WO2025129039A1 - Biosensor detection of breast cancer staging in saliva biomarkers - Google Patents

Abstract

Various examples are provided related to biosensor detection of breast cancer staging in saliva biomarkers. In one example, a method includes providing a bio fluid or tissue sample to a functionalized sensing area disposed between two electrodes of a disposable test strip. The method further includes generating two synchronized voltage pulses, one voltage pulse applied to a functionalized electrode of the disposable test strip thereby inducing charges to appear on another electrode of the disposable test strip, which is connected to a gate of a transistor, and a second voltage pulse applied to a load resistor which is connected to a drain of the transistor. The functionalized sensing area detects and a drain voltage output of the transistor provides an indication of HER2, CA15-3 or CA 125 concentration in a range from 5 × 10^-4 g/mL to 5 × 10^-15 g/mL.

Description

BIOSENSOR DETECTION OF BREAST CANCER STAGING IN SALIVA BIOMARKERS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, co-pending U.S. provisional application entitled “Biosensor Detection of Breast Cancer Staging in Saliva Biomarkers” having serial no. 63/609,979, filed December 14, 2023, the entirety of which is hereby incorporated by reference.

BACKGROUND

[0001] Breast cancer, the most frequently detected cancer globally, had over 2.3 million new cases and resulted in approximately 685,000 deaths in the year 2020. In the US alone, in 2022, approximately 287,850 new cases of invasive breast cancer and 51 ,400 cases of ductal carcinoma in situ (DCIS) were diagnosed among women, leading to 43,250 female deaths from breast cancer. The future impact of breast cancer is expected to escalate, with projected figures estimating over 3 million new cases and approximately 1 million deaths in the year 2040. Imaging techniques such as mammography, ultrasound, and/or magnetic resonance imaging (MRI), and biopsies are the gold standard procedures for breast cancer diagnosis. However, these methods have many disadvantages such as high cost, limited accessibility, inaccuracies in detecting early-stage cancer in young women with denser breast tissue, invasiveness, low-dose radiation exposure, especially for patients who are sensitive to radiation. In addition, there is a need for specialized equipment and additional support from technologists and radiologists.

SUMMARY

[0002] Aspects of the present disclosure are related to biosensor detection of breast cancer staging in saliva biomarkers. In one aspect, among others, a method, comprises providing a bio fluid sample to a functionalized sensing area disposed between two electrodes of a disposable test strip, the functionalized sensing area configured to detect a concentration of HER2, CA15-3 or CA 125 in a range from 5 x 10⁴ g/mL to 5 x w¹⁵ g/mL in the bio fluid sample; and generating two synchronized voltage pulses, a first voltage pulse applied to a first functionalized electrode of the disposable test strip thereby inducing charges to appear on a second electrode of the disposable test strip, which is connected to a gate of a transistor, and a second voltage pulse applied to a load resistor which is connected to a drain of the transistor, where a drain voltage output of the transistor is a function of the concentration of HER2, CA15-3 or CA 125 in the bio fluid sample and provides an indication of HER2, CA15-3 or CA 125 concentration in a range from 5 x 10⁴ g/mL to 5 x ¹⁵ g/mL.

[0003] In one or more aspects, the bio fluid sample can comprise saliva, blood, serum, sweat, urine, or tear fluid. In some aspects the sensing area can be functionalized with an anti-HER2/ERBB2, CA 15-3 or CA 125 monoclonal antibody. In other aspects, the sensing area can be functionalized with a CA15-3 monoclonal antibody. The sensing area can be functionalized with a recombinant anti-MUC16 antibody. Further in some aspects, the sensing area is functionalized with a monoclonal mouse anti-human SP1 antibody. The sensing area can be functionalized with a CEA Monoclonal antibody. In other aspects, the sensing area is functionalized with an anti-CA 27-29 monoclonal antibody.

[0004] In various aspects, the functionalized sensing area can be configured to detect a concentration of HER2, CA15-3 or CA 125 of less than 10¹¹ g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2 CA15-3 or CA 125 concentrations of less than 10¹¹ g/mL. The functionalized sensing area can be configured to detect a concentration of HER2, CA15-3 or CA 125 of less than 10'¹² g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2 CA15-3 or CA 125 concentrations of less than 10¹² g/mL. Furthermore, the functionalized sensing area can be configured to detect a concentration of HER2 or CA15-3 CA15-3 or CA 125 of less than 10 ¹³ g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2 CA15-3 or CA 125 concentrations of less than 10¹³ g/mL. In some aspects, the functionalized sensing area can be configured to detect a concentration of HER2, CA15-3 or CA 125 less than 10 ¹⁴ g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2, CA15-3 or CA 125 concentrations of less than 10¹⁴ g/mL.

[0005] In one or more aspects, the bio fluid sample can be provided to the functionalized sensing area via an opening in the disposable test strip. The disposable test strip can be configured for single-use, and the transistor can be electrically coupled to the two electrodes through a detachable connection. In various aspects, the indication of the HER2, CA15-3 or CA 125 concentrations can be an average of a plurality of synchronized gate and drain pulse measurements. In some aspects, the plurality of synchronized gate and drain pulse measurements can comprise 10 consecutive pulse measurements. Further in some aspects, the indication of the HER2, CA15-3 or CA 125 concentrations can be provided in 15 msec or less.

[0006] 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 dependent claims, 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

[0007] 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.

[0008] FIGS. 1A-1C illustrate examples of a test strip (with (left) and without (right) cover) and printed circuit board (PCB) and circuit design to generate a digital reading, in accordance with various embodiments of the present disclosure.

[0009] FIGS. 2A and 2B illustrate examples of output drain voltage waveforms for pure artificial saliva and HER2 protein diluted in saliva from 10⁷ g/mL to 10¹⁵ g/mL and output digital readings from PCB under different HER2 protein concentrations, respectively, in accordance with various embodiments of the present disclosure. The limit of detection is 1 O’ ¹⁵ g/mL while the sensitivity is 70/dec.

[0010] FIGS. 3A and 3B illustrate examples of output digital reading results from the human sample test with strips functionalized by HER2 antibody and conversion of the output reading from human samples into exact HER2 protein concentration, respectively, in accordance with various embodiments of the present disclosure.

[0011] FIG. 4 illustrates an example of average output digital reading from PCB with different CA15-3 protein concentrations, in accordance with various embodiments of the present disclosure. The limit of detection is 10’¹⁵ g/mL while the sensitivity is 30/dec.

[0012] FIGS. 5A and 5B illustrate examples of output digital reading results from the human sample test with strips functionalized by CA15-3 antibody and conversion of the output reading from human samples into exact CA15-3 protein concentration, respectively, in accordance with various embodiments of the present disclosure.

DETAILED DESCRIPTION

[0013] Disclosed herein are various examples related to biosensor detection of breast cancer staging in saliva biomarkers. Breast cancer is the most diagnosed cancer among women. Salivary biomarkers offer a non-invasive approach to detecting this cancer. Commercially available disposable based strips, like the commonly used glucose detection strips, can be utilized and functionalized to detect breast cancer using associated biomarkers such as HER2 and CA15-3. The results demonstrate limits of detection (LCD) for these two biomarkers reaching levels as low as 1 fg/mL, which are much lower than levels detected by conventional enzyme-linked immunosorbent assay (ELISA) in the range of 1~4 ng/mL. Both HER2 and CA15-3 biomarkers exhibit an exceptional LCD as low as 1 fg/mL, surpassing ELISA LCD by four orders of magnitude. This improved LCD facilitates the distinction of HER2-negative cases. 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.

[0014] ELISA based testing of HER2 and/or CA15-3 in serum are essential biomarkers used in breast cancer diagnosis, treatment selection, and monitoring response to targeted therapies used to screen or monitor breast cancer in conjunction with those invasive methods mentioned above. HER2 (ERBB2, HER-2/neu) is a protein that plays a significant role in normal cell growth and division. However, in approximately 15-20% of breast cancer cases, there is an overexpression or amplification of the HER2 gene, leading to an increased amount of HER2 protein on the surface of cancer cells. This overexpression is associated with aggressive tumor growth, higher recurrence rates, and poorer prognosis. CA15-3 is a tumor-associated antigen that can be detected in the blood of some breast cancer patients.

[0015] Elevated levels of CA15-3 may indicate the presence of cancer cells and can be used as a complementary tool alongside other diagnostic tests and imaging techniques. The concentrations of HER2 and CA15-3 in saliva can be correlated to their concentrations in serum, thus saliva samples can also be employed for breast cancer detection. However, the ELISA based detections of HER2 and CA15-3 require trained technicians and one to two weeks to obtain results. Furthermore, the limit of detection of ELISA is only around 10⁸ to about IO ¹⁰ g/mL. A more efficient and cost-effective alternative is the utilization of biosensors for the detection of breast cancer tumor markers.

[0016] Field-effect transistors (FETs) based bio-sensors have received attention for biomolecular detection due to their high sensitivity, label-free detection, and rapid real-time options. In addition, detection devices based on FETs can provide both qualitative and quantitative results in a short time. There are several kinds of FETs, such as silicon nanowire FET (SiNW-FET), graphene FET (gFET), Si metal-oxide-semiconductor field-effect transistors (MOSFETs) and tunneling FET (TFET) that have been used as part of biosensor devices to amplify the detected signals.

[0017] Instead of using the transistors as the sensors, which need to be disposed of after each use, a system with a reusable printed circuit board (PCB) containing a MOSFET and disposable test strips can be employed. In this approach, synchronized double-pulses can be applied at the gate and drain terminals of the transistor to ensure that the channel charge does not accumulate. Additionally, there is no need to reset the drain and gate paths to mitigate the charge accumulation at the gate and drain of the sensing transistor for sequential testing. With the double-pulse approach, it only takes a few seconds to show the result of the test, due to the rapid response of the functionalized test strips and the resulting electrical signal output. As an example, the limits of detection (LOD) have been demonstrated to reach 10‘¹⁵ g/mL and the sensitivity to 78/dec for COVID-19 detection. Similar approaches have been used to detect cerebrospinal fluid (CSF), cardiac troponin I, and Zika virus.

[0018] In this disclosure, a double-pulse measurement approach is used to detect HER2 and CA15-3 in saliva samples collected from healthy volunteers and breast cancer patients. The voltage output responses of the transistor correlated to the HER2 and CA15-3 concentrations, detection limits and sensing sensitivity were determined. By employing a synchronized double-pulse method to apply, e.g., ten 1.2 msec voltage pulses to the sensing strip electrode and transistor drain electrode for amplifying the detected signal, the sensing platform can be separated from the detection platform. The detected signal is the average of 10 digital output readings corresponding to the 10 voltage pulses. The sensor sensitivities achieved were approximately 70/dec for HER2 and 30/dec for CA15-3. Furthermore, the test time takes less than 15 msec and only needs 3 pL of saliva to complete. The technique is easy to operate and has the potential for widespread public use.

Material and methods

[0019] Commercially available test strips used for glucose tests, as shown in FIG. 1A, were functionalized and used for the testing of the breast cancer biomarkers. The strips were made by Luvnshare Biomedical Inc. in Hsinchu, Taiwan. The method of functionalizing the test strips is described in detail in “Fast SARS-CoV-2 virus detection using disposable cartridge strips and a semiconductor-based biosensor platform” by M. Xian et al. (J. Vac. Sci. Technol. B:Nanotechnol. Microelectron. 2021 , 39 (3), 033202) and “Digital biosensor for human cerebrospinal fluid detection with single-use sensing strips” by M. Xian et al. (J. Vac. Sci. Technol. B:Nanotechnol. Microelectron. 2022, 40 (2), 023202). Briefly, the first step is to plate the carbon electrodes with gold by connecting to the gate pulse source on the PCB board. The strips can then be immersed into 10mM thioglycolic acid (TGA) solution for 4 hours to form strong Au-S bonds on the gold plated electrode. After this, the strips can be soaked in N, N’-dicyclohexylcarbodi-imide (0.1 mM) and N-hydroxysuccinimide (0.1 mM) in acetonitrile for 2 hours. 20 pg/mL anti-HER2/ ERBB2 monoclonal antibody (Sino Biological Inc., Chesterbrook, PA) can then be injected into the microchannel after which the strips can be stored in a sealed disk for 18 hours under 4°C. For the test of CA15-3 (MUC1), the same steps can be followed for the HER2 process, with the exception of using CA15-3 monoclonal antibody (Sino Biological Inc. Chesterbrook, PA) for the last step. Lastly, ethanolamine can be used to terminate the un-functionalized groups to prevent interference. To relate the results into real concentration numbers, the HER2 and CA15-3 proteins were diluted serially in saliva and stored at 4°C before use. All the antibodies employed in this study went through the validation process to show their specificity of binding to the specific protein.

[0020] An antigen is any molecule or part of a molecule that can be recognized by the immune system. An antibody is a Y-shaped protein produced by the immune system to specifically recognize and bind to a corresponding antigen. Each antibody has a unique region, known as the variable region, that is structurally adapted to bind to a specific epitope (a small, unique portion of the antigen). The specificity of the antigen-antibody interaction depends on the epitope of the antigen and the paratope of the antibody. The epitope is the specific part of the antigen that is recognized by the antibody. In the case of an anti- HER2/ERBB2, CA 15-3 or CA 125 monoclonal antibody, the antibody will recognize specific amino acid sequences or structural features of the corresponding protein that are unique to the molecule. These epitopes may be located on the protein’s extracellular domain or glycosylated regions. The paratope is the region of the antibody that binds to the epitope. It is typically located within the variable region of the antibody and is made up of specific amino acid residues that create a complementary binding pocket for the epitope. The complementarity between the paratope (antibody) and the epitope (antigen) results in a highly specific interaction, often described as a "lock and key" model. The antibody's paratope has a shape and charge distribution that matches the epitope on the antigen.

[0021] The antigen-antibody binding interaction is a precise and specific molecular event, involving the recognition of epitopes on, e.g., the anti-HER2/ERBB2, CA 15-3 or CA 125 monoclonal antigen by the paratope of the antibody. This interaction is driven by a range of non-covalent forces and leads to various immune responses that can target and kill tumor cells. The use of anti-HER2/ERBB2, CA 15-3 or CA 125 monoclonal antibodies harnesses this precise binding to improve cancer detection. This also applies to recombinant anti- MUC16 antibodies, monoclonal mouse anti-human SP1 antibodies, CEA Monoclonal antibodies, anti-CA 27-29 monoclonal antibody, and others.

[0022] A printed circuit board (PCB), as illustrated in FIG. 1 B, was designed to convert the detected voltage signals related to the strips into digital readings. A MOSFET (STMicroelectronics STP200N3LL) was used to amplify the detected signal from the test strip. Synchronous voltage pulses can be sent to both the electrode of the strip connecting to the gate and drain electrodes of the MOSFET. The drain pulse can be applied, e.g., for around 1.1 msec at a constant voltage. The gate pulse can start, e.g., at 40 s after the drain pulse and end, e.g., at 40ps before the end of the drain pulse. A variable resistor is connected to the drain as the load resistor. FIG. 1C is a schematic diagram illustrates an example of circuitry of the PCB of FIG. 1 B. Additional details of sensor circuitry can be found in US Patent Application Pub. No. 2021/0003528, which is hereby incorporated by reference in its entirety.

[0023] In addition to obtaining a calibration curve from a series of diluted proteins, 17 human saliva samples from both breast cancer patients and 4 control samples from healthy volunteers were obtained. These samples were collected from patients and were preserved in a -78°C freezer. These de-identified samples all came with corresponding diagnoses, which were confirmed through biopsies as part of the patients’ routine care. After defrosting, the saliva samples were applied to the microfluidic channel directly without dilution, filtration or centrifugation. Based on the histologic type, the human samples were classified into three groups: (1) healthy control; (2) in situ breast cancer; and (3) invasive breast cancer. Among the invasive breast cancer samples, one of them was HER2 positive, while the rest of the samples were HER2 negative tested through biopsy results using immunohistochemistry (IHC). All the samples were tested with two different types of strips, which were functionalized with either HER2 or CA15-3. All of the output digital readings were averaged from ten pulse measurements, which took around 15 msec. The p-values of test results were analyzed with both Kruskal-Wallis test and pairwise Wilcox test.

Result and Discussion

[0024] To verify the authenticity of the sensor and its ability to detect HER2 protein, the bio-functionalized strips initially underwent testing using pure HER2 protein (Sino Biological Inc. Chesterbrook, PA) that was successively diluted to different concentrations. The dilutions ranged from pure artificial saliva (Pickering Laboratories Inc., Mountain View, CA), to a minimum dilution of 1 x 10^-15 g/mL in artificial saliva. FIG. 2A depicts dynamic drain output voltage waveforms at various concentrations ranging from 1 fg/ml to 10 pg/ml during each gate pulse. To convert the analog voltage signals to digital signals, the drain voltage levels were extracted at fixed 750 s from each curve and transformed into digital signals through a voltage-controlled oscillator (VCO) on the PCB board.

[0025] As shown in FIG. 2B, a sensitivity of 70/dec was achieved with a limit of detection (LOD) of 10 ¹⁵ g/mL, which was six to seven orders lower than the gold standard ELISA test, which is around 10⁸ to 10⁹ g/mL, used to measure these biomarkers. To ensure the repeatability and reliability of the measured results, the curve was refined by averaging ten consecutive identical pulse measurements. The total measurement time of 10 pulses is under 15 msec, hence, this technique holds promise for real-time point-of-service applications. Previously described and modeled, the antigen-antibody complexes undergo stretching and contracting, akin to double springs, in response to a pulsed gate electric field. This motion across the antibody-antigen structure, corresponding to the pulse voltage applied on the test strip, induces an alteration in the protein's conformation, resulting in a time-dependent electric field applied to the MOSFET gate. Consequently, a spring-like pattern emerges in the drain voltage waveform due to the external connection between the sensor strip and the MOSFET's gate electrode. In FIG. 2B, an observable trend emerges where increased spike antigen concentration of HER2, induces a consistent reduction in drain output voltage and a subsequent decrease in the digital reading.

[0026] FIG. 3A shows output digital readings of 21 human saliva samples, where there are clear differences among healthy, in-situ and invasive breast cancer cases. In-situ ductal carcinoma breast cancer is a type of cancer confined in a milk duct, which eventually grows into the rest of the breast tissue. Invasive breast cancer is a type of cancer which has spread into the surrounding breast tissue. Table 1 shows the median and the range of digital readings by disease status and overall p-value using Kruskal-Wallis test to examine if there exists statistically significant distinctions among two or more groups. The overall p-value is significant while the value for HER2 is 0.002, indicating that this sensor technology is an efficient way to detect HER2 biomarkers in saliva.

Table 1. Median (range) by disease status. P-values are the results of Kruskal-Wallis tests (overall).

Healthy Volunteers In Situ Breast Cancer Invasive Breast p value

(N=4, 19%) (N=3, 14%) Cancer

HER2 3226 (3105, 3318) 3053 (2974, 3142) 2879 (2058, 3025) 0.002

CA15-3 2620 (2589, 2673) 2496 (2378, 2623) 2356 (2108, 2575) 0.005

[0027] Immunohistochemistry (IHC), which was the test used to determine HER2 status on the patients, is a special staining process performed on fresh or frozen breast cancer tissue removed during biopsy to show whether or not the cancer cells have too much HER2 receptors and/or hormone receptors on their surface. IHC is a qualitative test based loosely off eye scored counting and gives a score of 0 to 3+ for the amount of HER2 receptor protein on the surface of cells in a breast cancer tissue sample. For example 0 to 1 + is HER2 negative, 2+ is borderline and is confirmed positive using fluorescence in situ hybridization (FISH) and, 3+ is HER2 positive. Among 17 saliva samples, there is only one HER2 positive sample and the rest of the 16 samples were HER2 negative, 0 or +1 . For HER2-positive cases, cancers tend to grow and spread faster than breast cancers that are HER2-negative, but are much more likely to respond to treatment with drugs that target the HER2 protein. By using sensor technology presented in this work, no invasive biopsy is required to determine the HER2 concentration. [0028] FIG. 3B shows the digital readings corresponding to the calibrated HER2 concentrations in the human samples, and the LOD of HER2 with gold standard ELISA kit is also labelled in the figure. ELISA can only be used to confirm the cancer status of the HER2 positive sample but not the HER2 negative samples because this test is not sensitive enough to detect the HER2 antibody for the majority of HER2 negative samples. This data clearly shows that this sensor technology has the potential to be used to identify the presence of breast cancer regardless of whether the samples are HER2 negative or positive.

[0029] Another cancer antigen, CA15-3, is used as a surrogate marker to monitor metastatic breast cancer patients undergoing treatment and for the preclinical detection of tumor recurrence. Levels of CA 15-3 have a significant relationship to outcome in patients with early breast cancer and is commonly used to detect breast cancer or monitor the effectiveness of cancer treatments. Detection of both CA15-3 and HER2 at the same time to ascertain breast cancer progression was strongly suggested. FIG. 4 illustrates the calibration curve for the CA15-3 biomarker and a LOD of 10‘¹⁵ g/mL with a sensitivity of 30/dec was demonstrated. The sensitivity of detecting CA15-3 is less than half of the sensitivity for HER2, which is 70/dec. This is due to the molecular weight of CA15-3 protein, 250~350 kDa, which is much larger than that of the HER2 protein, 185 kDa. According to the double spring model, in order to simulate the output voltage responses, the disparity in size between the HER2 molecule and the CA15-3 molecule would produce a smaller spring constant for the CA15-3 molecule and diminish the detection sensitivity of CA15-3.

[0030] In FIG. 5A, the test results for detecting CA15-3 of the human samples are shown. The digital reading decreases from the healthy group to the invasive breast cancer group, indicating an increase in CA15-3 concentration. FIG. 5B depicts the conversion from the test results of the human samples to the actual CA15-3 protein concentration. Similar DOL results as the HER2 detection, showing the CA15-3 DOL concentration is around 5 x 10'¹⁰ to 4 x 10'⁹ g/mL, which is slightly higher than the DOL of ELISHA. However, there was only invasive breast cancer sample (a star and located on the left side of CA15-3 DOL region in FIG. 5B) with the CA 15-3 concentration lower that the DOL. Unlike the HER2 cases, there were a bunch of stars marked on the left side of HER2 DOL region, as shown in FIG. 3B. This confirmed that CA15-3 is more sensitive to patients with early breast cancer, but ELISA based CA15-3 screening is not sensitive enough for in-situ breast cancer samples. The median, the range by disease status and overall p-value analyzed with the Kruskal- Wallis test for CA15-3 test are listed in Table 1. The overall p-value for CA15-3 is 0.005, indicating that this device provides an efficient way to detect the salivary biomarkers related to breast cancer.

[0031] An effective approach for identifying salivary biomarkers linked to breast cancer has been demonstrated. The method employed a hand-held size PCB to provide synchronized voltage pulses applied to a commercially available test strip, similar to the glucose detecting strip and the drain electrode of the transistor on the PCB, while the transistor on the board was used to amplify the detected signal. This saliva based non- invasive test, which leveraged biomarkers like HER2 and CA15-3, revealed impressive limits of detection (LOD) and sensitivity. Both HER2 and CA15-3 biomarkers exhibited an exceptional LOD as low as 1 fg/mL, surpassing ELISA kits by four orders of magnitude. This improved LOD facilitates the distinction of HER2-negative cases. To calibrate our output measurements to actual protein concentrations, a range of diluted proteins were employed. HER2 sensitivity was determined to be 70/dec, and CA15-3 sensitivity was 30/dec with diluted proteins. These established relationships allowed the test results to be converted into precise protein concentrations. Moreover, the non-invasive test was performed 10 times within 15 msec and the whole test took less than a minute to perform, including applying 3-5 L saliva sample on the strip. The method is user-friendly and holds significant promise for widespread use by the general public.

[0032] 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.

[0033] 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.

[0034] 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

CLAIMS Therefore, at least the following is claimed:

1. A method, comprising: providing a bio fluid sample to a functionalized sensing area disposed between two electrodes of a disposable test strip, the functionalized sensing area configured to detect a concentration of HER2, CA15-3 or CA 125 in a range from 5 x 10⁴ g/mL to 5 x w¹⁵ g/mL in the bio fluid sample; generating two synchronized voltage pulses, a first voltage pulse applied to a first functionalized electrode of the disposable test strip thereby inducing charges to appear on a second electrode of the disposable test strip, which is connected to a gate of a transistor, and a second voltage pulse applied to a load resistor which is connected to a drain of the transistor, where a drain voltage output of the transistor is a function of the concentration of HER2, CA15-3 or CA 125 in the bio fluid sample and provides an indication of HER2, CA15-3 or CA 125 concentration in a range from 5 x 1 o ⁴ g/mL to 5 x 10 '¹⁵ g/mL.

2. The method of claim 1 , wherein the bio fluid sample comprises saliva, blood, serum, sweat, urine, or tear fluid.

3. The method of claim 1 , wherein the sensing area is functionalized with an anti- HER2/ERBB2, CA 15-3 or CA 125 monoclonal antibody.

4. The method of claim 1 , wherein the sensing area is functionalized with a CA15-3 monoclonal antibody.

5. The method of claim 1 , wherein the sensing area is functionalized with a recombinant anti-MUC16 antibody.

6. The method of claim 1 , wherein the sensing area is functionalized with a monoclonal mouse anti-human SP1 antibody.

7. The method of claim 1 , wherein the sensing area is functionalized with a CEA Monoclonal antibody.

8. The method of claim 1 , wherein the sensing area is functionalized with an anti-CA 27-29 monoclonal antibody.

9. The method of claim 1 , wherein the functionalized sensing area is configured to detect a concentration of HER2, CA15-3 or CA 125 of less than 10¹¹ g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2 CA15-3 or CA 125 concentrations of less than 10¹¹ g/mL.

10. The method of claim 9, wherein the functionalized sensing area is configured to detect a concentration of HER2, CA15-3 or CA 125 of less than 10¹² g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2 CA15-3 or CA 125 concentrations of less than 10¹² g/mL.

11 . The method of claim 10, wherein the functionalized sensing area is configured to detect a concentration of HER2 or CA15-3 CA15-3 or CA 125 of less than 10¹³ g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2 CA15-3 or CA 125 concentrations of less than 10 ¹³ g/mL.

12. The method of claim 11 , wherein the functionalized sensing area is configured to detect a concentration of HER2, CA15-3 or CA 125 less than 10^-14 g/mL in the bio fluid sample and the drain voltage output of the transistor provides indications of HER2, CA15-3 or CA 125 concentrations of less than 10¹⁴ g/mL.

13. The method of claim 1 , wherein the bio fluid sample is provided to the functionalized sensing area via an opening in the disposable test strip.

14. The method of claim 1 , wherein the disposable test strip is configured for single-use, and the transistor is electrically coupled to the two electrodes through a detachable connection.

15. The method of claim 1 , wherein the indication of the HER2, CA15-3 or CA 125 concentrations is an average of a plurality of synchronized gate and drain pulse measurements.

16. The method of claim 15, wherein the plurality of synchronized gate and drain pulse measurements comprise 10 consecutive pulse measurements.

17. The method of claim 15, wherein the indication of the HER2, CA15-3 or CA 125 concentrations is provided in 15 msec or less.