Biosensor and method for manufacturing the same

Abstract

The present application relates to a kind of biochip and its manufacturing method and application.The biochip includes: substrate;Multiple pixel units are arranged on the substrate, and each pixel unit includes the microelectrode array with working electrode, counter electrode and dielectric structure;Wherein, the surface of the dielectric structure has surface modification layer, and the surface modification layer includes interlinked first surface modification structure and second surface modification structure;The first surface modification structure includes the alkoxysilane modification layer bonded with the surface of the dielectric structure and the functional modification layer connected with the alkoxysilane modification layer by sulfide bond;And the second surface modification structure includes the alkoxysilane modification layer bonded with the surface of the dielectric structure.The biochip surface has region-selective and functionally differentiated functional structure, and it is of great significance to promote the development of high-throughput, programmable and strong bio-compatible chip technology.

Description

Technical Field

[0001] This invention belongs to the field of biochip technology, specifically relating to a biochip and its manufacturing method, the biochip prepared by the method, and the application of the biochip in high-throughput synthesis of oligonucleotides or biological detection. Background Technology

[0002] Surface modification technology plays a crucial role in the fabrication of micro / nano devices and biochips. Traditional surface modification methods, such as dip-coating and drop-coating, are typically based on non-specific adsorption mechanisms, making it difficult to achieve precise modification of specific areas on the chip surface. This lack of spatial resolution presents significant limitations on chip platforms integrating multiple materials and functions, especially in electrode array chips.

[0003] Electrode array chips are typically composed of multiple materials, such as metal electrodes, dielectric insulating layers, and encapsulation layers, with different material regions performing different functions. In certain applications, such as electrochemical high-throughput oligonucleotide synthesis, single-molecule detection, or localized sensing, selective modification of only specific regions (such as metal electrodes or dielectric layers) is often required. For example, to functionalize initiators or trapping probes on metal electrodes while keeping the surrounding dielectric layer uncontaminated, the ability to selectively modify materials must be achieved.

[0004] On the other hand, electrode array chips consist of a large number of electrode units, with spatial distribution between different electrode units reaching the micrometer or even nanometer level. As chip integration density increases, different types of functionalization processing is often required on electrode units at different locations to endow the array with diverse functions, such as multi-channel parallel synthesis, molecular barcode encoding, and local sensor arrays. Therefore, how to achieve high spatial resolution, material selectivity, and functionally differentiated point-by-point modification has become a core technical challenge in the design and application of electrode array chips.

[0005] To address the aforementioned challenges, developing a chip structure capable of achieving regional selectivity and functional differentiation on the chip surface with microarray electrodes is of great significance for promoting the development of high-throughput, programmable, and biocompatible chip technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a biochip that utilizes different regions of the dielectric structure surface of a microelectrode array with different surface modification layers, thereby improving the regional functionalization differences and synthetic selectivity of the biochip.

[0007] According to a first aspect of the present invention, a biochip and a substrate are provided;

[0008] Multiple pixel units are disposed on the substrate, and each pixel unit includes a microelectrode array having a working electrode, a counter electrode, and a dielectric structure;

[0009] The dielectric structure has a surface modification layer on its surface, and the surface modification layer includes a first surface modification structure located in a first region of the dielectric structure and a second surface modification structure located in a second region of the dielectric structure.

[0010] The first surface-modified structure includes an alkoxysilane-modified structure bonded to the dielectric surface of the first region and a thioether-modified structure connected to the alkoxysilane-modified structure via a thioether bond; and

[0011] The second surface-modified structure includes the alkoxysilane-modified structure that is surface-bonded to the dielectric structure of the second region.

[0012] According to some embodiments, the surface modification layer has the structure shown in Formula I:

[0013] Formula I

[0014] Where n is an integer from 1 to 3;

[0015] m is an integer from 1 to 3;

[0016] R1 is C 1-4 alkyl groups; and

[0017] R2 is a hydroxyl, carboxyl, or tert-butoxycarbonyl amino group.

[0018] According to some embodiments, the surface modification layer has the structure shown in Formula II:

[0019] Formula II

[0020] R2 is a hydroxyl, carboxyl, or tert-butoxycarbonyl amino group.

[0021] According to some embodiments, the dielectric structure is made of a material selected from silicon oxides and silicon nitrides.

[0022] According to a second aspect of the present invention, a method for manufacturing a biochip is provided, the biochip comprising a substrate and a plurality of pixel units disposed on the substrate, the pixel units comprising a microelectrode array having a working electrode, a counter electrode, and a dielectric structure; the biochip having a two-electrode system consisting of a working electrode and a counter electrode, or a three-electrode system consisting of a working electrode, a counter electrode, and a reference electrode, characterized in that the method comprises the following steps:

[0023] The surface of the biochip is modified with an alkenyl-containing siloxane compound so that the surface of the biochip is attached with an alkoxysilane modified structure containing an alkenyl end group.

[0024] An electrolyte containing a thiol source is applied to the surface of a biochip with the alkoxysilane-modified structure attached, and a first potential is applied to the electrolyte to generate thiol radicals from the thiol source; and

[0025] A second potential is applied to the microarray electrodes within a predetermined region of the biochip to allow the thiol radicals to diffuse into the predetermined region and form a thioether structure bonded to the alkenyl end group of the alkoxysilane modified structure above the dielectric structure within the predetermined region.

[0026] According to one embodiment, the application of the first potential to the electrolyte is performed using cyclic voltammetry.

[0027] According to another embodiment, the selective surface modification step is achieved by applying the second potential in a manner that involves multiple potential step cycles;

[0028] Each potential step cycle includes a higher level phase and a lower level phase. The higher level phase initiates an electrochemical reaction, causing the thiol radical to diffuse into the predetermined region and react with the alkenyl group in the predetermined region. The lower level phase stops the electrochemical reaction, quenching the thiol radical and terminating the reaction between the thiol radical and the alkenyl group.

[0029] According to another embodiment, when the chip has a two-electrode system, the potential window of the cyclic voltammetry is 0.5~2.8V, the scan speed is 50~200mV / s, and the number of cycles of the cyclic voltammetry is 10~20 times.

[0030] According to another embodiment, when the chip has a three-electrode system, the potential window of the cyclic voltammetry is 0~1.2V, the scan rate is 50~200mV / s, and the number of cycles of the cyclic voltammetry is 10~20 times.

[0031] According to another embodiment, the potential of the high-level phase is at least 100mV higher than the potential at the peak current position of the cyclic voltammetry phase, and the on-time is 1~3s.

[0032] The low-level phase has a potential of 0V, or at least 100mV lower than the peak potential of the cyclic voltammetry phase, and lasts for 1-3 seconds; and

[0033] The period of the multiple potential step cycles is 200 to 400 high-level stage-low-level stage cycles.

[0034] According to yet another embodiment, in the electrolyte containing the thiol source,

[0035] The electrolyte concentration is 0.05~0.20 mol / L; and

[0036] The mercapto source accounts for 0.1% to 1% of the volume of the electrolyte.

[0037] According to yet another embodiment, the thiol-containing electrolyte includes a thiol source, an electrolyte, and a first solvent;

[0038] The thiol source is selected from tert-butoxycarbonyl mercaptoethylamine and mercaptoethanol;

[0039] The electrolyte is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, tetrabutylammonium hexafluorophosphate, and tetrabutylammonium p-toluenesulfonate; and / or

[0040] The first solvent comprises a mixture of water and acetonitrile.

[0041] According to another embodiment, when the electrolyte is selected from sodium dihydrogen phosphate and disodium hydrogen phosphate, the volume ratio of water to acetonitrile in the first solvent is (8~4):1.

[0042] When the electrolyte is selected from tetrabutylammonium hexafluorophosphate and tetrabutylammonium p-toluenesulfonate, the volume ratio of acetonitrile to water in the first solvent is (100~4):1.

[0043] In the first solvent, the volume ratio of water is less than or equal to 20%.

[0044] According to yet another embodiment, chemically modifying the chip surface includes immersing the chip in a chemical modification treatment solution, the chemical modification treatment solution comprising an acid, an alkenyl-containing siloxane compound, and a second solvent.

[0045] According to yet another embodiment, the acid is selected from one or both of glacial acetic acid and hydrochloric acid;

[0046] The alkenyl-containing siloxane compound is selected from allyltrimethoxysilane and allyltriethoxysilane; and / or

[0047] The second solvent is toluene.

[0048] According to yet another embodiment, the volume ratio of the second solvent, the alkenyl-containing siloxane compound, and the acid is 100:(1~10):(0.1~0.2).

[0049] According to another aspect of the present invention, a biochip is provided having a surface modified by the above method.

[0050] According to a third aspect of the present invention, the above-described biochip is provided for applications such as high-throughput synthesis of oligonucleotides or biological detection.

[0051] The biochip according to the present invention has different modification structures in different regions of the dielectric structure, realizing functional differences in the modified regions, thereby enabling selective synthesis at the micron or even nanoscale, with high spatial resolution, and is of great significance for promoting the development of high-throughput, programmable, and biocompatible chip technology. Attached Figure Description

[0052] The specific embodiments will be described below with reference to the accompanying drawings. The scope of the present invention is not limited thereto, wherein:

[0053] Figure 1 A schematic diagram of the microarray electrode structure of the biochip according to the present invention is shown;

[0054] Figure 2 and Figure 3 The CV cycle curve and the piecewise linear diagram of the current change over time in the multi-step step power-up method according to Embodiment 1 of the present invention are shown respectively.

[0055] Figure 4 and Figure 5 Fluorescence characterization images of the chip before and after deprotection according to Embodiment 1 of the present invention are shown respectively;

[0056] Figure 6 and Figure 7 The CV cycle curve and the piecewise linear diagram of the current change over time in the multi-step step power-up method according to Embodiment 2 of the present invention are shown respectively.

[0057] Figure 8 and Figure 9 Fluorescence characterization images of the chip before and after deprotection according to Embodiment 2 of the present invention are shown respectively;

[0058] Figure 10 and Figure 11 The CV cycle curve and the piecewise linear diagram of the current change over time in the multi-step step power-up method according to Embodiment 3 of the present invention are shown respectively.

[0059] Figure 12 A comparison of fluorescence characterization images of the modified chips according to Embodiments 1 and 3 of the present invention is shown;

[0060] Figure 13 The comparison of fluorescence characterization images of chips modified under different power conditions according to Embodiment 4 of the present invention is shown.

[0061] Figure 14 A CV cycle curve diagram according to Comparative Example 1 of the present invention is shown;

[0062] Figure 15 and Figure 16Fluorescence characterization images of the chip before and after deprotection according to Comparative Example 1 of the present invention are shown respectively;

[0063] Figure 17 The CV cycle curve of Comparative Example 2 according to the present invention is shown;

[0064] Figure 18 and Figure 19 Fluorescence characterization images of the chip before and after deprotection according to Comparative Example 2 of the present invention are shown respectively.

[0065] Explanation of reference numerals in the attached figures:

[0066] 1-Working electrode; 11-Ring working electrode; 12-Array working electrode; 2-Counter electrode; 3-Dielectric structure; 31-First sub-dielectric structure; 32-Second sub-dielectric structure. Detailed Implementation

[0067] Biochips are a technology that miniaturizes and integrates biochemical analysis systems onto a tiny chip. They enable high-throughput, parallel processing and analysis of biomolecules (such as DNA, proteins, and cells) and are widely used in biological science research and medical diagnostics.

[0068] Biochips typically include a substrate (e.g., a silicon wafer) and an electrode structure formed on the substrate. This electrode structure may include a working electrode, a counter electrode, and a reference electrode. Further, the working electrode, the counter electrode, and the dielectric structure located between them form a microarray electrode structure. In the biochip used in this invention, the working electrode and the counter electrode may be Pt electrodes, and the reference electrode may be Ag / Ag. + electrode.

[0069] Biochips typically require activation and / or surface modification before use in biosynthesis or biodetection. The surface properties of the chip after activation and / or surface modification have a significant impact on the efficiency and reliability of subsequent synthesis or detection.

[0070] biochip

[0071] This invention provides a biochip, comprising a microelectrode array and a dielectric structure. See details. Figure 1 The microelectrode array includes a working electrode 1, a counter electrode 2, and a dielectric structure 3. The working electrode 1 may include an array working electrode 12 and an annular working electrode 11 surrounding the array working electrode 12; the counter electrode 2 may surround the annular working electrode 11; the dielectric structure 3 may include a first sub-dielectric structure 31 located between the working electrodes, and a second sub-dielectric structure 32 located between the annular working electrode 11 and the counter electrode 2.

[0072] The working electrode 1 can be formed of a noble metal material, such as a Pt electrode. The counter electrode 2 can be formed of the same or different noble metal material as the working electrode 1, preferably also a Pt electrode. The dielectric structure can be made of silicon oxide (e.g., silicon dioxide) and / or silicon nitride (SiN). x Made from ( ).

[0073] Furthermore, the aforementioned dielectric structure undergoes surface modification to have a surface-modified layer, enabling it to be used as a synthesis region for oligonucleotide synthesis. Specifically, the first sub-dielectric structure 31 located between the plurality of array working electrodes 12 and between the plurality of array working electrodes 12 and the ring working electrode 11, and the second sub-dielectric structure located between the ring working electrode 11 and the counter electrode 2 constitute the aforementioned synthesis region.

[0074] The surface modification layer located on the surface of the dielectric structure 3 may include a first surface modification structure located in a first region of the dielectric structure and a second surface modification structure located in a second region of the dielectric structure.

[0075] The first surface-modified structure includes an alkoxysilane-modified structure surface-bonded to the dielectric structure 3 of the first region and a thioether-modified structure connected to the alkoxysilane-modified structure via thioether bonds. The second surface-modified structure includes an alkoxysilane-modified layer surface-bonded to the dielectric structure of the second region. Specifically, the thioether-modified structure of the dielectric structure 3 of the first region serves as the active region for subsequent synthesis steps.

[0076] According to one specific embodiment, the above-mentioned surface modification layer has the structure shown in Formula I:

[0077] Formula I

[0078] Where n can be an integer from 1 to 3;

[0079] m can be an integer from 1 to 3;

[0080] R1 can be C 1-4 The alkyl group may be, for example, methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, preferably methyl; and

[0081] R2 can be a hydroxyl group, a carboxyl group, or a tert-butoxycarbonyl amino group, preferably a tert-butoxycarbonyl amino group.

[0082] According to another specific embodiment, the above-mentioned surface modification layer has the structure shown in Formula II:

[0083] Formula II

[0084] R2 is a hydroxyl, carboxyl, or tert-butyloxycarbonyl amino group, for example, it can be tert-butyloxycarbonyl amino group.

[0085] In Formulas I and II above, R2 serves as the active site for synthesis.

[0086] Furthermore, the aforementioned surface modification layer may have the structure shown in Formula III:

[0087] Formula III.

[0088] After modification, the above-mentioned biochips have functional groups such as hydroxyl, carboxyl or tert-butoxycarbonyl amino groups on at least part of their surface, which can be used as the starting point for subsequent oligonucleotide synthesis, and can also be used for gene detection or hybridization analysis.

[0089] Methods for manufacturing biochips

[0090] The present invention provides a method for manufacturing a biochip, wherein the biochip includes a substrate and a plurality of pixel units disposed on the substrate, each pixel unit including a microelectrode array having a working electrode, a counter electrode and a dielectric structure; the biochip has a two-electrode system consisting of a working electrode and a counter electrode, or a three-electrode system consisting of a working electrode, a counter electrode and a reference electrode.

[0091] The above-mentioned method for manufacturing biochips includes a surface chemical modification step, an electrochemical reaction step, and a selective modification step.

[0092] The above-mentioned surface chemical modification steps may include modifying the surface of the biochip with alkenyl-containing siloxane compounds to attach an alkenyl-terminated alkoxysilane modified structure to the surface of the biochip.

[0093] According to one specific embodiment, chemically modifying the chip surface may include immersing the chip in a chemical modification solution, wherein the chemical modification solution may contain an acid, an alkenyl-containing siloxane compound, and a solvent. According to a specific embodiment, the volume ratio of the solvent, the alkenyl-containing siloxane compound, and the acid may be 100:(1~10):(0.1~0.2), preferably 100:(3~7):(0.1~0.2), for example, 100:5:0.15.

[0094] In this step, acid is used to rapidly hydrolyze the Si-OR1 groups in the alkenyl-containing siloxane compound to generate highly reactive Si-OH, which then undergoes dehydration condensation with the Si-OH on the chip surface to form stable Si-O-Si covalent bonds, thereby "anchoring" the alkenyl functional group to the electrode surface. The presence of acid significantly accelerates the hydrolysis process of Si-OR1 groups to Si-OH, and under acidic conditions, silanol groups preferentially bond to the chip surface, forming a relatively ordered and dense monolayer, and reducing the condensation reaction between silanol molecules in the solution. If the amount of acid is too small, the catalytic effect of the acid on the above hydrolysis process is insufficient, the reaction rate of silanol bonding to the chip surface is slow, and the surface bonding is incomplete. If the amount of acid is too large, the hydrolysis rate is too fast, the bonding reaction on the chip surface is difficult to control, and the condensation reaction between silanol molecules in the solution is enhanced, making it difficult to control the formation of a monolayer on the chip surface.

[0095] The aforementioned acid can be glacial acetic acid, hydrochloric acid, or a combination of glacial acetic acid and hydrochloric acid, preferably glacial acetic acid. The aforementioned alkenyl-containing siloxane compound can be allyltrialkoxysilane, preferably allyltri(C 1-4 Alkoxysilane, for example, one or both of allyltrimethoxysilane and allyltriethoxysilane. The solvent may be toluene.

[0096]

[0097] Through the interaction between the acid and allyltrialkoxysilane in the above surface chemical modification steps, a monolayer containing alkenyl end groups is formed on the entire surface of the chip. This surface chemical modification step achieves non-specific modification of the chip surface.

[0098] The above-mentioned electrochemical reaction steps may include applying an electrolyte containing a thiol source to the surface of the biochip that has been chemically modified and has an alkoxysilane-modified structure, and applying a first potential to the electrolyte to cause the thiol source to undergo an electrochemical reaction to generate thiol free radicals.

[0099] The electrolyte containing a thiol source may include a thiol source, an electrolyte, and a first solvent. The thiol source provides a source of thiol radicals for the electrochemical reaction and comprises a thiol group and a functional group with a protecting group (e.g., amino, hydroxyl, or carboxyl group). The thiol group is used to generate thiol radicals in the electrochemical reaction, and the functional group with the protecting group, after removal of the protecting group, can be used for subsequent functional modification of the chip structure. The thiol source may be, for example, one or both of tert-butyloxycarbonylthioethylamine (Boc thioethylamine) and mercaptoethanol, preferably tert-butyloxycarbonylthioethylamine. In the electrolyte, the volume fraction of the thiol source may be 0.1% to 1%, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or any value between two values.

[0100] The electrolyte described above provides conductivity to the electrolyte solution and may be a conjugate ammonium salt and / or phosphate of an inorganic acid or organic acid, such as one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, tetrabutylammonium hexafluorophosphate, and tetrabutylammonium p-toluenesulfonate, preferably tetrabutylammonium hexafluorophosphate. In the electrolyte solution described above, the concentration of the electrolyte may be 0.05~0.20 mol / L, for example, 0.07 mol / L, 0.10 mol / L, 0.13 mol / L, 0.16 mol / L, or any intermediate value between any two of the aforementioned values.

[0101] The first solvent mentioned above includes a mixture of water and acetonitrile. The water content in the solvent affects the oxidation behavior of the thiol source in the solution. On the one hand, the higher the water content, the weaker the oxidation behavior of the thiol source. On the other hand, a small amount of water can inhibit the self-deprotection of the Boc protecting group.

[0102] According to one embodiment, the first solvent is acetonitrile as the main solvent, and the first solvent is a mixture of acetonitrile and water with a volume ratio of (100~4):1. Preferably, the volume ratio of water in the first solvent is less than or equal to 20%, for example, 15%, 12%, 10%, 8%, 5%, 4%, or an intermediate value between any two of the aforementioned values. According to another embodiment, the first solvent is a mixture of water and acetonitrile with a volume ratio of (10~4):1. According to yet another embodiment, the first solvent is a mixture of water and acetonitrile with a volume ratio of (100~80):1 (e.g., 95:1, 90:1, or 85:1).

[0103] According to a preferred embodiment, when the electrolyte is selected from sodium dihydrogen phosphate and disodium hydrogen phosphate, the volume ratio of water to acetonitrile in the first solvent is (8~4):1, for example, 7:1, 6:1 or 5:1.

[0104] When the electrolyte is selected from tetrabutylammonium hexafluorophosphate and tetrabutylammonium p-toluenesulfonate, the volume ratio of acetonitrile to water in the first solvent is (100~4):1, for example, 95:1, 90:1 or 85:1.

[0105] As shown in schematic IV below, the thiol compound is electrochemically initiated at the first potential to generate a highly unstable thiol radical cation (R-SH). + • In the presence of a solvent, a deprotonation reaction occurs immediately, transforming the mercapto radical into a neutral mercapto radical. This means that the electron transfer and proton dissociation involved are a coordinated proton-coupled electron transfer process. In this electrochemical reaction step, the formation of the mercapto radical occurs on the surface of the working electrode.

[0106] Formula IV

[0107] In the electrochemical reaction step, a potential is applied to the electrolyte using the corresponding first potential application method, depending on the electrode system of the biochip. Cyclic voltammetry (CV) can be used for this purpose.

[0108] Specifically, when the biochip has a two-electrode system, i.e., the electrode system consists of a working electrode and a counter electrode, the potential window of the cyclic voltammetry is 0.5~2.8V, for example, 0.8V, 1.2V, 1.5V, 1.8V, 2.1V or 2.5V, the scan rate is 50~200mV / s, for example, 80mV / s, 120 mV / s, 160 mV / s or 180 mV / s, and the number of cycles is 10~20 times, for example, 12 times, 14 times, 16 times or 18 times.

[0109] When a biochip has a three-electrode system, i.e., the electrode system consists of a working electrode, a counter electrode, and a reference electrode, the potential window of the cyclic voltammetry is 0~1.2V, preferably 0.1~1.0V, for example, 0.4V, 0.7V or 0.9V, the scan rate is 50~200mV / s, and the number of cycles is 10~20 times, for example, 12 times, 14 times, 16 times or 18 times.

[0110] In the method of the present invention, the current-potential curve is obtained by scanning the cyclic voltammetry, and the peak current potential and the peak potential of the electrode modification system of the present invention are determined. Based on these two potential values, the higher level potential and the lower level potential of the potential step cycle stage are obtained by expanding them by more than 100mV.

[0111] Therefore, this electrochemical reaction step generates thiol radicals through cyclic voltammetry scanning, activates the pixel region, and determines the higher and lower potential levels of the potential step cycle.

[0112] The aforementioned selective surface modification step can be achieved by applying a second potential in a series of potential step cycles. Each potential step cycle includes a higher-level phase and a lower-level phase. The higher-level phase initiates an electrochemical reaction, causing thiol radicals to diffuse into a predetermined region and react with alkenyl groups within that region. The lower-level phase stops the electrochemical reaction, quenching the thiol radicals and terminating the reaction between the thiol radicals and alkenyl groups.

[0113] According to one implementation, the potential of the higher-level phase is at least 100mV higher than the potential at the peak current position of the cyclic voltammetry phase, for example, 150mV, 200mV, 250mV or 300mV higher, and the on-time is 1 to 3 seconds, for example 2 seconds.

[0114] The potential of the lower level phase can be 0V, or at least 100mV lower than the peak potential of the cyclic voltammetry phase, for example, 150mV, 200mV, 250mV or 300mV lower, and the duration is 1 to 3s, for example 2s.

[0115] The period of the above-mentioned multiple potential step cycles can be 200 to 400 cycles of higher level stage-lower level stage, preferably 250 to 350, for example 300.

[0116] The method of the present invention uses a multi-step step-electrode method to modify the surface of a biochip.

[0117] The aforementioned selective surface modification step may include applying a second electric field to the microarray electrodes within a predetermined region of the biochip, causing thiol radicals to diffuse to the predetermined region on the chip surface, and forming a thioether structure bonded to the alkenyl end group of the aforementioned alkoxysilane-modified structure above the dielectric structure within the predetermined region, thereby achieving surface modification of the biochip surface with alkenyl functional groups. Referring to Formula IV above, the thiol radicals generated in the electrochemical reaction step diffuse to the predetermined region under the action of the second electric field and selectively react with the alkenyl group formed in the chemical modification step within the predetermined region, forming a functionally modified structure with functional groups such as amino, carboxyl, or hydroxyl groups. This functionally modified structure serves as a site for subsequent functional synthesis. The range of this selective surface modification is determined by the diffusion range of the electrochemically initiated thiol radicals on the working electrode, and the diffusion range (i.e., the modification range) can be controlled by the electrochemical reaction kinetics (such as intermittent or continuous electrochemical reactions) on the electrode, thereby enabling selective site-specific modification at the micron or even nanoscale with high spatial resolution.

[0118] The method of this invention generates thiol radicals through an electrochemical reaction, overcoming the limitation that alkenyl groups in surface-modified structures grafted onto the dielectric material surface of a chip via chemical modification steps are difficult to react with functional groups such as carboxyl, amino, or hydroxyl groups under conventional conditions. The reaction between thiol radicals and alkenyl groups is a rapid click chemistry reaction with high specificity. Furthermore, cyclic voltammetry is used to precisely determine the potential window for the electrochemical reaction that generates thiol radicals, and the diffusion range of thiol radicals is controlled by cyclically controlling the voltage application through potential steps. This enables selective and differentiated functional modification of specific chip surface regions. By selecting thiol sources with different functional groups, modifications to the chip surface with different functionalities can be achieved.

[0119] Applications of biochips

[0120] The present invention also provides applications of the above-mentioned biochip, which can be used for high-throughput synthesis of oligonucleotides or biological detection.

[0121] The features and implementation methods of the present invention will be described below with reference to specific embodiments, but the present invention is not limited thereto.

[0122] Example 1 (ACN:H2O=4:1)

[0123] Selective modification:

[0124] The functional modification of the SiO2 dielectric layer near the electrochemically synthesized microarray electrode is carried out through the following steps.

[0125] 1) Modify the entire large-size microporous chip with an allyltrimethoxysilane monolayer. The specific steps include: cleaning the chip, which includes a three-electrode system (the working electrode and the counter electrode are both Pt electrodes, and the counter electrode is Ag / AgCl), and then plasma treating it for 5 min; immersing the chip in a solution of 2 mL toluene, 100 μL allylsilane and 3 μL glacial acetic acid overnight, then taking it out, rinsing it and drying it for later use.

[0126] 2) Activate the pixels in the predetermined region. Utilize Boc mercaptoethylamine as the amino functional material to electrochemically generate mercapto radicals. These mercapto radicals diffuse into the predetermined region and undergo a mercapto-alkene click chemical reaction with an allyltrimethoxysilane monolayer within the diffusion region. Specifically, in an electrolyte containing 0.1 M tetrabutylammonium hexafluorophosphate, 8 mL acetonitrile, 2 mL water (acetonitrile:water = 4:1), and 20 μL Boc mercaptoethylamine, apply a potential to the electrodes of the pixels in the activated region for CV scanning. The potential window for CV scanning is 0–1.2 V, the scan rate is 100 mV, and the number of cycles is 20. Then, apply a constant voltage of 1.0 V intermittently (1.0 V 2.0 sec, 0 V 2.0 sec, 100 cycles). The current-potential diagram of the CV scan is shown below. Figure 2As shown, the multi-step step power-up method is as follows: Figure 3 As shown.

[0127] Modification verification:

[0128] Fluorescence characterization of the modified Boc mercaptoethylamine:

[0129] 1) Immerse the chip in 4M dioxane hydrochloric acid solution to remove the Boc protecting group. After 30 minutes, remove the chip, rinse it with pure water and blow it dry.

[0130] 2) Dissolve phosphoramide-biotin in acetonitrile at a mass-volume ratio of 1:20, add an equal volume of 5-ethylthiotetrazole (ETT), and immerse the chip.

[0131] 3) After 2 hours, remove the chip, rinse with acetonitrile, dry it, and then proceed with the iodine oxidation step;

[0132] 4) Immerse the chip in trichloroacetic acid (TCA) to remove the phosphoramidite groups, then rinse and dry.

[0133] 5) Dilute streptavidin-Cy3 fluorescent dye (SA-cy3) at a volume ratio of 1:100 in Tris pH 7.5 buffer;

[0134] 6) Observe the fluorescence of cy3 using a fluorescence display to determine the modified area.

[0135] The fluorescence signal on the surface of the chip without the Boc protection group removal step (1) was observed, and the resulting fluorescence image is shown below. Figure 4 As shown. Observe the fluorescence signal on the modified chip surface, and the obtained fluorescence image is as follows. Figure 5 As shown.

[0136] like Figure 2 As shown, within a potential window of 0V to 1.2V, the electrolyte containing Boc-mercaptoethylamine exhibits a strong and sharp irreversible oxidation peak. This result indicates that an irreversible electrochemical reaction can occur within a potential window of 0~1.2V, demonstrating that the electrochemical system disclosed in this paper can generate thiol radicals and drive surface click reactions. Figure 3 The current-time variation curves of the multi-step step power-up method used in this embodiment are shown.

[0137] Figure 4 The image shows a small amount of Cy3 fluorescence detected in the Pt electrode and the surrounding SiO2 dielectric region before deprotection, indicated by a faint orange halo. This suggests that very few amino Boc protecting groups naturally detach from the chip surface before deprotection, resulting in very few prematurely exposed amino groups. These amino groups will adsorb functional groups in non-target areas during subsequent functional modification steps, thus reducing the selectivity of modification.

[0138] Figure 5 The results show that after deprotection, strong Cy3 fluorescence (bright orange rings in the image) was detected in the activated pixel region within the Pt electrode where an electrical signal was applied and the surrounding SiO2 dielectric region, while no fluorescence signal was detected in the Pt electrode and the surrounding SiO2 region where no electrical signal was applied (dark area on the left side of the image). This result demonstrates that the method of this invention generates thiol radicals electrochemically and precisely controls the diffusion range of the radicals through a multi-step step-addition method, forming a functionally modified structure with clear boundaries. This successfully achieves selective and site-specific modification of selected electrodes and their adjacent dielectric regions while maintaining the chemical inertness of unactivated regions, exhibiting excellent spatial resolution.

[0139] Example 2 (ACN:H2O=100:1)

[0140] The modification was carried out in the same manner as in Example 1, except that the electrolyte composition was 0.1 M tetrabutylammonium hexafluorophosphate, 10 mL acetonitrile, 100 μL water (acetonitrile:water = 100:1), and 20 μL Boc mercaptoethylamine. The current-potential graph obtained from the CV scan is shown below. Figure 6 As shown, the multi-step step power-up method is as follows: Figure 7 As shown.

[0141] Fluorescence characterization was performed on the chips before and after deprotection, and the resulting fluorescence patterns are shown below. Figure 8 and Figure 9 As shown.

[0142] Depend on Figure 8 and Figure 9 As can be seen, fluorescence characterization revealed that before deprotection, Figure 8 The image shown is generally dark, indicating that the amino Boc protecting group did not detach naturally. Figure 9 The images shown after deprotection exhibit weak fluorescence in the unprotected pixels, indicating the presence of thiol radical diffusion. This demonstrates that water influences thiol radical diffusion. This suggests that under the electrolyte conditions of this embodiment, unprotected pixels do not automatically deprotect from Boc, but a small amount of thiol radical diffusion occurs. In other words, water prolongs the survival time of thiol radicals and slows down their quenching.

[0143] Example 3 (ACN:H2O=4:1)

[0144] The same modifications were made as in Example 1, except that the electrode system was a two-electrode system, the potential window for CV scanning was 0~2.5V, the scan rate was 100mV, the number of cycles was 20, and then a constant voltage of 2.4V was applied intermittently (2.4V 2.0sec, 0V 2.0sec, 100 cycles). The current-potential diagram of the CV scan is shown below. Figure 10 As shown, the multi-step step power-up method is as follows: Figure 11 As shown.

[0145] Compare Figure 3 and Figure 11 The CV diagram shown is as follows. Figure 11 The two-electrode system of Example 3 shown requires a higher reaction potential, and the overall waveform does not... Figure 3 The waveform of the three-electrode system in Embodiment 1 is steep, and the overall current integration area is also smaller than that of the three-electrode system.

[0146] The modified chips according to Examples 1 and 3 were compared in terms of fluorescence characterization; that is, the deprotected chips were subjected to fluorescence characterization respectively, and the results were as follows: Figure 12 The fluorescence images shown are from Example 1 (right side is the image from Example 1, left side is the image from Example 3). Comparing the fluorescence characterization image of the deprotected chip according to Example 1 (right side), it can be seen that functional modification of the dielectric structure can be successfully achieved under both electrode systems. Furthermore, the functionally modified dielectric structure obtained from the chip using the three-electrode system of Example 1 exhibits stronger fluorescence, indicating a better modification effect.

[0147] Example 4 (ACN:H2O=100:1)

[0148] Using the same chip, three-electrode system, and electrolyte as in Example 2, surface modification was performed under the electrode conditions shown in Table 1 below. The modified chips were then characterized by fluorescence, and the resulting images are shown below. Figure 13 As shown.

[0149] Table 1

[0150]

[0151] like Figure 13 As shown in the diagram, from the left to the right, with the increase of CV cycles, the fluorescence on the modified chip surface becomes brighter and more uniform, until sample #3 in the right diagram completely fills the entire disk on the chip surface. This is because as the CV cycle lengthens, the thiol radicals in the electrochemical reaction system exist for a longer time during repeated step-by-step electrochemical reactions, allowing the modification reaction to proceed more fully.

[0152] Specifically, sample #1 in the left figure only underwent 2 CV cycles, which was too few to allow for a sufficient reaction; sample #2 in the middle figure underwent 20 CV cycles, which allowed for a reaction in the vicinity of the on-electrode, but not in the far regions; sample #3 in the right figure underwent 20 CV cycles and applied multiple potential steps, which allowed for a sufficient modification reaction.

[0153] Comparative Example 1 (ACN:H2O=1:4)

[0154] The modification was carried out in the same manner as in Example 1, except that the electrolyte composition was: PBS 8.0 0.1M, water 8 mL, acetonitrile 2 mL, and Boc mercaptoethylamine 20 μL. The current-potential graph obtained from CV scanning is shown below. Figure 14 As shown, fluorescence characterization was performed on the chip before and after deprotection, and the resulting images are shown below. Figure 15 and Figure 16 As shown.

[0155] Compared to Example 1, the solvent system contains a larger relative amount of water (water:acetonitrile = 4:1). Figure 14 The later the peak appears in the CV scan, the narrower the peak and the smaller the integrated area. This indicates that the oxidation behavior of the system is the weakest, the number of free radicals generated is small, but the free radical diffusion is relatively easy.

[0156] Depend on Figure 15 and Figure 16 The fluorescence characterization diagram shown indicates that under PBS-based electrolyte conditions, a significant spontaneous electrochemical deprotection of Boc occurs, generating a certain amount of free amino groups that adsorb onto the chip surface. Therefore, the fluorescence characterization before deprotection... Figure 15 It also exhibits some fluorescence emission.

[0157] Comparative Example 2 (without H2O)

[0158] The modification was carried out in the same manner as in Example 1, except that the electrolyte composition was: 0.1 M tetrabutylammonium hexafluorophosphate, 10 mL ACN, and 20 μL Boc mercaptoethylamine. The current-potential graph obtained from the CV scan is shown below. Figure 17 As shown, fluorescence characterization was performed on the chip before and after deprotection, and the resulting images are shown below. Figure 18 and Figure 19 As shown.

[0159] Compared to Example 1, the solvent system does not contain water and is a pure acetonitrile system. Figure 17 The peaks in the CV scan shown appear significantly earlier, with wider peak shapes, higher peak values, and larger integrated areas, indicating that the system exhibits the strongest oxidation behavior and generates a significantly greater number of free radicals.

[0160] Depend on Figure 18 and Figure 19 The fluorescence characterization diagram shown indicates that a strong spontaneous electrochemical deprotection of Boc occurs in anhydrous acetonitrile solvent system, generating a large number of free amino groups that adsorb onto the chip surface. Therefore, the fluorescence characterization before deprotection is crucial. Figure 18 There was also strong fluorescence emission. This indicates that the embodiments under these conditions could not achieve selective modification at all.

[0161] Comparing Example 1 with Comparative Examples 1 and 2, fluorescence characterization revealed strong electrochemical deprotection of Boc under both pure acetonitrile solvent and PBS-based electrolyte conditions, resulting in the adsorption of numerous free amino groups on the surface. Therefore, using acetonitrile as the primary solvent and a small amount of water as an auxiliary solvent can suppress Boc deprotection.

Claims

1. A method for manufacturing a biochip, the biochip comprising a substrate and a plurality of pixel units disposed on the substrate, the pixel units comprising a microelectrode array having a working electrode, a counter electrode, and a dielectric structure; the biochip having a two-electrode system consisting of a working electrode and a counter electrode, or a three-electrode system consisting of a working electrode, a counter electrode, and a reference electrode, characterized in that, The method includes the following steps: The surface of the biochip is modified with an alkenyl-containing siloxane compound so that the surface of the biochip is attached with an alkoxysilane modified structure containing an alkenyl end group. An electrolyte containing a thiol source is applied to the surface of a biochip with the alkoxysilane-modified structure attached, and a first potential is applied to the electrolyte using cyclic voltammetry to generate thiol radicals from the thiol source; and A second potential is applied to the microarray electrodes within a predetermined region of the biochip to allow the thiol radicals to diffuse into the predetermined region. The second potential is applied in a cyclic manner with multiple potential steps to achieve the formation of a thioether structure bonded to the alkenyl end group of the alkoxysilane modified structure above the dielectric structure within the predetermined region. Each potential step cycle includes a higher level phase and a lower level phase, and the higher level potential of the higher level phase and the lower level potential of the lower level phase are determined by scanning the cyclic voltammetry.

2. The method according to claim 1, wherein, The higher-level phase initiates the electrochemical reaction, causing the thiol radicals to diffuse to a predetermined region of the biochip and react with alkenes within that region. The lower-level phase stops the electrochemical reaction, quenching the thiol radicals and terminating the reaction between the thiol radicals and alkenes.

3. The method according to claim 1, wherein, When the biochip has a two-electrode system, the potential window of the cyclic voltammetry is 0.5~2.8V, the scan rate is 50~200mV / s, and the number of cycles of the cyclic voltammetry is 10~20.

4. The method according to claim 1, wherein, When the biochip has a three-electrode system, the potential window of the cyclic voltammetry is 0~1.2V, the scan rate is 50~200mV / s, and the number of cycles of the cyclic voltammetry is 10~20.

5. The method according to claim 1, wherein, The potential of the higher level phase is at least 100mV higher than the potential at the peak current position of the cyclic voltammetry phase, and the turn-on time is 1~3s. The potential of the lower level phase is 0V, or at least 100mV lower than the peak potential of the cyclic voltammetry phase, and the duration is 1~3s; and The period of the multiple potential step cycles is 200 to 400 cycles of higher level phases followed by lower level phases.

6. The method according to claim 1, wherein, The electrolyte containing a thiol source comprises an electrolyte and the thiol source. The concentration of the electrolyte is 0.05~0.20 mol / L; and The mercapto source accounts for 0.1% to 1% of the volume of the electrolyte.

7. The method according to claim 1 or 6, wherein, The electrolyte containing a thiol source includes a thiol source, an electrolyte, and a first solvent; The thiol source is selected from tert-butoxycarbonyl mercaptoethylamine and mercaptoethanol; The electrolyte is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, tetrabutylammonium hexafluorophosphate, and tetrabutylammonium p-toluenesulfonate; and / or The first solvent comprises a mixture of water and acetonitrile.

8. The method according to claim 7, wherein, When the electrolyte is selected from sodium dihydrogen phosphate and disodium hydrogen phosphate, the volume ratio of water to acetonitrile in the first solvent is (8~4):

1.

9. The method according to claim 7, wherein, When the electrolyte is selected from tetrabutylammonium hexafluorophosphate and tetrabutylammonium p-toluenesulfonate, the volume ratio of acetonitrile to water in the first solvent is (100~4):

1.

10. The method according to claim 1, wherein, The step of modifying the surface of the biochip with an alkenyl-containing siloxane compound includes immersing the biochip in a chemical modification solution comprising an acid, an alkenyl-containing siloxane compound, and a second solvent.

11. The method of claim 10, wherein, The acid is selected from one or both of glacial acetic acid and hydrochloric acid; The alkenyl-containing siloxane compound is selected from allyltrimethoxysilane and allyltriethoxysilane; and / or The second solvent is toluene.

12. The method according to claim 10, wherein, The volume ratio of the second solvent, the alkenyl-containing siloxane compound, and the acid is 100:(1~10):(0.1~0.2).

13. A biochip, prepared according to any one of claims 1 to 12.

14. The biochip according to claim 13, in, The surface of the dielectric structure has a surface modification layer, and the surface modification layer includes a first surface modification structure located in a first region of the dielectric structure and a second surface modification structure located in a second region of the dielectric structure; The first surface-modified structure includes an alkoxysilane-modified structure surface-bonded to the dielectric structure of the first region and a thioether-modified structure connected to the alkoxysilane-modified structure via a thioether bond; and The second surface-modified structure includes the alkoxysilane-modified structure that is surface-bonded to the dielectric structure of the second region.

15. The biochip according to claim 14, wherein, The surface modification layer has the structure shown in Formula I: Equation I Where n is an integer from 1 to 3; m is an integer from 1 to 3; R1 is C 1-4 alkyl groups; and R2 is a hydroxyl, carboxyl, or tert-butoxycarbonyl amino group.

16. The biochip according to claim 15, wherein, The surface modification layer has the structure shown in Formula II: Formula II R2 is a hydroxyl, carboxyl, or tert-butoxycarbonyl amino group.

17. The biochip according to any one of claims 13 to 16, wherein, The dielectric structure is made of a material selected from silicon oxides and silicon nitrides.

18. The biochip according to any one of claims 13 to 17 for use in high-throughput synthesis of oligonucleotides or in biological detection.

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