CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/337,871 , entitled “Impedimetric Biosensor Device for Detection and Quantification of Biochemical and Biological Reactions or Interactions to Quantify and Identify Biomarkers” and filed on May 3, 2022. The complete disclosure of said provisional application is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable

BACKGROUND

The advances made by medicine are evident, including diagnostic techniques derived from modern clinical and biological analysis equipment. Among these advances is the glucometer which, with its portability, reduced size and ease of use, popularized glucose dosage and improved the quality of life of millions of people with diabetes.

Currently, diagnostic technologies point to the development of analysis systems with the capacity to work with small amounts of sample, miniaturization, cost reduction, usability and accessibility of any analytical system. As they allow the automation of analytical processes, these small devices are the solution to solve problems of miniaturization and scalability of sensors developed for specific purposes. Point-of-care tests (POCT) are simple medical tests that can be performed at the bedside. Their simplicity is based on new technologies developed in recent years. The idea behind POCT is to bring the test conveniently and immediately to the patient. This increases the likelihood that the patient, physician, and care team will receive the results quicker, which allows for better immediate clinical management decisions to be made.

A biosensor is a device in which the material of biological origin, such as a protein, enzyme, organelle, animal or plant tissue, microorganism, antigen or antibody, nucleic acids, lectin, among others, is immobilized next to a suitable transducer. Based on the transducer used, the biosensor can be classified as electrochemical (potentiometric, amperometric and conductometric), optical (luminescence measurement, fluorescence, ellipsiometry, etc.), or mass detector (relates the oscillation of the frequency of piezoelectric crystals with variation of the dough). Depending on the type of interaction that occurs between the substance to be determined and the biological material, the biosensor is classified as catalytic or affinity.

An immunosensor is a type of biosensor based on the immunological reaction, and the antigen or antibody is immobilized on the surface of the transducer. Thus, several types of immunosensors can be built based on the type of transducer used. Biological markers are very useful in the diagnosis, treatment and monitoring of disease evolution. They can be used to monitor a certain clinical condition of inflammation or to assess the coevolution of the disease in relation to the treatment applied. They can still be used in predictive medicine and are widely used in determining when a patient will suffer a heart attack or stroke, as in the case of monitoring cytokine IL-6 and IL-10 in elderly patients. An impedimetric biosensor is one type of biosensor. In an impedimetric biosensor, the change in resistance of the sensor surface is measured before and after the reaction or interaction with the biological target. The electrical measurement is made by applying an alternating current signal to the surface and measuring the change in the phase of the generated alternate current. This change is called impedance and is an indirect measure of the surface resistivity.

There is a demand for the development of rapid, quantitative tests with high sensitivity and specificity for the quantification of molecular markers (e.g., cytokines) in the blood of patients with chronic diseases (e.g., autoimmune diseases). Patients with these diseases are treated with various therapeutic modalities based on the clinical conditions of the patients, which are extremely variable. The design of a portable, easy-to-use, highly sensitive and specific immunosensor will contribute to direct more effective treatment of autoimmune diseases in which therapies with monoclonal antibodies are used to target inflammatory cytokines. In addition to the high cost of these immunobiological drugs, the administration of inadequate doses makes the patient immunosuppressed and subject to infections and other serious pathologies during treatment. Accordingly, fast and low-cost POCT quantification methods can be the key to a fast, personalized and quality treatment for several diseases. DISCLOSURE OF THE INVENTION

The present invention is directed to an impedimetric biosensor device for detecting and/or quantifying biomarkers of medical, pharmaceutical, biotechnological and research interest such as amino acids, nucleic acids, microRNAs, carbohydrates, proteins, enzymes, antibodies, cytokines, interleukins, growth factors, cancer biomarkers, bacteria, viruses, and any kind of cells, biological tissues, and biological molecules that may indicate the presence of an infection, a cancer or a toxic substance. The device aims to assist in the diagnosis, prognosis, and treatment of diseases with detection in samples or in situ of total blood, blood plasma, blood serum, saliva, tears, sweat, urine and any kind of liquid from human beings through reactions or interactions with the biological element layer on the biosensor. One of the biological layers that can be used is an antibody layer which can be immobilized on the surface of the sensor strip and react or interact with the target. The biosensor device measures the change in the electrochemical impedance of the surface of a sensor strip after its reaction with the target. The sensor strips are inserted into the biosensor device and a drop of the sample is dripped onto the sensor region of the strip. The embedded software transforms the electrical signal measured into the concentration and/or the presence of the target. The applications already developed are: the interleukins human IL-1 -beta and human IL-6, the cytokine human TNF-alpha, the cytokine human INF-y, and the protein biomarker of COVID-19 infection — the Spike (S) protein from the SARS-COV-2 virus. More specifically, in one embodiment, the present invention is directed to an impedimetric biosensor device that comprises an electronic analysis unit and a test strip. The test strip is configured to receive a drop of a sample and to bind a target if present in the sample. The electronic analysis unit is configured to apply a current to the test strip and to measure a first impedance of the test strip and a second impedance of the test strip. The electronic analysis unit is operable to convert a measured difference between the first impedance and the second impedance to a concentration of the target in the sample.

In another embodiment, the present invention is directed to a method of detecting a concentration of a target in a sample. The method includes the steps of inserting a test strip into an electronic analysis unit, applying a first current from the electronic analysis unit to the test strip and measuring a first impedance of the test strip by the electronic analysis unit, adding a drop of the sample to the test strip, applying a second current from the electronic analysis unit to the test strip and measuring a second impedance of the test strip by the electronic analysis unit, and converting by the electronic analysis unit a measured difference between the first impedance and the second impedance to a concentration of the target in the sample.

These and other features, objects and advantages of the present invention will become better understood from a consideration of the following detailed description of the preferred embodiments and appended claims in conjunction with the drawings as described following: BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating the steps in the production of the strip using a carbon-paste electrode. Step (i) is creating a carbon-paste electrode modified with linkage agent, Step (ii) is immobilization of a specific biological layer (e.g., a specific antibody), Step (iii) is the addition of the blocking agent to prevent non-specific binding; and Step (iv) is addition of the sample.

FIG. 2 is an illustration of the biosensor device of the present invention.

FIGS. 3A-3E are schematics of the electrical circuit of the biosensor of the present invention.

FIG. 4 is a hardware block diagram of the biosensor of the present invention.

FIG. 5 is a software block diagram of the biosensor of the present invention.

FIG. 6 is a potential versus current graph for three non-modified commercial screen-printed electrodes (SPE).

FIG. 7 is a potential versus current graph for three nanocarbon/glutaraldehyde modified screen printed electrodes (SPE+G).

FIG. 8 is a potential versus current graph of a reversibility study on three nonmodified commercial screen-printed electrodes (SPE).

FIG. 9 is a potential versus current graph of a reversibility study on the nanocarbon/glutaraldehyde screen-printed electrodes (SPE+G).

FIGS. 10A-10B are graphs showing the relationship between ip and the square root of the scanning speed in non-modified commercial screen-printed electrodes (FIG. 10A) and nanocarbon/glutaraldehyde screen-printed electrodes (FIG. 10B). FIG. 11 is an electrochemical impedance spectroscopy (EIS) graph of the different layers of the electrode strip.

FIG. 12 is an EIS graph showing formation of the blocking layer with BSA at several reaction times.

FIG. 13 is an EIS graph showing the impedance for the modified electrode strip with the addition of saliva and different amounts of S proteins.

FIG. 14 is a graph showing the electrical impedance as additional S protein is added to the electrode strip.

FIG. 15 illustrates the Standard Curve for S Protein with an increase in the Z’(Q) as the concentration increases

FIG. 16 is a graph showing the change in resistance over time upon the addition of a positive saliva sample.

FIG. 17 is a graph showing the change in resistance over time upon the addition of a negative saliva sample.

FIG. 18 is an illustration of an embodiment of the biosensor device of the present invention.

FIG. 19 is an illustration of an embodiment of the biosensor device of the present invention.

FIG. 20A is an image of a sensor strip of the present invention.

FIG. 20B is an image of an embodiment of the biosensor device of the present invention.

FIG. 21 is a schematic showing the process of using the biosensor device and test strip of the present invention with saliva without dilution. A test kit may include the biosensor device, test strip, and a swab. The patient rubs the swab in their mouth and then the swab is rubbed onto the strip. The strip is inserted into the biosensor device and the analysis is performed. Additional swabs and strips may be used with the same biosensor device when additional testing is desired.

FIG. 22 is a schematic showing the process of using the biosensor device and test strip of the present invention with saliva and a dilution step. A test kit may include the biosensor device, test strip, swab, and extracting solution. The patient rubs the swab in their mouth and then dips the swab several times into the extraction solution. A few drops of the extraction solution containing the sample is poured on the strip. The strip is inserted into the biosensor device and the analysis is performed. The process is repeated with a new swab, new strip, and new extracting solution when the next test is performed.

FIG. 23 is a schematic showing the process of using the biosensor device and test strip of the present invention with blood. A test kit may include the biosensor device, test strip and a lancet. The patient pricks their finger with the lancet to draw blood. A drop of blood is placed on the strip. The strip is inserted into the biosensor device and the analysis is performed. Additional strips may be used with the same biosensor device when additional testing is desired.

FIG. 24 is a flowchart showing the step-by-step process for determining the concentration of a target in a sample using the biosensor device and test strip of the present invention. DETAILED DESCRIPTION OF THE INVENTION

With reference to FIGS. 1 -24, the preferred embodiments of the present invention may be described. The present invention is directed to an impedimetric biosensor device for detection and quantification of biological reactions and/or interactions between a target and the bioactive material layer of the nanocarbon/glutaraldehyde electrode test strip. The biosensor does not require a laboratory, trained personnel, or expensive equipment. Instead, it can be used by the health professional or by the patient himself. The biosensor offers better precision, accuracy and faster results than conventional methods, such as PCR and ELISA. For each particular biological layer, a specific alternating voltage signal and frequency is applied to the strip and impedance is measured as a function of time at a constant AC voltage frequency.

The biosensor 10 includes an electronic analysis device (or unit) 12 and an electrode test strip 14, as shown in FIG. 2. The electronic analysis device includes a transducer and electronics for processing and converting the electrical signal applied to an electrical response. The electronic analysis device also includes a display for showing the results of the analysis. The applied signal consists of an alternating current (AC) of controlled voltage on the electrode surface generating an electrical response signal. This generated signal suffers an opposition that a surface circuit presents to a current when a voltage is applied, which is a phenomenon known as impedance. The electronic circuit measures the phase difference of the applied wave and translates this as surface resistance. Each modification of the surface of the electrode test strip caused by the presence of different concentrations of analyte causes a change in the surface resistance value that can be read as concentration using an embedded software. The electronic analysis device works by connecting to a power supply through an interface/connector. In one embodiment, the power supply is a cellphone but the device also has it own battery for power. The device is turned on and the calibration is activated by pressing a button or alternatively moving the calibration switch to the upward position. The device performs the calibration next to an internal resistor. After the calibration, the display of the device shows the message “Calibrated System.” After calibration, 10uL of the sample is added on the surface of the strip after the strip is slid into the analysis device, the start button is pressed, and the analysis is performed. After the analysis is performed, the analysis device displays the value of the measured substance in pg/mL or another appropriate unit.

The biosensor device works by analyzing impedance changes in the surface of the strip after the contact with the sample using three electrode systems. The device performs the application of alternating electrical voltage with a specific amplitude. To calculate the electrical value for determining the concentration, the total Z (Q) value (electrical impedance) calculated in the data collection of the value corresponding to the largest negative phase angle is used. The total Z (Q) value is applied to the standard curve, which is saved in the device's programming, for the analyzed cytokine and the concentration value is shown in the device display in pg/mL or another appropriate unit.

A flowchart showing the step-by-step process for determining the concentration of a target in a sample using the biosensor device of the present invention is shown in FIG. 24. The electrode test strip is first slid into the electronic device. An electrical signal is then applied by the electronic device to the surface of the test strip and the initial surface impedance of the test strip is measured. During this time, the display of the electronic device shows that the device is calibrating. After the calibration step, a drop of the sample (e.g., blood, saliva or any extraction solution) is added to the test strip. The electronic device applies an electrical signal again to the surface of the test strip and a new impedance value is measured. Embedded software in the electronic device, which uses a pre-programmed analytical curve, calculates the concentration of the target in the sample based on the change in impedance measured. The concentration of the target is then showed on the display of the electronic device.

Hardware: The biosensor device includes embedded hardware in the electronic analysis device. The device uses a sine wave signal generated by a AD5933 impedance converter system. Since the inventors only have control over the frequency value, they must manipulate the signal generated by the integrated circuit through amplifiers to obtain the desired amplitude for each type of reading. A schematic description of the electrical circuit in the device is shown in FIGS. 3A-3E. A hardware block diagram of the device is shown in FIG. 4, and a list of hardware components are listed below:

ATMEGA328P - 1 unit - Microcontroller AD5933 - 1 unit - Direct Digital Synthesizer

DS1077LZ-40 - 1 unit - ECONOSCILLATOR/DIVIDER NE5532D - 2 units - Operational Amplifier ADG1604 - 1 unit - Analog Multiplexer Single LMC7660 - 1 unit - Charge Pump LM7805 - 1 unit - Standard Regulator LM1117 - 1 unit - LDO Regulator BS138 - 2 units - Jump Oscillator 16MHz - 1 unit SERIAL MODULE - 1 unit Display LCD - 1 unit GreenLED 5mm - 2 units Resistor 1 k - 4 units Resistor 10k - 8 units Resistor 20k - 2 units Resistor 47k - 4 units

Capacitor 10OnF - 7 units Capacitor 10uF - 4 units Capacitor 22pF - 2 units Capacitor 100uF - 1 units

Software: The Arduino IDE was used to program the Atmel ATMEGA328P microcontroller by utilizing I2C serial communication for establishing a channel between the microcontroller and the AD5933. Then, the ATMEGA328P microcontroller is programmed via address registers, which were assigned an initial frequency, increment frequency and number of increments. As a result, a response as a Real value and an Imaginary value were obtained. Based on the values returned by the AD5933 direct digital synthesizer, the impedance value of what was measured was calculated.

Screen printed electrode strip: The biosensor device uses a sensor strip on which the biological active layer interacts with target in the sample . The sensor strip is specific to the target. For example, the strip must be modified to detect the S protein from Sars-Cov-2. In order to demonstrate the innovativeness of the biosensor, two applications for the device are discussed below.

Potential window: Potential window is the range of useful potential of the electrode (i.e. , the range where there is no surface degradation or parallel reactions). To characterize the commercial electrode potential window, the experiments described herein were conducted. Solutions of 100mL of 0.1 M potassium ferricyanide, 100mL of 0.1 M potassium ferrocyanide, 100mL of 0.5 M sulfuric acid, and 100mL of 0.1 M glutaraldehyde were prepared. To prepare a carbon paste, 0.1 g of nanoparticulate carbon (e.g., Vulcan carbon powder) was added to a small 10 mL beaker on an analytical balance. After weighing the carbon, the mixture was macerated with a glass stick until it formed a fine powder and then 100 uL of 5% glutaraldehyde solution was added. Then mineral oil was added every 4 drops until it was incorporated into the carbon powder and formed a paste. Only 8 drops of mineral oil should be added for each 0.1 g of carbon.

The carbon paste was then added over the working electrode on a commercial electrode. Three non-modified commercial electrodes and three nanocarbon/glutaraldehyde modified electrodes described above were tested. The experimental conditions were as follows: Initial Potential of 0V, Potential range of -1 to 1 V, Step potential of 0.025V, Scan rate of 0.05V/S, and Scan number of 4 scans.

The results of the testing of the three non-modified commercial screen-printed electrodes (SPE) are shown in FIG. 6, which show that the SPEs have no significant electrochemical reaction on the range of 0.3 V to 1.0 V. The results of the testing of the three nanocarbon/glutaraldehyde modified screen printed electrodes (SPE+G) are shown in FIG. 7, which shows that the SPE+Gs have no significant electrochemical reaction on the range of 0.3V to 1 V. This means that there are no parallel reactions.

Reversibility and Active Area of the Modified Electrode: In order to determine the electrode area and whether the electrode can keep the reaction of the ferri- ferrocyanide redox pair reversible, which indicates that the migration, diffusion and double electrical layer phenomena are occurring correctly over the electrode surface, the three non-modified commercial electrodes and three nanocarbon/glutaraldehyde modified electrodes were utilized in the tests. The electrode being reversible means that the layers are sensible to only the analyte. The experimental conditions were as follows: Initial Potential of 0V, Potential range of -0 to 1V, Step potential of 0.005V, Scan rate of 0.005 V/s, 0.01 V/s, 0.025 V/s, 0.05 V/s, 0.075 V/s, 0.1 V/s, and 0.15 V/s, and Scan number of 2 scans.

The results of the reversibility study on the three non-modified commercial screen-printed electrodes (SPE) are shown in FIG. 8, which shows that the reaction was reversible in all scan rates and there were no changes in the reaction redox potential. The results of reversibility study on the nanocarbon/glutaraldehyde screen-printed electrodes (SPE+G) are shown in FIG. 9, which also shows that the reaction was reversible in all scan rates and there were no changes in the reaction redox potential. Both results show potential difference between reduction and oxidation peaks of less than 59/2mV and IPC and IPA ratios that are close to one, which demonstrates a reversible reaction. It was observed that the potentials occur at 0.25V (oxidation) and 0.35V (reduction). As a result, the inventors used the 0.25V as potential applied during measurement and 0.1V as potential amplitude in the impedance experiments. In both FIG. 8 and FIG. 9, the bottom line at 0.25V is the 150mV/s line, the second line from the bottom is the 100mV/s line, the third line from the bottom is the 75 mV/s line, the fourth line from the bottom is the 50 mV/S line, the fifth line from the bottom is the 25 mV/s line, the sixth line from the bottom is the 10 mV/s line, and seventh line from the bottom is the 5 mV/s line. Conversely, the top line at 0.35V is the 150mV/s line, the second line from the top is the 100mV/s line, the third line from the top is the 75 mV/s line, the fourth line from the top is the 50 mV/S line, the fifth line from the top is the 25 mV/s line, the sixth line from the top is the 10 mV/s line, and seventh line from the top is the 5 mV/s line.

The curves obtained by the relation between ip and the square root of the scanning speed in non-modified commercial screen-printed electrodes and nanocarbon/glutaraldehyde screen-printed electrodes are shown in FIGS. 10A and 10B, respectively. The linearity of the results demonstrates the reversibility of the reaction. By using the curve angular coefficient, it is possible to calculate the surface area using the Randles-Sevcik equation: ip = ± 2.686x1 O ⁵ n ^3/2 CoD ^1/2 v ^1/2 A, where ip is current peak, n is the number of electrons in the reaction, A is the surface area, D is the diffusion coefficient, Co is the species concentration, and v is the scan rate. The equation may be rearranged as: where iplv ^1/2 is the angular coefficient obtained. The electrode area is 100.17 cm ² in the non-modified commercial screen-printed electrodes and 135.73 cm ² in the Vulcan nanocarbon/glutaraldehyde screen-printed electrodes. This increase in the electrode area shows the modification of the commercial screen-printed electrodes into an active electrode functionalized with glutaraldehyde with the capacity to bind and/or immobilize biological molecules or structures, such as amino acids, nucleic acids, microRNAs, carbohydrates, proteins, enzymes, antibodies, cytokines, interleukins, growth factors, cancer biomarkers, bacteria, viruses, and any kind of cells, biological tissues, and biological molecules. Also, knowing the electrode area is useful to determine the amount or size of a biological active layer to be added to the surface.

Electrode characterization by impedance: The electrode strips were also tested to determine their electrode impedimetric behavior. First, 50pL of Ferri- Ferrocyanide solution was added to the surface as an electrochemical probe of the non-modified SPE electrode. A 0.25V DC potential was applied changing the frequency from 10Hz to 100kHz. The nanocarbon/glutaraldehyde modified electrode was prepared by the addition of the carbon paste as described above. After the initial characterizations of functionality of the strips, the specific reactions to turn the nanocarbon/glutaraldehyde electrode strip into a biosensor specific to reactions with S Protein of SARS-COV-2 were made. In the first step, the immobilization was made through the reaction on the surface of the nanocarbon/glutaraldehyde modified electrode strip with the addition of 50 uL of a solution with monoclonal antibody against S protein of SARS-COV-2 at the concentration of 100 ug/mL with 30 minutes of reaction. In the second step, to avoid unspecific reactions or more immobilization of nonspecific proteins or biological substances in the step of analyses of real samples, the surface of the nanocarbon/glutaraldehyde electrode strip was blocked through the reaction with BSA (bovine serum albumin) by the addition of 50 pL of 0.5% and 5% BSA protein with the reaction time of 10, 20 and 60 minutes. All layers were characterized by impedance by the addition of 50pL of Ferri-Ferrocyanide solution on the electrode surface. A 0.25V DC potential was applied changing the frequency from 10Hz to 100kHz.

FIG. 11 is an electrochemical impedance spectroscopy (EIS) graph of the different layers of the electrode chip. SPE+C+G+A refers to the modified electrode with an active layer of antibodies, while SPE+C+G+A+B refers to the modified electrode with active layer of antibodies and an active blocking agent layer. The increase of the semi-circle shape in the top line (denoted by downward pointing triangles) shows the increment of surface resistance after the antibody active layer addition. It occurs because the antibody links in a covalent reaction with the nanocarbon/glutaraldehyde active layer. The blockade layer with BSA protein shows an impedimetric behavior showing no Warburg behavior. FIG. 12 is an EIS graph showing formation of the blocking layer with BSA at several reaction times.

Standard curve by protein spike in saliva: The inventors sought to determine whether there was a linear analytical response between S protein concentration and impedance. For the linear analytical response studies, the inventors used saliva samples from individuals not infected with COVID. Using a swab, a smear of the gum region is obtained from the individuals. The swab was rinsed in a phosphate- buffered saline (PBS) solution. Solutions of 1 , 10, 100 and 1000 pg/mL of S protein were prepared using the saliva solution to imitate a sample from COVI D-positive individual. The EIS experiments were performed by the addition of 50pL of Ferri- Ferrocyanide solution on the electrode surface. A 0.25V DC potential was applied changing the frequency from 10Hz to 100kHz.

FIG. 13 is an EIS graph showing the impedance for the modified electrode strip with the addition of saliva and different amounts of S proteins. In the impedance spectra, a significant increase in the response with the addition of saliva on the electrode surface was observed. This is due to the proteins contained in the samples that increase the resistivity of the surface. When adding S protein to the electrode surface, a significant decrease in the resistivity of the electrode surface was observed. In other words, a qualitative response of the electrode to S protein was observed. In FIG. 13, at 6000 Ohms on the x-axis, the bottom line is the SPE+C+G+A+Saliva l OOpgmL line, the second line from the bottom is the SPE+C+G+A+Saliva 1 pgmL line, the third line from the bottom is the SPE+C+G+A+Saliva 1000 pgmL line, the fourth line from the bottom is the SPE+C+G+A+Saliva 10 pgmL. The top line is the SPE+C+G+A+Saliva line.

Standard Curve of SARS-COV-2 biosensor: The inventors then performed another method of analyses to obtain the standard curve. The standard curve is used to mathematically transform the impedance value measured to a concentration. This calculation is made by the software embedded in the electronic biosensor device and made available on the reader of the device. The inventors used a fixed frequency measured through time to analyze the electrode strip. The test used EDC 0.25V, EAC 0.1 V and a frequency of 1000 Hz. The tests were conducted between 180 seconds and 3600 seconds. To determine the Standard Curve, 50uL of Ferri- Ferrocyanide solution was added to the electrode surface and the measurements were then conducted. The inventors waited around 100 seconds to stabilize the system, and then a 1 uL of solution of 10 fg/mL of PBS solution with S Protein from SARS-COV-2 was added to the electrode surface. After waiting about 60 more seconds, the inventors added 1 uL more each time until 26 uL was reached. FIG. 14 is a graph showing the electrical impedance as additional S protein is added to the electrode strip. Each downward spike (i.e. , up-down) corresponds to 1 uL of S protein added. The increase of impedance Z’(O) shows the increase of surface resistance after the S Protein solution additions. It occurs because the antibody reacts with the S Protein. After the experiments the inventors extracted the data and made the Standard Curve correlations.

FIG. 15 illustrates the Standard Curve for S Protein with an increase in the Z’(Q) as the concentration increases and with a correlation coefficient of R ² =0.9904 and an equation correlation as follows: y = 0.0274X + 1371 , where y corresponds to the response of the change on the surface sensor and x corresponds to the concentration. Alternatively,

Response = 0.0274*(Concentration) + 1371 Concentration = (Response -1371 )/0, 0274

Validation of the sensor in real samples: To test the electrode strips, 40 saliva samples were used. All the saliva samples were mixed 1 :1 in a solution of 10% SDS to inactivate the virus. The inventors then added 50uL of Ferri-Ferrocyanide solution to the electrode surface and waited 60 seconds for the electrode surface to become stable. After the stabilization, the inventors added 5uL of saliva sample and the impedance surface change was measured.

FIG. 16 is a graph showing the change in resistance over time. In this particular sample (Sample 5 - a positive control), 5uL of saliva sample was added to the electrode strip at 153 seconds. The increase of impedance Z’(Q) shows the increase of surface resistance after the additions of the sample. It occurs because the antibody on the electrode strip reacts with the S Protein from the saliva or nasopharyngeal sample and makes a more resistive system.

FIG. 17 is a graph showing the change in resistance over time. In this particular sample (Sample 23 - a negative control), 5uL of saliva sample was added to the electrode strip at 258 seconds and the signal decreases. The decrease in the Z’(Q) indicates that the surface resistance has changed, and this behavior indicates a negative sample. The electrode strip is now less stable and is becoming more conductive.

From extracting the data from sample 5, it was possible to determine from the Standard Curve of FIG. 15 that a value of 27,445.2 ag/mL corresponds to 27.44 fg/mL of S protein in the sample. Taking in consideration that the samples were diluted to 1 :1 to a solution to inactive the virus, it was determined that the actual concentration was 54.88 fg/mL.

The 40 saliva samples were divided into three groups. The first group with 15 samples was made of positive RT-PCR patients with symptoms at the time of collection. The second group was made of 21 patients that had negative RT-PC results but had mild symptoms, and the third group was made of 4 patients with negative RT-PCR results with no symptoms. The first sample group presented a positive result for RT-PCR, positive for the test of the present invention, and positive for the IgG tests and, in some cases, the IgM tests. In the second sample group, 21 samples showed a negative RT-PCR result, but a positive response to the test of the present invention with an intensity response signal 10-20 times lower than the first group with positive RT-PCR samples. Two IgG tests and/or IgM tests also showed a positive result. Thus, the inventors believe that these samples had a low viral load, which can generate false-negative results for RT-PCR. The third group showed negative results in RT-PCR, a negative response to the test of the present invention, and a positive response to IgG and/or IgM.

APPLICATIONS OF THE PRESENT INVENTION

Application #1 : Biosensor device for monitoring the evolution of the inflammation of patients infected with COVID-19 through the detection and quantification of inflammatory cytokines. The mechanisms of COVID-19 disease involve the infection of cells responsible for the chain response of inflammation in the human organism, thereby leading to severe inflammation. The main biomarkers that indicate the correlation of COVID-19 with the clinical condition of patients are TNF-a, IL-6 and IL-1 [3. These inflammatory markers correlate in the activation of the inflammatory response and in the activation of the body's defense cells. Therefore, fast and low-cost quantification methods can be the key to a fast, personalized and quality treatment for COVID-19. It therefore is an objective to develop a point-of- care biosensor device for quantifying biomarkers to assist in prognosis and treatment COVID-19. This device of the present invention can quantify the cytokines TNF-alpha, IL-1 (3 and IL-6 and, through the application of a mathematical model, generate decisive information in the prognosis and treatment Covid-19. The functionality and benefits of the device are shown in FIG. 18.

Basic Principles of the Device: The biosensor device consists of an electrochemical platform with embedded systems and disposable sensor strips that quantify the three cytokines in a single sample. Tests results come in less than one minute. The device is tested and validated in the laboratory. A discussion of the hardware, software and sensor strips are discussed above. An illustration of the device is shown in FIG. 19. An image of a sensor strip is shown in FIG. 20A, and an image of the device is shown in FIG. 20B.

How can the device help? COVID-19 disease, caused by infection with SARS-CoV-2, is related to a series of physiopathological mechanisms that mobilize a wide variety of biomolecules, mainly immunological in nature. In the most severe cases, the prognosis can be markedly worsened by the hyperproduction of mainly proinflam matory cytokines, such as IL-1 , IL-6, IL-12, IFN-y, and TNF-a. Cytokines, such as interleukin-1 -b, interleukin-6 and tumor necrosis factor, guide neutrophils from the blood vessels to the infected tissue. These cytokines can increase heartbeat, elevate body temperature, trigger blood clots that trap the pathogen and stimulate the neurons in the brain to modulate body temperature, cause weight loss, and elicit other physiological responses that have evolved to kill the virus. When the production of these same cytokines is uncontrolled, immunologists describe the situation as a “cytokine storm.” During a cytokine storm, the blood vessels widen further (vasolidation), leading to low blood pressure and widespread blood vessel injury. The storm triggers a flood of white blood cells to enter the lungs, which in turn summon more immune cells that target and kill virus-infected cells. The result of this battle is a stew of fluid and dead cells, and subsequent organ failure. The cytokine storm is a centerpiece of the COVID-19 pathology with devastating consequences for the host. When the cells fail to terminate the inflammatory response, production of the cytokines make macrophages hyperactive. The hyperactivated macrophages destroy the stem cells in the bone marrow, which leads to anemia. Heightened interleukin 1 b results in fever and organ failure. The excessive tumor necrosis factor causes massive death of the cells lining the blood vessels, which become clotted. At some point, the storm becomes unstoppable and irreversible. One strategy behind the treatments for COVID is based in part on breaking the vicious cycle of the “cytokine storm.” This can be done by using antibodies to block the primary mediators of the storm, like IL-6, or its receptor, which is present on all cells of the body. Knowing exactly the concentration of inflammatory or anti-inflammatory cytokines can bring key information about the evolution of the disease, the effect of treatments, and the mechanisms of action of the virus. In addition to helping with the COVID inflammatory process, the biosensor of the present invention can help with autoimmune and/or inflammatory diseases that originate or are correlated with cytokine imbalance, such as cancer, bacterial infections, viral infections, parasitic infections, sepsis, Type 1 and 2 diabetes, Lupus, Systemic Lupus Erythematosus (SLE), psoriasis/psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, Addison’s disease, Graves’ disease, Sjogren’s Syndrome, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease, among others.

Application #2: Biosensor device for indirect detection of SARS-COV-2 by measuring the levels of spike protein. Real-time reverse transcription- polymerase chain reaction (RT-PCR) is the primary means of diagnosis of SARS- COV-2. Cases of asymptomatic COVID-19 transmission have been reported. Molecular diagnosis using real-time RT-PCR takes at least 3 hours and needs a specific machine, trained technicians and a laboratory. In addition, the RT-PCR RNA preparation step can affect diagnostic accuracy. Thus, it is very important to develop sensitive diagnostic methods that directly detect viral antigens in clinical samples without sample preparation in a fast and accurate diagnosis of COVID-19. It therefore is an object to develop a point-of-care biosensor device for quantifying S protein to indirectly detect the SARS-COV-2.

Basic Principles: The spike (S) protein is the sole viral membrane protein responsible for cell entry. It binds to the receptor on the target cell and mediates subsequent virus-cell fusion. Each virus has approximately 100 units of S protein. As described above, the inventors developed a system that consists of an immunosensor device with embedded software that reads the changes in the surface of the sensor strip due to the presence of protein S in the sample. Each reading lasts two minute and uses a sample of saliva removed with a cotton swab and solubilized in an extraction solution. The device is prepared to be point-of-care with one sample reading at a time but can also be adapted to read several samples. The device is able to quantify S protein in amounts at atto-gram per milliliter of samples, thereby indirectly detecting a very small concentration of viruses such as that found in asymptomatic patients. The ease of testing and its point-of-care feature meets the need for mass testing. The process of using the device is shown in FIG. 21.

Novelty of the Biosensor Device: The benefits of the biosensor device of the present invention include: (1 ) the device uses impedance measurement; (2) the device does not need an electrochemical impedance spectroscopy measurement; (3) the device provides results in minutes; (4) no sample preparation is required before using the device; (5) the device s a point-of-care device; (6) no training is required to operate the device; (7) the device alleviates the need for a laboratory; (8) the device can be used at home, office or beside the bed in the hospital; (9) the device does not require any reagents; and (10) the device uses a specific signal amplitude for each different substance.

Advantages of the Biosensor Device Over Prior Art: The biosensor device also includes the following advantages of the prior art: (1 ) much lower cost by measure; (2) more sensitive; (3) less susceptible to cross reactions; (4) uses impedance instead of amperometry which does not degrade the reaction surface during measurement; and (5) simultaneously determines the presence of three cytokines.

The present invention has been described with reference to certain preferred and alternative embodiments that are intended to be exemplary only and not limiting to the full scope of the present invention as set forth in the appended claims.