each sensor unit will be manufactured from silicon wafers on a sapphire, quartz, BSO, or GSO crystal base substrate, such as those currently used for manufacture of microchips. All frontal surfaces will be used to produce and store energy.

The second type of these sensors would be a biological based sensor falling into two categories; bio pore sensors and the optical based sensors. Bio pore sensors are micro-miniature pools made up of pores containing substances (ligands) preferably in gels or other substances, and electro-sensing technologies. These bio pores contain the ligand in gel resting on electrodes that will react based on the presence of one simple molecule of a target. During the reaction, the bio pore will produce an electric signature pulse and static electricity, which will be analyzed and trigger an alert if a particular target is present. This analyzation would include comparing the electric signature pulse with a plurality of electro signature pulses stored in the memory of the DSP. This technology will require biological data sets documenting the reactive ligand for each target. This data will be used to choose the gel substances for the bio pores. In

all other ways, including data acquisition, data processing, and data communication, operational implementations are identical for any target.

Biological optical based sensors will have much in common with bio pore sensors. The mam difference m their design is the integration of light-sensing micro-systems to detect and discriminate the sequence of photon bursts generated at the interaction of the target and ligand. These photon-bursts would be in the form of electro-optical signature pulses, compared to a plurality of electro-optical signature pulses stored in the memory of the DSP.

A particularly useful biological sensor configuration relies on the use of an array of bio pore sensors that is capable of detecting various antigens of Type A viruses that cause Type A influenza, such as the H5N1 (hemagglutinin and neuraminidase) strain of the avian flu virus. The array comprises at least one bio pore array element that is coated with a gel containing H5 antibodies (in the form of ligands) that are capable of generating a unique H5 electrostatic pulse signature signal when one or more of the H5 antigens attach to one or more of the H5 antibodies. The array also comprises at least one bio pore array element that is coated with a gel containing Nl antibodies (for example, in the form of ligands) that are capable of generating a unique Nl electrostatic pulse signature signal when one or more Nl antigens attach to one or more of the Nl antibodies. Thus, the presence of the H5N1 avian flu virus in close proximity to the two sensor array elements causes the H5 antigen to be captured by and bind with the H5 antibody and the Nl antigen to be captured by and bind with the Nl antibody such that a unique H5 electrostatic pulse signature signal is generated by the array element that captures the H5 antigen, and a unique Nl electrostatic pulse signature signal is generated by the array element that captures the Nl antigen. By detecting and storing each of these unique electrostatic pulse signature signals and comparing these signals to a library of stored electrostatic

pulse signature signals, and particularly a stored H5 electrostatic pulse signature signal and a stored Nl electrostatic pulse signature signal within the library, the presence of the H5N1 avian Flu virus may be readily detected. The process is similar for the detection of other toxins and diseases. For example, an antibody (for example, in the form of a ligand) for the P24 core protein can be used in the same way to detect the antigen HIV-I. Any disease or toxin in which an antibody exists (in the form of a ligand) that will bind with the corresponding antigen for that target disease or toxin may be detected using the sensor system of this invention.

The present system for determining the presence of a biological target comprises a sensor array comprising at least one sensor element, each sensor element further comprising: a plurality of ligands of at least one ligand type applied to a coating on the sensor element; an electrostatic output signal generated by the sensor element from an interaction when one of the plurality of ligands of the at least one ligand type binds to at least one biological target type; an electrostatic sensing surface, positioned in proximity to the at least one ligand for detecting the electrostatic output signal; and a measurement means for measuring the detected electrostatic output signal. The system further comprises a controller for controlling a sequencing of the selection of sensor elements within the array to sequentially select a column of the sensor array, to sequentially select each of the rows of the array and then to sequentially select and input a sample of the electrostatic output signal of each sensor element. The present invention further comprises a means for converting the electrostatic output signal of the sensor element from an analog signal to a digital equivalent signal by a digital-to-analog converter, the digital equivalent signal being stored m the controller. The digital equivalent sample signal is combined with other digital equivalent sample signals from the same sensor element and is stored in the controller to form a digital signature signal. The

digital signature signal is transmitted to a digital signal processor which compares the digital signature signal with a library of pre-stored signature signals representing known biological targets. An alert notification is generated when the digital signature signal matches a pre-stored signal type in the library of pre-stored signature signals that represents a signature generated when the at least one ligand type binds with the at least one biological target.

The system for determining the presence of a biological target in accordance with further comprises the binding of the at least one ligand with the at least one biological target, the detecting and measuring of the electrostatic output signal, the controlling of the sequencing of the selection of sensor elements within the array and selecting of the electrostatic output signal of each sensor element, the converting of the electrostatic output signal to the digital equivalent signal, the transmitting and the comparing of the digital equivalent signal to the library of pre-stored signature signals representing known biological targets, and said binding, detecting, measuring, controlling, converting, transmitting and comparing occurs in real-time.

The system comprises at least one ligand type that is an antibody for a protein hemagglutinin subtype H5, or at least one ligand type that is an antibody for a protein neuraminidase subtype Nl, or at least one ligand type that is an antibody for a protein P24 or a similar toxin.

The system may have at least one ligand type of a first sensor element selected from the group consisting of an antibody for a protein hemagglutinin and at least one ligand type of a second sensor element selected from the group consisting of an antibody for a protein neuraminidase.

The system may have a sensor element within the sensor array comprising at least one ligand type selected from the group consisting of antibodies for a protein P24.

The system further comprises at least one biological target type that may be antigens of the protein hemagglutinin

subtype H5 that bind with the at least one ligand type antibody for the protein hemagglutinin subtype H5.

The system further comprises at least one biological target type that may be antigens of the protein neuraminidase subtype Nl that bind with the at least one ligand type antibody for the protein neuraminidase subtype Nl.

The system further comprises at least one biological target type that may be antigens of the protein p24 that bind with the at least one ligand type antibody for the protein p24.

The system further comprises a first sensor element of the sensor array wherein the first sensor element comprises a plurality of ligands of a first ligand type that is an antibody for hemagglutinin subtype H5, a second sensor element of the sensor array wherein the second sensor element comprises a plurality of ligands of a second ligand type neuraminidase subtype Nl, means for detecting a first electrostatic output signal when an influenza virus containing an hemagglutinin subtype H5 antigen is introduced in proximity to the first sensor element, means for detecting a second electrostatic output signal when an influenza virus containing a neuraminidase subtype Nl antigen is introduced in proximity to the second sensor element, means for controlling the sequencing of the selection of the first and second sensor elements to read samples of the first and second electrostatic output signals, means for converting the first and second electrostatic output signal samples to first and second digital equivalent sample signals, means for combining a plurality of first digital equivalent sample signals from a same sensor element to form a first digital signature signal and combining a plurality of second digital equivalent sample signals from a same sensor element to form a second digital signature signal, means for transmitting the first and second digital equivalent signals to a digital signal processor which compares the first and second digital signature signals to the library of pre-stored signature signals containing a first pre-stored signature signal representing a binding event when the first

ligand type for hemagglutinin subtype H5 binds with a hemagglutinin subtype H5 antigen and a second pre-stored signature signal representing a binding event when the second ligand type for neuraminidase subtype Nl binds with a neuraminidase subtype Nl antigen, and means for generating an alert upon matching both the first digital signature signal with the first pre-stored signature signal and the second digital signature signal with the second pre-stored signature signal.

The system further comprises a first sensor element of the sensor array wherein the first sensor element comprises a plurality of ligands of a first ligand type that is an antibody for hemagglutinin subtype H5, a second sensor element of the sensor array wherein the second sensor element comprises a plurality of ligands of a second ligand type that is an antibody for neuraminidase subtype Nl, means for detecting a first electrostatic output signal when ctn influenza virus containing an hemagglutinin subtype antigen selected from the group consisting of Hl through H4 and H6 through H16 is introduced in proximity to the first and second sensor elements, means for detecting a second electrostatic output signal when an influenza virus containing a neuraminidase subtype antigen selected from the group consisting of N2 through N9 is introduced m proximity to the first and second sensor elements, means for controlling the sequencing of the selection of the first and second sensor elements to read samples of the first and second electrostatic output signals, converting the first and second electrostatic output signals to first and second digital equivalent sample signals, means for combining a plurality of first digital equivalent sample signals from a same sensor element to form a first digital signature signal and combining a plurality of second digital equivalent sample signals from a same sensor element to form a second digital signature signal, means for transmitting the first and second digital signature signals to a digital signal processor which compares the first and second digital signature signals to a library of pre-stored signature

signals containing binding events representing a binding of an antibody for hemagglutinin subtype selected from the group consisting of Hl through H4 and H6 through H16 and representing a binding of an antibody for neuraminidase subtype selected from the group consisting of N2 through N9, and means for indicating that the antigen hemagglutinin subtype selected from the group consisting of Hl through H4 and H6 through H16 and the antigen neuraminidase subtype selected from the group consisting of N2 through N9 have been detected.

The system further comprises a first sensor element of the sensor array wherein the first sensor element comprises a plurality of ligands of a first ligand type that is an antibody for hemagglutinin subtype H5, a second sensor element of the sensor array wherein the second sensor element comprises a plurality of ligands of a second ligand type that is an antibody for neuraminidase subtype Nl, a third sensor element of the sensor array wherein the third sensor element comprises a plurality of ligands of a ligand type that is an antibody for hemagglutinin with a subtype selected from the qroup consisting of Hl through H4 and H6 through H16, a fourth sensor element of the sensor array wherein the fourth sensor elerrent comprises a plurality of ligands of a ligand type that is an antibody for neuraminidase with a subtype selected from the group consisting of N2 through N9, means for detecting multiple electrostatic output signals when an influenza virus containing a protein of hemagglutinin subtype selected from the group consisting of Hl through H16 is introduced m proximity to the sensor elements and an influenza virus containing a protein neuraminidase subtype selected from the group consisting of Nl through N9 is introduced in proximity to the sensor elements, means for controlling the sequencing of the selection of the sensor elements to read samples of the electrostatic output signals, means converting the electrostatic output signal samples to digital equivalent sample signals, means for combining a plurality of digital equivalent sample signals from each sensor element to form digital signature

signals; means for transmitting the digital signature signals to a digital signal processor which compares the digital signature signals to a library of pre-stored signature signals containing binding events representing a binding of an antibody for hemagglutinin with a hemagglutinin antigen with a subtype selected from the group consisting of Hl through H16 and representing a binding of an antibody for neuraminidase antigen with a subtype selected from the group consisting of Nl through N9, and means for using the comparison to the library of pre- stored signature signals to identify the antigen that has been detected selected from the group consisting of hemagglutinin Hl through H16 and the antigen that has been detected selected from the group consisting of neuraminidase Nl through N9.

The system further comprising a first sensor element of the sensor array wherein the first sensor element comprises a plurality of ligands of a p24 ligand type representing an antibody of a p24 antigen, means for detecting a first electrostatic output signal when the p24 antigen is introduced m proximity to the first sensor element, means for controlling the sequencing of the selection of the first sensor element to read samples of the first electrostatic output siαnal, means for converting the first electrostatic output signals to a first digital equivalent sample signal, means for combining a plurality of digital equivalent sample signals from each sensor element to form digital signature signals; means for transmitting the first digital signature signal to a digital signal processor which compares the first digital signature signal to a library of pre- stored signature signals containing a first signature signal representing a binding event when the p24 ligand type binds with the p24 antigen, and generating an alert upon ma t ching the first digital signature signal with the first pre-stored signature signal representing a p24 binding event.

The system for determining the presence of a biological target further comprises at least one sensor element, each sensor element further comprising: a plurality of ligands of at least

one ligand type applied to a coating on the sensor element, an electrostatic output signal generated by the sensor element from an interaction when one of the plurality of ligands of the at least one ligand type binds to at least one biological target type, an electrostatic sensing surface, positioned in proximity to the at least one ligand for detecting the electrostatic output signal, and a measurement means for measuring the detected electrostatic output signal. The system further comprises a controller to select and input the electrostatic output signal samples of each sensor element, means for converting the electrostatic output signal samples of the sensor element from an analog signal to a digital equivalent signal sample by a digital- to-analog converter, the digital equivalent signal sample being stored in the controller, means for combining a plurality of digital equivalent sample signals from each sensor element to form digital signature signals; means for transmitting the digital equivalent signal to a digital signal processor which compares each digital signature signal with a library of pre- stored signature signals representing known biological targets, and means for generating a notification alert when the digital signature signal matches a pre-stored signal in the library of pre-stored signature signals that represents a signature generated when the at least one ligand type binds with the at least one biological target type.

The system further comprises a system in which the digital signature signal is a time domain signature signal which is compared to pre-stored time domain signature signals using cross-correlation techniques to determine a match. The system further comprises a system in which the digital signature signal is converted to a frequency spectrum and then compared to pre- stored frequency spectrum signature signals using cross- correlation techniques to determine a match.

An embodiment of the present invention comprises at least one sensor element where each sensor element further comprises a plurality of ligands of at least one ligand type

applied to a coating on the sensor element, a voltage change generated by the sensor element from an interaction when one of the plurality of ligands of the at least one ligand type binds to at least one biological target type, a sensing surface positioned in proximity to the at least one ligand for detecting the voltage change and a detection means for detecting the voltage change. The system further comprises a controller to select and input samples of the voltage change of each sensor element and means for indicating that a biological target has been detected when the voltage change of a sensor element exceeds a threshold voltage. The detection and measurement means may be an FET. In another embodiment of the present invention, the sensor elements may be an array of sensor elements.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing generalized description of the invention will be better understood from the following detailed description of preferred embodiments of the invention with reference to the drawings that include the following:

Figure IA is a diagram of an acoustical based sensing unit;

Figure IB is a diagram of the acoustical based sensing unit of Figure IA in the collecting mode;

Figure 1C is a diagram of the acoustical based sensing unit of Figure IA in the analyzation mode;

Figure ID is a diagram of the acoustical based sensing unit of Figure IA in the cleaning mode;

Figure IE is a diagram of two acoustical based sensing units, each in a different mode of operation;

Figure 2 is a diagram illustrating a single bio pore sensing unit;

Figure 3 is a diagram of several bio pore sensing units and a field effect transistor (FET) that form a basic structure of a bio pore sensing element used to sense a reaction between one or more ligand and one or more specific target;

Figure 4 shows the basic structure of a bio pore sensing element of Figure 3 with ligands encased in a gel;

Figure 5 shows an alternate embodiment of the bio pore sensing element illustrated in Figures 3 and 4;

Figure 6 illustrates another alternate embodiment of a io pore sensing element having two electrodes;

Figure 7 illustrates another alternate embodiment of a bio pore sensing element having multiple nanotubes;

Figure 8 represents a top view of a bio pore sensing element having nanotubes formed between two electrodes;

Figure 9 is a side view of the bio pore sensing element shown in Figure 8;

Figure 10 illustrates a biologically optical based sensing unit;

Figure 11 illustrates a typical DSP and its relationship to other elements of a sub-system, according to the present invention;

Figure 12 illustrates an embodiment of a system according to the present invention;

Figure 13A illustrates a sensor array in a system configuration where the elements in the array may be selected from one of the embodiments of the sensor elements shown in Figure 2 through Figure 9 above;

Figure 13B shows an example of how ligands may be distributed on the sensor array shown in Figure 13A;

Figure 14A - Figure 14E show typical responses from bio pore sensing elements coated with H5, Nl And P24 antibodies and being subjected to Hl, Nl, N5, H5 and P24 antigens;

Figure 15 shows an alternate structure of a bio pore sensing element of Figures 2-9 with multiple ligand types encased in a gel; and

Figure 16 illustrates process steps required to implement an embodiment of the present invention.

Figure 17 illustrates an embodiment of a sensor array in a system configuration where the elements an the array may be selected from one of the embodiments of the sensor elements shown in Figure 2 through Figure 9 above.

Figure 18 illustrates process steps required to implement an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Each of the chips used as a sensor unit will be manufactured from silicon wafers on a sapphire, quartz, or BSO or GSO crystal base substrate or similar material, such as those currently used for manufacture of microchips. All frontal surfaces of the sensor units (except the bio pores) will be used to produce and store energy. The operationally integrated sensor units will act as a massively distributed sensor network. This network will function as a monolithic unit, providing a de-facto three dimensional real time sensing of the presence of biological substances. For instance, some clusters of sensor units could form a synchronous group executing on the same working cycles, thereby increasing the sensitivity and reliability of the system, and creating special features such as a distributed antenna. The invention lends itself to customization, and is readily adaptable to diverse operational configurations. For example, clusters of units could be aligned to monitor a statically charged air pump

which will move air, which could include targets, in one specific direction. This will increase the sensor sensitivity because the target particles will be brought into closer proximity with the sensor units. This system exhibits reliable capabilities to sort all targets using static electricity. This invention capitalizes on the fact that all target particles saturated in the air do not react equally to the polarity of the static electricity charge. Groups of the sensor units will change polarity together, generating additional information about the target distribution in air, such as how the air in a ventilation system is moving. This will create the basis for relational databases mapping the nature of the target to atmospheric conditions.

Referring to the drawings, Figure IA illustrates an acoustical based sensor 1 having a sensor unit 2 comprising a plurality of micro resonators 3 on the surface of the sensor unit, thereby forming an oscillating web. Figures IB, 1C and ID illustrate the operation of the acoustical based sensor 1 in the collecting mode shown in Figure IB, the analyzation mode illustrated in Figure 1C and the cleaning mode detailed in Figure ID. The acoustical based sensors as well as all of the other sensors described in the present invention are adapted to be applied to the surface of a product, such as medical equipment, clothing or food or are adapted to be air borne. Regardless of whether the acoustical based sensors or biological based sensors are affixed to an object or are drifting m air, the purpose is to detect the presence of one or more of a plurality of biological substances denoted as "targets" 24 which would be harmful to humans and/or animals. These targets 24 would generally be air borne along with various other floating matter such as protein strings 5 and dust particles 6. The acoustical based sensor 1 would be characterized as a sensor unit 2 having a surface onto which the various particles 5, 6 and 24 would settle. The surface unit would be connected to a DC current source 7 having a battery 8. Two switches 9a, 9b would be

attached in parallel to the source of electricity. Therefore, in the collecting mode as shown in Figure IB, electricity would be applied to the surface of the sensor unit 2, allowing the sensor unit to oscillate at a first frequency. The switches would be in the position shown in Figure IB to apply a first current level to the surface of the sensor unit to allow that sensor unit to oscillate at that first frequency and power. As shown by the arrows attached to each of the air borne elements 5, 6 and 24 shown in Figure IB, these air borne elements would become attracted to the surface of the sensor unit.

Once these particles 5, 6 and 24 become attached, or rest upon the surface 2 of the sensor unit, switch 9a moves to the position shown in the analyzation mode shown in Figure 1C, thereby removing the source of electricity from the surface of the sensor unit. At this time, the air borne particles which would include target particles 24 would begin to oscillate at a second frequency, different than the frequency in which the surface of the sensor unit would oscillate in Figure IB. A DSP including a bidirectional wireless communication, an internal power supply as well as a memory would sense the particular resonating frequency. This frequency would be compared to frequencies stored in the memory of the DSP, indicative of particular targets. If a match is made between the oscillating frequency of the target or targets and the oscillating frequency stored in the memory of the DSP, this match would be noted and stored in the memory of the DSP. At that time, or at a later time, this information would be transmitted utilizing the particular communications capacity of the sensors to adjoining sensors, to one or more routers, or to a centralized station m which a decision regarding the presence of toxic biological substances, indicative of a bio terrorist attack would then create the appropriate alert.

Once the analyzation step is complete as illustrated in Figure 1C, the surface of the sensor unit 2 would be cleaned by

moving switch 9b to the position shown in Figure ID. At this point, the surface of the sensor unit would oscillate at a third frequency, thereby ejecting all of the air borne material 5, 6 and 24 from the surface of the sensor unit as shown by the arrows included in Figure ID.

Figure IE shows two adjacent sensor units in differing phases such as the cleaning phase or the collection phase. The collection phase is illustrated by the sensor unit on the left and the cleaning phase is illustrated by the sensor unit on the right. The inclusion of the cleaning phase shown in Figure ID would result in enabling repeated use of the acoustical base sensor unit.

Figure 2 shows a single bio pore sensing unit including a ligand 22 in the presence of a biological target 24. Additionally, an optional biological amplification unit 20 can be affixed to a non-sensing surface of the ligand 22. The ligand 22 may be an ion or molecule that reacts to form a complex with another molecule. The target 24 may be the molecule bound specifically by the ligand. Each ligand operates in conjunction with a specific target, of which there are a multitude of possible ligand/target pairs. The target may be a single molecule such as a protein, glycoprotein, saccharide, or lipid. The target may also be an organism such as bacteria or its spore, a virus, fungus, mold, or yeast. The ligand 22 and target 24 bind together with high affinity and specificity. Examples of ligand/target pairs are an antibody and whatever macromolecule the antibody was generated against, a cellular receptor and whatever substance specifically binds and activates the receptor, or a surface feature on a microorganism such as hemagglutinin on an influenza virus and an antibody or molecule (such as sialic acid in the influenza example) that binds the surface feature. It is important to note that a target will only completely attach itself to only one type of ligand. An interaction by the ligand with a target to which it should not bind completely, would

result in, at best, only a partxal binding, for an instant of time .

Interactions between a ligand and its target arise from mtermolecular attractions that include complementary conformations, charges, polarities, Van der Waals interactions, and reordering of the water molecules m the surrounding millieu. These attractive forces are cooperative and accumulate as the target and ligand come in proximity. Each target/ligand interaction has a specific kinetic and thermodynamic signature that can be characterized and quantified:

Ligand + Target ~ Ligand/Target Complex (1) ω k f f

The equilibrium constant is derived from the relation of the on and off constants:

K or = k r /k rff (2)

K= j is related to free energy by λG = δGE + RT In K er , and at equilibrium δG = 0, so:

δG = -RT In K - j

For K Pq = 1, δGE = 0

For K Qq = 10, δGE = -1.4 Kcal/mole

For K oq = 10% δGE = -7 Kcal/mole (3)

with R = universal gas constant

T = temperature (Kelvin Scale)

The K= q for avidm-biotm interaction is approximately 10 1 M ""1 , and for a "typical" antigen-antibody interaction is approximately 10 1^ M 1 . Thus the energy released from a mole of avidin-biotm interaction is approximately 21Kcal/mole and for

antigen-antibody approximately 16 Kcal/mole. The unique pattern of energy release is a function of the interaction signature for each ligand/target pair.

The single bio pore sensing unit shown in Figure 2 is based on micro-miniature pores of ligands generally, but not necessarily embedded within aqueous gels on sensing element surfaces with electro-sensing technologies cumulatively called bio pores. Each bio pore is filled with one or more ligands in gel and will react to the presence of one single molecule of a specific target for that ligand. During reaction, the reaction between the ligand and the specific target molecule will produce an electric pulse signature and static electricity which will be analyzed and trigger an alert if the proper target is present. This technology will require biological data sets describing the electrostatic signature generated by binding of each ligand/target pair. This data will be used to differentiate among targets. In all other ways, including data acquisition, data processing, and data communication, all implementations may be identical to other ligand/target pairs.

The materials and methods disclosed herein provide an effective manner for the mass production of uniform micro fabricated units. To customize a deployment of units to a particular targets of interest (Hepatitis C, Salmonella, Anthrax, etc.), the bio pores will contain the appropriate and unique reactive ligands. More specifically, each sensor element comprising a sensing unit of the present invention comprises a signal-converting element, a transducer, a responsive element, and the ligand shown in the sensing unit of Figure 2. Conversion circuits will include electron sensitive circuits, single electron transistors, photosensitive based circuits, acoustic sensitive based circuits, and inductivity sensitive detection circuits, based upon the type of sensor utilized. Depending on the application, specific bio-amplification elements may be used. The signal-converting element is comprised of an active moiety and signal-transforming domain. The ligand-specific moiety

specifically recognizes a selected target. A sensing unit used with the ligand shown in Figure 2 would require software and hardware to monitor and detect specific targets. Depending on the preliminary detector conversion circuits, the bio-amplification or device 20 may or may not be used. For instance, in some cases, when dealing with an extremely low energy ligand/target interaction, a sensing unit with amplifier 20 such as enzymatic fluorescence or chemiluminescence generation, with a photon- sensitive detector can be employed. In this case, after detection by the sensing unit, an electrical pulse will be converted to a photon stream, which will be detected by a sensitive photo-detector.

Figure 3 represents the use of a field effect transistor (FET) 30 with a sensing gate 32 as a measurement device awaiting integration of the gel and molecules of ligand 31. The ligand 31 is placed on or close to the gate 32 as possible, such that any ligand/target interaction will generate a current from the source area 34 through the gate 32 to a drain area 36. The FET is provided on a semiconductor base 38. A layer of insulation 39 is provided over the gate 32, the source 34 and the drain 36. This FET structure will be implemented in several formats as will be discussed. The FET structure can take the form of a miniature extra sensitive field effect transistor (ESFET) .

Figure 4 depicts the FET 30 with gel 33 incorporated in the design. The gel utilized should exhibit the properties of remaining moist, having optical sensitivity and allowing the targets to pass through the gel and to bind to the ligand. There are several ways to place the ligand in operational proximity to the gate area. For instance, the surface of the gate 32 can be coated with ammosilane. The ligand is tethered to the amino groups via a variety of cross linkers, for example, disuccinimidyl suberate, Bhydroxy disuccmimidyl suberate, etc. The cross linkers can be chosen with specificity to selected

functional groups on the ligand to achieve the desired orientation .

Figure 5 depicts an alternative embodiment of the FET approach. This FET 40 includes a silicon base 48 on which a source area 46 and a dram area 44 are provided. A gate 50 is provided on an insulator 42. A number of ligands 52, 54, 56, 58 and 60 are associated with the FET 40. These ligands are captured with a DC field produced by a DC current source 62 and an electrode 64. AS was true with the FET shown in Figure 4, a similar gel 66 will be incorporated in the design. This facilitates orientation of the sensing elements to provide optimal sensing capability.

Figure 6 depicts an alternative approach FET 70 to facilitate orienting the ligands 72, 74, 76, 78 and 80, electrostatically prior to introduction of the gel 90. Besides orienting the ligands, the dual electrode configuration including a DC current source 92, an upper electrode 94 and a lower electrode 96 m proximity to the gate area 98 will facilitate movement of the ligands to the gate area 98, ultimately attaching them to the lower electrode 96 in the area of the gate area. The sensor unit includes a silicon base 82, a source area 84 and a dram area 86 and a layer of insulation 88. The lower electrode 96 will then completely dissolve, permitting the FET to function normally. Alternatively, the lower electrode will be only partially dissolved, facilitating a bias feedback capability.

Figure 7 depicts an advanced FET sensor 100 incorporating one or more catalyst islands 120 positioned on the FET gate electrode 110 in the area at the gate 122. The catalyst island is capable of growing nanotubes 114, 116, 118. The FET 100 includes a silicon base 102, a drain area 104, a source area 106 and an insulation coating 108. The catalyst island 120 consists of chemical ingredients which form a base for growing the nanotubes. Nanotubes typically grow in a chaotic manner.

Their ultimate quantity and volume are managed by controlling time and temperature. The responsiveness to time and temperature are dependent on the ingredients of the catalyst. Generally, multiple nanotubes will be grown. The surface of the nanotubes can be customized using alternative methods to modify their properties. Modification can be achieved using chemical solutions to etch the nanotubes surfaces. Alternatively, the nanotubes can be coated with chemicals. The primary configuration for this invention will include coating the nanotubes with conductive or semi-conductive materials. This will be followed by application of the gel. This dramatically increases the surface area for target detection without increasing the linear surface of the detector. Operationally, after the ligand/target interaction, the signal will come through the surface of the nanotubes to the gate of the FET. Since the nanotubes are indirectly in contact with the gate of the FET, and the ligands would adhere to the surface of the walls of the nanotubes, more ligands would indirectly be in contact with the measurement device, i.e. the gate area. Operation then proceeds as previously described.

Figure 8 presents one possible implementation of the bio pore sensor 130. In this case, the pore 132 has been created on the silicon chip surface. In the bio pore, nanotubes 134, 136, 138 generally extend between two electrodes 140 and 142. All surfaces of the nanotubes will be covered with metal (clayed or plaque) . The result is a dense electrode mesh. The pore is filled with many ligand elements connected to the nanotubes. When contact between a ligand and a target is achieved, a signal will be propagated over the nanotube mesh and to the electrodes 140, 142. Electrodes are connected to the registration circuits (not shown) .

Figure 9 depicts a side view of the bio pore 132 and a multidimensional perspective of the relative locations of the electrodes 140, 142 and the nanotubes within the pore. There are

multiple configurations for the various components that constitute a bio pore. The optimal configuration is a function of the planned deployment. These configurations will not be limited by availability of materials. It has been shown that available materials retain their film-forming properties even when non-latex water-soluble components (e.g., proteins, enzymes, polysaccharides such as agarose, or synthetic polymers) comprise up to approximately 25o by weight of the material. This alleviates a significant consideration related to a micro fabrication process for the production of biosensors; the established film adheres effectively to a planar substrate even in the presence of large amounts of additives (i.e., enzymes). Particle latex materials have been used traditionally to immobilize all manner of biologically active materials. Thus, the biosensor units of the present invention provide a flexible, generic system that can be adapted to recognize any selected biological substances.

A biological optical based sensor is shown in Figure 10. It is based on micro-miniature bio pores made up of pores of gel 152 containing ligands 154, 156, 158 and a light-sensing detector 160. During the interaction of the target with the ligand, a sequence of photon bursts or signatures will be generated and detected by the light-sensing micro-system including detector 160. The micro-systems will be built based on Avalanche Diodes type, Charge Coupled Devices (CCD) , or other light-sensing technologies. Upon detection, a comparative analysis of the newly observed data and data stored in the DSP memory will be performed in the manner previously described with respect to the acoustical based and the non-optical based biological sensors.

Optical techniques have been successfully used in the field of sensors, monitoring reactions by measuring changes in absorption, fluorescence, scatter, and refractive index. In particular, for the biological optical based sensor, a layer

which undergoes an optical change is integrated onto the surface of the device so that the evanescent field of the light penetrates the sensing layer. Monoclonal antibodies may be used as the sensing layer, with high specificity to defined targets, then changing the sensing layer composition. Any reactions occurring at the sensing layer affect the evanescent field and hence the optical properties of the device.

This biological optical based sensor will take advantage of interaction energy conversion to fluorescence, detecting the emitted light after interaction. The gel and the ligands in this detector will be located based on descriptions accompanying Figures 5 and 6.

As previously described, each of the various types of sensor units would be provided with a DSP 170 as shown m Figure 11.

Each sensor unit has a dedicated input/output channel 202 for initial power-up, charging the main storage capacitor, programming, and performance of test procedures. Connection to this channel will be dore over dedicated devices, during initial test procedures. The input/output channel allows communication from each of the sensor units, such as the bio pore or bio optical sensor 172 and the acoustical sensor to a CPU 176, through a communication controller 204. Each unit has three additional channels: a near range (NR) communication channel including an acoustical antenna 208, a radio frequency (RF) channel including an RF antenna 206; and an optical channel including an optical antenna 210. The NR communication channel has an ultrasonic transmitter/receiver. This communication channel allows each sensor unit to communicate with nearby sensor units. In other words, the sensor units start to sense each other, exchange data packets, and even convey information data packets, as well as to coordinate the various operational modes employed by the acoustical based sensor unit.

The RF channel is intended to be used for middle range communication and cluster definition. This channel is faster and can convey more information in a given period of time. In some circumstances this channel could be used to communicate between sensor units, thus it is anticipated incorporating an RF processor to manage the data flow between sensor units.

The optical channel is mainly intended to partially, or in some circumstances, totally substitute for the main RF channel during long-range communication with the router or with large cluster-to-cluster communications as well as to the centralized station. If RF spectrum pollution is experienced, this channel, along with the NR channel, becomes the communication media.

Based upon the distances between the sensor units, the router and the centralized station including a computer, each of the aforementioned manner of connections can be used to disseminate information between the sensor units, the router and the centralized station computer.

A non-alterable memory read only memory (ROM) or an EEPROM 190 is provided in the DSP and consists of Programmed Logical Matrix (PLM) and controlling circuits. The primary intended use for the memory is to hold all operational programs and instructions. Additionally, the memory will hold some sample signature patterns of a number of targets. These signature patterns can be tailored to the type of sensor unit employed, or could include all of the possible signature patterns, regardless of the sensor unit.

A random access memory (RAM) 188 is also included m the DSP. The RAM 188 is used to hold variables, acquired data, temporary data, temporary variables, and other miscellaneous data

A flash memory (not illustrated) is provided in the DSP. It is divided into functional groups including: a stack and stack pointer, variables and current states, additional program files, and data files. This memory is mainly used by an

arithmetic logic unit (ALU) 182 for internal operations of the DSP. The ALU 182 can be used along with the EEPROM 190 and the RAM 188 to compare a measured signature with the signatures contained in the EEPROM 190.

The sensor units have some potential sources of interruption provided in the DSP. These sources of interruption include a watchdog timer 194, a wake-on-change 196; a real-time clock, various counters such as time counters 198 and a program counter 186, and overflow interrupts 196. Each of the above- mentioned events generates a special signal to interrupt program flow and switch to the respective special attention functions. The watchdog timer 194 is the first tier of defense if an irresolvable DSP situation or any other event causes an unpredicted condition. This would be expected to occur most frequently if the processor is overwhelmed with different tasks and the power source capacity would not allow it to perform all functions simultaneously. Conceivably, the DSP could become trapped m an infinite loop with no normal manner to extricate itself. In this case the watchdog timer 194 will generate a high level interrupt to stop the loop and restart the DSP. Sensors and I/O channels produce a wake-on-change interrupt even during the power-saving sleep mode to allow the DSP to wake-up from an energy saving mode and assume the full operational mode. Overflow interrupts occur if corresponding flags in a special function register are enabled. The real-time clock is the main source of time synchronization. This interrupt allows performance of sequential operations with the DSP, its peripheral.

The sensor unit contains a 4-bit or 8-bit general- purpose ALU 182 performing arithmetic and Boolean functions between data m a working registers 184 and any register file such as instruction register 192.

The register files are divided into two functional groups consisting of special function registers and general- purpose registers. The special function registers are used by

the DSP and peripheral components to control the operation of the device. The special function registers include the working register, a timer register, the program counter 186 and I/O registers. In addition, special function registers are used to control the I/O port configuration. The general-purpose registers are used for data and control information under command of the instructions.

The functions of the macro access controller (MAC) will be performed by the DSP. This will save power and space on the crystal, to optimize timing and avoid communication delays.

A bus 200 is included in the CPU 176 to allow for transfer of data to and from the components therein as well as to communicate with the I/O channel 202.

An RF processor in communication with the DSP provides synchronous and asynchronous communication modes for each sensor unit. The RF processor receives an RF synchronization sequence, determines the required action, adjusts receiving and transmission parameters, and receives and transmits data. The RF processor also optimizes power acquisition procedures.

Primarily for purposes of energy conservation, all RF related circuits are designed based on resonance based ideology, and are incorporated in close proximity on the chip. The current design includes compatible or semi-compatible spectrum and frequency requirements, as per IEEE 802. Ixx standard, which will allow use of existing communication capabilities. There will be additional advantages for power acquisition in the given frequency range.

All amplification of signals is done at the minimum levels necessary to receive and transmit signals. Since there are strict power limitations, we assume all data transmissions include some data-loss. All data correction will be done within the DSP and its software. Thus, power conservation is the cornerstone of all operation and design.

The antenna field on each unit is symmetrical and occupies all available space on the chip's surface. Likewise, the antenna assumes the shielding function for all internal sub units. The size of the antenna and its geometry are functions of the frequency spectrum, proposed sensitivity, and transmission power level. The transmission power level will be in the range of microwatts, thus thick antenna metallic layers will not be required. Thicknesses are expected to be in the range of 5 to 10 nm. Recent developments in surface etching show promise for the use of multilayer antenna wiring, which will increase antenna surfaces many fold. Switching facilities will facilitate low power, low loss, and CMOS types of serial/parallel switches to achieve extremely low energy loss. Considering the low power required for switching, power requirements are optimized (minimized) through fast switching capabilities Even separate elements of the same antenna facilities will have incorporated switches for multiple segment switching. This allows optimization of total antenna capacitance and inductivity resulting in transmitting and achieving high quality resonance reception. Cumulatively, this leads to power conservation.

As previously indicated, information is communicated between individual sensor units, between the sensor units and one or more router units and also between a centralized system computer and the routers and the centralized system computer. During a communication cycle, each data package will consist of a preamble, data, and signature. If the package is not designated, it is directed to the centralized system computer. If the centralized system computer does not send confirmation in the established time frame, the centralized system computer will try to transmit the package via nearby sensor units. In this case, the end of the transmitted package will have a designation mark for chain communication. This mark will trigger any nearby sensor units to receive the package, and immediately retransmit with the same designation mark. In this way, the centralized system computer will receive the package by multiple paths, from

other sensor units, and perhaps many times. After receiving the first package, if no errors are present, the centralized system computer will form and transmit a response package with specific information as to which package has been successfully received. This will interrupt all other transmissions of the same package. All units will then switch to the normal operating mode.

For long-range communication each sensor unit can communicate with any and all sensor units. During initial handshake procedures, the sensor units are synchronized and are capable of generating and transmitting data packages simultaneously, forming phase antenna fields on the carrier frequency. During the transmission process, while data is being acquired by one sensor unit, all sensor units from the group will be involved. Before transmission, all members of the group will be assigned unique group numbers. After transmission, the first unit of the group will form a package of data, consisting of preamble, data, and signature. Then, each sensor unit provides package encryption and adds a designation descriptor. The sensor chip transmits these packages to other sensor chips. When another sensor unit(s) receives a package with a destination mark, the mark will be analyzed. If the destination mark prescribes a data package to be transmitted via the long-range communication mode, each sensor unit from the group will receive and place the data package in a special holding queue. All group members then start the RF synchronization cycle and when synchronization is achieved; all group members will transmit one single data package simultaneously, thus increasing the communication distance. After initial data from one of the units is transmitted, the second unit of the group will transmit their own package with a designation signature to all group members and the cycle will then repeat, until all data from all group members has been successfully transmitted. The main receiving unit will form and transmit a confioation receipt for each package transmitted by the group. If any errors are acquired, the

package will be retransmitted a reasonable number of times until error free transmission is achieved.

The power facilities are distributed over and among different circuits. They include antenna facilities; receive, with all distributed amplification; RF processor; power management facilities; and power storage devices.

Each sensor unit has a unique input/output channel for initial power-up, charging the main storage capacitor, programming, and performing test procedures, some of which are activated through a power recovery and storage unit 212. Connection to this port will be accomplished during initial test procedures. During normal operation, meaning operation m an open environment, the sensor unit will not be connected to any external power source for charging and operations. For power acquisition, the sensor unit collects power from the environment, including, but not limited to a solar battery 218. The sensor unit is designed specifically to allow optimal use of unit volume and all system properties for acquisition, storage, and power management. The mam power source is the electromagnetic radiation available in the complete radio frequency range received by RF receiver 216. This type of energy is widely available in all places where there is human activity. These sources include radio transmitters m all AM/FM bands; radio receivers, because their converter circuits generate RF waves; police radar-based speed detectors; military or civilian radar; computer monitors, which are a significant near-field RF source; computer networks; and wires within the power grid. Secondary sources of energy are also available and each unit has designated facilities to acquire that energy. Mainly there are X-ray band and Gamma band sources as shown by receiver 220, which are widely available in medical facilities screening facilities in airports, railroad and train stations, etc. Another source of repeatable energy may be motion of the object or surface upon which the unit is installed. An ultrasonic receiver 214 such as a piezoelectric

genomic element, will absorb this type of energy. Scenarios locating the unit on a surgical glove or surgical dressing could incorporate these ultrasonic receivers capable of absorbing temperature gradients and producing other health status parameters .

The RF band will be used as following: Power acquisition begins with the idle cycle of the main DSP processor.

The DSP will advise the RF processor to open all receiving circuits and start to acquire signals in the wide spectrum. The RF processor will search the complete frequency range and attempt to determine the available energy. If any is available, all input circuits will be optimized on that specific frequency range. Detection and storage of the energy is done by multiple stages of detection and charging of the main capacitors. An optical sensor is the ideal because it collects any energy in the optical or close range bands. This additional function will not degrade main sensor functionality. Energy collected in the x-ray and Gamma-ray bands will be used on the reverse side of the unit.

The chip volume in this scenario works like a massive filter of optical rays, allowing detection of only x-ray or Gamma rays. These rays freely penetrate silicon substances. An additional benefit of such a detector and power acquisition element is that the sensor unit will collect information about radiation background and/or radiation bursts.

The mam storage capacitors are located on the lower layer of the sensor unit. The capacitors are configured in large fields of non-electrolyte, dry capacitors.

Power management facilities incorporate on/off and hibernation functionality. These circuits are principally designed for monitoring the mam load circuits, stages of power consumption, and facilitating a power consumption prediction algorithm. Together with the main software on the DSP, power management software modules will detect the shortfalls of stored power and will re-allocate depending on power cycles. This allows decreased peak consumption and power-related heat

consumption. Additionally, the power management unit allows determination of maximum power storage peaks and allocates the maximum consumption at that specific moment, to maximize output transmitting performance. Information about power status is included in each block of data, and in this way the main unit can determine when it needs to run the main charging cycle to restore (replenish) power.

In the case of a new sensor unit or a sensor unit which has totally lost power, all circuits are designed such that receiving circuits switch to maximum power and the power storage cycle is active. In this way, if an operator or the main unit initiates unit activation, they are ready to acquire energy and recharge their power facilities. The replenishment cycle will be postponed until all capacitors are fully charged, and power management facilities will then initiate first wake-up procedures. During wake-up procedures, the DSP runs a simple self-test and then performs a testing of peripheral elements. After the test is successful, the DSP will initiate a short transmission session to check the RF channel. After all this is complete, a status code will be recorded in the memory along with the date and time. If the wake-up status is allowed, the DSP will switch to the normal acquisition and analysis phase. If the wake-up procedures generate a different code, that code will be sent to the main unit for further analysis and subsequent operational instructions. To enhance energy saving during the normal functioning modes, the power management system will power- up only those sensors and systems, needed at that particular moment. In the mode "collect or wait for an event", most of the system is in the power-saving mode. If some facilities are damaged during transportation or from improper previous usage, all possible codes will be stored in the unit memory for detailed scanning. Scanning can be performed with an external device to determine overall power status.

Power conservation is explicitly integrated in the operational power system. All circuits in the sensor unit allow

power management in a multiple stage conservation process. The circuits of the sensor units will be monitored for excessive power consumption. If this happens, a status flag of excessive power consumption will be generated and the centralized computer will further analyze that event.

The low power consumption stage is mainly designed to switch non-critical processes to low power, which will make execution time longer, but will provide enhanced power.

A super low power consumption stage will be activated when absolutely non-critical scenarios are encountered. The performance cycles will switch to the minimum possible operating level for very slow continuous operations, with minimum operations needed for survival of the chip, but not crucial for that specific environment. An example of such an event could be long-term survival, when no RF povver sources are available, but there is a need to maintain operations to acquire possible energy bursts .

Hibernation of all circuits is not related to power conservation but will reduce the amount of consumed power. Usually hibernation is predictable, controllable, and will often be used during normal operation.

Each of the sensor units will be in the power-off stage when delivered from the factory. There is insufficient power to initiate operational and initialization tests. during this stage all power facilities are oriented to collect and conserve power.

No calculations or transmissions are executed.

Figure 12 illustrates the system of the present invention in which a plurality of groups of sensor units 230 are dispersed in various locations. As previously indicated, each of the sensor units within each group 230 can transmit and receive information from any of the sensor units within that group. Each of the sensor units within each of the sensor groups or clusters 230 would also be in communication with a router 232. This communication is generally wireless m nature and would utilize

the three types of transmitting technologies previously described. Some of the routers are provided with a switch 234 and a server 236 for transmitting information wirelessly or through an internet, VPN or internet system 238 to a centralized computer system 240. This centralized computer system would receive and transmit data to and from the routers, as well as the individual sensor units. Based upon the information received by the centralized computer system 240, a decision is made as to whether toxic biological substances are prevalent in one or more areas as well as whether this would constitute a bio terrorist attack. This decision making process is done either automatically utilizing an appropriate computer, or m conjunction with individuals reviewing the output of the centralized computer based upon information received from the groups of sensor units 230.

Figure 13A illustrates a sensor array 310 in a typical system configuration 300, where the elements in the array may be selected from one of the embodiments of the sensor elements shown in Figure 2 through Figure 9 above or may be some other type of sensor element such as a single electron transistor. The controller 360 controls the sequencing of the selection of sensor elements within the array 310 by controlling the demultiplexer 370 to sequentially select one of four columns 330, 332, 334, 336 of the sensor array 310. When one of the four columns 330, 332, 334, 336 is selected, the controller 360 commands the multiplexer 340 to sequentially select each of the four rows 320, 322, 324, 326 of the array 310. As each row 320, 322, 324, 326 is selected by the multiplexer 340, a sample of the output of the sensor element located at the selected row and selected column is selected by the multiplexer 340 and sent to a digital-to-analog (D/A) converter 350 for conversion to a digital equivalent signal sample 355 that is stored in the controller 360. As this sequence progresses, the output of each sensor element is

digitized and stored in the controller 360. This process of sampling and digitizing outputs from the sensors and reconstructing a digital signature signal using time division multiplexing is well-known to those skilled in the relevant art of digital signal processing. The controller 360 combines a plurality of digital equivalent signal samples from each sensor element in the sensor array 310 to form a digital signature signal for each element in the array 310. The digital signature signal 365 from each sensor element is then transmitted to a digital signal processor (DSP) 390, which compares each digitized sensor output signature signal 365 with a library of pre-stored signature signals 380 representing known targets that may match the ligand coating on each sensor element in the array 310. In this manner, any detected target that matches any one of the ligand coatings on at least one of the sensor elements is sensed and processed in real-time. When a match is found by the DSP 390 between a digitized sensor signature signal 365 and at least one of the pre-stored signature signals in the signal library 380, a notification and alert are generated 395 for notification of appropriate personnel.

When the DSP 390 receives a digitized sensor signature signal 365, it may process the signals using several alternate process embodiments. One process embodiment is to sequentially compare each of the time domain digitized sensor signature signals 365 with each of the pre-stored time domain signature signals in the signal library 380 using cross-correlation techniques to determine a match. Another process embodiment is to sequentially convert each received digitized sensor signature signal 365 to a frequency spectrum and then sequentially compare each of the frequency domain digitized sensor signature signals with each of the pre-stored frequency domain signature signals in the signal library 380 using cross-correlation techniques to determine a match.

An example of how ligands or antibodies may be distributed on a sensor array 310 is shown in Figure 13B. As a first example, assume that the sensor element located at column 1 330 row 1 320 of the sensor array 310 is coated with an H5 antibody (ligand) . If the sensor array 310 were exposed to an Hl antigen, a response from the sensor located at column 1 330 row 1 320 shown in Figure 14A would result. Figure 14A shows a negative signature response characteristic indicating that an H5 antigen was not detected. If the sensor array 310 were exposed to an H5 antigen, a response from the sensor located at column 1 330 row 1 320 shown in Figure 14B would result. Figure 14B shows a positive signature response characteristic indicating that an H5 antigen was detected.

As a second example, assume that the sensor element located at column 4 336 row 3 324 of the sensor array 310 is coated with an Nl antibody. If the sensor array 310 were exposed to an N5 antigen, a response from the sensor element located at column 4 336 row 3 324 shown in Figure 14C would result. Figure 14C shows a negative signature response characteristic indicating that an Nl antigen was not detected. If the sensor array 310 were exposed to an Nl antigen, a response from the sensor element located at column 4 336 row 3 324 shown in Figure 14D would result. Figure 14D shows a positive signature response characteristic indicating that an Nl antigen was detected.

Another example is where the sensor element located at column 2 332 row 1 320 of the sensor array 310 JS coated with a P24 antibody. If the sensor array 310 were exposed to a P24 antigen, a response from the sensor element located at column ? 332 row 1 320 shown in Figure 14E would result. Figure 14E shows a positive signature response characteristic indicating that an P24 antigen was detected.

It should be noted, for example, that simultaneous positive responses from a sensor element coated with H5

antibodies and a sensor element coated with Nl antibodies would indicate a presence of the H5N1 avian flu virus.

It should also be noted that although the sensor array 310 shown in Figures 13A and 13B is a four by four (4 by 4) square array, an array according to the present invention may take on numerous elements and array configurations. For example, an array may be a square array, a rectangular array, a three dimensional array, a circular array and the like. The array may also include any number of array elements.

Figure 14A - Figure 14E show typical responses from bio pore sensing elements coated with H5, Nl And P24 antibodies and being subjected to Hl, Nl, N5, H5 and P24 antigens. If one of the sensor elements shown in Figures 2-9 and 13 were coated with H5 antibodies and exposed to an Hl antigen, a response from the sensoi shown in Figure 14A would result. Figure 14A shows a negative signature response characteristic indicating that an H5 antigen was not detected. If one of the sensor elements shown in Figures 2-9 and 13 were coated with H5 antibodies and exposed to an H5 antigen, a response from the sensor shown in Figure 14B would result. Figure 14B shows a positive signature response characteristic indicating that an H5 antigen was detected. If one of the sensor elements shown in Figures 2-9 and 13 were coated with Nl antibodies and exposed to an N5 antigen, a response from the sensor shown in Figure 14C would result. Figure 14C shows a negative signature response characteristic indicating that an Nl antigen was not detected. If one of the sensor elements shown in Figures 2-9 and 13 were coated with Nl antibodies and exposed to an Nl antigen, a response from the sensor shown in Figure 14D would result. Figure 14D shows a positive signature response characteristic indicating that an Nl antigen was detected. If one of the sensor elements shown m Figures 2-9 and 13 were coated with P24 antibodies and exposed to a P24 antigen, a response from the sensor shown m Figure 14E would result. Figure 14E shows a positive signature response

characteristic indicating that a P24 antigen was detected. It should be noted, for example, that simultaneous positive responses from a sensor element coated with H5 antibodies and a sensor element coated with Nl antibodies would indicate a presence of the H5N1 avian flu virus.

Figure 15 shows an alternate structure of a bio pore sensing element 400 of Figures 2-9 with multiple ligand or antibody types 440-448 encased in a gel 420. Figure 15 depicts the FET 410 with gel 420 incorporated in the design. The FET includes a gate region 430, a source 432, a dram 434, a silicon base 436, an insulator 438 over the source 432, drain 434 and gate region 430, and cross-linkers 412 over the gate region 430. The gel 420 utilized should exhibit the properties of remaining moist, having optical sensitivity and allowing the target antigens 450 -456 to pass through the gel and to bind to the ligands or antibodies 440-448. There are several ways to place the ligands or antibodies 440-448 in operational proximity to the gate area 430. For instance, the surface of the gate 430 can be coated with ammosilane. The ligands are tethered to the amino groups via a variety of cross linkers 412, for example, disuccinimidyl suberate, Bhydroxy disuccinimidyl suberate, etc. The cross linkers 412 can be chosen with specificity to selected functional groups on the ligands or antibodies 440-448 to achieve the desired orientation. The multiple antibody bio pore sensor element may replace the sensor array 310 shown in Figure 13A in some applications. Note that with multiple ligand or antibody types 440-448, multiple target antigens 450-456 may be sensed. Therefore, the bio pore sensing element 400 having a coating of H5 and Nl antibodies would be capable of sensing the H5N1 avian flu virus. The resultant signature signal output from such a sensor element upon sensing the H5N1 virus would be a superposition of the H5 signature signal shown m Figure 14B and the Nl signature signal shown in Figure 14D, which could be

easily stored in the pre-stored signature signal library 380 shown in Figure 13 A.

Figure 16 illustrates process steps 500 required to implement a system embodiment of the present invention. The process is started 510 by cleaning the surface of the sensor 515. This may be accomplished by mechanical chiseling, laser cleaning, chemically cleaning or thermally cleaning, so as not to affect the effectiveness of the sensor elements. The surface of the sensor elements are then treated with cross-linkers 520 to provide an appropriate orientation to the ligands or antibodies. The surface of the sensor elements are then coated with a selected ligand or antibody 525 capable of uniquely sensing a specific antigen, such as an H5 antibody and an Nl antibody 530 and may be suspended in a gel. The system is then deployed to expose the sensor elements to harmful antigens 535. The system then looks for an output signature signal from the sensor elements 540. If an output signature signal is detected, it is measured 545 and converted to a digital representation 550. The output signature signal is then compared to a library of pre- stored signature signals 555 to determine if there exists a match to a known antigen 560. If no match exists 560, the system returns to sensing an output signal from the sensor elements 540. If a match is found between the output signature signal and one or more pre-stored signature signals in the library 560, an alert and notification is generated and sent to appropriate authorities 565. It must then be determined if it is necessary to clean the sensor surface 570. If the sensor surface requires cleaning 570, the process then returns to the beginning for cleaning the sensor surface 515. If the sensor surface does not require cleaning 570, the system returns to exposing the sensor elements to harmful antigens 535.

The real time detection of biological substances, to include pathogens, allergens, and microorganisms in multiple diverse environments requires the integration of several

scientific bodies of knowledge. As described, the present invention incorporates multiple technologies, demonstrates multiple functions, and has multiple applications.

Figure 17 illustrates a sensor array 710 in a typical system configuration 700, where the elements in the array may be selected from one of the embodiments of the sensor elements shown in Figure 2 through Figure 9 above or may be some other type of sensor element such as a single electron transistor. The controller 760 controls the sequencing of the selection of sensor elements within the array 710 by controlling the demultiplexer 770 to sequentially select one of four columns 730, 732, 734, 736 of the sensor array 710. When one of the four columns 730, 732, 734, 736 is selected, the controller 760 commands the multiplexer 740 to sequentially select each of the four rows 720, 722, 724, 726 of the array 710. As each row 720, 722, 724, 726 is selected by the multiplexer 740, a sample of the output at the sensor element located at the selected row and selected column is selected by the multiplexer 740. If a ligand binds with its corresponding biological target, the sample output will be a change in voltage. The sample output 755 in the form of a voltage change is sent to a threshold detector 750 to determine if the voltage change that is detected exceeds a voltage threshold which indicates that a particular biological substance is preset. In an alternate embodiment, the threshold detector logic is contained in the controller. As this sampling of the sensor elements sequence progresses, the output of each sensor element is examined to determine if a biological target is present. This process of sampling outputs from the sensors is well-known to those skilled in the relevant art of signal sample processing. In this manner, any detected voltage change that exceeds a threshold voltage indicates that a ligand and target have interacted and that the biological target is present. This process of sampling the sensor elements and indicating a threshold has been exceeded is sensed and processed in real-time.

When the threshold is exceeded a notification is sent 780 and notification and alert are generated 795.

Figure 18 illustrates process steps 800 required to implement a system embodiment of the present invention. The process is started 810 by cleaning the surface of the sensor 815. This may be accomplished by mechanical chiseling, laser cleaning, chemically cleaning or thermally cleaning, so as not to affect the effectiveness of the sensor elements. The surface of the sensor elements are then treated with cross-linkers 820 to provide an appropriate orientation to the ligands or antibodies. The surface of the sensor elements are then coated with a selected ligand or antibody 825 capable of uniquely sensing a specific antigen, such as an H5 antibody and an Nl antibody 530 and may be suspended in a gel. The system is then deployed to expose the sensor elements to harmful antigens and biological substances 835. The system then looks for a change in voltage from the sensor elements 840. If voltage change is detected, it is then compared to a threshold for that sensor element 845. If the sensor meets or exceeds the threshold 850, an alert and notification is generated 860 indicating a biological substance has been detected. If the threshold has not been exceeded 860, the next sensor element is selected and processing continues at step 840. It must then be determined if it is necessary to clean the sensor surface 870. If the sensor surface requires cleaning 870, the process then returns to the beginning for cleaning the sensor surface 815. If the sensor surface does not require cleaning 870, the system returns to exposing the sensor elements to harmful antigens and biological substances 835.

The multiple technologies may include micro miniature integrated circuitry with embedded sensing technologies that capitalize on the uniquely defining characteristics of the biological substances at hand. These characteristics include biochemical, electrochemical, physical, or thermodynamic

phenomenon. To enhance the sensitivity, nanotubes are grown in some units as an adjunct to electrodes upon which rest the ligands associated with the selected biological substances. After detection and discrimination, an alert is passed via the integrated circuitry to external receiving devices enabling a digitized alert of the biological substances' presence.

The units are multifunctional. Their functions include: detection, discrimination, amplification, digitizing, filtering, discrimination, energy acquisition from the environment, communication between units and to external routers and controllers, and network based sharing of information. This multiple functionality is possible because state-of-the-art biochemistry, information technology, and integrated circuitry are combined in such a way as to build a synergistic system oriented to the defining characteristics of the biological substances .

As can be appreciated, the individual sensor units and groups of sensor units can be utilized in many different types of environments and can be affixed to many different types of objects. These environments and objects could include their use in blood transfusion operations and blood plasma collection and storage operations as well as being employed with syringe needles. The sensor units could be attached to various types of gloves, such as used in surgery and drawing blood made from rubber and rubber substitutes. Similarly, condoms constructed from rubber and rubber substitutes and other pregnancy prevention devices could also have sensor units being attached thereto.

Various objects provided in a patient's room affixed to bedside point-of-care diagnostics, intensive care locations and hallways could also be utilized as a base for the individual sensor units. Furthermore, various HVAC ventilation systems and equipment could be provided with a plurality of sensor units as well as sensor unit groups. This would also include air moving equipment as well as local air filtration equipment, patient clothing and dressings, bed services, benches and other furniture

as well as face masks used by clinicians and patients. Furthermore, the present invention could be employed in toilet facilities for real time urine and excrement analysis or applied to the service or inside of dental and other human prosthetic fixtures. Furthermore, the present invention could be utilized in the animal or pet as well as fish environment.

The present invention has application in the food handling industry to include services of food processing equipment, conveyors, processing rooms, containers, silverware and other equipment including the inside surfaces of cans and containers, storage facilities and transportation equipment. The present invention has application in all aspects of the food chain, such as farms, food sources, waste management and packing houses .

The present invention has application in conjunction with organic materials used to manufacture produces such as leather products, cloth products and plastic products.

The present invention further has application in monitoring places in which the population gathers, such as train stations, airports, bus stations, offices, tunnels, bridges, terminals, distribution centers, stadiums, cafeterias, restaurants, bars and governmental facilities. The present invention would have application to be used in tickets, badges or passports or other identification documentation.

The present invention would also have application with units used in cabins of airplanes, train carriages, water craft, hovercraft, cars, trucks, and similar types of conveyances.

Given this disclosure, alternative equivalent embodiments as well as other uses will become apparent to those skilled in the art. These embodiments and further uses are also within the contemplation of the invention.