阅读说明：本技术 微珠芯片及其电镀入孔式制备方法 (Microbead chip and electroplating hole-entering type preparation method thereof ) 是由 李智 刘超钧 许心意 于 2020-12-14 设计创作，主要内容包括：本发明公开了微珠芯片及其电镀入孔式制备方法。微珠芯片的电镀入孔式制备方法,包括如下步骤：步骤一,硅板表面预处理；将光刻胶层均匀旋涂到硅板的表面致使形成一层均匀的光刻胶层,利用光刻机将掩膜板上的图案转移到光刻胶层上；步骤二,硅板固定到硅板固定装置上,所述硅板固定装置将硅板、透明中空隔膜以及ITO导电玻璃三者从下至上堆叠并固定,使得硅板与ITO导电玻璃之间形成流通间隙；步骤三,装载二氧化硅微球：并将注射泵与硅板固定装置上的流通间隙导通；硅板与ITO导电玻璃分别接上脉冲直流电源的导电正极以及导电负极,注射泵与脉冲直流电源同时打开；步骤四,清洗去除剩余光刻胶层。(The invention discloses a microbead chip and an electroplating hole-entering preparation method thereof. The electroplating hole-entering type preparation method of the microbead chip comprises the following steps: firstly, pretreating the surface of a silicon plate; uniformly spin-coating the photoresist layer on the surface of the silicon plate to form a uniform photoresist layer, and transferring the pattern on the mask plate to the photoresist layer by using a photoetching machine; fixing the silicon plate on a silicon plate fixing device, wherein the silicon plate fixing device stacks and fixes the silicon plate, the transparent hollow diaphragm and the ITO conductive glass from bottom to top, so that a circulation gap is formed between the silicon plate and the ITO conductive glass; step three, loading silica microspheres: and the injection pump is communicated with the circulation gap on the silicon plate fixing device; the silicon plate and the ITO conductive glass are respectively connected with a conductive anode and a conductive cathode of a pulse direct-current power supply, and the injection pump and the pulse direct-current power supply are simultaneously turned on; and step four, cleaning and removing the residual photoresist layer.)

1. The electroplating hole-entering type preparation method of the microbead chip is characterized by comprising the following steps of: the method comprises the following steps:

firstly, pretreating the surface of a silicon plate;

uniformly spin-coating the photoresist layer on the surface of the silicon plate to form a uniform photoresist layer, transferring the pattern on the mask plate to the photoresist layer by using a photoetching machine, and etching densely-arranged small hole structures on the surface of the silicon plate by using a plasma etching technology at the hollow part of the photoresist layer;

fixing the silicon plate;

the silicon plate is fixed on the silicon plate fixing device, and the silicon plate fixing device stacks and fixes the silicon plate, the transparent hollow diaphragm and the ITO conductive glass from bottom to top, so that a circulation gap is formed between the silicon plate and the ITO conductive glass;

the photoresist layer of the silicon plate faces the ITO conductive glass;

the thickness of the transparent hollow diaphragm is 100-110 μm;

step three, loading silica microspheres:

washing the silicon dioxide microspheres with a conductive solution with the conductivity of 40uS/cm-100 muS/cm, and diluting to a solution to be injected with the solid concentration of 0.1% -0.2%;

putting the solution to be injected into an injection pump, and communicating the injection pump with a circulation gap on the silicon plate fixing device;

the silicon plate and the ITO conductive glass are respectively connected with a conductive anode and a conductive cathode of a pulse direct-current power supply, the injection pump and the pulse direct-current power supply are simultaneously turned on for 30-180 seconds, and the flow rate of liquid in the injection pump is ensured to be between 3uL/s and 15 uL/s;

and step four, cleaning and removing the residual photoresist layer.

2. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: and in the third step, conducting ultrasonic treatment on the conductive liquid mixed with the silicon dioxide microspheres for more than 3 minutes, then placing the conductive liquid in a vortex oscillator for 30 seconds for cleaning, and centrifuging the conductive liquid at the rotating speed of 3000g for 6 minutes by using a centrifugal machine for solid-liquid separation.

3. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: the silicon plate fixing device comprises a bottom plate for bearing a silicon plate, and the bottom plate is hollow;

the silicon plate fixing device also comprises a clamping mechanism, the clamping mechanism is used for clamping and fixing the ITO conductive glass, the transparent hollow diaphragm, the silicon plate and the bottom plate which are sequentially stacked and arranged from top to bottom, and a small hole structure is etched on the upper surface of the silicon plate;

the length of the through hole of the transparent hollow diaphragm in the left and right directions is larger than that of the bottom plate and the ITO conductive glass, and the ITO conductive glass is exposed out of the left end and the right end of the through hole of the transparent hollow diaphragm;

the ITO conductive glass, the transparent hollow diaphragm and the silicon plate enclose the circulation gap, and areas, exposed out of the ITO conductive glass, at the left end and the right end of the through hole of the transparent hollow diaphragm are respectively an inlet and an outlet of the circulation gap;

the silicon plate fixing device also comprises a conveying channel for conveying a microbead solution, wherein the microbead solution is a conductive liquid mixed with silicon dioxide microbeads, an inlet of the conveying channel is communicated with an injection pump, an outlet of the conveying channel is communicated with the inlet, and the outlet is communicated with a recovery tank;

and the conductive positive electrode of the pulse direct-current power supply is used for penetrating through the hollow area of the bottom plate to abut against the silicon plate, and the conductive negative electrode of the pulse direct-current power supply abuts against the ITO conductive glass.

4. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: the silica microspheres couple negatively charged oligonucleotide chains.

5. The method for preparing a microbead chip by electroplating according to claim 4, wherein the method comprises the following steps: a method of coupling silica microspheres to oligonucleotide chains, comprising the steps of:

A. amino group modified on surface of silicon dioxide microsphere

Preparing the silica microspheres into 10mg/mL-200mg/mL xylene suspension; adding a silanization reagent, wherein the volume concentration of the silanization reagent in the mixed solution is less than 10%, and fully reacting to modify the surface of the silicon dioxide microsphere with amino;

B. activated silica microspheres

Suspending the silicon dioxide microspheres with the surface modified with amino groups obtained in the step A in acetonitrile, wherein the mass concentration of the microspheres is 10mg/mL-200 mg/mL; adding diisopropylethylamine and cyanuric chloride, and shaking for reaction; washing with acetonitrile to remove excessive reactants, suspending the silica microspheres in a sodium borate buffer solution, and adjusting the pH value to 7.5-8.5;

C. covalent linking of silica microspheres to oligonucleotide chains

Dissolving 100nmol of oligonucleotide chain dry powder to be connected by using 2M sodium chloride solution, mixing with the silica microsphere sodium borate suspension obtained in the step B, wherein the content of the silica microspheres is 10-100mg, carrying out centrifugal treatment at 1000-3000rpm after carrying out oscillation reaction for 5-8 hours, and keeping supernatant; and (5) cleaning and drying.

6. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: the pH value of the conductive liquid is less than 9 and greater than 5.

7. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: the conductive liquid comprises 4.2-4.8mM of tris (hydroxymethyl) aminomethane, 4.2-4.8mM of boric acid and 0.01-0.03% of Triton X-100 by mass percentage.

8. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: the diameter of the silica microspheres is between 0.5um and 5 um.

9. The method for preparing a microbead chip by electroplating according to claim 1, wherein the method comprises the following steps: the voltage of the pulse direct current power supply is 1.0V-10.0V, the frequency is 1Hz-20Hz, and the cycle period is 5% -40%.

10. The microbead chip prepared by the electroplating hole-in-hole preparation method according to any of claims 1-9.

Technical Field

The invention relates to the technical field of semiconductors, in particular to a microbead chip and a preparation method thereof.

Background

Compared with sequencing technology, the high-density biochip has the advantages of low cost, large amount of detection sites, uniform format, quick analysis and the like, and gene libraries of large-scale people are established in the United kingdom and the United states by using the biochip technology.

Under the background of new coronary pneumonia epidemic situation, Europe and the United states start the group genome detection of new coronary patients, and adopt the biochip technology to research the relationship between genes and new coronary susceptibility.

The silicon plate is the main carrier substrate of the biochip. The surface of the silicon plate has more than 3 hundred million pores, each pore is used for fixing a micro-bead, and the pores are arranged in a honeycomb shape.

How to increase the hole-access rate of the microbeads is one of the difficulties in the biochip production process.

Patent document 1(CN111282604A) discloses a method for entering microspheres of a biochemical chip, which utilizes bubbles generated by a foaming agent to regulate the range of microspheres that have entered the biochemical chip. The method mainly comprises the steps of taking a biochemical chip, putting the biochemical chip into a microsphere solution, and fixing microspheres on the inner surface of the biochemical chip through chemical connection or a physical mode; and then introducing a foaming agent solution with bubbles, removing the microspheres close to the inner surface by using the bubbles, and reserving the microspheres in the prefabricated microstructure on the inner surface of the biochemical chip. The method has the defects that 1) after the microspheres enter, bubbles are needed to be removed, and the steps are complicated. 2) The number of microspheres and the number of pit surfaces are 2: 1, the entry rate is about 80%, the consumption of the microbeads is high, and the utilization rate is low.

Therefore, the technical problem faced at present is to develop a method for improving the hole-entering rate and the utilization rate of the microbeads.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an electroplating hole-entering type preparation method of a microbead chip, which aims to solve the technical problems of low hole-entering rate and low utilization rate in the prior art.

The invention provides a microbead chip, which aims to solve the technical problems of low hole-access rate and low utilization rate of the conventional microbead chip.

The technical scheme of the invention is as follows: the electroplating hole-entering type preparation method of the microbead chip is characterized by comprising the following steps of:

firstly, pretreating the surface of a silicon plate;

uniformly spin-coating the photoresist layer on the surface of the silicon plate to form a uniform photoresist layer, transferring the pattern on the mask plate to the photoresist layer by using a photoetching machine, and etching densely-arranged small hole structures on the surface of the silicon plate by using a plasma etching technology at the hollow part of the photoresist layer;

fixing the silicon plate;

the silicon plate is fixed on the silicon plate fixing device, and the silicon plate fixing device stacks and fixes the silicon plate, the transparent hollow diaphragm and the ITO conductive glass from bottom to top, so that a circulation gap is formed between the silicon plate and the ITO conductive glass;

the photoresist layer of the silicon plate faces the ITO conductive glass;

the thickness of the transparent hollow diaphragm is 100-110 μm;

step three, loading silica microspheres:

washing the silicon dioxide microspheres with a conductive solution with the conductivity of 40uS/cm-100 muS/cm, and diluting to a solution to be injected with the solid concentration of 0.1% -0.2%;

putting the solution to be injected into an injection pump, and communicating the injection pump with a circulation gap on the silicon plate fixing device;

the silicon plate and the ITO conductive glass are respectively connected with a conductive anode and a conductive cathode of a pulse direct-current power supply, the injection pump and the pulse direct-current power supply are simultaneously turned on for 30-180 seconds, and the flow rate of liquid in the injection pump is ensured to be between 3uL/s and 15 uL/s;

and step four, cleaning and removing the residual photoresist layer.

This patent is through the structure of optimizing the silicon board, has improved microballon hole rate and utilization ratio.

When the silicon plate is etched, the surface of the pure silicon wafer is processed by a photoresist layer, and a photoresist layer (with extremely poor conductivity) of about 1um is formed on the surface of the silicon plate. When the surface pattern of the mask plate is transferred to the photoresist layer on the wafer, full exposure is ensured, so that monocrystalline silicon on the exposed pattern is exposed in the air (the photoresist layer of the pattern part is fully reacted), thus the surface of the small hole pattern part is monocrystalline silicon with good conductivity, and the surface of the non-small hole pattern part is still the photoresist layer with poor conductivity. During the electroplating process, the potential difference from the conductive glass to the micro-holes will be much higher than the potential difference to the photoresist layer.

This patent is still through the fixed mode of optimizing the silicon chip, and the conducting solution through mixing the microballon forms the electric field in through the circulation clearance, and microballon solution flows through between the electrode, and the lorentz force makes the microballon entering hole downthehole, and then has improved microballon access hole rate and utilization ratio.

More preferably, in the third step, the conductive liquid mixed with the silica microspheres is subjected to ultrasonic treatment for more than 3 minutes, then the conductive liquid is placed in a vortex oscillator for 30 seconds to be cleaned, and a centrifuge is used for solid-liquid separation after centrifugation for 6 minutes at the rotating speed of 3000 g.

Further preferably, the silicon plate fixing device comprises a bottom plate for bearing the silicon plate, and the bottom plate is hollowed out;

the silicon plate fixing device also comprises a clamping mechanism, the clamping mechanism is used for clamping and fixing the ITO conductive glass, the transparent hollow diaphragm, the silicon plate and the bottom plate which are sequentially stacked and arranged from top to bottom, and a small hole structure is etched on the upper surface of the silicon plate;

the length of the through hole of the transparent hollow diaphragm in the left and right directions is larger than that of the bottom plate and the ITO conductive glass, and the ITO conductive glass is exposed out of the left end and the right end of the through hole of the transparent hollow diaphragm;

the ITO conductive glass, the transparent hollow diaphragm and the silicon plate enclose the circulation gap, and areas, exposed out of the ITO conductive glass, at the left end and the right end of the through hole of the transparent hollow diaphragm are respectively an inlet and an outlet of the circulation gap;

the silicon plate fixing device also comprises a conveying channel for conveying a microbead solution, wherein the microbead solution is a conductive liquid mixed with silicon dioxide microbeads, an inlet of the conveying channel is communicated with an injection pump, an outlet of the conveying channel is communicated with the inlet, and the outlet is communicated with a recovery tank;

and the conductive positive electrode of the pulse direct-current power supply is used for penetrating through the hollow area of the bottom plate to abut against the silicon plate, and the conductive negative electrode of the pulse direct-current power supply abuts against the ITO conductive glass.

This patent is through forming the electric field in the gap, and the microballon solution flows through between electrically conductive positive pole and the electrically conductive negative pole, and the lorentz force makes the microballon entering downthehole. Due to the arrangement of the circulation gaps, the hole access rate and the utilization rate of the microbeads are improved.

Further preferably, the silica microspheres couple negatively charged oligonucleotide chains. The oligonucleotide chain is negatively charged by hydrolytic ionization. When charged microspheres pass through the surface of the silicon plate with the small hole patterns, the electric field force applied to the microspheres is larger, and the Lorentz force applied to the surfaces of the monocrystalline silicon in the direction of the microspheres is larger, so that the microspheres can be preferentially gathered in micropores, and the hole access rate of the microspheres is improved.

A method of coupling silica microspheres to oligonucleotide chains, comprising the steps of:

A. amino group modified on surface of silicon dioxide microsphere

Preparing the silica microspheres into 10mg/mL-200mg/mL xylene suspension; adding a silanization reagent, wherein the volume concentration of the silanization reagent in the mixed solution is less than 10%, and fully reacting to modify the surface of the silicon dioxide microsphere with amino;

B. activated silica microspheres

Suspending the silicon dioxide microspheres with the surface modified with amino groups obtained in the step A in acetonitrile, wherein the mass concentration of the microspheres is 10mg/mL-200 mg/mL; adding diisopropylethylamine and cyanuric chloride, and shaking for reaction; washing with acetonitrile to remove excessive reactants, suspending the silica microspheres in a sodium borate buffer solution, and adjusting the pH value to 7.5-8.5;

C. covalent linking of silica microspheres to oligonucleotide chains

Dissolving 100nmol of oligonucleotide chain dry powder to be connected by using 2M sodium chloride solution, mixing with the silica microsphere sodium borate suspension obtained in the step B, wherein the content of the silica microspheres is 10-100mg, carrying out centrifugal treatment at 1000-3000rpm after carrying out oscillation reaction for 5-8 hours, and keeping supernatant; and (5) cleaning and drying.

Further preferably, the silicon plate is a silicon plate after the photoresist layer is etched;

the upper surface of the silicon plate is provided with a photoresist layer of 0.9-1.1 μm, the small hole structure is positioned on the photoresist layer, and the small hole structure penetrates through the photoresist layer, so that the silicon plate is partially exposed.

The structural design of the silicon plate is beneficial to electroplating. When the silicon plate is etched, the surface of the pure silicon wafer is processed by a photoresist layer, and a photoresist layer (with extremely poor conductivity) of about 1um is formed on the surface of the silicon plate. When the surface pattern of the mask plate is transferred to the photoresist layer on the wafer, full exposure is ensured, so that monocrystalline silicon on the exposed pattern is exposed in the air (the photoresist layer of the pattern part is fully reacted), thus the surface of the small hole pattern part is monocrystalline silicon with good conductivity, and the surface of the non-small hole pattern part is still the photoresist layer with poor conductivity. During the electroplating process, the potential difference from the conductive glass to the micro-holes will be much higher than the potential difference to the photoresist layer.

Further preferably, the conductivity of the conductive liquid is between 40uS/cm and 100uS/cm, and the pH value is less than 9 and greater than 5.

Further preferably, the conductive liquid comprises 4.2-4.8mM of tris (hydroxymethyl) aminomethane, 4.2-4.8mM of boric acid and 0.01% -0.03% of Triton X-100 by mass percentage.

Further preferably, the pH value of the conductive liquid is 8.5-8.7, and the conductivity is 60-62 muS/cm.

Further preferably, the conductive liquid comprises 4.5mM tris, 4.5mM boric acid and 0.01% Triton X-100. The pH was 8.7 and the conductivity was 62. mu.S/cm.

Further preferably, the silica microspheres have a diameter between 0.5um and 5 um. CV of silica microspheres is < 5.0%.

Further preferably, the voltage of the pulse direct current power supply is 1.0V-10.0V, the frequency is 1Hz-20Hz, and the cycle period is 5% -40%. The frequency of the pulse direct current power supply is 1Hz, the cycle period is 5 percent, namely 5 percent of the time in 1 second is supplied with power, and the rest time is not supplied with power. The frequency of the pulse direct current power supply is 20Hz, the cycle period is 40 percent, namely, 40 percent of the time in 20 seconds is supplied with power, and the rest time is not supplied with power.

The electroplating of the microbead chip is performed into the microbead chip prepared by the hole-type preparation method.

Has the advantages that: 1) and the preparation steps are optimized, and the photoresist is removed immediately after the traditional photoresist is etched. This patent photoresist washs behind the microballon hand-hole and gets rid of. Thus, the small hole structure on the surface of the silicon plate is monocrystalline silicon with good conductivity, and the non-small hole structure area is still a photoresist layer with poor conductivity due to the retention of the photoresist. During the electroplating process, the potential difference from the conductive glass to the micro-holes will be much higher than the potential difference to the photoresist layer. Is favorable for improving the inlet hole of the micro-bead.

2) An electric field is formed in the conductive liquid mixed with the microbeads through the circulation gaps, the microbead solution flows between the electrodes, and the Lorentz force enables the microbeads to enter the holes, so that the hole access rate and the utilization rate of the microbeads are improved.

3) The porosity of the microspheres reaches 99.5 to 99.9 percent.

4) The microspheres have high utilization rate and cannot be left on the surface for subsequent treatment.

5) In the method of coupling silica microspheres to oligonucleotide chains, the concentration of the silylating agent and the concentration of the cyanuric chloride respectively ensure the density of amino groups on the surface of the microspheres and the density of the linking groups for final connection with the oligonucleotide chains. The connection efficiency can reach 1000-10000 oligonucleotide chains/square micron, the manufacturing cost is greatly saved, and a stable silicon dioxide microsphere product with covalently bonded oligonucleotide chains and long preservation time is provided for the subsequent manufacturing of biochips.

Drawings

FIG. 1 is a flow chart of a method for electroplating and hole-in-hole fabrication of a microbead chip according to the present invention;

FIG. 2 is an exploded view of the silicon plate holding apparatus of the present invention;

FIG. 3 is a cross-sectional view of a silicon plate holding apparatus of the present invention;

FIG. 4 is a microscopic view of a product prepared in the embodiment 1 of the present invention;

FIG. 5 is a microscopic view of a product prepared in the embodiment 2 of the present invention;

FIG. 6 is a microscope photograph of a product prepared in specific example 3 of the present invention;

FIG. 7 is a microscopic image of a product prepared in comparative Experimental example 1 of the present invention;

FIG. 8 is a microscopic image of a product prepared in comparative test example 2 of the present invention;

FIG. 9 is a microscopic image of a product prepared in comparative test example 3 of the present invention.

In the figure: the device comprises a base plate 1, a silicon plate 2, a transparent hollow partition plate 3, ITO conductive glass 4 and a clamping mechanism 5.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Referring to fig. 1, embodiment 1, a method for preparing a plated hole of a microbead chip includes the following steps:

firstly, pretreating the surface of a silicon plate;

placing the silicon plate on a spin coating instrument with the rotating speed of 2000rpm for selecting the coating time of 30s, so that a layer of photoresist layer is attached on the silicon plate, further transferring the pattern on the mask plate onto the silicon plate by using a stepping motor, and forming a micropore structure on the surface of the silicon plate;

fixing the silicon plate; the silicon plate fixing device is stacked and fixed from bottom to top according to the sequence of the bottom plate, the silicon plate (the photoresist layer faces the transparent hollow diaphragm), the transparent hollow diaphragm and the ITO conductive glass, so that a circulation gap is formed between the silicon plate and the ITO conductive glass;

step three, loading silica microspheres:

the silica microspheres are coupled with oligonucleotide chains.

The diameter of the silica microspheres is 0.5um, and the CV of the silica microspheres is less than 5.0%. The silica microspheres were washed with a conductive liquid and diluted to a solution to be injected in a state of a solid concentration of 0.1%. The specific steps are that the conductive liquid mixed with the silicon dioxide microspheres is subjected to ultrasonic treatment for 3 minutes, the conductive liquid is placed in a vortex oscillator for 30 seconds to be cleaned, and a centrifugal machine is used for carrying out solid-liquid separation after being centrifuged for 6 minutes at the rotating speed of 3000 g. The solid concentration is a mass percentage concentration.

The conductive liquid comprises 4.5mM of tris (hydroxymethyl) aminomethane, 4.5mM of boric acid and 0.02% of Triton X-100 by mass percentage. The pH was 8.7 and the conductivity was 62. mu.S/cm.

Putting the solution to be injected into an injection pump, and communicating the injection pump with a circulation gap on the silicon plate fixing device;

the silicon plate and the ITO conductive glass are respectively connected with a conductive anode and a conductive cathode of a voltage supply system, the injection pump and the pulse direct-current power supply are simultaneously turned on, 30 seconds are taken as one injection period, and the injection is circulated for three times, so that the flow rate of liquid in the injection pump is ensured to be between 3 uL/s;

the voltage of the pulse direct current power supply is 1.0V, the frequency is 1Hz, and the cycle period is 5 percent;

and step four, cleaning and removing the photoresist layer on the surface of the silicon plate.

Referring to fig. 4, the obtained microbeads had a porosity of about 87% and a utilization rate of more than 80% by the method of this example.

In specific embodiment 2, an electroplating method for improving a bead porosity by using an electroplating hole-entering preparation method for a bead chip includes the following steps:

firstly, pretreating the surface of a silicon plate;

placing the silicon plate on a spin coating instrument with the rotating speed of 2000rpm for selecting the coating time of 30s, so that a layer of photoresist layer is attached on the silicon plate, further transferring the pattern on the mask plate onto the silicon plate by using a stepping motor, and forming a micropore structure on the surface of the silicon plate;

fixing the silicon plate; the silicon plate fixing device is stacked and fixed from bottom to top according to the sequence of the bottom plate, the silicon plate (the photoresist layer faces the transparent hollow diaphragm), the transparent hollow diaphragm and the ITO conductive glass, so that a circulation gap is formed between the silicon plate and the ITO conductive glass;

step three, loading silica microspheres:

the silica microspheres are coupled with oligonucleotide chains.

The diameter of the silica microspheres is 5um, and the CV of the silica microspheres is less than 5.0%. Washing the silicon dioxide microspheres with a conductive solution and diluting to a solution to be injected with a solid concentration of 0.2%;

the conductive liquid comprises 4.5mM of tris (hydroxymethyl) aminomethane, 4.5mM of boric acid and 0.02% of Triton X-100 by mass percentage. The pH was 8.7 and the conductivity was 62. mu.S/cm.

Putting the solution to be injected into an injection pump, and communicating the injection pump with a circulation gap on the silicon plate fixing device;

the silicon plate and the ITO conductive glass are respectively connected with a conductive anode and a conductive cathode of a voltage supply system, the injection pump and the pulse direct-current power supply are simultaneously turned on, 180 seconds are taken as one injection period, and the injection is circulated for three times, so that the flow rate of liquid in the injection pump is ensured to be between 15 uL/s;

the voltage of the pulse direct current power supply is 10.0V, the frequency is 20Hz, and the cycle period is 40 percent;

and step four, cleaning and removing the photoresist layer on the surface of the silicon plate.

Referring to fig. 5, the obtained microbeads had a porosity of about 99.8% and a utilization rate of 80% or more by the method of this example.

In specific embodiment 3, an electroplating method for improving a bead porosity by electroplating a bead chip in a hole-type preparation method includes the following steps:

firstly, pretreating the surface of a silicon plate;

placing the silicon plate on a spin coating instrument with the rotating speed of 2000rpm for selecting the coating time of 30s, so that a layer of photoresist layer is attached on the silicon plate, further transferring the pattern on the mask plate onto the silicon plate by using a stepping motor, and forming a micropore structure on the surface of the silicon plate;

fixing the silicon plate; the silicon plate fixing device is stacked and fixed from bottom to top according to the sequence of the bottom plate, the silicon plate (the photoresist layer faces the transparent hollow diaphragm), the transparent hollow diaphragm and the ITO conductive glass, so that a circulation gap is formed between the silicon plate and the ITO conductive glass;

step three, loading silica microspheres:

the silica microspheres are coupled with oligonucleotide chains.

The diameter of the silica microspheres is 3um, and the CV of the silica microspheres is less than 5.0%.

Washing the silicon dioxide microspheres with a conductive solution and diluting to a solution to be injected with a solid concentration of 0.15%;

the conductive liquid comprises 4.5mM of tris (hydroxymethyl) aminomethane, 4.5mM of boric acid and 0.02% of Triton X-100 by mass percentage. The pH was 8.7 and the conductivity was 62. mu.S/cm.

Putting the solution to be injected into an injection pump, and communicating the injection pump with a circulation gap on the silicon plate fixing device;

the silicon plate and the ITO conductive glass are respectively connected with a conductive anode and a conductive cathode of a voltage supply system, the injection pump and the pulse direct-current power supply are simultaneously turned on, 100 seconds are taken as one injection period, and the injection is circulated for three times, so that the flow rate of liquid in the injection pump is ensured to be between 9 uL/s;

the voltage of the pulse direct current power supply is 5.0V, the frequency is 10Hz, and the cycle period is 22 percent;

and step four, cleaning and removing the photoresist layer on the surface of the silicon plate.

Referring to fig. 6, the obtained microbeads had a porosity of about 93% and a utilization rate of 80% or more by the method of this example.

Comparative test example 1: otherwise, as in example 3, only in the first step, after the micro-porous structure is formed on the surface of the silicon plate, the photoresist layer is cleaned and removed.

Referring to fig. 7, the method of the present embodiment is adopted to obtain a microbead porosity of more than 93%, and a microbead utilization rate of more than 40%.

Comparative test example 2: otherwise, the difference from example 3 is only that in the second step, the conductivity of the conductive liquid was 30 uS/cm. The conductive liquid comprises 4.5mM of tris (hydroxymethyl) aminomethane, 4.2mM of boric acid and 0.02% of Triton X-100 by mass percentage.

Referring to fig. 8, the obtained microbeads had a porosity of about 50% and a utilization rate of more than 80% by the method of this example.

Comparative test example 3: otherwise, the difference is only that in step three, the liquid flow rate in the syringe pump is 60 uL/s.

Referring to fig. 9, the obtained microbeads had a porosity of about 60% and a utilization rate of more than 80% by the method of this example.

	Pore-entering rate of micro-beads	Utilization rate of micro-beads
			Detailed description of the preferred embodiment 1	≈87％	>＝80％
Specific example 2	≈99.8％	>＝80％
			Specific example 3	≈93％	>＝80％
Comparative Experimental example 1	≈93％	≈40％
			Comparative experiment example 2	≈50％	>＝80％
Comparative experiment example 3	≈60％	>＝80％

In the above specific embodiments 1, 2, and 3 and the comparative test examples, the silicon plate fixing device includes a bottom plate 1 for bearing the silicon plate 2, and the bottom plate 1 is hollowed out; the silicon plate fixing device also comprises a clamping mechanism 5, the clamping mechanism 5 is used for clamping and fixing the ITO conductive glass 4, the transparent hollow diaphragm 3, the silicon plate 2 and the bottom plate 1 which are sequentially stacked and arranged from top to bottom, and a small hole structure is etched on the upper surface of the silicon plate 2; the thickness of the transparent vacuum diaphragm is 110 μm, the length of the through hole of the transparent hollow diaphragm 3 in the left and right direction is larger than that of the bottom plate 1 and the ITO conductive glass 4, and the ITO conductive glass 4 is exposed out of the left and right ends of the through hole of the transparent hollow diaphragm 3; the areas of the left and right ends of the through hole of the transparent hollow diaphragm 3 exposed out of the ITO conductive glass 4 are respectively an inlet and an outlet communicated with the circulation gap;

the device also comprises a conveying channel for conveying the microbead solution, wherein the microbead solution is a conductive liquid mixed with silicon dioxide microbeads, an inlet of the conveying channel is communicated with the injection pump, an outlet of the conveying channel is communicated with the inlet, and an outlet of the conveying channel is communicated with a recovery tank;

and the conductive anode of the pulse direct-current power supply is used for penetrating through the hollow area of the bottom plate to abut against the silicon plate, and the conductive cathode of the pulse direct-current power supply abuts against the ITO conductive glass.

The bottom plate 1 is an insulating piece, and the bottom plate 1 is equipped with strip protruding including the bracing piece that sets up around and the connecting rod that is used for the joint support pole, the inboard of bracing piece, and strip protruding flushes with the top of connecting rod. The clamping mechanism 5 is a metal clamping piece, the metal clamping piece comprises clamping arms which are arranged in a left-right mirror symmetry mode, the clamping arms are used for clamping the front side and the rear side of the bottom plate 1, a downward concave portion is arranged in the center of the metal clamping piece, and the left end and the right end of the downward concave portion are connected with the clamping arms respectively. The lower concave part is abutted against the conductive glass.

In specific example 5, the silica microspheres are coupled to negatively charged oligonucleotide chains. When charged microspheres pass through the surface of the silicon plate with the small hole patterns, the electric field force applied to the microspheres is larger, and the Lorentz force applied to the surfaces of the monocrystalline silicon in the direction of the microspheres is larger, so that the microspheres can be preferentially gathered in micropores, and the hole access rate of the microspheres is improved.

A method of coupling silica microspheres to oligonucleotide chains, comprising the steps of:

A. amino group modified on surface of silicon dioxide microsphere

The raw material is solid silicon dioxide microspheres with silicon hydroxyl on the surface, the particle size is 3um, and the solid silicon dioxide microspheres are prepared into xylene suspension of 100 mg/mL. A silylating agent (3-aminopropyltrimethoxysilane) capable of aminating the surface thereof was added, and the concentration of the silylating agent in the mixed solution was 1.0%. The mixed solution is shaken for 1 hour at room temperature, and after the full reaction is completed, the surfaces of the microspheres are provided with amino groups.

B. Activated silica microspheres

Suspending microspheres with amino groups on the surface in acetonitrile, wherein the mass concentration of the microspheres is 100 mg/mL. Adding diisopropylethylamine and cyanuric chloride, wherein the concentration of the diisopropylethylamine in the mixed solution is 0.3M, the concentration of the cyanuric chloride is 0.1M, shaking the mixed solution for 1 hour at room temperature, and carrying out condensation reaction on the cyanuric chloride and amino on the surface of the microsphere, so that the surface group activity of the microsphere is greatly enhanced. Washing with acetonitrile for 3-5 times to remove excessive reactants and products, washing with 2M sodium borate buffer solution for 3-5 times, suspending the microspheres in the sodium borate buffer solution, and adding appropriate amount of hydrochloric acid to adjust the pH value of the suspension to 7.5-8.5.

C. Covalent linking of silica microspheres to oligonucleotide chains

Dissolving 100nmol of oligonucleotide chain dry powder to be connected with a 2M sodium chloride solution (the length of the oligonucleotide chain is 15 mers, and the tail end is modified with amino), mixing the oligonucleotide chain dry powder with a proper amount of sodium borate microsphere suspension, wherein the content of the microspheres is 50mg, and oscillating the mixed solution at room temperature for 5-8 hours to ensure that the oligonucleotide chain is fully contacted with the microspheres and reacts with active groups on the surfaces of the microspheres. After the ligation reaction was completed, the mixture was centrifuged at 1000-. Washing the microspheres with ultrapure water for 3-5 times, removing redundant reactants and products, drying into dry powder, and storing for later use.

The microspheres coupled with the oligonucleotide chains were hybridized with a target (complementary to the oligonucleotide chains and having FAM fluorescent groups at the ends), incubated at 20 ℃ lower than the melting temperature of the corresponding oligonucleotide chains for 30 minutes, and the fluorescence intensity was measured. The microsphere concentration is 2mg/mL during detection, and the excitation/emission filter is 488nm/520 nm. All fluorescence intensities mentioned in the present invention were measured under the above conditions. The conversion of fluorescence intensity to concentration used a standard curve (obtained by measuring fluorescence values from formulated standards): c-8.797 e-7I-0.0055. Wherein C is the concentration of the fluorescent group, the unit is nmol/mL, and I is the fluorescence intensity. The concentration of the fluorescent groups can be obtained from the fluorescence intensity, and the quantity of the microspheres is known, so that the number of the fluorescent groups on the unit surface area can be calculated. Thereby calculating the amount of the oligonucleotide chain participating in the ligation reaction and the ligation efficiency of the microspheres to the oligonucleotide chain. Through multiple experiments, the connection efficiency can reach about 9500-10500 oligonucleotide chains/square micron (the microsphere particle size is 3um, and the fluorescence intensity is 40048, 42619 and 39890 through multiple measurements).

Has the advantages that: 1) the porosity of the microspheres reaches 99.5 to 99.9 percent.

2) The microspheres have high utilization rate and cannot be left on the surface for subsequent treatment.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.