Coded suspension chip, surface modification method and application thereof

Abstract

This application discloses a coded suspension chip, a surface modification method, and applications thereof. The surface modification method comprises sequentially performing amino modification and initiator modification on a silica-based coded suspension chip, then uniformly mixing the modified coded suspension chip with an acrylate, a crosslinking agent, a reducing agent, a catalyst, and a solvent, and performing an atom transfer radical polymerization reaction to obtain a polymer-modified coded suspension chip. The surface modification method for the coded suspension chip provided in this application is easy to operate and can effectively improve the storage stability and probe molecule loading capacity of the coded suspension chip while maintaining excellent detection specificity, sensitivity, and resistance to nonspecific adsorption, with broad application prospects.

Description

Technical Field

[0001] The present application relates to a coded suspension chip, and in particular to a surface modification method of a coded suspension chip. Background Art

[0002] Suspension chip technology is a new chip technology developed in recent years. It primarily utilizes the specific interaction between sensory materials immobilized on coded microparticles and the sample to be tested to perform multi-target detection and analysis in fluids. Compared to traditional planar microarray technology, suspension chip technology offers advantages such as high sensitivity, high throughput, multiplex detection, and automation.

[0003] When using multi-factor detection technology based on coded suspension chips, there are problems such as difficulty in long-term storage and insufficient stability of the coded suspension chips, which leads to greater uncertainty in the detection of complex samples such as serum and plasma. For example, after one month of storage at 4°C, the signal of some existing coded suspension chips drops by nearly 30%. Some researchers believe that modifying the coded suspension chips with polymer macromolecular structures is expected to improve this situation. However, existing solutions for modifying coded suspension chips with polymers still have some shortcomings. For example, the modified coded suspension chips are often subject to strong interference with the detection specificity of the target substance. Summary of the Invention

[0004] The main purpose of this application is to provide a coded suspension chip, a surface modification method and application thereof, so as to overcome the deficiencies of the prior art.

[0005] To achieve the aforementioned invention objectives, the technical solutions adopted in this application include:

[0006] One aspect of the present application provides a surface modification method for a coded suspension chip, comprising:

[0007] S1. Providing a silicon dioxide-based coded suspension chip, and modifying the surface of the coded suspension chip with amino groups;

[0008] S2. Covalently linking an initiator to the coded suspension chip treated in step S1, wherein the initiator is selected from a halogenated compound having a conjugated stabilizing group;

[0009] S3, uniformly mixing the coded suspension chip treated in step S2 with a polymerization monomer, a cross-linking agent, a reducing agent, a catalyst, and a solvent, and performing an atom transfer radical polymerization reaction to obtain a polymer-modified coded suspension chip;

[0010] Wherein, the polymerizable monomer is selected from acrylate.

[0011] Another aspect of the present application provides a method for preparing a coded suspension chip, comprising:

[0012] 1) Providing a silicon dioxide-based coded suspension chip, and performing surface modification on the coded suspension chip using any of the aforementioned surface modification methods to obtain a polymer-modified coded suspension chip;

[0013] 2) Connecting the probe molecules to the polymer-modified coding suspension chip.

[0014] Another aspect of the present application provides a coded suspension chip, which is treated by any of the aforementioned surface modification methods or is prepared by the aforementioned preparation method.

[0015] Another aspect of the present application further provides the use of the coded suspension chip in preparing a kit or performing substance analysis.

[0016] Compared with the existing technology, the surface modification method of the coded suspension chip provided in this application is easy to operate, can effectively improve the storage stability and probe molecule loading capacity of the coded suspension chip, while also maintaining excellent detection specificity, sensitivity and resistance to nonspecific adsorption, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a schematic structural diagram of a coding suspension chip in a typical implementation case of this application;

[0019] Figure 2 This is a schematic diagram of the surface modification process of the coded suspension chip in Example 1 of the present application;

[0020] Figure 3A-3B The bright field and fluorescence images of the fluorescence quality inspection of the blank group sample after antibody coupling in Example 1 of the present application are shown respectively;

[0021] Figure 3C-3D The bright field and fluorescence images of the fluorescence quality inspection of the sample after coupling with the antibody in Example 1 of the present application are shown respectively;

[0022] Figure 4 The figure shows the changes in the detection signals of the blank group samples and the sample-coupled probe molecules before and after storage in Example 1 of the present application;

[0023] Figure 5A schematic diagram of the process of multiple suspension chip sandwich detection in one embodiment of the present application is shown;

[0024] Figure 6 The specific cross-experimental results of the example samples in Example 1 of the present application after being coupled with antibodies are shown. DETAILED DESCRIPTION

[0025] In view of the shortcomings of the existing technology, the inventors of this case have proposed the technical solution of this application after long-term research and practice. In summary, this application provides a coded suspension chip, which includes a silica coded suspension chip (hereinafter referred to as the chip) as a substrate and a polymer layer that induces atom transfer radical polymerization on the surface of the substrate; at the same time, this application also provides a surface modification method for the coded suspension chip, which mainly involves first modifying the surface of the chip with amino groups, then fixing an initiator for initiating polymerization on the chip surface, and then obtaining a polymer-modified chip through atom transfer radical polymerization. Finally, by coupling probe molecules, etc., the chip can be used for in vitro detection, immunoassay, etc. The following will further explain this technical solution, its implementation process and principles.

[0026] Some embodiments of the present application provide a surface modification method for a coded suspension chip, including:

[0027] S1. Providing a silicon dioxide-based coded suspension chip, and modifying the surface of the coded suspension chip with amino groups;

[0028] S2. Covalently linking an initiator to the coded suspension chip treated in step S1, wherein the initiator is selected from a halogenated compound having a conjugated stabilizing group;

[0029] S3, uniformly mixing the coded suspension chip treated in step S2 with a polymerization monomer, a cross-linking agent, a reducing agent, a catalyst, and a solvent, and performing an atom transfer radical polymerization reaction to obtain a polymer-modified coded suspension chip;

[0030] Wherein, the polymerizable monomer is selected from acrylate.

[0031] In one embodiment, the acrylate includes sodium acrylate, sodium methacrylate, and the like, but is not limited thereto.

[0032] In one embodiment, step S1 specifically includes dispersing the encoded suspension chip in an aminosilane solution at room temperature and allowing the solution to react sufficiently, thereby modifying the surface of the encoded suspension chip with amino groups; the aminosilane solution contains 5-10% aminosilane by volume, and the solvent used includes ethanol. The chip treated in step S1 can be referred to as an amino chip.

[0033] Furthermore, the aminosilane includes amino-terminated polydimethylsiloxane (APDMS), aminopropyltriethoxysilane (APTES), etc., but is not limited thereto.

[0034] In one embodiment, step S2 specifically includes dispersing the coded suspension chip treated in step S1 in an organic solution containing 5-10 V / V% triethylamine and 5-10 V / V% initiator, and allowing the mixture to react at -5°C to 5°C, thereby covalently attaching the initiator to the coded suspension chip; wherein the initiator is bromoisobutyryl bromide. The chip treated in step S1 can be referred to as an initiator chip.

[0035] In step S2, adding triethylamine to the reaction system is beneficial to promoting the reaction to proceed in the forward direction.

[0036] Furthermore, the organic solvent used in step S2 includes toluene, etc., but is not limited thereto.

[0037] In one embodiment, step S3 specifically includes dispersing the coded suspension chip treated in step S2 in a mixed solution containing 10-500 mmol / L of a polymerizable monomer, 2-100 mmol / L of a cross-linking agent, 10-100 mmol / L of a reducing agent, and 5-10 μmol / L of a catalyst, and allowing the mixture to react fully at room temperature, thereby obtaining a polymer-modified coded suspension chip, which may be referred to as a polymer-modified chip.

[0038] Wherein, the molar ratio of the cross-linking agent to the polymer monomer is less than 50:1.

[0039] In one embodiment, the catalyst comprises Cu 2+ Complexes formed with organic ligands.

[0040] The organic ligand includes pentamethyldiethylenetriamine or bipyridine, but is not limited thereto.

[0041] In one embodiment, the solvent used in the mixed solution in step S3 includes water, but is not limited thereto.

[0042] Furthermore, the solvent used in the mixed solution in step S3 also includes ethanol, but is not limited thereto.

[0043] In one embodiment, the cross-linking agent includes one or more of polyethylene glycol dimethacrylate and N,N'-methylenebisacrylamide, but is not limited thereto.

[0044] In one embodiment, the reducing agent includes ascorbic acid or ascorbate (such as sodium ascorbate), but is not limited thereto.

[0045] This application utilizes atom transfer radical polymerization (ATRP) to form a polyacrylic acid modified layer on the surface of a coded suspension chip with silica as the main component through a one-step reaction. The reaction is easy to implement, the conditions are mild, and subsequent probe molecules can be directly coupled, making the operation convenient. In particular, in this application, by adding a reducing agent and the like to cooperate with the acrylate polymerization monomer, it is unnecessary to remove water and oxygen from the atom transfer radical polymerization reaction system in advance, and the formed polymer layer carries a large number of functional groups for coupling probe molecules (especially carboxyl groups that are easy to couple to proteins), which can effectively improve the storage stability and probe molecule loading capacity of the coded suspension chip, while also maintaining excellent detection specificity, sensitivity, and resistance to nonspecific adsorption.

[0046] Some embodiments of the present application provide a coded suspension chip that has been treated with any of the aforementioned surface modification methods. Figure 1 The surface of the coded suspension chip is modified with a cross-linked polymer layer.

[0047] Some embodiments of the present application provide a method for preparing a coded suspension chip, including:

[0048] 1) Providing a silicon dioxide-based coded suspension chip, and performing surface modification on the coded suspension chip using any of the aforementioned surface modification methods to obtain a polymer-modified coded suspension chip;

[0049] 2) Connecting the probe molecules to the polymer-modified coding suspension chip.

[0050] In one embodiment, step 2) specifically includes: washing the polymer-modified coded suspension chip with MES buffer, then dispersing the polymer-modified coded suspension chip in MES buffer, then mixing and reacting with MES buffer containing EDC and NHS, and then mixing and reacting with NaAc-HAc buffer containing probe molecules at 4°C to room temperature; wherein the MES buffer and NaAc-HAc buffer are both weakly acidic.

[0051] In a more specific embodiment, step 2) specifically includes:

[0052] The polymer-modified coded suspension chip can be washed once with 0.1 mol / L MES buffer (pH = 4.7), and then the polymer-modified coded suspension chip can be dispersed in 0.1 mol / L MES buffer (pH = 4.7) and adjusted to weak acidity to form a chip suspension;

[0053] An activation solution containing 130 mmol / L EDC and 326 mmol / L NHS was prepared using 0.1 mol / L MES buffer (pH = 4.7);

[0054] The chip suspension and activation solution were mixed and reacted at 4°C to room temperature for about 30 minutes, then washed, and then mixed and reacted with the probe molecule solution (solvent: 0.1 mol / L NaAc-HAc buffer (pH = 5.0) at 4°C to room temperature for 6h to 12h.

[0055] In one embodiment, the probe molecule includes proteins, polypeptides, nucleic acids, etc., but is not limited thereto.

[0056] Some embodiments of the present application provide a coded suspension chip prepared by any of the aforementioned preparation methods.

[0057] Some embodiments of the present application provide a kit comprising any of the aforementioned coded suspension chips.

[0058] Obviously, the kit may further include a buffer solution, instructions, etc. for use with the encoded suspension chip. This is easily conceivable by those skilled in the art and will not be described in detail here.

[0059] Some embodiments of the present application provide a substance analysis method comprising: dispersing the coded suspension chip in a liquid phase system containing a target substance, and maintaining the coded suspension chip in a suspended state; then allowing probe molecules on the coded suspension chip to fully bind to the target substance in the liquid phase system; then removing the coded suspension chip from the liquid phase system and imaging it in an optical channel of a set wavelength to perform qualitative or quantitative analysis of the target substance in the liquid phase system.

[0060] The following describes the technical solution of the present application in more detail with reference to the accompanying drawings and several examples. However, it should be understood that the following examples are merely for the purpose of explaining and illustrating the technical solution and do not limit the scope of the present application. Furthermore, unless otherwise specified, the various raw materials, reaction equipment, detection equipment, and methods used in the following examples are all known in the art.

[0061] Example 1, please refer to Figure 2 A surface modification method for a coded suspension chip comprises the following steps:

[0062] 1) Select 2×10 5 The particle coded suspension chip (mainly composed of silicon dioxide, hereinafter referred to as "chip") was dispersed in 1000 μL of 5% aminosilane (APDMS) ethanol solution (prepared with 95% ethanol), fully reacted at room temperature for 30 minutes and then washed to obtain the amino chip.

[0063] 2) Suspend the amino chip in 1 mL of toluene, add 50 μL of triethylamine (TEA), and gradually add 60 μL of bromoisobutyryl bromide (BiBB) dropwise, mixing thoroughly. React at 0°C for 16 hours, then wash to obtain an initiator chip with an initiator immobilization efficiency exceeding 90%. This initiator immobilization efficiency can be determined indirectly, for example, by reacting the amino chip and initiator chip with glutaraldehyde, then coupling them with the same concentration of fluorescent protein. The bromine initiator conversion efficiency (%) is calculated by measuring the decrease in fluorescence value using the following formula:

[0064]

[0065] The amino chip signal value MFI was measured NH2 =47184, signal value MFI after fixing the initiator sample =3994, so the bromination efficiency is 91.5%.

[0066] 3) 1×10 5 The initiator chip obtained in step 2) is placed in a microcentrifuge tube, centrifuged and the supernatant is discarded.

[0067] 4) Reagent preparation:

[0068] a. Catalyst: Cu(II)PMDETA ligand ethanol solution: 85.2 mg CuBr2, 140 μL pentamethyldiethylenetriamine (PMDETA) and 812 μL ethanol were mixed to form.

[0069] b. Reducing agent: Ascorbic acid solution: formed by mixing 352 mg of ascorbic acid and 2.5 ml of ethanol.

[0070] c. Cross-linking agent: Bisacrylamide solution: formed by mixing 77 mg N, N'-methylenebisacrylamide and 1 ml ethanol.

[0071] 5) Weigh 20 mg of sodium methacrylate and add it to the reaction tube containing the initiator chip treated in step 3), add 1 ml of ultrapure water to completely dissolve it, add 20 μL of the prepared ascorbic acid solution, add 100 μL of the prepared bisacrylamide solution, and finally add 2 μL of the prepared Cu(II)PMDETA ligand ethanol solution. After fully reacting at room temperature for 5 hours, wash it to obtain a cross-linked sodium polyacrylate in situ modified suspension chip (i.e., a polymer-modified chip), which can be defined as an example sample. By analyzing the Fourier infrared spectrum of the example sample, the ~1100 cm -1 Si-O-Si antisymmetric stretching vibration peak, ~750cm -1 Symmetric stretching vibration peak of Si-O bond, ~1550cm -1 It is the symmetrical stretching vibration peak of C=O bond, ~3000cm-1 It is the -CH2- symmetric stretching vibration peak, indicating that sodium polyacrylate has been successfully modified on the chip surface.

[0072] Example 2 A surface modification method for a coded suspension chip comprises the following steps:

[0073] 1) Select 5×10 5 The coded suspension chip (the same as in Example 1) was dispersed in 1000 μL of a 5% aminosilane (APDMS) ethanol solution (prepared with 95% ethanol), fully reacted for 30 minutes, and then washed to obtain an amino chip.

[0074] 2) Suspend the amino chip in 1 mL of toluene, add 80 μL of triethylamine (TEA), and gradually add 80 μL of bromoisobutyryl bromide (BiBB) dropwise to mix thoroughly. Incubate in an ice bath at 0°C for 16 hours, then rinse to obtain the initiator chip.

[0075] 3) 1×10 5 The initiator-coded suspension chip obtained in step 2) is placed in a microcentrifuge tube, centrifuged and the supernatant is discarded.

[0076] 4) Reagent preparation:

[0077] a. Catalyst: Cu(II)PMDETA ligand ethanol solution: 85.2mg CuBr2, 140μL PMDETA and 812μL 20% ethanol were mixed to form.

[0078] b. Reducing agent: Ascorbic acid solution: 352 mg of ascorbic acid was mixed with 2.5 ml of 20% ethanol.

[0079] c. Cross-linking agent: Poly(ethylene glycol) dimethacrylate solution: 75 mg of poly(ethylene glycol) dimethacrylate having a weight average molecular weight of 750 was mixed with 1 ml of 20% ethanol.

[0080] 5) Weigh 10 mg of sodium acrylate and add it to the reaction tube containing the initiator chip treated in step 3), add 1 ml of ultrapure water to completely dissolve it, add 20 μL of the prepared ascorbic acid solution, add 500 μL of the prepared poly (ethylene glycol) dimethacrylate solution, and finally add 2 μL of the prepared Cu (II) PMDETA ligand solution. The mixture is fully reacted at room temperature for 10 hours and then washed to obtain a polymer-modified chip.

[0081] Example 3 A surface modification method for a coded suspension chip comprises the following steps:

[0082] 1) Select 2×10 5The coded suspension chip (the same as in Example 1) was dispersed in 1000 μL of a 5% aminosilane (APDMS) ethanol solution (prepared with 95% ethanol), fully reacted for 30 minutes, and then washed to obtain an amino chip.

[0083] 2) Suspend the amino chip in 1 mL of toluene, add 50 μL of triethylamine (TEA), and gradually add 60 μL of bromoisobutyryl bromide (BiBB) dropwise to mix. Incubate in an ice bath at 0°C for 12 hours, then rinse to obtain the initiator chip.

[0084] 3) 1×10 5 Place the initiator-coded suspension chip in a microcentrifuge tube, centrifuge and discard the supernatant.

[0085] 4) Reagent preparation:

[0086] a. Catalyst: Cu(II)PMDETA ligand ethanol solution: formed by mixing 85.2 mg CuBr2, 140 μL PMDETA and 812 μL ultrapure water.

[0087] b. Reducing agent: Ascorbic acid solution: 352 mg of ascorbic acid was mixed with 2.5 ml of ultrapure water.

[0088] c. Cross-linking agent: Poly(ethylene glycol) dimethacrylate solution: 75 mg of poly(ethylene glycol) dimethacrylate having a molecular weight of 750 was mixed with 1 ml of ultrapure water.

[0089] 5) Weigh 10 mg of sodium acrylate and add it to the reaction tube containing the initiator chip treated in step 3), add 1 ml of ultrapure water to completely dissolve it, add 20 μL of the prepared ascorbic acid solution, add 50 μL of the prepared poly (ethylene glycol) dimethacrylate solution, and finally add 2 μL of the prepared ethanol solution of Cu (II) PMDETA ligand. React at room temperature for 4 hours and then wash to obtain a polymer-modified chip.

[0090] Comparative Example 1: The surface modification method of a coded suspension chip is basically the same as that of Example 1, except that:

[0091] In step (5), glycidyl methacrylate is used instead of sodium methacrylate, and N,N'-methylenebisacrylamide is not used.

[0092] The product finally obtained in Comparative Example 1 can be defined as the Comparative Example 1 sample.

[0093] Comparative Example 2: The surface modification method of a coded suspension chip is basically the same as that of Example 1, except that:

[0094] In step (5), no ascorbic acid solution is added to the reaction system.

[0095] The product finally obtained in Comparative Example 2 can be defined as Comparative Example 2 sample.

[0096] The sample of Comparative Example 2 was tested using infrared spectroscopy and Raman spectroscopy, and it was found that the modification of the polymer was not completed.

[0097] Application Example 1

[0098] The example sample was dispersed in acetic acid / sodium acetate buffer solution at pH 5 containing 10 μg / mL TNF-α Capture Antibody for coupling overnight and stored in PBST solution at 37° C. for one month.

[0099] As a control, TNF-α Capture Antibody was coupled to a chip without polymer modification (blank sample) in the same manner and stored in PBST solution at 37° C. for one month.

[0100] See also Figure 3A-3B Shown are bright field and fluorescence images of the blank sample after antibody conjugation. Figure 3C-3D Bright field and fluorescence images of the example samples after antibody conjugation are shown.

[0101] See also Figure 4 It can be seen that after storage, the TNF-α binding performance of the blank group samples decreased significantly, while the TNF-α binding performance of the example samples did not decrease. The coupled antibody detection signal was three times higher than that of the blank group samples, the antibody loading capacity was significantly improved, and the storage stability was significantly enhanced.

[0102] Application Example 2

[0103] in accordance with Figure 5 The principle shown is that seven freshly prepared example samples with different codes are coupled to detection antibodies (i.e., probe molecules) of IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ, respectively, and then mixed into the same microtube and reacted with 1 ng / mL standard (std) of the seven factors, respectively. Figure 6 ,The results showed that seven chips specifically bound to the corresponding cytokines, and the ,cross-results were less than 3%.

[0104] The sample of Comparative Example 1 was tested in the same manner. The results showed that the ability of the sample of Comparative Example 1 to resist nonspecific adsorption was reduced, and the final detection sensitivity was at least one order of magnitude lower than that of the example sample.

[0105] The polymer-modified chip prepared in the above embodiments of the present application has significantly improved storage stability (stored at 37°C for 47 days, the signal value fluctuates within a 20% error range without a significant decrease). At the same time, the IL-1β detection sensitivity of the polymer-modified chip can reach up to 0.1 pg / mL, and the errors caused by nonspecific adsorption capacity and multiple cytokine cross-talk are both below 10%.

[0106] The above embodiments are merely preferred embodiments for fully illustrating the present application, and the scope of protection of the present application is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present application are all within the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

Claims

1. A substance analysis method, characterized in that: include: Preparation of a coded suspension chip, comprising: S1. Dispersing a silica-based coded suspension chip in an aminosilane solution at room temperature and allowing the solution to react sufficiently, thereby modifying the surface of the coded suspension chip with amino groups, wherein the aminosilane solution contains 5-10 V / V% aminosilane, and the solvent used includes ethanol; S2. Dispersing the coded suspension chip treated in step S1 in an organic solution containing 5-10 V / V% triethylamine and 5-10 V / V% initiator, and fully reacting at -5°C to 5°C, thereby covalently linking the initiator to the coded suspension chip, wherein the initiator is bromoisobutyryl bromide or ethyl bromoisobutyrate; S3, dispersing the coded suspension chip treated in step S2 in a mixed solution containing 10-500 mmol / L polymer monomer, 2-100 mmol / L cross-linking agent, 10-100 mmol / L reducing agent and 5-10 μmol / L catalyst, and performing an atom transfer radical polymerization reaction at room temperature to obtain a polymer-modified coded suspension chip, wherein the polymer monomer is selected from acrylate, the cross-linking agent includes N,N'-methylenebisacrylamide or polyethylene glycol dimethacrylate, the molar ratio of the cross-linking agent to the polymer monomer is less than 50:1, the solvent used in the mixed solution includes water, and the catalyst includes Cu 2+ A complex formed with an organic ligand, wherein the organic ligand includes pentamethyldiethylenetriamine or bipyridine; S4, washing the polymer-modified encoding suspension chip with MES buffer, then dispersing the polymer-modified encoding suspension chip in MES buffer, then mixing and reacting with MES buffer containing EDC and NHS, and then mixing and reacting with NaAc-HAc buffer containing probe molecules at 4°C to room temperature, wherein the MES buffer and NaAc-HAc buffer are both weakly acidic; Thereafter, the coded suspension chip is dispersed in a liquid phase system containing a target substance and maintained in a suspended state. The probe molecules on the coded suspension chip are then fully bound to the target substance in the liquid phase system. The coded suspension chip is then removed from the liquid phase system and imaged in an optical channel of a set wavelength to perform qualitative or quantitative analysis of the target substance in the liquid phase system.

2. The substance analysis method according to claim 1, characterized in that The reducing agent includes ascorbic acid or ascorbate.

3. The substance analysis method according to claim 1, characterized in that The solvent used in the mixed solution in step S3 also includes ethanol.

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