Method for preparing biochip and biochip prepared therefrom

Abstract

This disclosure provides a method for preparing a biochip and the prepared chip. The preparation method includes the following steps: coating the chip with a first solution containing biotin, a second solution containing avidin, and a third solution containing a biotinylated probe.

Description

Methods for preparing biochips and the prepared biochips This disclosure relates to a method for preparing a biochip, particularly a method for preparing a biochip with improved sensitivity, and the biochip prepared therefrom. A chip is formed from an integrated circuit (IC), which consists of transistors, capacitors and other electronic components on a small, flat wafer of semiconductor material. Chips are used to process electronic signals for various measurement results. One of the common transistors used in chips is the metal oxide semiconductor field-effect transistor (MOSFET), which controls the current flowing through an electric field. MOSFETs have advantages such as high sensitivity, high specificity, and instantaneous detection. Biochips consist of biological elements and chip elements. The surface of a biochip needs to be modified to accommodate specific probes for various detection targets. (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GA) are the most common modifiers and activators for surface functionalization of wafers. APTES is an amino-functionalized organosilicon. GA is a dialdehyde containing two aldehyde functional groups. The amino group from APTES readily reacts with the aldehyde group from GA. One of the aldehyde groups in GA can react with APTES, and the other aldehyde groups can react with the antibody of the probe (Udomsom et al., Coatings (2021), 11, 595). Other modifiers such as CPTES and VTES, and activators such as gelatin, EDC, and NHS, can be applied to wafer surface modification processes (Kujawa et al., Science of the Total Environment 801 (2021) 149647). Triethoxysilyllundecanal (TESUD) is an alternative modifier that can replace the two-step APTES-GA process (Lee et al., Sensors and Actuators B 175 (2012) 201-207). Because of the functionalization of the surface of the biochip, antibody probes can bind to the surface of the chip with the attached functional groups, and then analytes are added for detection (Sadighbayan et al., Trends in Analytical Chemistry (2020)). However, the number of functional groups carried by individual antibody probes varies, which can lead to inconsistent binding efficiencies. Furthermore, the random orientation of antibody probes binding to APTES or GA reduces the number of Fab (fragment, antigen-binding) regions that interact with the antigen, and decreases the antibody probe density (Trilling et al., Analyst (2013)). Biotin / avidin affinity systems are often used to address this issue. While this method can increase probe binding efficiency, sensitivity remains low. Therefore, it is necessary to prepare biochips with biotin / anti-biotin / biotinylated probes that improve sensitivity. The purpose of this invention is to provide a biochip with significantly improved sensitivity. To achieve the above objectives, the present invention provides a method for preparing a biochip, comprising the following steps: coating a chip with a first solution containing biotin to form a biotin-coated chip, wherein the biotin in the first solution has a first concentration ranging from 0.1 to 1 μg / ml; providing the biotin-coated chip and a second solution containing avidin to form an avidin / biotin-coated chip, wherein the avidin in the second solution has a second concentration ranging from 0.1 to 100 μg / ml; and providing the avidin / biotin-coated chip and a third solution containing a biotinylated probe to form a biochip, wherein the biotinylated probe in the third solution has a third concentration ranging from 1 to 3 μg / ml. In one embodiment, the chip is an extended gate field-effect transistor (EGFET). In one embodiment, the first concentration of biotin in the first solution is 0.1 μg / ml. In one embodiment, the second concentration of the avidin in the second solution is 30 μg / ml. In one embodiment, the third concentration of the biotinylated probe in the third solution is 1 μg / ml. In one embodiment, the first concentration of biotin in the first solution is 1 μg / ml. In one embodiment, the second concentration of the avidin in the second solution is 100 μg / ml. In one embodiment, the ratio of the first concentration, the second concentration, and the third concentration is 1:300:10. In one embodiment, the biotinylated probe is selected from the group consisting of biotinylated anti-Porcine reproductive and respiratory syndrome virus (PRRSV) antibody, biotinylated anti-Tau antibody, and biotinylated SARS-CoV-2 probe. To help understand the purpose, features and effects of this disclosure, detailed embodiments and accompanying drawings are provided. The chip disclosed herein is an extended gate field-effect transistor (EGFET) for sensing analytes (e.g., bioanalytes). A biosensing system can utilize a biosensing layer on a substrate (e.g., a conductive substrate or sensing surface region) coupled to a field-effect transistor (FET). The gate of the FET can be connected to the substrate on which the biosensing layer is located. The functionalized substrate can include well-defined regions (e.g., wells, matrices, or flat regions) on which a specific predetermined volume of fluid (e.g., liquid, colloid) is held. An external electrode can be immersed in the fluid and connected to a power source supplying a voltage, which can be a gate voltage or a power supply. The charge transfer change from the external electrode to the gate electrode of the FET due to the presence of target molecules in the biosensing layer modulates the source characteristics of the FET to drain characteristics. This modulation can then be correlated with the concentration of the target analyte (e.g., based on known correlations derived from previous calibration results or similar results), which is present in the sensing layer. This biosensing system isolates the sensing electronics from the potentially harmful environment of the fluid used for sensing (i.e., the FET element is isolated from the fluid containing the analyte). The system can include an external functionalized substrate connected to the gate of the FET. The FET also includes a source and a drain, along with a gate electrode accompanied by an insulator and the gate itself. The external electrode can contact an electrolyte, and a gate voltage is applied to the external electrode. The interaction between ions present in the electrolyte and functional groups present on the extended gate substrate causes a change in surface potential. This potential change results in a change in the current to the gate of the FET. Conversely, the gate current modulates the drain-source current in the FET. The regulation of the drain current due to the presence of biomolecules on the surface of the extended gate makes the FET a biosensor. The biotin coating concentration was determined using conjugate focal microscopy. EGFETs were treated with the following biotins in phosphate-buffered saline (PBS) for 16 hours to form biotin-coated wafers: biotin coupled with 0.1 μg / ml fluorescein isothiocyanate (FITC) (Thermo Scientific, #22030), biotin coupled with 0.5 μg / ml FITC, biotin coupled with 1 μg / ml FITC, and biotin coupled with 10 μg / ml FITC. Fluorescence density was then observed using a conjugate focal microscope (TCS SP8 STED, Leica), and the images are shown in Figure 1A. However, the surface of the biotin wafer was not flat, making focusing difficult. Therefore, conjugate focal images of the biotin-coated wafers treated with 1 μg / ml biotin-FITC were captured again. The results are shown in Figure 1B, which indicates strong fluorescence density at concentrations of 0.1 μg / ml and 1 μg / ml. Considering material cost, a concentration of 0.1 μg / ml of biotin coated on the wafer was chosen. Determine the appropriate concentration range for avidin. EGFETs were treated in phosphate-buffered saline (PBS) with the following avidin Alexa Fluor 488 (Thermo Scientific, #A21370) for 10 minutes to form avidin-coated wafers: 0.1 μg / ml, 1 μg / ml, 10 μg / ml, 30 μg / ml, and 50 μg / ml. The fluorescence density of the avidin-coated wafers was then observed using a conjugate focal microscope, and the images are shown in Figure 2. The results, as shown in Figure 2, indicate strong fluorescence density at concentrations of 0.1 μg / ml and 30 μg / ml. Subsequently, EGFETs were treated with biotin-NHS (Thermo Scientific, #20217) at a concentration of 0.1 μg / ml to form biotin-coated wafers. These wafers were then treated with the following avidin Alexa Fluor 488 concentrations for 10 minutes each to form avidin / biotin-coated wafers: Alexa Fluor 488 at 1 μg / ml, Alexa Fluor 488 at 30 μg / ml, and Alexa Fluor 488 at 100 μg / ml. The fluorescence density of the avidin / biotin-coated wafers was then observed using a conjugate focal microscope; the image is shown in Figure 3A. 30 μg / ml of avidin is equal to 2.7 x 10^14 avidin molecules, and 0.1 μg / ml of biotin is equal to 2.4 x 10^14 biotin molecules, meaning the ratio of avidin molecules to biotin molecules is 1:1. The results are shown in Figure 3A, which indicates that after treating biotin-coated wafers (treated with 0.1 μg / ml of biotin) with the following avidin Alexa Fluor 488 to form avidin / biotin-coated wafers, the avidin / biotin-coated wafers exhibited better fluorescence density at an avidin concentration of 30 μg / ml: 1 μg / ml, 30 μg / ml, and 100 μg / ml. Conversely, after treating biotin-coated wafers (with 1 μg / ml biotin) with the following avidin Alexa Fluor 488 to form avidin / biotin-coated wafers, the avidin / biotin-coated wafers exhibited better fluorescence density at an avidin concentration of 100 μg / ml: 1 μg / ml, 30 μg / ml, and 100 μg / ml, as shown in Figure 3B. 100 μg / ml of avidin is equal to 9 x 10^14 avidin molecules, and 1 μg / ml of biotin is equal to 2.4 x 10^15 biotin molecules; that is, the ratio of avidin molecules to biotin molecules is 1:2.66.30 μg / ml of avidin is equal to 2.7 x 10^14 avidin molecules, and 1 μg / ml of biotin is equal to 2.4 x 10^15 biotin molecules; that is, the ratio of avidin molecules to biotin molecules is 1:8.8. In Figure 3B, the fluorescence density from 1 μg / ml biotin and 100 μg / ml avidin is better than that from 1 μg / ml biotin and 30 μg / ml avidin. Determine the suitable concentration range of the biotinylated probe. Based on the above experiments, the combination of 0.1 μg / ml biotin and 30 μg / ml avidin and the combination of 1 μg / ml biotin and 100 μg / ml avidin were used in subsequent experiments to determine the optimal concentration of the biotinylated probe. The nucleic acid probe was modified to have biotin at the 5' end and fluorescein amidites (FAM) at the 3' end. A wafer coated with 0.1 μg / ml biotin, 30 μg / ml avidin, and then 1 μg / ml PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) probe (SEQ ID NO: 1) was observed to have better fluorescence intensity, as shown in Figure 4. Demonstrates the concentration range of biotinylated antibodies on the chip The concentration conditions obtained above are demonstrated based on changes in electrical signals. Changes in electrical signals were measured on wafers coated with 0.1 μg / ml biotin and 30 μg / ml avidin at different concentrations of biotinylated anti-PRRSV antibodies (0.1 μg / ml, 1 μg / ml, and 3 μg / ml), including PRRSV-142 VL (SEQ ID NO: 2) and PRRSV-142 HL (SEQ ID NO: 3). The threshold voltage (V) of the ID-VG curve for each concentration was obtained at a constant drain current. th The threshold voltage difference (ΔV) is obtained by calculating the difference between the baseline and the threshold voltage at each concentration. th When the concentration of avidin was increased to 30 μg / ml, ΔV th The increase is shown in Figure 5. When the avidin concentration increases to 30 μg / ml, ΔV... thIncreased to 6.19.1 μg / ml. The biotinylated PRRSV probe produced the maximum ΔV. th The value was 0.49. However, when the concentration of the biotinylated PRRSV probe increased to 3 μg / ml, ΔV... th It won't decrease, but it will become unstable. Demonstrating the concentration range of the target peptide on the chip Biotinylated anti-Tau antibodies were initially used to measure the detectability of target peptides to determine the sensitivity and detectability of pathogens in this biological system. A wafer was coated with 0.1 μg / ml biotin, 30 μg / ml avidin, and 1 μg / ml biotinylated anti-Tau antibody, comprising Tau-217-123 VL (SEQ ID NO: 4) and Tau-217-123 VH (SEQ ID NO: 5). Subsequently, various concentrations of the target peptide (i.e., Tau peptide (SEQ ID NO: 6)) were added. The results showed that the lowest detectable concentration of Tau peptide was 1 ag / ml, and the highest detectable concentration of Tau peptide on the wafer was 50 pg / ml, as shown in Figure 6. Demonstrates the concentration range of the target nucleic acid on the chip. The sensitivity of this biological system to pathogen detection was tested using biotinylated SARS-CoV-2 probes comprising probe-F (SEQ ID NO:7), probe-P1 (SEQ ID NO:8), and probe-R (SEQ ID NO:9) in a 1:1:1 ratio. The wafer was coated with 0.1 μg / ml biotin, 30 μg / ml avidin, and then 1 μg / ml biotinylated SARS-CoV-2 probes. The initial concentration of total SARS-CoV-2 RNA was 2.68 x 10⁻⁶. 6 TCID50. Then, different concentrations of SARS-CoV-2 total RNA were added, 2.68 x 10⁻⁶. -6 2.68x10 -4 2.68x10 -2 2.68, 2.68x10 2 and 2.68x10 3Total SARS-CoV-2 RNA with TCID50. Results indicate that the sensitivity of the chip for detecting total SARS-CoV-2 RNA reached 10. -6 TCID50 (Ct:48), and there are 10 -6 TCID50 to 10 3 The linear range of TCID50 is shown in Figure 7. In this disclosure, the preferred concentrations of biotin, avidin, and biotinylated anti-PRRSV antibody used for coating the wafer are 0.1 μg / ml, 30 μg / ml, and 1 μg / ml, respectively. The preferred concentration ratio of biotin, avidin, and biotinylated anti-PRRSV antibody is 1:300:10. This ratio has also been demonstrated on the wafer to generate the highest voltage signal value. This ratio can be applied to the detection of p-Tau 217 and SARS-CoV-2. Phosphorylated Tau protein in cerebrospinal fluid (CSF) is an important biomarker for Alzheimer's disease (AD) (García-Chamé et al., Biosensors and Bioelectronics 159 (2020)). p-Tau 181, p-Tau 217, and p-Tau 231 are key biomarkers for the pathogenesis of AD (Leuzy et al., EMBO Mol Med (2022) 14: e14408). For early detection of AD in plasma, p-Tau 217 outperforms p-Tau 181 (Janelidze et al., Nature Communications (2020) 11:1683). Enzyme-linked immunosorbent assay (ELISA) is the most widely used immunoassay method in biomarker research. However, its relatively low sensitivity leads to missed detections. Alternative analytical methods based on techniques such as electrochemiluminescence (ECL) immunosensors, single-molecule array (SIMOA) analysis, immunomagnetic reduction assay (IMR), and mass spectrometry (MS) are suitable for detecting plasma Tau protein (Park et al., Clin Neurol (2022) 18(4):401-409). SIMOA is currently the most sensitive detection method. The limit of quantification (LLOQ) of p-Tau217 in plasma obtained by SIMOA analysis is 1.8 pg / ml. The LLOQ of p-Tau217 in plasma obtained by ECL-based immunosensors is 34 pg / ml, and the detection range is 0.1 to 100 ng / ml (Jalili et al., Molecules (2022) 27, 431). The sensitivity of the p-Tau217 ELISA kit ranges from 1 pg / ml to 80 pg / ml (OmnimAbs. OM641673). When compared to all the techniques mentioned in the preceding paragraphs, the biotin / avidin wafer system disclosed herein exhibits a sensitivity that is 180 times higher than the SIMOA system and 340 times higher than the ECL-based system. The limit of detection (LOD) of this biotin / avidin wafer system is 1 ag / ml (equivalent to 0.001 fg / ml) and shows a linear range from 10 fg / ml to 50,000 fg / ml. The average concentration of p-Tau217 in normal control plasma is 0.13 pg / ml (equivalent to 130 fg / ml) (Barthélemy et al., J. Exp. Med. (2020) Vol. 217 No. 11), which falls entirely within the linear detection range of current biotin / avidin wafer systems. Furthermore, this system requires only a small amount of plasma for detection, and the operating procedure is relatively simple. Therefore, this disclosure provides useful methods and tools for detecting p-Tau217 in plasma at an early stage of AD diagnosis. For the detection of SARS-CoV-2, real-time PCR is the gold standard method for qualitative and quantitative detection of viral nucleic acids. However, this method requires operation in an accredited laboratory, skilled technical personnel, and a considerable period before data analysis. Due to the COVID-19 pandemic, many new detection methods have been developed, such as graphene-FET biosensors and silicon nanowire-based viral spike protein detection. The graphene-FET biosensor device was able to detect 16 pfu / ml in culture medium and 2.42 × 10⁻⁶ pfu / ml in clinical samples. 2 Copies / ml (Sadighbayan et al., Trends in Analytical Chemistry (2020)). The valence of 0.7 pfu that can be estimated is theoretically equal to 1 TCID50 (Pourianfar et al., Indian J Virol. (2012)). Therefore, 16 pfu / ml equals 22.8 TCID50. The detection limit of the current biotin / avidin chip system is 10 copies / ml. -6The TCID50 (Ct:48) sensitivity of the biotin / avidin chip system disclosed herein is superior to that of graphene-FET biosensors. The biotin / avidin chip system of this disclosure uses nucleic acids as probes for nucleic acid detection without amplification, which shortens detection time and eliminates the need for highly skilled technical personnel. When the viral load is very low and still in the incubation period, the biotin / avidin chip system of this disclosure can rapidly and accurately detect SARS-CoV-2, enabling early and real-time monitoring of SARS-CoV-2. Although the present disclosure has been described through specific embodiments, various modifications and variations can be made by those skilled in the art without departing from the scope and spirit of the present disclosure as set forth in the appended claims. none Figure 1A shows an image captured by conjugate microscopy, illustrating the fluorescence density of biotin-FITC coated on a wafer. Figure 1B shows an image captured by conjugate microscopy, illustrating the fluorescence density of biotin-FITC coated on a wafer. Figure 2 shows an image captured by conjugate microscopy, illustrating the fluorescence density of avidin Alexa Fluor 488 coated on a wafer. Figure 3A shows an image captured by conjugate microscopy, illustrating the fluorescence density of avidin Alexa Fluor 488 on a wafer treated with 100 ng / ml biotin-NHS. Figure 3B shows an image captured by conjugate microscopy, illustrating the fluorescence density of avidin Alexa Fluor 488 on a wafer treated with 1 μg / ml biotin-NHS. Figure 4A shows an image captured by conjugate microscopy, illustrating the fluorescence density of the PRRSV probe-FAM on a wafer treated with 100 ng / ml biotin followed by 30 μg / ml avidin. Figure 4B shows an image captured by conjugate microscopy, illustrating the fluorescence density of the PRRSV probe-FAM on a wafer treated with 1 μg / ml biotin followed by 100 μg / ml avidin. Figure 5A is a graph showing the electrical signals of 1 ng / ml, 1 μg / ml, and 30 μg / ml avidin on a wafer treated with 0.1 μg / ml biotin. Figure 5B is a graph showing the electrical signals of 0.1 μg / ml, 1 μg / ml, and 3 μg / ml biotinylated antibody on a wafer treated with both biotin and avidin. Figure 6 is a graph demonstrating the concentration range of Tau peptide on the wafer. Figure 7 is a graph demonstrating the concentration range of SARS-CoV-2 RNA on the wafer. none

Claims

1. A method for preparing a biochip, comprising: coating a chip with a first solution containing biotin to form a biotin-coated chip, wherein the biotin in the first solution has a first concentration ranging from 0.1 to 1 μg / ml; providing a second solution containing avidin to the biotin-coated chip to form an avidin / biotin-coated chip, wherein the avidin in the second solution has a second concentration ranging from 0.1 to 100 μg / ml; and providing a third solution containing a biotinylated probe to the avidin / biotin-coated chip to form the biochip, wherein the biotinylated probe in the third solution has a third concentration ranging from 1 to 3 μg / ml.

2. The method as described in request item 1, wherein, The chip is an extended gate field-effect transistor (EGFET).

3. The method as described in request item 1, wherein, The first concentration of biotin in the first solution is 0.1 μg / ml.

4. The method as described in request item 3, wherein, The second concentration of the avidin in the second solution is 30 μg / ml.

5. The method as described in request item 4, wherein, The third concentration of the biotinylated probe in the third solution was 1 μg / ml.

6. The method as described in request item 1, wherein, The first concentration of biotin in the first solution is 1 μg / ml.

7. The method as described in claim 6, wherein, The second concentration of the avidin in the second solution is 100 μg / ml.

8. The method as described in request item 7, wherein, The third concentration of the biotinylated probe in the third solution was 1 μg / ml.

9. The method as described in claim 1, wherein, The ratio of the first concentration, the second concentration and the third concentration is 1:300:

10.

10. The method as described in claim 1, wherein, The biotinylated probe was selected from a group consisting of biotinylated anti-porcine reproductive and respiratory syndrome virus antibody, biotinylated anti-Tau antibody, and biotinylated SARS-CoV-2 probe.

11. A biochip prepared by a method comprising: coating a chip with a first solution containing biotin to form a biotin-coated chip, wherein the biotin in the first solution has a first concentration ranging from 0.1 to 1 μg / ml; providing a second solution containing avidin to the biotin-coated chip to form an avidin / biotin-coated chip, wherein the avidin in the second solution has a second concentration ranging from 0.1 to 100 μg / ml; and providing a third solution containing a biotinylated probe to the avidin / biotin-coated chip to form the biochip, wherein the biotinylated probe in the third solution has a third concentration ranging from 1 to 3 μg / ml.

12. The biochip as claimed in claim 11, wherein, The chip is an extended gate field-effect transistor (EGFET).

13. The biochip as claimed in claim 11, wherein, The first concentration of biotin in the first solution is 0.1 μg / ml.

14. The biochip as claimed in claim 13, wherein, The second concentration of the avidin in the second solution is 30 μg / ml.

15. The biochip as claimed in claim 14, wherein, The third concentration of the biotinylated probe in the third solution was 1 μg / ml.

16. The biochip as claimed in claim 11, wherein, The first concentration of biotin in the first solution is 1 μg / ml.

17. The biochip as claimed in claim 16, wherein, The second concentration of the avidin in the second solution is 100 μg / ml.

18. The biochip as claimed in claim 17, wherein, The third concentration of the biotinylated probe in the third solution was 1 μg / ml.

19. The biochip as claimed in claim 11, wherein, The ratio of the first concentration, the second concentration and the third concentration is 1:300:

10.

20. The biochip as claimed in claim 11, wherein, The biotinylated probe was selected from a group consisting of biotinylated anti-porcine reproductive and respiratory syndrome virus antibody, biotinylated anti-Tau antibody, and biotinylated SARS-CoV-2 probe.

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