Method for preparing biochip and biochip prepared therefrom

Abstract

The present disclosure provides a method for preparing a biochip. The method includes coating a chip with a first solution of biotin to form a biotin-coated chip, wherein the biotin in the first solution is at a first concentration; subjecting the biotin-coated chip to a second solution of neutravidin to form a neutravidin / biotin-coated chip, wherein the neutravidin in the second solution is at a second concentration; and subjecting the neutravidin / biotin-coated chip to a third solution of a biotinylated probe to form the biochip, wherein a first molar ratio of the first concentration to the second concentration is 1 : 1, 1 :0.25, or 1 :2.

Description

METHOD FOR PREPARING BIOCHIP AND BIOCHIP PREPAREDTHEREFROMCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to US Provisional Patent Application No.63 / 699,825 filed on September 27, 2024, and the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (sequencelisting. xml; size:7,181 bytes; and date of creation: September 11, 2025) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION 1. Field of the Invention

[0003] The present disclosure relates to a method for preparing a biochip, and in particular to a method for preparing a biochip with improved sensitivity and a biochip prepared therefrom.2. Description of the Related Art

[0004] With the emergence of novel infectious diseases and the advancement of precision medicine, there is an increasing need for point-of-care tests (POCT) that can rapidly and accurately detect disease-related biomarkers. Among biosensing platforms, field-effect transistor (FET) biosensors (bioFET) are leading candidates for POCT due to their portability, sensitivity, and ease of manufacturing (Pullano et al., Sensors 18, 4042 (2018); Chen and Bashir, Nanotechnology 34, 492002 (2023)). The basic components of a bioFET include a source electrode connected to a drain electrode via a functionalized semiconductor gate, along with a reference electrode.The voltage difference between the drain and source electrodes (Vd) generates a drain current (Id), which is modulated by the conductance of the gate. The gate surface is functionalized with a system designed to capture target biomolecules. When the charged target biomolecules bind to the gate surface, they alter the surface potential and gate conductance, resulting in a change in the drain current (Id) (Sung and Koo, Biomed. Eng. Lett. 11, 85-96 (2021)).

[0005] Since the bioFET detection mechanism measures the change in current after target molecules bind to the capture system on the gate surface, the design of thesensing surface and capture system is crucial for bioFET performance. The most common strategy for functionalizing the metallic gate surface involves silanization with bifunctional silanes, such as (3-aminopropyl)triethoxysilane (APTES) (Wang et al., Chem. Soc. Rev. 50, 6507-6540 (2021)). After functionalization, probes for target molecules can be immobilized on the surface using linkers. A common linker for conjugating probes to an APTES-functionalized surface is the biotin-avidin linkage system. The interaction between biotin and avidin is among the strongest non-covalent bonds in nature, with a dissociation constant (Kd) of approximately 10’15M; thus, biotin-avidin systems are widely used as probes or affinity systems (Haugland and You, Methods Mol. Biol. Clifton NJ 418, 13-24 (2008)). Biotin, a small molecule consisting of a tetrahydrothiophene ring fused to a tetrahydroimidizalone ring, can be conjugated to APTES after modification with N- hydroxysuccinimide ester (NHS) (Miller et al., Peptides 18, 1585-1595 (1997); Jain and Cheng, J. Control. Release Off. J. Control. Release Soc. 245, 27-40 (2017)). Avidin, a tetrameric protein with four biotin-binding sites, acts as a bidirectional linker, with one end attached to the APTES-biotin surface and the other linking biotinylated molecules (Cho et al., Anal. Biochem. 365, 14-23 (2007)). APTES- biotin-avidin linker systems serve as versatile platforms for conjugating variousprobes, including biotinylated DNA / RNA and biotinylated antibodies. Although these systems have been used in several biosensor platforms, the optimal concentrations and proportions of biotin and avidin for the APTES-biotin-avidin linker system in a bioFET remain unclear.

[0006] Although the biotin / avidin affinity system has been commonly used in bioFET systems, the sensitivity of the bioFET systems is still low.

[0007] Thus, it is desirable to prepare a biochip having biotin / avidin / biotinylated probe with improved sensitivity. BRIEF SUMMARY OF THE INVENTION

[0008] It is an object of the present disclosure to provide a biochip with significantly improved sensitivity.

[0009] To achieve at least the above object, the present disclosure provides a method for preparing a biochip, including coating a chip with a first solution of biotin to form a biotin-coated chip, wherein the biotin in the first solution is at a first concentration; subjecting the biotin-coated chip to a second solution of neutravidin to form a neutravidin / biotin-coated chip, wherein the neutravidin in the second solution is at a second concentration; and subjecting the neutravidin / biotin-coatedchip to a third solution of a biotinylated probe to form the biochip, wherein a first molar ratio of the first concentration to the second concentration is 1 : 1, 1 :0.25, or 1 :2.

[0010] In an embodiment, the chip is an extended gate field-effect transistor (EGFET).

[0011] In an embodiment, the biotin in the first solution is at the first concentration of 0.1 pg / ml or 1 pg / ml.

[0012] In an embodiment, when first concentration is 0.1 pg / ml, the first concentration, the second concentration, and the third concentration are present in a second molar ratio of 1 : 1 :3, 1 : 1 :0.3, 1 :2:0.3, or 1 :2:3.

[0013] In an embodiment, when first concentration is 1 pg / ml, the first concentration, the second concentration, and the third concentration are present in a second molar ratio of 1 : 1 :3, 1 : 1 :0.3, 1 :0.25 :0.3, or 1 :0.25 :3.

[0014] In an embodiment, the biotinylated probe is specific to one of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) DNA, Tau protein, and E. coli genomic DNA.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color.Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] FIG. 1 shows confocal images illustrating the coating of biotin on the biochip of the present disclosure.

[0017] FIGS. 2A and 2B show the fluorescence intensity quantification results for the images of FIG. 1.

[0018] FIG. 3 is a graph showing voltage shifts in the electrical signal tests of the biochip coated with various concentrations of biotin according to the present disclosure.

[0019] FIG. 4A and FIG. 4B are confocal images illustrating showing the fluorescence detection results of the biochip prepared from various molar concentration ratios of biotin (1 pg / mL or 0.1 pg / mL): neutravidin: probe according to the present disclosure.

[0020] FIGS. 5A to 5D show the fluorescence quantification results of the images in FIG. 4A and FIG. 4B.

[0021] FIG. 6A is a graph showing voltage shifts in the electrical signal tests of the biochip prepared from various concentrations of biotin and neutravidin at the molar ratio of 1 : 1, and FIG. 6B is a graph showing voltage shifts in the electrical signaltests of the biochip prepared with different molarity ratios of biotin to neutravidin.

[0022] FIG. 7 is a graph showing the electrical signal test results of the biochip prepared from various molar concentration ratios of biotin (1 pg / mL) to neutravidin according to the present disclosure.

[0023] FIG. 8 is a graph showing the electrical signal test results of the biochip prepared from various molar concentration ratios of biotin (0. 1 pg / mL) to neutravidin according to the present disclosure.

[0024] FIG. 9A and FIG. 9B are confocal images showing the fluorescence detection results for various molar concentration ratios of biotin (1 pg / mL): neutravidin: probe according to the present disclosure.

[0025] FIGS. 10A to 10D show the fluorescence quantification results of the images in FIG. 9A and FIG. 9B.

[0026] FIG. 11A and FIG. 1 1B are confocal images showing the fluorescence detection results for various molar concentration ratios of biotin (0.1 pg / mL): neutravidin: probe according to the present disclosure.

[0027] FIGS. 12A to 12D show the fluorescence quantification results of the images in FIG. 1 1A and FIG. 11B.

[0028] FIG. 13A and FIG. 13B are graphs showing the electrical signal test resultsof the biochip prepared from various molar concentration ratios of biotin (1 pg / mL): neutravidin: probe according to the present disclosure.

[0029] FIG. 14A and FIG. 14B are graphs showing the electrical signal test results of the biochip prepared from various molar concentration ratios of biotin (0.1 pg / mL): neutravidin: probe according to the present disclosure.

[0030] FIG. 15 A and FIG. 15 B are graphs showing biosensing performances of the biochip for detecting E. coli genomic DNA (FIG. 15A) and p-Tau217 protein (FIG. 15B). DETAILED DESCRIPTION OF THE INVENTION

[0031] To facilitate understanding of the object, characteristics and effects of the present disclosure, embodiments together with the attached drawings for the detailed description of the present disclosure are provided.

[0032] The chip in the present disclosure is an extended gate field-effect transistor (EGFET) for sensing analytes (e.g., biological analytes). A biosensing system can utilize a bio-detection layer on a substrate (e.g., a conductive substrate or sensing surface area), which can be coupled to a field effect transistor (FET). The gate of the field effect transistor is connected to the substrate having the bio-detection layerthereon. The functionalized substrate can include a well-defined area (e.g., a well, matrix or a flat area) that can hold a specific, pre-determined volume of fluid (e.g., liquid, colloid) on it. An external electrode can be dipped in the fluid and can then be connected to a power source supplying a voltage, which can be the gate voltage or power source. The variations in the charge transfer from the external electrode to the gate electrode of the FET due to the presence of target molecules on the biodetection layer modulates the source to drain characteristics of the FET. These modulations can be then correlated to the concentration of the target analyte (e.g., based on an already known correlation due to a prior calibration or the like) that is present on the detection layer. Such a biosensing system allows for the isolation of the sensitive electronic components from the potentially detrimental environment of the fluids used for sensing (i.e., the FET components are isolated from the fluid containing the analyte). This system can include an external functionalized substrate that is connected to the gate of the FET. The FET also includes a source and a drain, along with the insulator and gate electrode of the gate. The external electrode can touch the electrolyte, and a gate voltage is applied across the external electrode. The interaction between the ions present in the electrolyte and the functional group present on the extended gate substrate causes a surface potential change. This changeof potential causes a change in the flow of current to the gate of the FET. In turn, the gate current modulates the drain-source current in the FET. The modulation of drain current due to the presence of biomolecules on the surface of the extended gate makes the FET as a biosensor.

[0033] Fabrication of the EGFET

[0034] The EGFET was constructed using an N-type FET with an aluminum extended gate. The entire circuit was covered by an oxide layer, except for the 10,000 pm2sensing area of the extended gate. The EGFET was fabricated according to standard CMOS procedures at United 5 Microelectronics (Hsinchu, Taiwan).

[0035] Chip cleaning and surface modification

[0036] The chips were washed with acetone (Echo Chemical, Miaoli, Taiwan) and 99.5% ethanol (Echo Chemical) to remove unwanted chemical compounds through sonication for 10 minutes, followed by heating in an oven at 120°C to eliminate excess ethanol. The chips were then treated with oxygen plasma for 1 min using a plasma cleaner (PDC-00; Harrick Plasma, NY, US) to form OH terminals on the chip surface. For surface modification, the chips were soaked in 2% APTES (Sigma- Aldrich, MO, US; diluted in 99.8% ethanol) for 30 minutes to introduce amine groups on the surface. The chips were subsequently washed with 99.5% ethanol throughsonication for 10 minutes and then dried in an oven.

[0037] Biotinylation of the APTES-functionalized surface

[0038] NHS-biotin (Thermo Scientific, MA, US) stock solution (10 pg / pL in DMSO) was diluted to a working solution using 10 mM Bis-Tris propane (BTP) buffer (Sigma-Aldrich). To biotinylate the APTES-functionalized surface, 100 pL of biotin solution was applied to the sample well and the chip surface was submerged for 16 hours at 4°C. The biotin solution was then removed, and the chip was rinsed five times with 70 pL of wash buffer [10 mM BTP with 0.05% Tween-20 (Sigma- Aldrich)] followed by five rinses with 70 pL of 10 mM 1 BTP buffer.

[0039] Fluorescence imaging of biotinylation of the APTES-functionalized surface

[0040] In total, 100 pL of fluorescent reagent (biotin-DyLight488; Thermo Scientific) were added to the well. The chip was placed in a dark box to avoid light exposure and incubated at 4°C overnight. After incubation, the reagent was removed, and 5 pL of mounting medium (S3023; Dako North America, CA, US) were applied to the chip surface. The chip was covered with a coverslip for fluorescence imaging using a confocal fluorescence microscope (TCS SP8X; Leica Microsystems, Wetzlar, Germany).

[0041] Neutravidin conjugation

[0042] Neutravidin (Thermo Scientific) stock solution (3 mg / mL in 10 mM BTP buffer) was diluted to the target concentration using 10 mM BTP buffer. Seventy microliters of the diluted neutravidin solution were applied to the sample well, and the chip surface was submerged for 10 minutes at room temperature. After submersion, the neutravidin solution was removed, and the chip surface was washed five times with 70 pL of wash buffer, followed by five washes with 70 pL of 10 mM BTP buffer.

[0043] Preparation and conjugation of biotinylated probes for E. coli genomic DNA and biotinylated antibody for p-Tau217

[0044] To detect E. coli genomic DNA, primers and probes with the following sequences were synthesized by Sigma-Aldrich based on Zhang et al., Sci. Rep. 1 1, 8771 (2021): biotinylated forward primer (SEQ ID NO: 1); biotinylated probe E (SEQ ID NO: 2); and biotinylated reverse primer (SEQ ID NO: 3).

[0045] The p-Tau217 antibody was developed in-house. Female BALB / c mice (6-8 weeks 3 old; National Laboratory Animal Center, Taipei, Taiwan) were immunized with the p-Tau217 peptide, wherein p-Tau217 peptide is a phosphorylated Tau peptide (SEQ ID NO: 7). After immunization, the spleens of these mice werecollected and fused with myeloma cells for hybridoma preparation and semi-solid selection (ClonaCell Hybridoma Kit, STEMCELL Technologies, Vancouver, Canada). High-affinity antibodies produced by the hybridoma were selected using protein G Sepharose resin (Cytiva, MA, US) and concentrated with Amicon Ultra-15 centrifugal filter units (Merck Millipore, MA, US). The p-Tau217 antibody includes Tau-217-123 VL (SEQ ID NO: 4) and Tau-217- 123 VH (SEQ ID NO: 5). The p- Tau217 antibody was biotinylated with EZ-Link Sulfo-NHS-Biotin (Thermo Scientific), in accordance with the manufacturer’ s protocol. Briefly, 5 mg / mL of purified p-Tau217 monoclonal antibody were dissolved in PBS. Biotin reagent solution was added to the antibody solution to a final concentration of 10 mM, with a 30-min incubation at room temperature. The labeled p-Tau217 monoclonal antibody was then purified for optimal performance and stability using desalting columns (Zeba Spin Desalting Columns; Sigma Aldrich).

[0046] To conjugate the biotinylated probes to the APTES-biotin-neutravidin system, 70 pL of the biotinylated p-Tau217 antibody [1 pg / mL in PBS (Sigma- Aldrich)] or E. coli probes (10 pg / mL in ddH2O) were added to the sample well, and the chip surface was submerged for 10 minutes at room temperature. The probe solution was then removed, and the chip surface was washed five times with 70 pLof wash buffer, followed by five washes with 70 pL of 10 mM BTP buffer.

[0047] Sample preparation

[0048] E. coli genomic DNA was kindly provided by Dr. Huang Chung-Guei atChang Gung Memorial Hospital, Taiwan. The p-Tau217 peptide was synthesized by BIOTOOLS (New Taipei City, Taiwan).

[0049] Laser intensity used for confocal microscope

[0050] Fluorescence intensity quantification of confocal image

[0051] The integrated morphometry analysis was performed using MetaMorph Offline software to quantify average intensity (average fluorescence intensity per unit area) and total intensity (total fluorescence intensity). For each fluorescence image acquisition, three positions were captured per experiment. The experiments were repeated three times, and the resulting data were combined for analysis to calculate the average value and standard deviation.

[0052] Electrical measurement

[0053] A well was adhered to a sensing area of the EGFET chip. Then, 100 pL of biotin solution was added into the well at 4°C overnight. Subsequently, the well waswashed with BTP buffer to remove the excess biotin solution, and then 100 pL ofBTP buffer was added and left for 10 minutes to allow ion stabilization. Electrical measurements were performed using the 512 NSOx GUI Application software, with biosensing conducted three times per cycle. The supernatant was removed, and 100 pL of neutravidin solution was added for a 10-minute reaction at room temperature. After removing the solution, the well was rinsed with BTP buffer. Then, 100 pL of BTP buffer was added and left for 10 minutes to allow ion stabilization, followed by measurement. Biosensing was performed three times per cycle.

[0054] The supernatant was removed, and 100 pL of the probe solution was added for a 10-minute reaction at room temperature. After removal of the solution, the well was rinsed with BTP buffer. Then, 100 pL of BTP buffer was added and left for 10 minutes to allow ion stabilization, followed by measurement. Biosensing was performed three times per cycle.

[0055] The supernatant was removed, and 100 pL of the blocking solution was added for a 30-minute reaction at room temperature. After removal of the solution, the well was rinsed with BTP buffer. Then, 100 pL of BTP buffer was added and left for 10 minutes to allow ion stabilization, followed by measurement. Biosensing was performed three times per cycle.

[0056] The supernatant was removed, and 100 pL of the control solution (blank) was added for a 10-minute reaction at room temperature. After removal of the solution, the well was rinsed with BTP buffer. Then, 100 pL of BTP buffer was added and left for 10 minutes to allow ion stabilization, followed by measurement. Biosensing was performed three times per cycle.

[0057] The supernatant was removed, and 100 pL of the sample solution was added for a 10-minute reaction at room temperature. After removal of the solution, the well was rinsed with BTP buffer. Then, 100 pL of BTP buffer was added and left for 10 minutes to allow ion stabilization, followed by measurement. Biosensing was performed three times per cycle.

[0058] The data from the three measurements were averaged to plot the ID-VG curve (Current-Voltage Gate curve). The shift of the curve (either left or right) was analyzed to evaluate the optimal concentration ratios of the solutions and to confirm the reproducibility of the experiment.

[0059] Biosensing procedure

[0060] The electric signals of the biochip at different sample concentrations were obtained using a customized reader that measured the drain voltage (Cd) and performed a gate voltage (Pg) sweep from 0 to 2 V. To establish the baseline, 70 pLof 10 mM BTP buffer were loaded in the sample well. The Vd-Vg curve was obtained after allowing the system to stabilize for 10 minutes. After the baseline measurement, the buffer was removed, and the chip was rinsed five times with 70 pL of 10 mM BTP buffer. Samples with different concentrations were then loaded sequentially using the same procedure to obtain the signal. The signal for each sample concentration was defined as the difference in Vgbetween the sample and the background when Vd = 1 V

[0061] Statistics and data analysis

[0062] All continuous data are presented as the mean ± standard deviation. Data were obtained from three replicates of each experiment. Statistical analysis and visualization were performed using Igor 7 software (WaveMetrics, OR, US). Multiple comparisons were made using analysis of variance followed by Tukey’s test, with the significance threshold regarded as 0.05. LODs were calculated using residuals from the linear regression plus three times the standard deviation of the residuals.

[0063] Determination of coating concentration of biotin via confocal microscopy

[0064] A well was adhered to a sensing area of the EGFET chip. Then, 100 pL of biotin solution at various concentrations (0.00001 pg / ml to 100 pg / ml) was addedinto the well, and the reaction well was incubated at 4°C overnight. Subsequently, the well was washed with BTP buffer to remove the excess biotin solution, and then 100 pL of BTP buffer was added to maintain moisture. 100 pL of fluorescent reagent was added into the well, and the chip was placed in a dark box to avoid light exposure, and incubated at 4°C overnight. The buffer was then removed, and the well was peeled off. Excess moisture was wiped away using lens paper. Then, 5 pL of mounting medium was applied onto the chip surface, and a coverslip was placed over it.

[0065] The fluorescence intensity was observed using the confocal microscope. The laser intensity was adjusted based on the fluorescence brightness. The images are shown in FIG. 1. The fluorescence signal intensity was analyzed using MetaMorph Offline software. As shown in FIG. 2A, the average intensity (average fluorescence intensity per unit area) of the control group was 6, while that of the experimental group ranged from 22 to 82. As shown in FIG. 2B, the total intensity (total fluorescence intensity) of the control group was 6.32* 106, whereas the total intensity of the experimental group ranged from 2.33 * 107to 8.66* 107. The results shown in FIG. 2A and FIG. 2B indicate that the fluorescence intensity was strong at concentrations of 0.1 pg / mL and 1 pg / mL.

[0066] Based on the electrical signal test results (as shown in Table 1 and FIG. 3), using 10 pg / mL of E. coli nucleic acid extract as the test sample, the results indicate that 0.1 pg / mL and 1 pg / mL with corresponding voltage shifts (steps) of 15 and 7, respectively, are the optimal concentrations of biotin. In the present disclosure, each step corresponds to 3.9 millivolts (mV), and thus the total shift for 0.1 pg / mL is 58.5 mV, and the total shift for 1 pg / mL is 27.3 mV.

[0067] Table 1. Electrical signal test results of the biochip coated with various concentrations of biotin

[0068] In Table 1, the ratios listed in the leftmost column represent biotin: neutravidin: probe in molar concentrations.

[0069] Determination of appropriate ratio of biotin to neutravidin

[0070] A well was adhered to a sensing area of the EGFET chip. Then, 100 pL of biotin solution (Ipg / mL, O. l pg / mL) was added into the well, and the reaction well was incubated at 4°C overnight. Subsequently, the well was washed with BTP buffer toremove excess biotin solution, followed by the addition of 100 pL of neutravidin solution for a 10-minute reaction at room temperature. Subsequently, the well was washed with BTP buffer to remove excess neutravidin solution, and then PRRSV 2 PROBE-BIOTIN(NA)-FAM was added for coating overnight. PRRSV 2 PROBE- BIOTIN (NA)-F AM was a PRRSV probe (SEQ ID NO: 6) biotinylated at the 5’ terminus and labeled with a FAM fluorophore at the 3 ’ terminus. Then, 100 pL of fluorescent reagent was added into the well. The EGFET chip was placed in a dark box to avoid light exposure and incubated at 4°C overnight. The well was washed with the 10 mM Bis-Tris propane (BTP) buffer to remove excess fluorescent reagent, and then 100 pL of the buffer was added to maintain moisture. The buffer was then removed, and the well was peeled off. Excess moisture was wiped away using lens paper. Then, 5 pL of mounting medium was applied onto the chip surface, and a coverslip was placed over it. The experiment was repeated three times.

[0071] The fluorescence intensity was observed using a confocal microscope. The laser intensity was adjusted based on the fluorescence brightness. The images were shown in FIG. 4A and FIG. 4B. The fluorescence signal intensity was analyzed using MetaMorph Offline software, and the results were shown in FIGS. 5A to 5D.

[0072] Due to the molecular weights of biotin (244.31 g / mol) and neutravidin(approximately 60,000 g / mol), their mass or volume concentration ratio is about1 :300 when they are present at a 1 : 1 molar ratio. 1 pg / mL biotin solution and 10 pg / mL probe solution were used. Only the concentration of neutravidin was adjusted to investigate the optimal ratio of biotin to neutravidin. The results were shown in FIG. 4A, FIG. 5A and FIG. 5B. The average intensity of the control group was 6, and the average intensities of the experimental groups ranged from 22 to 69. The total intensity of the control group was 6.62* 106, whereas the total intensities of the experimental groups ranged from 2.31 >< 107to 7.26* 107. The results show that when the concentration of biotin is 1 pg / mL, the molar concentration ratios of biotin to neutravidin ranging from 1 :0.1 to 1 :2 are all suitable for preparing the biochip with great sensitivity.

[0073] 0.1 pg / mL biotin solution and 1 pg / mL probe solution were used, and then the concentration of neutravidin was adjusted in the same manner for testing. The results were shown in FIG. 4B, FIG. 5C and FIG. 5D. The average intensity of the control group was 3, and the average intensities of the experimental groups ranged from 18 to 33. The total intensity of the control group was 3.25* 106, and the total intensities of the experimental groups ranged from 1.91 * 107to 3.47* 107. The results show that when the concentration of biotin is 0.1 pg / ml, the molar concentrationratios of biotin to neutravidin ranging from 1 :0.1 to 1 :2 are all suitable for preparing the biochip with great sensitivity.

[0074] As shown in FIGS. 4A, 4B and 5A to 5D, the optimal concentration ratios of the biotin (1 pg / mL, 0.1 pg / mL) to neutravidin were identified as 1 : 1 and 1 :0.25.

[0075] The sensitivity of the biochip is reflected in the voltage shift of the biochip when the biological target binds to the probe. Thus, the voltage shifts of the biochip were measured upon detection of 103copies of E. coli genomic DNA with different concentrations of biotin and neutravidin. The results were shown in FIG. 6 A and FIG. 6B. FIG. 6 A shows voltage shifts with different biotin-neutravidin concentrations under a 1 : 1 molarity ratio. FIG. 6B shows voltage shifts with different molar ratios between biotin and neutravidin.

[0076] 10 pg / mL of E. coli nucleic acid extract was used as the test sample. The optimal concentration ratios of biotin to neutravidin for sensitivity of the biochip were determined to be 1 :0.25 and 1 : 1, with corresponding voltage shifts (steps) of 1 1 and 19, respectively, as shown in Table 2 and FIG. 7. Each step corresponds to3.9 mV; therefore, the electrical shift at a biotin-to-neutravidin ratio of 1 :0.25 is42.9 mV, and the electrical shift at a ratio of 1 : 1 is 74.1 mV. These results clearly indicate that when the biotin concentration is 1 pg / mL, the optimal biotin-to-neutravidin concentration ratios are 1 :0.25 and 1 : 1.

[0077] Table 2. Electrical signal test results of the biochip prepared from various molar concentration ratios of biotin (1 pg / mL) to neutravidin

[0078] In Table 2, the ratios listed in the leftmost column represent biotin: neutravidin: probe in molar concentrations.

[0079] 0.1 pg / mL biotin solution and 1 pg / mL probe solution were used, and the concentration of neutravidin was adjusted in the same manner for testing. 10 pg / mL of E. coli nucleic acid extract was used as the test sample. The optimal concentration ratios of biotin to neutravidin for sensitivity of the biochip were determined to be 1 : 1 and 1 :2, with corresponding voltage shifts (steps) of 14 and 18, respectively, as shown in Table 3 and FIG. 8. The electrical shift at a biotin-to-neutravidin ratio of1 : 1 is 54.6 mV, and the electrical shift at a ratio of 1 :2 is 70.2 mV. These results clearly indicate that when the biotin concentration is 0.1 pg / mL, the optimal biotin- to-neutravidin concentration ratios are 1 : 1 and 1 :2.

[0080] Table 3. Electrical signal test results of the biochip prepared from variousmolar concentration ratios of biotin (0.1 pg / mL) to neutravidin

[0081] In Table 3, the ratios listed in the leftmost column represent biotin: neutravidin: probe in molar concentrations.

[0082] Determination of appropriate ratio of biotin: neutravidin: probe

[0083] 1 pg / mL biotin solution and 300 pg / mL neutravidin solution were used, and the concentration of probe was adjusted to determine the appropriate ratios of biotin: neutravidin: probe, wherein the probe was PRRSV 2 PROBE-BIOTIN(NA)-FAM. According to the fluorescence detection results (FIGS. 9A, 10A and 10B), the average intensity of the control group was 6, and the average intensity of the experimental groups ranged from 27 to 91. The total intensity of the control group was 6.62x 10®, and the total intensity of the experimental groups ranged from 2.85 x l07to 9.61 X 107.

[0084] Then, 1 pg / mL biotin solution and 75 pg / mL neutravidin solution were used, and the concentration of probe was adjusted for testing, wherein the probe wasPRRSV 2 PROBE-BIOTIN(NA)-FAM. According to the fluorescence detection results (FIGS. 9B, IOC and 1OD), the average intensity of the control group was 6, and the average intensity of the experimental groups ranged from 28 to 74. The total intensity of the control group was 6.62x 10®, and the total intensity of the experimental groups ranged from 2.97x l 07to 7.79x l07. These results show that the molar concentration ratios biotin: neutravidin: probe — from 1 : 1 :0.03 and 1 :0.25 :0.03 for low concentrations to 1 : 1 :30 and 1 :0.25 :30 for high concentrations — are all suitable for preparing the biochip with great sensitivity.

[0085] Then, 0.1 pg / mL biotin solution and 30 pg / mL neutravidin solution were used, and the concentration of probe was adjusted for testing, wherein the probe was PRRSV 2 PROBE-BIOTIN(NA)-FAM. According to the fluorescence detection results (FIGS. 1 1 A, 12A and 12B), the average intensity of the control group was 3, and the average intensity of the experimental groups ranged from 22 to 41. The total intensity of the control group was 3.25 x 10®, and the total intensity of the experimental groups ranged from 2.34x l07to 4.36x l 07.

[0086] Then, O. l pg / mL biotin solution was used and 60 pg / mL neutravidin solution were used, and the concentration of probe was adjusted for testing, wherein the probe was PRRSV 2 PROBE-BIOTIN(NA)-FAM. According to the fluorescencedetection results (FIGS. 11B, 12C and 12D), the average intensity of the control group was 3, and the average intensity of the experimental groups ranged from 22 to 34. The total intensity of the control group was 3.25 x 10®, and the total intensity of the experimental groups ranged from 2.34x l 07to 3.58 x l07. These results show that the molar concentration ratios of biotin: neutravidin: probe — from 1 : 1 :0.03 and 1 :2:0.03 for low concentrations to 1 : 1 :30 and 1 :2:30 for high concentrations — are all suitable for preparing the biochip with great sensitivity.

[0087] According to the electrical signal tests of the biochip prepared from the various molar concentrations of biotin to neutravidin, I pg / mL biotin solution was used, 300 pg / mL or 75 pg / mL neutravidin solution was used, the concentration of probe was adjusted to determine the optimal molar concentration ratios of biotin: neutravidin: probe. The results were shown in Table 4 and FIG. 13. 10 pg / mL of E. coli nucleic acid extract was used as the test sample. The optimal concentration ratios of biotin: neutravidin: probe for sensitivity of the biochip were determined to be 1 : 1 :0.3, 1 : 1 :3, 1 :0.25 :0.3 and 1 :0.25 :3, with corresponding voltage shifts (steps) of19, 31, 12 and 17, respectively, as shown in Table 4 and FIG. 13. Each step corresponds to 3.9 mV; therefore, the electrical shifts at biotin: neutravidin: probe being 1 : 1 :0.3 and 1 : 1 :3 are 74.1 mV and 120.9 mV, respectively, and the electricalshifts at biotin: neutravidin: probe being 1 :0.25:0.3 and 1 :0.25:3 are 46.8 mV and66.3 mV, respectively. These results clearly indicate that when the biotin concentration is 1 pg / mL, the optimal biotin-to-neutravidin concentration ratios are 1 :0.25 and 1 : 1.

[0088] Table 4. Electrical signal test results of the biochip prepared from various molar concentration ratios of biotin (1 pg / mL): neutravidin: probe

[0089] In Table 4, the ratios listed in the leftmost column represent biotin: neutravidin: probe in molar concentrations.

[0090] The results shown in Table 4 and FIG. 13 indicate that the optimal molar concentration ratios of biotin (1 pg / mL): neutravidin: probe for sensitivity of the biochip are 1:1:0.3, 1:1:3, 1:0.25:0.3 and 1:0.25:3.

[0091] 0.1 pg / mL biotin solution was used, 30pg / mL or 60pg / mL neutravidin solution was used, and the concentration of probe was adjusted for testing. The results were shown in Table 5 and FIG. 14. 10 pg / mL of E. coli nucleic acid extract was used as the test sample. The optimal concentration ratios of biotin: neutravidin: probe for sensitivity of the biochip were determined to be 1:1:0.3, 1:1:3, 1:2:0.3 and 1:2:3, with corresponding voltage shifts (steps) of 9.5, 5, 6.5 and 6.5, respectively, as shown in Table 5 and FIG. 14. The electrical shifts at biotin: neutravidin: probe being 1: 1:0.3 and 1:1:3 are 37.05 mV and 19.5 mV, respectively, and the electrical shifts at biotin: neutravidin: probe being 1 :2:0.3 and 1 :2:3 are both 25.35 mV.

[0092] Table 5. Electrical signal test results of the biochip prepared from various molar concentration ratios of biotin (0.1 pg / mL): neutravidin: probe

[0093] In Table 5, the ratios listed in the leftmost column represent biotin: neutravidin: probe in molar concentrations.

[0094] The results shown in Table 5 and FIG. 14 indicate that the optimal molar concentration ratios of biotin (0.1 pg / mL): neutravidin: probe for sensitivity of the biochip are 1:1:0.3, 1:1:3, 1:2:0.3 and 1:2:3.

[0095] In the method for preparing the biochip of the present disclosure, the optimal concentrations of biotin are 0.1 pg / mL and 1 pg / mL; the optimal molar concentration ratios of biotin to neutravidin are 1:0.25, 1:1 and 1:2; when the concentration of biotin is 1 pg / mL, the optimal molar concentration ratios of biotin: neutravidin: probe are 1:1:0.3, 1:1:3, 1:0.25:0.3, and 1:0.25:3; and when the concentration of biotin is0.1 pg / mL, the optimal molar concentration ratios of biotin: neutravidin: probe are 1:1:0.3, 1:1:3, 1:2:0.3, and 1:2:3.

[0096] In the present disclosure, a biotinylated DNA probe was conjugated to E. coli genomic DNA and a biotinylated antibody was conjugated to p-Tau217,separately, on the biochip, and then the biosensing performance of the biochip was evaluated. As shown in FIG. 15 A and FIG. 15B, the biochip of the present disclosure demonstrated the great sensing performance for both DNA and protein targets, with linearities of 0.96 (DNA) and 0.99 (protein), and an approximately 10,000-fold dynamic range in concentration. The estimated LODs were 3.5 copies for E. coli genomic DNA and 0.3 fg / mL for p-Tau217. The biochip of the present disclosure has the following advantages. For DNA detection, it does not require DNA amplification via polymerase chain reaction. For protein detection, the sensitivity of the biochip prepared in the present disclosure surpasses that of conventional enzyme-linked immunosorbent assays, which are typically around the pg / mL level.

[0097] In the present disclosure, a versatile, highly sensitive biochip using our inhouse extended-gate field-effect transistor (EGFET) platform was constructed and optimized. Unlike a conventional FET, an EGFET exhibits a physically separated sensing domain (referred to as the extended gate) connected to the gate via a metallic wire, enabling the separation of wet (biological material) and dry (chips) environments. In the present disclosure, the EGFET platform was fabricated using the standard complementary metal-oxide-semiconductor (CMOS) process with an N- type FET and an aluminum extended gate. The APTES-biotin-neutravidin linkersystem is conjugated to the extended gate. Neutravidin, a deglycosylated version of chicken avidin, displays fewer non-specific interactions than avidin (Marttila et al., FEBS Lett. 467, 31-36 (2000)). Sensing performance with varying proportions of biotin and neutravidin was evaluated using fluorescence imaging. In the present disclosure, the biochip can be used for detecting both DNA and protein targets. For DNA sensing, the lower limit of detection (LOD) was approximately 3.5 copies for E. coli genomic DNA, a common pathogen in sepsis patients. The same linkage configuration produced a sensitivity of 0.3 fg / mL for tau protein phosphorylated at threonine 217 (p-Tau217), a key biomarker for Alzheimer’ s disease (Ashton et al., Neurology (2023)).

[0098] While the present disclosure has been described by means of specific embodiments, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope and spirit of the present disclosure set forth in the claims.

Claims

WHAT IS CLAIMED IS :

1. A method for preparing a biochip, comprising: coating a chip with a first solution of biotin to form a biotin-coated chip, wherein the biotin in the first solution is at a first concentration; subj ecting the biotin-coated chip to a second solution of neutravidin to form a neutravidin / biotin-coated chip, wherein the neutravidin in the second solution is at a second concentration; and subj ecting the neutravidin / biotin-coated chip to a third solution of a biotinylated probe to form the biochip, wherein a first molar ratio of the first concentration to the second concentration is 1 :2, or 1 :0.25.

2. The method according to claim 1, wherein the chip is an extended gate field-effect transistor (EGFET).

3. The method according to claim 1 , wherein the biotin in the first solution is at the first concentration of 0.1 pg / ml or 1 pg / ml.

4. The method according to claim 3, wherein when first concentration is 0.1 pg / ml, the first concentration, the second concentration, and the third concentration are present in a second molar ratio of 1 :2:0.3, or 1 :2:3.

5. The method according to claim 3, wherein when the first concentration is 1 pg / ml, the first concentration, the second concentration, and the third concentration are present in a third molar ratio of 1 :0.25 :0.3, or 1 :0.25 :3.

6. The method according to claim 1, wherein the biotinylated probe is specific to one of PRRSV DNA, Tau protein, and E. coli genomic DNA.

7. A method for preparing a biochip, comprising: coating a chip with a first solution of biotin to form a biotin-coated chip, wherein the biotin in the first solution is at a first concentration; subj ecting the biotin-coated chip to a second solution of neutravidin to form a neutravidin / biotin-coated chip, wherein the neutravidin in the second solution is at a second concentration; and subj ecting the neutravidin / biotin-coated chip to a third solution of a biotinylated probe to form the biochip, wherein a first molar ratio of the first concentration to the second concentration is 1 : 1 .

8. The method according to claim 7, wherein the chip is an extended gate field-effect transistor (EGFET).

9. The method according to claim 7, wherein the biotin in the first solution is at thefirst concentration of 0.1 pg / ml or 1 pg / ml.

10. The method according to claim 9, wherein the first concentration, the second concentration, and the third concentration are present in a second molar ratio of 1 : 1 :0.3, or 1 : 1 :

3.

11. The method according to claim 7, wherein the biotinylated probe is specific to one of PRRSV DNA, Tau protein, and E. coli genomic DNA.

12. A biochip, prepared by a method comprising: coating a chip with a first solution of biotin to form a biotin-coated chip, wherein the biotin in the first solution is at a first concentration; subj ecting the biotin-coated chip to a second solution of neutravidin to form a neutravidin / biotin-coated chip, wherein the neutravidin in the second solution is at a second concentration; and subj ecting the neutravidin / biotin-coated chip to a third solution of a biotinylated probe to form the biochip, wherein a first molar ratio of the first concentration to the second concentration is 1 : 1 , 1 :2, or 1 :0.25.

13. The biochip according to claim 12, wherein the chip is an extended gate fieldeffect transistor (EGFET).

14. The biochip according to claim 12, wherein the biotin in the first solution is at the first concentration of 0.1 pg / ml or 1 pg / ml.

15. The biochip according to claim 14, wherein when the first concentration is 0.1 pg / ml, the first concentration, the second concentration, and the third concentration are present in a second molar ratio of 1 : 1 :0.3, 1 : 1 :3, 1 :2:0.3, or 1 :2:3.

16. The biochip according to claim 14, wherein when the first concentration is 1 pg / ml, the first concentration, the second concentration and the third concentration are present in a third molar ratio, which is 1 : 1 :0.3, 1 : 1 :3, 1 :0.25 :0.3, or 1 :0.25 :3.

17. The biochip according to claim 12, wherein the biotinylated probe is specific to one of PRRSV DNA, Tau protein, and E. coli genomic DNA.

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