Oligonucleotide coupling in-situ monitoring system and detection method

Abstract

The invention provides an oligonucleotide coupling in-situ monitoring system and a detection method.The monitoring system is internally provided with a red light LED, an objective lens, a diaphragm, a doublet lens, a polaroid, a first cylindrical lens, a cylindrical prism, a flow cell chip, a second cylindrical lens, a first imaging lens, a second imaging lens and a CMOS camera in sequence in the light beam direction. The incident light path and the reflected light path are arranged at an angle, the flow cell chip and the cylindrical prism are subjected to refractive index matching through coupling liquid, reflected light spots are collected by analyzing the CMOS camera, the corresponding spectral resonance angle change of a detection area is obtained in real time, and then the conjugate change of the detection area is represented. The monitoring system utilizes the surface plasmon resonance effect to sensitively analyze the substance binding condition on the surface of a sensitive film layer, and in-situ characterization and multi-channel and array real-time detection of the oligonucleotide coupling process are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to an oligonucleotide coupling in-situ monitoring system and a detection method. Background Art

[0002] DNA synthesis is one of the key enabling technologies for synthetic biology, and independent innovation in DNA synthesis equipment is an important factor in promoting the advancement of life sciences and medical technology. Coupling efficiency is a method of measuring whether a DNA synthesis device can accurately add new bases to a DNA chain. It is crucial to the synthesis yield and chain length. When synthesizing long chains, even if the coupling efficiency is reduced by only 1%, the loss is huge. Therefore, accurate detection of coupling efficiency can help to make further adjustments to specific technical details such as DNA synthesis equipment, reagents, and processes to increase the final yield of synthesis.

[0003] At present, coupling efficiency detection generally adopts offline optical detection methods. Among them, the most commonly used and representative one is the DMT absorbance detection method in standard oligonucleotide synthesis. However, the detection limit of absorbance detection is high, and it is difficult to guarantee the detection accuracy at low sample concentrations and / or oligonucleotide surface coverage. In addition, fluorescent end labeling can also be used, but its application scenarios are still limited to exploratory experiments on the synthesis process, and the introduction of dye labels is complicated and costly for actual synthesis. Although the second-generation solid-phase chip synthesis has been developed for more than ten years, there is still a lack of solutions that can integrate in-situ online detection of coupling efficiency into DNA synthesizers.

[0004] Surface plasmon resonance sensing is a type of sensing method that can stimulate a rapidly decaying evanescent field to detect material changes and interactions at the molecular level. By making specific functional modifications to the sensing film layer, a target and its binding process can be detected. This detection method has a nanomolar detection limit without using a signal amplification strategy, and surface plasmon resonance sensing with imaging capabilities can present arrayed, high-throughput analysis results. The surface plasmon resonance sensor has a simple structure, and the instrument and chip architecture are compatible with existing DNA chips and synthesizers. Combining the above factors, the sensing method based on surface plasmon resonance is expected to become a new means of single-base resolution and real-time in situ detection of coupling efficiency. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an oligonucleotide coupling in-situ monitoring system.

[0006] Another technical problem to be solved by the present invention is to provide a detection method using the above oligonucleotide-coupled in-situ monitoring system.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] An oligonucleotide coupling in-situ monitoring system, wherein a red light LED, an objective lens, an aperture, a double glued lens, a polarizer, a first cylindrical lens, a cylindrical prism, a circulation cell chip, a second cylindrical lens, a first imaging lens, a second imaging lens and a CMOS camera are sequentially arranged in the optical path system along the light beam direction, the incident light path and the reflected light path are arranged at an angle, the circulation cell chip comprises a base glass, an adhesive layer, a sensitive film layer, a cover plate and a liquid path interface, the circulation cell chip comprises a base glass, an adhesive layer, a sensitive film layer, a cover plate and a liquid path interface, the base glass is fixedly connected to the cover plate to form a cavity as a circulation cell, the adhesive layer and the sensitive film layer are placed in the cavity, the base glass is fixed with an adhesive layer, and the adhesive layer is fixed with a sensitive film layer , a liquid path interface is provided on the cover plate; wherein, the flow cell chip is located above the cylindrical prism, and there is a gap between the two for filling the refractive index matching liquid, the CMOS camera is connected to the computer, the divergent light emitted by the red light LED is focused on the aperture through the objective lens, and is collimated through the double-glued lens (the light is changed from divergent light to parallel light), the parallel light passes through the polarizer, and its polarization direction changes from natural polarization to p-polarization, and is focused on the flow cell chip through the first cylindrical lens and excites the surface plasma resonance of the sensitive film layer, the reflected light carrying the resonance peak information is converted into parallel light through the second cylindrical lens, and is imaged on the photosensitive image surface of the CMOS camera through the first imaging lens and the second imaging lens, and the grayscale information of each pixel is sent to the computer for further processing.

[0009] Preferably, in the above oligonucleotide coupling in situ monitoring system, the angle between the incident light path and the reflected light path is 120°.

[0010] Preferably, in the above oligonucleotide coupling in situ monitoring system, the focusing angle of the light beam after being focused by the first cylindrical lens is 15°-20°, and the incident angle is 50°-70°.

[0011] Preferably, in the above oligonucleotide-coupled in-situ monitoring system, the polarization direction of the polarizer is p-polarization.

[0012] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the cylindrical prism is made of high refractive index heavy flint glass SF11 or sapphire glass.

[0013] Preferably, in the above oligonucleotide coupled in situ monitoring system, the flow cell chip is prepared by the following method:

[0014] 1) Pre-treating the substrate glass: immersing the substrate glass in anhydrous ethanol and performing ultrasonic cleaning to remove surface contaminants;

[0015] 2) Covering the substrate glass with a mask and preparing an adhesive layer by magnetron sputtering or vacuum evaporation;

[0016] 3) preparing a sensitive film layer on the adhesive layer by magnetron sputtering or vacuum evaporation;

[0017] 4) The bonding surface of the base glass and the flow cell cover is treated by oxygen plasma cleaning, and then the two are aligned and bonded to form a cavity as a flow cell, so that the sensitive membrane layer is located in the flow cell, and finally pressure is applied to complete the bonding.

[0018] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the substrate glass material is high refractive index heavy flint glass SF11 or sapphire glass, and the thickness of the substrate glass is 0.3-1 mm.

[0019] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the thickness of the substrate glass is 0.5 mm.

[0020] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the material of the bonding layer is metal chromium or oxide titanium dioxide, and the thickness of the bonding layer is 2-5 nm.

[0021] Preferably, in the above oligonucleotide-coupled in-situ monitoring system, the thickness of the adhesive layer is 3 nm.

[0022] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the material of the sensitive film layer is metal gold, metal silver or a mixture of the two, and the thickness of the sensitive film layer is 45-52 nm.

[0023] Preferably, in the above oligonucleotide-coupled in-situ monitoring system, the thickness of the sensitive membrane layer is 47-48 nm.

[0024] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the adhesive layer and the sensitive film layer are rectangular or circular, and are arranged on the substrate glass in a linear array.

[0025] Preferably, in the above oligonucleotide coupling in-situ monitoring system, the flow cell chip is bonded to the cylindrical prism via a refractive index matching liquid with a refractive index of 1.78 and clamped using a clamp.

[0026] Preferably, in the above-mentioned oligonucleotide coupling in situ monitoring system, the clamp design is customized according to the geometric shapes of the cylindrical prism and the circulation pool chip to be clamped, and the clamp includes a U-shaped clamp body and a crossbeam, the crossbeam is respectively connected to the top and bottom of the clamp body, and the crossbeam is provided with a crossbeam hole, the top of the clamp body is provided with a prism fixing hole and a crossbeam fixing hole, the bottom of the clamp body is provided with a through hole, and one side of the clamp body is provided with a chip card slot and a prism card slot.

[0027] The preferred material of the above-mentioned fixture in the 3D printing process is nylon, and the preferred material in the CNC numerical control processing process is aluminum alloy.

[0028] The detection method of the above-mentioned oligonucleotide coupling in-situ monitoring system is applied, and the refractive index change of the surface of the sensitive film layer is detected by surface plasmon resonance spectroscopy. The reflected surface plasmon resonance spectrum is uploaded to the computer user program interface through a CMOS camera. The image data collected by the CMOS camera is filtered and peak-finding is performed to obtain the surface plasmon resonance spectrum, and a curve of the change of the resonance peak position and time is established. The change of the position of the resonance peak is used to characterize the coupling of the analyte on the sensitive film layer. The larger the resonance peak shift, the more analytes are coupled to the surface.

[0029] Preferably, the above detection method, when it is necessary to detect the coupled monolayer, specifically comprises the following steps:

[0030] 1) Passing an ethanol solution of mercaptoalkanoic acid into the flow cell chip and reacting overnight in the dark to form a tightly covered self-assembled monolayer on the sensitive film layer, followed by washing with ethanol and deionized water in turn and drying with a nitrogen stream;

[0031] 2) PBS buffer was introduced and the baseline position of the surface plasmon resonance peak (SPR resonance peak) was recorded;

[0032] 3) introducing EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) in MES solution (4-morpholineethanesulfonic acid, used as a buffer) and NHS (N-hydroxysuccinimide) in MES solution, wherein the molar ratio of EDC to NHS is 4:1, and reacting for 15-20 minutes to activate the carboxyl group;

[0033] 4) Pass 400 μg / mL streptavidin acetate solution and react for 15-20 minutes to fix streptavidin on the sensitive membrane layer through amide condensation coupling; wash with PBS buffer;

[0034] 5) Pass 5 μM biotin-modified polythymidine primer (Oligo(dT) Primer, for example, oligonucleotide chain 5'-Biotin-TTTTTTTTTTTTTTTTT-3') solution, react for 10-20 minutes, couple the single chain to the sensitive membrane layer through the specific binding of biotin and streptavidin, pass PBS buffer, record the surface plasmon resonance peak after coupling, and end the resonance peak recording.

[0035] Preferably, the above detection method, when it is necessary to detect stepwise coupling, comprises the following specific steps:

[0036] (1) Gold film modification, continuous flow of 6-mercapto-1-hexanol (MCH) to form a self-assembled monolayer of hydroxyl groups on its surface;

[0037] (2) measuring the spectrum in acetonitrile and recording the resonance peak baseline;

[0038] (3) introducing an active phosphoramidite tetrazole active intermediate for coupling;

[0039] (4) measuring the resonance peak position after extending one base in an acetonitrile environment;

[0040] (5) introducing capping reagent;

[0041] (6) introducing iodine-containing tetrahydrofuran solution for oxidation;

[0042] (7) introducing trichloroacetic acid deprotection reagent for deprotection;

[0043] (8) The next synthesis cycle is carried out starting from step (3) until the desired sequence is obtained.

[0044] Preferably, in the above detection method, the sampling device used for sampling is a syringe pump or a peristaltic pump.

[0045] Beneficial effects:

[0046] The above-mentioned oligonucleotide coupling in situ monitoring system uses the surface plasmon resonance effect to sensitively analyze the binding of substances on the surface of the sensitive membrane layer. The flow cell chip is coupled into the optical path system through refractive index matching with the cylindrical prism through coupling liquid. The reflected light spot is collected by analyzing the CMOS camera to obtain the change of the spectral resonance angle corresponding to the detection area in real time, and then the change of the conjugate in the detection area is characterized in situ. The design of the optical path and the flow cell chip enables the system to realize multi-channel, arrayed real-time in situ detection, and is compatible with the in situ characterization of oligonucleotide coupling under the chemical and enzymatic chip synthesis systems of DNA, and has the ability to realize the integration of DNA synthesis and detection applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a schematic structural diagram of the oligonucleotide coupling in situ monitoring system of the present invention;

[0048] Figure 2 It is a schematic diagram of the structure of the flow cell chip of the oligonucleotide coupling in situ monitoring system of the present invention;

[0049] Figure 3 Schematic diagram of the structure of the clamp used in the embodiment;

[0050] Figure 4 It is a surface plasmon resonance spectrum obtained by the oligonucleotide coupling in situ monitoring system of the present invention;

[0051] Figure 5 is the resonance peak change curve of activated coupled streptavidin;

[0052] Figure 6 It is a curve diagram of the resonance peak change of the coupled oligonucleotide monolayer.

[0053] In the figure: 1-red light LED, 2-objective lens, 3-aperture, 4-double glued lens, 5-polarizer, 6-first cylindrical lens, 7-cylindrical prism, 8-circuit cell chip, 9-second cylindrical lens, 10-first imaging lens, 11-second imaging lens, 12-CMOS camera, 101-base glass, 102-adhesive layer, 103-sensitive film layer, 104-cover plate, 105-liquid path interface, 201-through hole, 202-chip card slot, 203-prism card slot, 204-prism fixing hole, 205-crossbeam, 206-crossbeam fixing hole, 207-crossbeam hole. DETAILED DESCRIPTION

[0054] The oligonucleotide coupling in situ monitoring system and detection method of the present invention are described below with reference to the examples and drawings.

[0055] Example 1

[0056] like Figure 2 As shown, the flow cell chip 8 includes a base glass 101, an adhesive layer 102, a sensitive film layer 103, a cover plate 104 and a liquid path interface 105. The flow cell chip 8 includes a base glass 101, an adhesive layer 102, a sensitive film layer 103, a cover plate 104 and a liquid path interface 105. The base glass 101 is fixedly connected to the cover plate 104 to form a cavity. The adhesive layer 102 and the sensitive film layer 103 are placed in the cavity. The base glass 101 is fixed with an adhesive layer 102, the adhesive layer 102 is fixed with a sensitive film layer 103, and the cover plate 104 is provided with a liquid path interface 105. The specific steps of preparing the flow cell chip 8 are as follows:

[0057] (1) Pretreatment of substrate glass: The substrate was immersed in anhydrous ethanol and ultrasonically cleaned for 10 min to remove surface contaminants, then cleaned with deionized water and dried with nitrogen.

[0058] (2) A mask is attached to the substrate glass, and metal chromium is deposited on the substrate glass by magnetron sputtering to enhance the adhesion of the film to the substrate. The thickness of the metal chromium is 2-3 nm. Metal chromium absorbs visible light. An overly thick chromium film reduces the overall intensity of the surface plasmon resonance reflection spectrum.

[0059] (3) Replace the magnetron sputtering target and deposit a gold film on the bonding layer as a sensitive film layer.

[0060] (4) The bonding surface of the base glass 101 and the flow cell cover plate 104 is treated by oxygen plasma cleaning, and then the two are aligned and bonded to form a cavity as a flow cell, so that the sensitive film layer 103 is located in the flow cell, and finally the base glass 101 and the flow cell cover plate 104 are bonded and pressure is applied to complete the bonding. The specific method is:

[0061] The flow cell chip cover is bonded to the surface of the base glass by oxygen plasma treatment or piranha solution treatment, and the base glass and the flow cell chip cover are placed in a plasma cleaning device for plasma treatment. The power is 300W and the treatment time is 45s. After the treatment is completed, the base glass and the flow cell chip cover are attached and pressed for 30s, and then the base glass is placed face down on a heating plate and heated at 80°C for 5min to further increase the bonding strength.

[0062] The specific steps for preparing the cover plate 104 in the flow cell chip are as follows:

[0063] (1) Mold making: A positive mold of the flow cell chip structure is prepared by photolithography, 3D printing technology or CNC processing. The positive mold material can be a silicon wafer, a photosensitive resin or other materials that can form a micron-scale structure. The structure of the flow cell chip cavity is etched or printed on the positive mold.

[0064] (2) Preparation of PDMS prepolymer: PDMS prepolymer and cross-linking agent were mixed in a mass ratio of 10:1, stirred evenly, and then degassed in a vacuum degassing device until the bubbles completely disappeared.

[0065] (3) PDMS casting and curing: Pour the degassed PDMS prepolymer evenly into the prepared positive mold to ensure that the flow cell chip cavity is completely filled with PDMS; then place the mold in an oven at 85°C for curing for 1 hour.

[0066] (4) Demolding: After curing, the PDMS is carefully peeled off from the mold to obtain a PDMS body with a flow cell chip cavity structure.

[0067] (5) Cutting and punching: Use a blade to cut out the circulation pool chip cavity and the bonding part in the PDMS body, and use a micro drill or laser punching equipment to punch holes at appropriate positions of the PDMS body according to the coating pattern array to form a liquid path interface. The pore size is adjusted according to the design requirements of the fluid channel, usually 1-1.2 mm.

[0068] The mask is prepared by laser engraving technology. The material is PMMA (polymethyl methacrylate) (or stainless steel) and the thickness is 0.5-1 mm.

[0069] The fixture is manufactured by 3D printing technology (or CNC machining technology), and the fixture design is customized according to the geometric shapes of the cylindrical prism 7 and the flow cell chip 8 to be clamped. By applying appropriate coupling pressure, the prism and the flow cell chip are closely fitted to prevent the loss of coupling liquid and reduce the influence of external vibration on the surface plasmon resonance spectrum. Figure 3As shown, the design of the clamp is customized according to the geometric shapes of the cylindrical prism 7 and the circulation pool chip 8 to be clamped, and the clamp includes a U-shaped clamp body and a crossbeam 205, and the crossbeam 205 is respectively connected to the top and bottom of the clamp body by fixing screws, and a crossbeam hole 207 is provided on the crossbeam 205. The top of the clamp body is provided with a prism fixing hole 204 and a crossbeam fixing hole 206, and the bottom of the clamp body is provided with a through hole 201, and one side of the clamp body is provided with a chip card slot 202 and a prism card slot 203.

[0070] The preferred material of the fixture in the 3D printing process is nylon, and the preferred material in the CNC numerical control processing process is aluminum alloy.

[0071] The use of 3D printing technology can ensure that the fixture can maintain lightweight while having sufficient mechanical strength and structural complexity, which has advantages in manufacturing efficiency and production costs; CNC machining technology has highly precise subtractive manufacturing capabilities and can cut various metal materials with micron-level accuracy, ensuring that the fixture has good wear resistance and strength and a long service life.

[0072] Example 2

[0073] like Figure 1As shown, the oligonucleotide coupling in-situ monitoring system has a light path system in which a red light LED 1, an objective lens 2, an aperture 3, a doublet lens 4, a polarizer 5, a first cylindrical lens 6, a cylindrical prism 7, the flow cell chip 8 described in Example 1, a second cylindrical lens 9, a first imaging lens 10, a second imaging lens 11 and a CMOS camera 12 are sequentially arranged along the light beam direction. The incident light path and the reflected light path are arranged at an angle of 120°. The polarization direction of the polarizer 5 is p polarization. The focusing angle of the light beam after being focused by the first cylindrical lens 6 is 15°-20°, and the incident angle is 50°-70°. The flow cell chip 8 includes a base glass 101, an adhesive layer 102, a sensitive film layer 103, a cover plate 104 and a liquid path interface 105. The base glass 101 is made of high-refractive-index heavy flint glass SF11 (or sapphire glass), and the thickness of the base glass 101 is 0.5 mm. The material of the adhesive layer 102 is metal chromium ( The thickness of the adhesive layer 102 is 3 nm, the material of the sensitive film layer 103 is metal gold (or metal silver or a mixture of the two), the thickness of the sensitive film layer 103 is 48 nm, the adhesive layer 102 and the sensitive film layer 103 are rectangular or circular, and are arranged on the base glass 101 in a linear array, the base glass 101 is fixedly connected to the cover plate 104 to form a cavity as a flow cell, the adhesive layer 102 and the sensitive film layer 103 are placed in the cavity, the adhesive layer 102 is fixed on the base glass 101, the sensitive film layer 103 is fixed on the adhesive layer 102, and the cover plate 104 is provided with a liquid path interface 105; wherein the flow cell chip 8 is located above the cylindrical prism 7, and there is a gap between the two for filling the refractive index matching liquid, the material of the cylindrical prism 7 is high refractive index heavy flint glass SF11 (or sapphire glass), the CMOS camera 12 is connected to the computer, and the red light LED The divergent light emitted by 1 is focused on the aperture 3 through the objective lens 2 and collimated by the doublet lens 4 (the light is changed from divergent light to parallel light). The parallel light passes through the polarizer 5, and its polarization direction is changed from natural polarization to p-polarization. It is focused on the flow cell chip 8 through the first cylindrical lens 6 and excites the surface plasma resonance of the sensitive film layer 103. The reflected light carrying the resonance peak information is converted into parallel light through the second cylindrical lens 9, and is imaged on the photosensitive image surface of the CMOS camera 12 through the first imaging lens 10 and the second imaging lens 11. The grayscale information of each pixel is sent to the computer for further processing.

[0074] The above oligonucleotide coupling in-situ monitoring system is connected to the liquid path interface 105 using a polytetrafluoroethylene tube with an inner diameter of 0.8 mm and an outer diameter of 1.2 mm. The interface is sealed with epoxy glue or light-curing glue. The sampling device used for sampling is a syringe pump (or peristaltic pump).

[0075] When clamping the circulation cell chip 8 and the cylindrical prism 7, one side of the cover plate 104 of the circulation cell chip is placed in the rectangular through hole 201 of the fixture, and the base glass 101 is limited by the chip slot 202. The refractive index matching liquid (commercially available product) with a refractive index of 1.78 is added to one side of the base glass 101 to make it consistent with the refractive index of the cylindrical prism 7 clamped in the prism slot 203. The cylindrical prism 7 is limited by the prism slot 203. A thread with a specification of M4 is provided at the prism fixing hole 204, and the cylindrical prism 7 is pressed tightly by a rubber head screw. The crossbeam 205 is fixedly connected to the fixture body by screw connection through the upper and lower fixed fixing holes 206 with a specification of M3, and the coupling pressure is increased by tightening the rubber head screw matching the crossbeam hole 207.

[0076] Example 3

[0077] The method for detecting oligonucleotide monolayer coupling using the oligonucleotide coupling in situ monitoring system described in Example 2 specifically comprises the following steps:

[0078] (1) Before bonding the PDMS cover plate to the coated glass, the coated glass was rinsed with deionized water and anhydrous ethanol in turn, and dried with nitrogen gas. It was then immersed in a 5 mM ethanol solution of mercapto undecanoic acid (MUA) at 4 °C for 24 h in a dark environment. Mercapto undecanoic acid was fixed on the surface of the sensitive film through a gold-sulfur bond to form a self-assembled molecular layer. The glass was then rinsed with deionized water and anhydrous ethanol in turn, and dried with a nitrogen gas stream.

[0079] (2) Place the bonded flow cell chip into the fixture in the optical path, add refractive index matching liquid to the glass substrate of the flow cell chip, and then place the cylindrical prism into the fixture. Adjust the rubber head screw of the fixture to make the flow cell chip and the cylindrical prism fit tightly. Pass PBS buffer into the flow cell chip, and record the corresponding stable spectral resonance peak position in the user program interface as the baseline of this detection signal. Figure 4 As shown, when the metal film deposition thickness is 50nm, the surface plasmon resonance peak in the reflection spectrum has a good decline depth. The curve can be smoothed by filtering, which is beneficial to the resonance peak positioning.

[0080] (3) 0.4 M EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.1 M NHS (N-hydroxysuccinimide) were dissolved in 0.1 M MES buffer, and the MES solution of EDC and the MES solution of NHS were mixed in equal volumes to prepare an activation solution, which was passed into the flow cell chip at a flow rate of 20 μL / min for 10 min to activate the carboxyl groups on the gold film surface. EDC reacts with carboxyl groups to generate an intermediate product, 0-acylurea, which has high reactivity and can undergo nucleophilic addition reaction with amino groups to form a stable amide bond. However, this intermediate product is unstable and needs to be converted into a more stable NHS ester by NHS for reaction.

[0081] (4) Dissolve streptavidin in acetate solution to a final concentration of 400 μg / mL, and pass it into the flow cell chip at a flow rate of 10 μL / min to cross-link the amino groups on the streptavidin with the carboxyl groups on the surface of the gold film. After reacting for 15-20 minutes, wash with PBS buffer. Figure 5 As shown in the figure, due to the activation of EDC and NHS, the self-assembled monolayer formed NHS esters, and the increase in surface mass caused the refractive index to change, causing the resonance peak to shift to the right by 10.0 pixels compared to the baseline. Streptavidin was bound to the gold film surface through amide bonds and gradually saturated, causing the resonance peak to shift to the right by 13.5 pixels compared to the baseline.

[0082] (5) The biotin-modified Oligo(dT) Primer dry powder primer is centrifuged, PBS solution is added to a final concentration of 5 μM, and then the mixture is passed into the flow cell chip 8 at a flow rate of 10 μL / min to couple the biotin-modified oligonucleotide with the streptavidin on the gold film. The streptavidin-biotin system is a non-covalent coupling method, and one molecule of streptavidin can quickly and highly specifically bind to four molecules of biotin stably. Figure 6 As shown, 0ligo was coupled to the gold film surface through its modified biotin group and showed a saturation trend, causing the resonance peak to shift rightward by 2.2 pixel units compared to the baseline.

[0083] Example 4

[0084] The oligonucleotide coupling in situ monitoring system described in Example 2 is used to monitor the stepwise coupling of oligonucleotides. The specific steps are as follows:

[0085] (1) Preparation of synthetic reagents: phosphoramidite monomers protected by DMT (dimethoxytrityl) groups of four bases A, T, C and G are respectively dissolved in anhydrous acetonitrile to prepare a base protection solution with a concentration of 0.2 M; a tetrazole activator such as ETT (5-ethylthiotetrazolyl) is dissolved in anhydrous acetonitrile to prepare an activator solution with a concentration of 0.25 M; elemental iodine is dissolved in tetrahydrofuran to prepare an oxidant solution with a concentration of 0.1 M; trichloroacetic acid is prepared in anhydrous dichloromethane as a solvent to prepare a 3% deprotection reagent solution; acetic anhydride is prepared in anhydrous acetonitrile as a solvent to prepare a 10% capping reagent A; and N-methylimidazole is prepared in anhydrous acetonitrile as a solvent to prepare a 10% capping reagent B.

[0086] (2) Surface modification: Before packaging the flow cell chip, the substrate glass was immersed in MCH (D6-mercapto-1-hexanol) at room temperature for 12 h to form a self-assembled monolayer covered with hydroxyl groups on the surface of the gold film, and then dried with nitrogen.

[0087] (3) Synthesis monitoring: The packaged chip is clamped in the DNA coupling detection system, and the spectrum resonance peak is recorded. When anhydrous acetonitrile is introduced, the baseline is recorded, and the following phosphite amide solid phase chemical synthesis method is used to circulate in the flow cell chip until the oligonucleotide reaches the synthetic sequence length:

[0088] (4) Activation coupling: The phosphoramidite monomer is mixed with an activator to obtain an active intermediate phosphoramidite tetrazole, which is then coupled with a solid support or the 5′ hydroxyl group of the preceding nucleoside to form a phosphorous triester, thereby extending the oligonucleotide chain by one base. After the activation coupling is completed, the spectral resonance peak of the coupling reaction is recorded when anhydrous acetonitrile is introduced.

[0089] (5) Capping: The mixed capping reagents A and B are introduced, and the unsuccessful oligonucleotide single strands are blocked by acetic anhydride acylation reaction, so that these byproducts will no longer be extended in the subsequent synthesis cycle.

[0090] (6) Oxidation: An oxidant solution is introduced to convert the unstable phosphite bonds in the oligonucleotide into stable pentavalent phosphate bonds.

[0091] (7) Deprotection: A deprotection reagent is introduced to remove the DMT protecting group attached to the 5′ end of the terminal nucleotide of the sequence on the solid support, exposing the 5′ end hydroxyl group to obtain a coupling site.

[0092] (8) The next round of synthesis cycle is carried out from step (3), starting from the 3' end until the desired oligonucleotide sequence (5'-ATGCTCGC-3') is obtained.

[0093] After each step of the reaction in the above cycle, the reaction chamber was cleaned with anhydrous acetonitrile. After completing the above steps, the product was cut from the chip with fresh concentrated ammonia. The surface plasmon resonance peak shift is related to the amount of analytes coupled to the surface of the sensitive membrane layer. The coupling effect was evaluated by combining the spectral resonance peak change curve of each cycle of the coupling process and the prior data to calculate the coupling efficiency. The coupling efficiency of each synthesis round is shown in Table 1.

[0094] Due to the capping step, the unreacted single strands are no longer extended in the subsequent rounds. The changes in the spectral resonance peaks before and after coupling characterize the successfully coupled single strands. The step-by-step coupling efficiency calculation method P i =(spectral shift of resonance peak in the i-th round of synthesis / spectral shift of saturated coupling in prior data)×100%.

[0095]

[0096] Table 1

[0097] Round 1 2 3 4 5 6 7 8 Coupling efficiency 98.7％ 98.2％ 98％ 97.5％ 97.4％ 97.3％ 97.0％ 96.5％

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An oligonucleotide coupling in situ monitoring system, characterized in that: The optical path system is provided with a red light LED (1), an objective lens (2), an aperture (3), a doublet lens (4), a polarizing plate (5), a first cylindrical lens (6), a cylindrical prism (7), a flow cell chip (8), a second cylindrical lens (9), a first imaging lens (10), a second imaging lens (11) and a CMOS camera (12) in sequence along the direction of the light beam. The incident light path and the reflected light path are arranged at an angle. The flow cell chip (8) comprises a base glass (101), an adhesive layer (102), a sensitive film layer (103), a cover plate (104) and a liquid path interface (105). The flow cell chip (8) comprises a base glass (101), an adhesive layer (102), a sensitive film layer (103), a cover plate (104) and a liquid path interface (105). 02), a sensitive film layer (103), a cover plate (104) and a liquid path interface (105); the base glass (101) and the cover plate (104) are fixedly connected to form a cavity as a flow cell; the adhesive layer (102) and the sensitive film layer (103) are placed in the cavity; the base glass (101) is fixed with an adhesive layer (102), the adhesive layer (102) is fixed with a sensitive film layer (103), and the cover plate (104) is provided with a liquid path interface (105); wherein the flow cell chip (8) is located above the cylindrical prism (7), and there is a gap between the two for filling with a refractive index matching liquid; and the CMOS camera (12) is connected to a computer.

2. The oligonucleotide coupling in situ monitoring system according to claim 1, characterized in that: The angle between the incident light path and the reflected light path is 120°, the focusing angle of the light beam after being focused by the first cylindrical lens (6) is 15°-20°, the incident angle is 50°-70°, and the polarization direction of the polarizer (5) is p polarization.

3. The oligonucleotide coupling in situ monitoring system according to claim 1, characterized in that: The cylindrical prism (7) is made of high-refractive-index heavy flint glass SF11 or sapphire glass.

4. The oligonucleotide coupling in situ monitoring system according to claim 1, characterized in that: The circulation cell chip (8) is prepared by the following method: 1) Pre-treating the substrate glass (101): immersing the substrate glass (101) in anhydrous ethanol for ultrasonic cleaning to remove surface contaminants; 2) Covering the substrate glass (101) with a mask, and preparing an adhesive layer (102) by magnetron sputtering or vacuum evaporation; 3) preparing a sensitive film layer (103) on the adhesive layer (102) by magnetron sputtering or vacuum evaporation; 4) The bonding surfaces of the base glass (101) and the flow cell cover (104) are treated by oxygen plasma cleaning, and then the two are aligned and bonded to form a cavity as a flow cell, so that the sensitive film layer (103) is located in the flow cell, and finally pressure is applied to complete the bonding.

5. The oligonucleotide coupling in situ monitoring system according to claim 4, characterized in that: The base glass (101) is made of high-refractive-index heavy flint glass SF11 or sapphire glass, and the thickness of the base glass (101) is 0.3-1 mm; the bonding layer is made of metal chromium or oxide titanium dioxide, and the thickness of the bonding layer (102) is 2-5 nm; the sensitive film layer (103) is made of metal gold, metal silver or a mixture of the two, and the thickness of the sensitive film layer (103) is 45-52 nm.

6. The oligonucleotide coupling in situ monitoring system according to claim 4 or 5, characterized in that: The adhesive layer (102) and the sensitive film layer (103) are rectangular or circular, and are arranged on the substrate glass (101) in a linear array.

7. The oligonucleotide coupling in situ monitoring system according to claim 1, characterized in that: The flow cell chip (8) is bonded to the cylindrical prism (7) via a refractive index matching liquid having a refractive index of 1.78 and is clamped using a clamp.

8. A detection method using the oligonucleotide coupled in situ monitoring system according to any one of claims 1 to 7, characterized in that: The surface plasmon resonance spectrum is used to detect the refractive index change on the surface of the sensitive film layer (103), and the reflected surface plasmon resonance spectrum is uploaded to a computer user program interface through a CMOS camera (12). The surface plasmon resonance spectrum is obtained by filtering and peak searching the image data collected by the CMOS camera (12), and a curve of the change of the resonance peak position and time is established. The change of the resonance peak position is used to characterize the coupling of the analyte on the sensitive film layer (103). The greater the resonance peak shift, the more analytes are coupled on the surface.

9. The detection method according to claim 8, characterized in that: The divergent light emitted by the red light LED (1) is focused on the aperture (3) through the objective lens (2) and collimated through the doublet lens (4). The parallel light passes through the polarizer (5), and its polarization direction is changed from natural polarization to p-polarization. It is focused on the flow cell chip (8) through the first cylindrical lens (6) and excites the surface plasma resonance of the sensitive film layer (103). The reflected light carrying the resonance peak information is converted into parallel light through the second cylindrical lens (9), and is imaged on the photosensitive image surface of the CMOS camera (12) through the first imaging lens (10) and the second imaging lens (11). The grayscale information of each pixel is sent to a computer for further processing.

10. The detection method according to claim 8 or 9, characterized in that: When it is necessary to detect the coupled monolayer, the following steps are specifically included: 1) introducing an ethanol solution of mercaptoalkanoic acid into a flow cell chip (8) and reacting overnight in the dark to form a tightly covered self-assembled monolayer on the sensitive film layer (103), followed by washing with ethanol and deionized water in turn, and blowing nitrogen gas; 2) PBS buffer was introduced and the baseline position of the surface plasmon resonance peak was recorded; 3) introducing an EDC-MES solution and an NHS-MES solution, wherein the molar ratio of EDC to NHS is 4:1, and reacting for 15-20 minutes to activate the carboxyl group; 4) introducing 400 μg / mL streptavidin acetate solution and reacting for 15-20 minutes to fix the streptavidin on the sensitive membrane layer (103) through amide condensation coupling; washing with PBS buffer; 5) introducing a 5 μM biotin-modified polythymidine primer solution, reacting for 10-20 minutes, coupling the single strand to the sensitive membrane layer (103) through the specific binding of biotin and streptavidin, introducing a PBS buffer, and recording the surface plasmon resonance peak after coupling, and ending the resonance peak recording; ||When step-by-step coupling is required, the specific steps are as follows: (1) Gold film modification, continuous flow of 6-mercapto-1-hexanol to form a self-assembled monolayer of hydroxyl groups on its surface; (2) measuring the spectrum in acetonitrile and recording the resonance peak baseline; (3) introducing an active phosphoramidite tetrazole active intermediate for coupling; (4) measuring the resonance peak position after extending one base in an acetonitrile environment; (5) introducing capping reagent; (6) introducing iodine-containing tetrahydrofuran solution for oxidation; (7) introducing trichloroacetic acid deprotection reagent for deprotection; (8) The next synthesis cycle is carried out starting from step (3) until the desired sequence is obtained.

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