Functionalization of antibodies on sensor strip for oral cancer detection

Abstract

Various examples are provided related to functionalization for sensing of antibodies. In one example, a method for functionalizing a test strip includes cleaning a gold surface of an electrode of a test strip; submerging the cleaned gold surface in a 3-Mercaptopropanyl-N-hydroxysuccinimide ester solution; cleaning channels of the test strip; injecting an antibody into the channels; sealing the injected antibody in the test strip; storing the sealed test strip at a controlled temperature; and deactivating unfunctionalized groups. In another example, a method includes connecting a functionalized test strip to a strip connector; generating a test pattern that is passed through the functionalized test strip and input into a gate terminal of a MOSFET in a readout block; converting a readout signal into a frequency signal; and counting the frequency signal at fixed intervals to generate a counter output, and displaying a sample concentration based upon the counter output.

Description

FUNCTIONALIZATION OF ANTIBODIES ON SENSOR STRIP FOR ORALCANCER DETECTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional application entitled “Functionalization of Antibodies on Sensor Strip for Oral Cancer Detection” having serial no. 63 / 639,036, filed April 26, 2024, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under DE025001 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Oral leukoplakia (OL), as defined oral by the World Health Organization, is a condition where a white patch of oral mucosa cannot be erased or classified otherwise, either clinically or histopathologically. OL is the most common oral potentially malignant disorders (OPTMs), defined as any oral mucosal abnormality that is associated with a statistically increased risk of developing oral cancer. Oral cancer stands as a considerable global public health concern, constituting a substantial proportion of overall cancer incidence. Annually, millions of individuals receive diagnoses of oral cancer, contributing significantly to the global cancer burden. The American Cancer Society reports an anticipated 54,540 new cases of oral cavity or oropharyngeal cancer in 2023, with an estimated 11,580 fatalities attributed to these specific cancer types in the same year. The utilization of tobacco and the consumption of alcohol are recognized as established risk factors for oral cancer, with approximately 75% of all reported cancer cases being linked to these specific exposures. Additional factors, including human papillomavirus (HPV) infection,dietary habits, nutritional considerations, gender, and exposure to radiation, contribute to an elevated likelihood of being diagnosed with oral cancer. Cancers also result in productivity losses, with oral cancer causing an economic impact of approximately $0.74 billion in India and $112,308 in South Africa.SUMMARY

[0004] Aspects of the present disclosure are related to functionalization for sensing of antibodies. In one aspect, among others, a method for functionalizing a test strip comprises cleaning a gold surface of an electrode of a test strip; submerging the cleaned gold surface of the electrode in a 3-Mercaptopropanyl-N-hydroxysuccinimide ester (NHS ester) solution for a predefined reaction period; cleaning channels of the test strip; injecting an antibody into the channels of the test strip; sealing the injected antibody in the test strip; storing the sealed test strip at a controlled temperature for a defined storage time; and deactivating unfunctionalized groups. In one or more aspects, the cleaned gold surface of the electrode can be submerged in the NHS ester from about 15 minutes to about 2 hours. The test strip can be submerged in the NHS ester.

[0005] In various aspects, the antibody can be a monoclonal CIP2A antibody, HER2, CA125, CA15-3, anti-SARS-CoV-2 spike glycoprotein RBD antibody, anti-Cardiac troponin I antibody, type I IFNs, dsRNA, BAFF, or IL6 / IL1 beta. The antibody can be injected at a concentration in a range from about 1 mg / mL to about 1 pg / mL. The controlled temperature can be 4°C and the defined storage time is 18 hours. Deactivating unfunctionalized groups can comprise treatment with ethanolamine. The electrode can be a gold-plated electrode. Cleaning the gold surface of the electrode can comprise ozone or oxygen plasma treatment of the gold surface. Cleaning the gold surface of the electrode can further comprise application of a diluted ammonium hydroxide (NH4OH) solution or a diluted hydrogen chloride (HCI) solution. Cleaning the gold surface of the electrode can further comprise rinsing with a deionized water and drying with nitrogen.

[0006] In another aspect, a method comprises connecting a functionalized test strip to a strip connector of a portable detection device, the functionalized test strip comprising a test sample; generating a test pattern by a pattern generator of the portable detection device, the test pattern passed through the functionalized test strip and input into a gate terminal of a metal-oxide-semiconductor field-effect transistor (MOSFET) in a readout block of the portable detection device; converting a readout signal of the readout block into a frequency signal; and counting the frequency signal at fixed intervals by a counter of the portable detection device to generate a counter output, and displaying a sample concentration on the portable detection device based upon the counter output. In one or more aspects, the readout signal can be converted to the frequency signal by a voltage-controlled oscillator (VCO). The frequency signal can correspond to a voltage level of the readout signal.

[0007] In various aspects, a series of test patterns can be generated by the pattern generator, passed through the functionalized test strip and input into the gate terminal of the MOSFET of the portable detection device; and the displayed sample concentration can be based upon an average of counter outputs associated with the series of test patterns. The gate terminal can be grounded between generation of each of the series of test patterns. The antibody can be a monoclonal CIP2A antibody. The antibody can be HER2, CA125, CA15-3, anti-SARS-CoV-2 spike glycoprotein RBD antibody, anti-Cardiac troponin I antibody, type I IFNs, dsRNA, BAFF, or IL6 / IL1 beta. The antibody can be injected at a concentration in a range from about 1 mg / mL to about 1 pg / mL.

[0008] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependentclaims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIG. 1 is an image illustrating an example of a test strip, in accordance with various embodiments of the present disclosure.

[0011] FIG. 2 illustrates an example of test strip functionalization, in accordance with various embodiments of the present disclosure.

[0012] FIG. 3 illustrates examples of sample collection without and with lysis, in accordance with various embodiments of the present disclosure.

[0013] FIGS. 4A and 4B illustrate an example of printed circuit board circuitry used for functionalized test strip sensing, in accordance with various embodiments of the present disclosure.

[0014] FIGS. 5A-5C illustrate examples of the effect of capacitance on the printed circuit board (PCB), in accordance with various embodiments of the present disclosure.

[0015] FIG. 6 illustrates an example of an output voltage pulse from the PCB with different concentrations of P90 protein, in accordance with various embodiments of the present disclosure.

[0016] FIG. 7 illustrates an example of the output digital reading under different P90 protein concentrations, in accordance with various embodiments of the present disclosure.

[0017] FIG. 8 illustrates an example of output digital reading results from human sample tests with test strips functionalized by P90 antibodies, in accordance with various embodiments of the present disclosure.

[0018] FIG. 9 is a boxplot illustrating distribution of the testing results, in accordance with various embodiments of the present disclosure.DETAILED DESCRIPTION

[0019] Disclosed herein are various examples related to functionalization for sensing of antibodies. Reference will now be made in detail to the description of the embodiments as illustrated in the drawings, wherein like reference numbers indicate like parts throughout the several views.

[0020] Early detection of oral cancer is important for successful treatment. The primary diagnostic methods for oral cancer involve clinical examination, biopsy, imaging studies (X- rays, CT scans, and MRI scans), and endoscopy. Clinical examination entails visual inspection by a healthcare professional to identify visible abnormalities. Biopsy is essential for confirming the presence of cancerous cells and determining cancer type and stage. Imaging studies, including X-rays and CT scans, provide detailed images aiding in tumor identification. These common diagnostic approaches facilitate early detection, crucial for effective treatment and improved outcomes in oral cancer cases. While there are variety of diagnostic methods, they have inherent limitations. Clinical examinations may be subjective and limited to surface observations, potentially missing deeper lesions. Biopsies, while definitive, are invasive and carry a risk of sampling errors due to the small tissue sample obtained. Imaging studies can produce false positives / negatives and expose patients to radiation. Acknowledging these limitations emphasizes the ongoing need for research to refine existing methods and explore complementary approaches for more accurate and comprehensive oral cancer diagnostics.

[0021] P90, alternatively identified as KIAA1524 and Cancerous Inhibitor of PP2A(CIP2A), exhibits elevated expression levels in both oral squamous cell carcinoma (OSCC) cell lines and tissues. This protein is of considerable interest as a promising therapeutic target or a potential diagnostic marker given the relatively low levels of CIP2A expression in normal tissues. There are additional reports that implicate a positive role for proteinphosphate 2A (PP2A) in inflammatory lung diseases like asthma and chronic obstructive pilmonary disease (COPD) and in heart function. These effects can be attributed to PP2A‘s inhibitory effects on the mediators of inflammation. For the analysis of CIP2A in saliva, enzyme-linked immunoassay (ELISA) test kits are readily accessible, and the development of immunosensors utilizing carbon nanotubes has commenced. However, growing the nanotubes is time-consuming and also quite expensive.

[0022] A method for detecting CIP2A showcases high sensitivity through a transistorbased biosensor system. The P90 protein is treated with a PBS solution in this process, achieving a low limit of detection at 1015g / mL. Notably, the sensitivity of this method surpasses that of commercially available ELISA test kits. While exhibiting high sensitivity and a low limit of detection, it is noteworthy that the standard calibration solution used in this method is PBS, rather than artificial saliva. Furthermore, human sample testing was not conducted. In contrast, the study established a calibration curve using P90 protein diluted in artificial saliva. A specially designed printed circuit board (PCB) was implemented for the detection of P90 protein concentrations. The accuracy of the newly developed method was demonstrated through testing human samples obtained from oral cancer patients.Materials and Methods

[0023] Commercially available glucose test strips (Luvnshare Biomedical Inc. in Hsinchu, Taiwan) were used in this study. FIG. 1 shows an example of a test strip. The tip of the test strip includes microfluidic channels for sample injection. A gold-plated electrode is present on the tip, undergoing a sequence of functionalization processes with the P90 antibody. The functionalization process is illustrated in FIG. 2. This functionalization enables the test strips to discern variations between samples. The initial step in the functionalization process is the ozone treatment of the strips for a duration of 15 minutes, which effectively removes any carbon residues. Subsequently, a diluted ammonium hydroxide (NH4OH) solution is used to eliminate gold oxide. Following this surface cleaning step, deionized (DI) water is employed to rinse the channels, and nitrogen is utilized for the drying process. The next phase involves preparing a 3-Mercaptopropanyl-N-hydroxysuccinimide ester (NHSester) solution, which is dissolved in ethanol. NHS ester has a three-carbon chain ending in a thiol group, with an attached N-hydroxysuccinimide ester. This compound is utilized for bioconjugation, offering a reactive site for selective coupling with amine-containing molecules. The strips are submerged in this solution and left to react for 2 hours. The channels are then cleaned using DI water and nitrogen. Monoclonal CIP2A antibody 2G10- 3B5 (Santa Cruz Biotechnology, Dallas, TX) at a concentration of 20 pg / mL was injected into the channel, and the strips were sealed and stored in a disk at 4°C for 18 hours. Lastly, ethanolamine was employed to deactivate un-functionalized groups, mitigating the risk of potential interference. Previous research provided confirmation of antibody functionalization through uniform methodologies, as indicated by current- voltage and capacitance measurements. CIP2A Protein (MyBioSource, San Diego, CA) were diluted into series of concentration with artificial saliva (Pickering Laboratories Inc., Mountain View, CA) to establish the calibration curve.

[0024] Seventeen human saliva samples and tissue samples were obtained from individuals, including both pre-oral cancer patients and healthy volunteers, through collaboration with the University of Florida Oral Pathology Clinic and Dental Clinical Research Unit. The age range of the healthy samples was from 20 to 80 years old, while samples from inflammatory oral lesions (clinically diagnosed as leukoplakia) ranged from 40 to 80 years old. Table 1 displays comprehensive information regarding the patients examined in this study. The aim was to investigate whether age influences the detection results. Brush kits (Andwin Scientific, Simi Valley, CA) were used in the collection of tissue samples. These specimens were carefully preserved in a deep-freeze storage unit at -78°C.Healthy . , ■Cno / x Leukoplakia p value(N 10, 59 / o) (N=7, 41%)Male 6 (60%) 3 (43%)Gender 0.637Female 4 (40%) 4 (57%)20-30 3 (30%) 0 (0%)Age 40-60 5 (50%) 2 (29%) 0.04560-80 2 (20%) 5 (71%)Asian 4 (40%) 1 (14%)Race 0.338White 6 (60%) 6 (86%)Table 1. Patient characteristics. P-values are the results of Fisher’s exact tests(categorical variables) and Mann-Whitney tests (continuous variables).

[0025] In this study, three groups of samples were tested: Group A consisted of saliva samples without cell lysis, Group B comprised lysed saliva samples, and Group C comprised lysed tissue samples. The native lysis buffer used for saliva and tissue samples was purchased from Thermo Fisher Scientific (Massachusetts, U.S.). The sample collection and cell lysis procedures for the three groups are shown in FIG. 3. Epithelium cells were obtained by turning the brushes against the mucosa inside the oral cavity. The head of the brushes were then cut and placed in a 1.5 mL microcentrifuge tube. 1 mL of 1x PBS buffer solution was added into the tube, and the tissue samples were suspended in the solution by vortex mixer. In the cell lysis procedure, the sample solution was initially combined with the native lysis buffer in a 1 :10 ratio in a 1.5 mL microcentrifuge tube. Here, a 50 pL (1 drop) sample solution was taken with 50 pL lysing agent, followed by thorough mixing using a vortex mixer. Subsequently, the mixture was incubated at room temperature for 10 minutes. Finally, the mixture was centrifuged at 14000 ref for 15 minutes at 4°C, separate the resulting supernatant and pellet, and were in the refrigerator for subsequent analysis.

[0026] An image of a printed circuit board (PCB) used in this study is shown in FIG. 4A. This portable detection device comprises a readout block (Readout), pattern generator (Pattern Gen.), digitalizer, strip connector, Arduino (microcontroller circuitry), display, control switch, and system clock and power management unit (CLK & PMU). FIG. 4B is a simplified circuit diagram of the PCB circuitry. The following explains the device operation and provides further details about the device.

[0027] For sensor readout, the test strip is first connected to the strip connector of the PCB while the Arduino is activated. The Arduino triggers the pattern generator to generate a test pattern for measurement of the functionalized sensor. The test pattern, passing through the test strip, produces output signals of different magnitudes. This signal is then inputted into the gate terminal of a metal-oxide-semiconductor field-effect transistor (MOSFET) in the readout block, where amplification occurs using the MOSFET and a drain-side potentiometer. The amplified readout signal is converted into a frequency signal by a voltage-controlled oscillator (VCO) in the digitalizer. This frequency signal is then counted at fixed intervals by the counter. When the VCO reads a higher voltage, resulting in a higher output frequency, the counter outputs a larger value. Conversely, when the VCO reads a lower voltage, resulting in a lower output frequency, the counter outputs a smaller value. Because the counter's output varies with the MOSFET's output voltage, this value can be used as a digital representation of the readout voltage. Finally, the counter's output is processed by the Arduino and displayed on the display, allowing users to directly read the numerical value to determine the concentration of the solution on the test strip.

[0028] In each measurement, the device outputs multiple test patterns for repeated measurements, and the results can be averaged to reduce measurement errors. To avoid the charge accumulation effect on the test strip during the measurement process, the gate terminal of the MOSFET can be grounded to release the accumulated charge on the strip and gate terminals after each test pattern measurement is completed. This ensures the accuracy of each measurement. Additionally, this device has high adjustability. For example, the control switch can adjust the length of the test pattern and control the timing of thedigitalizer's voltage reading. The Arduino can control the time interval and frequency of test pattern generation. The voltage of the test pattern can be adjusted by a potentiometer on the PCB. Moreover, the MOSFET on the PCB can use an active socket, allowing for the replacement of MOSFET. Multiple adjustable parameters can be optimized according to the type of strip, ensuring that measurement parameters fall within the optimal range.

[0029] Various characteristic tests were conducted on the printed circuit board (PCB), involving alterations in load resistance, gate voltage, and the incorporation of an external capacitor parallel to the MOSFET. The heightened concentration of the sample solution induces an elevation in capacitance, elucidated by the electric double layer theory. Consequently, commercial external capacitors were employed as the strip during the test to determine the optimal operational settings.Results and Discussion

[0030] The outcomes of these tests are illustrated in FIGS. 5A-5C, which illustrates the effect of capacitance on the board. FIG. 5A shows various load resistance with fixed gate voltage as 1 .5 V; FIG. 5B shows various gate voltage with fixed load resistance as 104 kQ; and FIG. 5C shows the addition of an external capacitor parallel to the MOSFET (setting RL= 104 kQ and VG= 1.5 V as reference). These results serve as a guide for optimizing the board's conditions to enhance the sensitivity of the detection method. In FIG. 5A, the impact of adjusting the load resistance connected to the VDD on the PCB is demonstrated. A lower load resistance creates more space for voltage drop, expanding the operational range for testing and enhancing sensitivity. However, due to constraints imposed by other components on the PCB, the lowest permissible condition is set at 104 kQ. Consequently, 104 kQ was selected as the load resistance for subsequent tests.

[0031] FIG. 5B presents the device's performance under varying gate voltages. Sensitivity increases with higher gate voltages, and although VG=1.9 exhibits the steepest slope for optimal sensitivity, it yields a smaller detection range compared to other gate voltages. Striking a balance between sensitivity and detection range, VG=1.5 V was employed in subsequent biomarker tests. The outcomes of incorporating an externalcapacitor parallel to the MOSFET are depicted in FIG. 5C. The total capacitance (Ctotal) of the strips and MOSFET can be expressed as:Here, CM0SFETis the MOSFET capacitance, Cexternalis the external capacitor capacitance, and Cstripis the strip capacitance. Notably, the digital reading exhibits significant differences only when the MOSFET capacitance approaches that of the strips. Consequently, when the sample's capacitance is substantial, the external capacitor proves beneficial in extending the detection range towards larger capacitance values.

[0032] The output voltage from the printed circuit board (PCB) with a series of P90 protein concentration standard solutions is depicted in FIG. 6. The pattern of the voltage pulse can be elucidated by the double spring model.

[0033] The digital output reading is obtained by integrating the area under the curve of the output voltage. FIG. 7 illustrates the calibration curve, showcasing a sensitivity of 147 / dec. This implies that the digital reading decreases by approximately 147 when the protein concentration increases by one order of magnitude. In addition to its commendable sensitivity, the detection method achieves a limit of detection (LOD) as low as 1015g / mL while the detection range of commercial ELISA kits is limited to 0.156 to 10 ng / mL.

[0034] In addition to evaluating the standard solution, human sample testing was conducted in this study. FIG. 8 illustrates the test results of human sample test with saliva and tissue samples after the cell lysis process. Experiments were conducted on three distinct sample groups. Group A comprises saliva samples without undergoing cell lysis processing. Group B underwent testing after saliva lysis, whereas Group C consisted of tissue samples subjected to lysis before testing. Different symbols were employed to distinguish age ranges in the figure: dots with white centers represent data from healthy volunteers aged 20 to 30 years, while solid dots represent individuals aged 40 to 80 years. Across groups A, B, and C, there was no significant difference in P90 expression, consistent with previous findings. Notably, group A utilized distinct PCB settings compared to groups Band C, as determined by the earlier capacitance study, aimed at optimizing detection sensitivity.

[0035] FIG. 9 illustrates a boxplot representing the distribution of testing results. Within the boxplot, the bottom corresponds to the 25th percentile (Q1), the middle line indicates the median, and the top of the box signifies the 75th percentile (Q3). The upper and lower branches represent the maximum and minimum values of the data, respectively, excluding outliers. Outliers are depicted as individual data points. An outlier, which is represented by the hollow dot in the figure, is defined as a data point that falls significantly outside the expected variation around the median of the remaining dataset. For the interpretation of the first column, focusing on the healthy group, the saliva before lysis readings show a median just over 3000, with Q1 at approximately 2950 and Q3 at about 3150. The highest nonoutlier observation was around 3200, while the lowest was approximately 2800. Notably, there was one observation at around 3450, which stands out as unusually high compared to the tightly clustered data around 3000. Thus, it was defined as the outlier. Similar interpretations apply to the second and third columns.

[0036] In group A, saliva samples were analyzed without cell lysis, revealing potential differences indicated by lower readings in pre-cancer samples and a significant p-value of 0.001. P-values were derived from Fisher’s exact tests for categorical variables and Mann- Whitney tests for continuous variables. Groups B and C had identical PCB settings to ensure comparable readings. As anticipated, all data points in group C were lower than those in group B, indicating a higher P90 concentration in tissue samples. The p-value for group B was 0.0001 , demonstrating the efficacy of the technique in a non-invasive manner. Furthermore, for group C, the p-value decreased even further to 0.0008. The low p-value indicates a low probability of a false-positive result, underscoring the technique's high accuracy. Detailed data analysis findings are summarized in Table 2.Healthy Oral Leukoplakia o-va ue(N=10, 59%) (N=7, 41%)H3049 (152); 2270 (529);Group A 3014 [2972, 3084]; 2238 (1832,2685]; 0.001(2870, 3430) (1630,2987)3336 (123); 2745 (306);Group B 3326 [3288, 3358]; 2922 [2608,2964]; 0.0001(3091 , 3545) (2170,2979)2867 (230); 1618 (241);Group C 2850 [2766, 3046]; 1609 [1570,1752]; 0.0008(2376, 3138) (1154,1921)Table 2. Analytical result of the test. Continuous variables presented as mean (standard deviation); median [Interquartile Range]; (range); categorical variables presented as N (row%). P-values are the results of Fisher’s exact tests (categorical variables) and Mann-Whitney tests (continuous variables).

[0037] Utilizing the calibration curve derived from the standard solution and the results of the human sample tests, these findings can be integrated to estimate the relative protein concentration in the human samples. Demonstrating a lower LCD compared to commercial ELISA kits, the sensor introduced in this study exhibits the capability to differentiate low- concentration samples and provides valuable insights for oral cancer detection.

[0038] This study marks a significant advancement in the realm of oral cancer diagnostics by introducing a transistor-based biosensor system for the detection of the P90 protein, this marker is involved in inflammatory processes and with oral squamous cell carcinoma. The data indicated that P90 may be associated with leukoplakia that may have a potential for progression to oral malignant transformation. The specially designed printed circuit board (PCB) optimizes the functionality of the biosensor, ensuring accurate measurements and enhanced sensitivity. The superior sensitivity, low limit of detection, and successful human sample testing underscore the potential of this biosensor technique as an invaluable tool in the early detection of oral cancer, ultimately contributing to improvedpatient outcomes and a reduction in the global burden of this prevalent and impactful disease. Human sample testing validates the biosensor's effectiveness in distinguishing samples after cell lysis. This study contributes to advancing accurate and cost-effective diagnostic approaches for oral pre cancer and cancer tissues.

[0039] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

[0040] The term "substantially" is meant to permit deviations from the descriptive term that don't negatively impact the intended purpose. Descriptive terms are implicitly understood to be modified by the word substantially, even if the term is not explicitly modified by the word substantially.

[0041] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. The term “about” can include traditional rounding according to significant figures of numerical values. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.

Claims

CLAIMSTherefore, at least the following is claimed:

1. A method for functionalizing a test strip, comprising: cleaning a gold surface of an electrode of a test strip; submerging the cleaned gold surface of the electrode in a 3- Mercaptopropanyl-N-hydroxysuccinimide ester (NHS ester) solution for a predefined reaction period; cleaning channels of the test strip; injecting an antibody into the channels of the test strip; sealing the injected antibody in the test strip; storing the sealed test strip at a controlled temperature for a defined storage time; and deactivating unfunctionalized groups.

2. The method of claim 1 , wherein the cleaned gold surface of the electrode is submerged in the NHS ester from about 15 minutes to about 2 hours.

3. The method of any of claims 1 or 2, wherein the test strip is submerged in the NHS ester.

4. The method of claim 1 , wherein the channels are cleaned using deionized water and nitrogen.

5. The method of any of claims 1-4, wherein the antibody is a monoclonal CIP2A antibody, HER2, CA125, CA15-3, anti-SARS-CoV-2 spike glycoprotein RBD antibody, anti-Cardiac troponin I antibody, type I IFNs, dsRNA, BAFF, or IL6 / IL1 beta.

6. The method of claim 5, wherein the antibody is injected at a concentration in a range from about 1 mg / mL to about 1 pg / mL.

7. The method of any one of claims 1 -6, wherein the controlled temperature is 4°C and the defined storage time is 18 hours.

8. The method of any one of claims 1 -7, wherein deactivating unfunctionalized groups comprises treatment with ethanolamine.

9. The method of any one of claims 1 -8, wherein the electrode is a gold-plated electrode.

10. The method of any of claims 1 -9, wherein cleaning the gold surface of the electrode comprises ozone or oxygen plasma treatment of the gold surface.11 . The method of claim 10, wherein cleaning the gold surface of the electrode further comprises application of a diluted ammonium hydroxide (NH4OH) solution or a diluted hydrogen chloride (HCI) solution.

12. The method of claim 11 , wherein cleaning the gold surface of the electrode further comprises: rinsing with a deionized water; and drying with nitrogen.

13. A method, comprising: connecting a functionalized test strip to a strip connector of a portable detection device, the functionalized test strip comprising a test sample; generating a test pattern by a pattern generator of the portable detectiondevice, the test pattern passed through the functionalized test strip and input into a gate terminal of a metal-oxide-semiconductor field-effect transistor (MOSFET) in a readout block of the portable detection device; converting a readout signal of the readout block into a frequency signal; and counting the frequency signal at fixed intervals by a counter of the portable detection device to generate a counter output, and displaying a sample concentration on the portable detection device based upon the counter output.

14. The method of claim 13, wherein the readout signal is converted to the frequency signal by a voltage-controlled oscillator (VCO).

15. The method of claim 14, wherein the frequency signal corresponds to a voltage level of the readout signal.

16. The method of any one of claims 13-15, wherein a series of test patterns are generated by the pattern generator, passed through the functionalized test strip and input into the gate terminal of the MOSFET of the portable detection device; and the displayed sample concentration is based upon an average of counter outputs associated with the series of test patterns.

17. The method of claim 16, wherein the gate terminal is grounded between generation of each of the series of test patterns.

18. The method of any of claims 13-17, wherein the antibody is a monoclonal CIP2A antibody.

19. The method of claim 18, wherein the antibody is injected at a concentration in a range from about 1 mg / mL to about 1 pg / mL.

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