A chip card, a molecular interaction analysis system based on the chip card, and an analysis method

Abstract

The present invention belongs to the field of detection technology, and more specifically, relates to a chip card, a molecular interaction analysis system based on the chip card, and an analysis method. The chip card includes a card body, a first groove opened on the surface of the card body, and a second groove opened at the bottom of the first groove; a sample inlet hole, a detection chip, and a waste liquid outlet hole are axially arranged in the second groove. A transparent cover body is embedded in the first groove, and the transparent cover body covers the surface of the second groove, so that the second groove becomes a closed groove. The design of the chip card of the present invention can avoid liquid leakage when detecting a liquid sample flowing in the chip card, and has good stability. The desktop molecular interaction analysis system obtained by connecting and assembling the chip card with a liquid path unit, an optical path unit, and a computer can be used to realize the affinity detection between different molecules. The analysis system is portable, low-cost, and the detection result is accurate and reliable.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology. Specifically, it relates to a chip card, a molecular interaction analysis system and an analysis method based on the chip card; more specifically, it relates to a chip card, a nano-plasmon resonance desktop molecular interaction analysis system and an analysis method based on the chip card. Background Art

[0002] The measurement of molecular interaction kinetics is becoming increasingly important in drug discovery, genetic screening and clinical diagnosis, because dynamic binding information can improve the understanding of diseases and thus provide new ideas for treatment.

[0003] Surface plasmon resonance (SPR) sensors, such as the commercial Biacore SPR biosensor system, can monitor kinetic biomolecular interactions in real time, without labeling and without being affected by a large amount of background. Since the commercial Biacore SPR biosensor system belongs to traditional SPR equipment, it requires special equipment and professional operation, resulting in a relatively high cost for purchasing or using this biosensor system.

[0004] Patent document CN111781370A discloses a method for measuring the molecular binding amount by using an enzyme-linked immunosorbent assay (ELISA) reader and an SPR sensor with a nano-columnar array. The specific SPR detection chip is placed in a bottomless well plate to detect a sample to be tested labeled with nano-gold particles. Although this method can minimize the complex system and equipment requirements of commercial biosensors, since the SPR chip is placed in the well plate, the sample to be tested needs to be added to the well plate for reaction and then detected by an ELISA reader, this detection method has the following defects: (1) When detecting, the sample to be tested is added to the well plate coated with protein and then detected by an ELISA reader. The intermolecular reaction is very fast, and the reaction has basically been completed when it is to be detected, resulting in the inability to obtain the interaction information at the reaction starting point; (2) The result obtained by detecting with an ELISA reader is a numerical value, and the kinetic map curve needs to be manually processed; (3) This document only tested the molecular binding part when detecting with an ELISA reader, without testing the molecular dissociation part, and the obtained equilibrium dissociation constant K D is not accurate enough; at the same time, if both the molecular binding and dissociation parts are tested, the molecular binding and dissociation steps need to be carried out separately and cannot be carried out continuously; (4) When detecting with an ELISA reader, the chip coated with protein and the buffer solution containing the sample to be tested are mixed and then detected under static conditions, resulting in a reduced reaction rate and lower sensitivity. Summary of the Invention

[0005] Aiming at the defects of the prior art, the purpose of the present invention is to provide a chip card for molecular interaction analysis, a nano-plasmonic resonance desktop molecular interaction analysis system and an analysis method of the chip card, so as to solve the technical problems existing in the prior art when using an enzyme-labeled instrument and an SPR sensor with a nano-columnar array to measure the molecular binding amount, such as the molecular interaction being too fast to detect the starting point of the interaction, the binding and dissociation needing to be tested separately, and the operation and data analysis steps being cumbersome.

[0006] To achieve the above purpose, the present invention provides a chip card for molecular interaction analysis, which includes a card body, a first groove opened on the surface of the card body, and a second groove opened at the bottom of the first groove;

[0007] A fluid first transmission hole, a detection chip and a fluid second transmission hole are axially arranged in the second groove; both the fluid first transmission hole and the fluid second transmission hole are through holes passing through the card body, and the hole surfaces of the fluid first transmission hole and the fluid second transmission hole are flush with the bottom surface of the second groove;

[0008] A transparent cover is embedded in the first groove, and the transparent cover covers the surface of the second groove, making the second groove a closed groove.

[0009] Preferably, the transparent cover is glass, a PET film or an acrylic plate.

[0010] Preferably, the thickness of the card body is 5 mm - 15 mm, the grooving depth of the first groove is 1 mm - 5 mm, the grooving depth of the second groove is 0.5 mm - 1.5 mm, and the width of the detection chip is 35 mm - 45 mm.

[0011] Preferably, a fluid third transmission hole is further arranged on the extension line of the detection chip and the fluid second transmission hole, a reference chip is arranged between the fluid third transmission hole and the fluid second transmission hole, the fluid third transmission hole is a through hole passing through the card body, and the hole surface of the fluid third transmission hole is flush with the bottom surface of the second groove.

[0012] Preferably, a first chip groove and a second chip groove are opened in the second groove, the first chip groove is used to accommodate and fix the detection chip, and the second chip groove is used to accommodate and fix the reference chip, so that the surfaces of the detection chip and the reference chip are flush or substantially flush with the bottom surface of the second groove.

[0013] Further preferably, a convex platform protruding relative to the bottom surface of the second groove is arranged on one side of the fluid second transmission hole facing the fluid third transmission hole, and a semi-circular bulge is formed on the side wall of the second groove relative to the position of the convex platform, so that the second groove forms a U-shaped fluid channel.

[0014] According to another aspect of the present invention, there is provided a molecular interaction analysis system based on the chip card, comprising an optical path unit, a liquid path unit, the chip card as described above, and a control unit; wherein:

[0015] The optical path unit is used to provide the light required for molecular interaction analysis for the chip card, and it comprises a light source, an optical fiber, and a detector;

[0016] The liquid path unit is used to provide a fluid passage required for molecular interaction analysis for the chip card. A fluid flow power device, a liquid pipeline, and a waste liquid collection device are arranged on the fluid passage; the sample is conveyed to the liquid pipeline by the fluid flow power device, flows through the chip on the chip card, and under the illumination provided by the optical path unit, molecular interaction analysis and detection are realized. The waste liquid flows into the waste liquid collection device for collection; the chip on the chip card is a plasmon resonance chip sensor with a nano-columnar array.

[0017] The control unit is a computer, which controls the detector and the optical fiber through a data transmission line. The light emitted by the light source enters the chip in the chip card through the optical fiber, plasmon resonance occurs at the chip, and the obtained feedback signal is collected by the detector and then transmitted to the computer, and the computer analyzes and processes the collected signal.

[0018] Preferably, in the second groove of the chip card, a first fluid transmission hole, a detection chip, and a second fluid transmission hole are axially arranged; wherein the first fluid transmission hole is a sample inlet hole, the second fluid transmission hole is a waste liquid outlet hole, and a buffer solution storage device, a quantitative component, a first multi-channel valve, the chip card, a fluid flow power device, and a waste liquid collection device are sequentially arranged on the fluid passage; wherein the first multi-channel valve is connected to the sample inlet hole, and the waste liquid outlet hole is connected to the fluid flow power device.

[0019] Preferably, in the second groove of the chip card, a first fluid transmission hole, a detection chip, a second fluid transmission hole, a reference chip, and a third fluid transmission hole are axially arranged; the first fluid transmission hole is a sample inlet hole, the second fluid transmission hole is a waste liquid outlet hole, the third fluid transmission hole is a second outlet hole, and a buffer solution storage device, a quantitative component, a first multi-channel valve, the chip card, a second multi-channel valve, a fluid flow power device, and a waste liquid collection device are sequentially arranged on the fluid passage; wherein the first multi-channel valve is connected to the sample inlet hole, and both the waste liquid outlet hole and the second outlet hole are connected to the second multi-channel valve; during use, by switching the opening direction of the second multi-channel valve, the waste liquid is made to flow out from the waste liquid outlet hole or the second outlet hole by the power provided by the fluid flow power device.

[0020] Further preferably, in the second slot of the chip card, a first fluid transfer hole, a detection chip, a second fluid transfer hole, a reference chip, and a third fluid transfer hole are arranged along the axial direction; the second fluid transfer hole is the first sample inlet hole, the third fluid transfer hole is the second sample inlet hole, and the first fluid transfer hole is the waste liquid outlet hole. A buffer solution storage device, a quantification component, a first multi-channel valve, a second multi-channel valve, the chip card, a fluid flow power device, and a waste liquid collection device are sequentially arranged on the fluid passage; wherein the first multi-channel valve is connected to the waste liquid outlet hole, and both the first sample inlet hole and the second sample inlet hole are connected to the second multi-channel valve; during use, by switching the opening direction of the second multi-channel valve, the sample is caused to enter from the first sample inlet hole or the second sample inlet hole by the power provided by the fluid flow power device.

[0021] According to another aspect of the present invention, a method for performing molecular interaction analysis using the analysis system as described above is provided, including the following steps:

[0022] (1) Inject an activation solution for activating the carboxylated chip into the quantification component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, and cause the activation solution to enter the chip card from the first fluid transfer hole along with the buffer solution and flow through the detection chip. The detection chip is a carboxylated detection chip to achieve the activation of the carboxylated detection chip; use the power provided by the fluid flow power device to cause the waste liquid generated during the activation process to flow out from the waste liquid outlet hole to the waste liquid collection device, obtaining an activated carboxylated detection chip;

[0023] (2) Inject a first molecular sample into the quantification component through the sample inlet of the first multi-channel valve, switch the opening direction of the first multi-channel valve, and cause the first molecule to enter the chip card from the first fluid transfer hole along with the buffer solution and flow through the activated carboxylated detection chip, achieving the fixation of the first molecule on the detection chip; use the power provided by the fluid flow power device to cause the waste liquid generated during the fixation process to flow out from the second fluid transfer hole to the waste liquid collection device; obtaining a detection chip with the first molecule fixed thereon;

[0024] (3) Inject the second molecular sample into the quantitative component through the sample injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the second molecule enters the chip card with the buffer solution from the first fluid transfer hole and flows through the detection chip fixed with the first molecule, and under the illumination provided by the optical path unit, make the second molecule interact with the first molecule fixed on the detection chip; Use the power provided by the fluid flow power device to make the detection waste liquid flow out from the second fluid transfer hole to the waste liquid collection device.

[0025] According to another aspect of the present invention, there is provided a method for analyzing molecular interaction using the analysis system as described above, including the following steps:

[0026] (1) Inject the activation solution for activating the carboxylated chip into the quantitative component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the activation solution enters the chip card with the buffer solution from the first fluid transfer hole and sequentially flows through the carboxylated detection chip and the carboxylated reference chip to activate the carboxylated detection chip and the carboxylated reference chip; Switch the opening direction of the second multi-channel valve, and use the power provided by the fluid flow power device to make the waste liquid generated during the activation process flow out from the third fluid transfer hole to the waste liquid collection device to obtain the activated carboxylated detection chip and the activated carboxylated reference chip;

[0027] (2) Inject the first molecular sample into the quantitative component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the first molecule enters the chip card with the buffer solution from the first fluid transfer hole and flows through the activated carboxylated detection chip to fix the first molecule on the detection chip; Use the power provided by the fluid flow power device to make the waste liquid generated during the fixation process flow out from the second fluid transfer hole to the waste liquid collection device;

[0028] (3) Inject the second molecular sample into the quantitative component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the second molecule enters the chip card with the buffer solution from the first fluid transfer hole and sequentially flows through the detection chip fixed with the first molecule and the activated carboxylated reference chip, and under the illumination provided by the optical path unit, make the second molecule interact with the first molecule fixed on the detection chip; Switch the opening direction of the second multi-channel valve, and use the power provided by the fluid flow power device to make the detection waste liquid flow out from the third fluid transfer hole to the waste liquid collection device.

[0029] According to another aspect of the present invention, there is provided a method for molecular interaction analysis using the analysis system as described above, comprising the following steps:

[0030] (1) Inject the activation solution for activating the carboxylated chip into the quantitative component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, and enable the activation solution to enter the chip card from the third fluid transfer hole along with the buffer solution and flow through the carboxylated reference chip and the carboxylated detection chip in sequence, so as to activate the carboxylated reference chip and the carboxylated detection chip; switch the opening direction of the second multi-channel valve, and use the power provided by the fluid flow power device to make the activated waste liquid flow out from the first fluid transfer hole to the waste liquid collection device, obtaining the activated carboxylated detection chip and the activated carboxylated reference chip;

[0031] (2) Inject the first molecular sample into the quantitative component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, and enable the first molecule to enter the chip card from the second fluid transfer hole along with the buffer solution and flow through the activated carboxylated detection chip, so as to fix the first molecule on the detection chip; use the power provided by the fluid flow power device to make the waste liquid generated during the fixing process flow out from the first fluid transfer hole to the waste liquid collection device;

[0032] (3) Inject the second molecular sample into the quantitative component through the sample injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, and enable the second molecule to enter the chip card from the third fluid transfer hole along with the buffer solution and flow through the activated carboxylated reference chip and the detection chip with the first molecule fixed thereon in sequence; under the illumination provided by the optical path unit, enable the second molecule to interact with the first molecule fixed on the detection chip; use the power provided by the fluid flow power device to make the detection waste liquid flow out from the first fluid transfer hole to the waste liquid collection device.

[0033] Generally speaking, compared with the prior art through the above technical solution conceived by the present invention, the following beneficial effects are achieved:

[0034] (1) The chip card for nano-plasmon resonance desktop molecular interaction analysis provided by the present invention comprises a card body, a first groove opened on the surface of the card body, and a second groove opened at the bottom of the first groove; a sample inlet hole, a detection chip, and a waste liquid outlet hole are axially arranged in the second groove. A transparent cover body is embedded in the first groove, and the transparent cover body covers the surface of the second groove, making the second groove a closed groove. The design of the chip card of the present invention can avoid liquid leakage when the detected liquid sample flows in the chip card, and has good stability.

[0035] (2) The chip card for nano - plasma resonance desktop molecular interaction analysis provided in the preferred embodiment of the present invention can be a single - channel detection chip card or a dual - channel detection chip card with a reference chip added on the basis of single - channel detection. By adding a reference chip, the background noise in the molecular interaction analysis detection process can be deducted, and the non - specific adsorption of the chip can be reduced. The present invention can also be repeatedly set multiple times on the basis of the above - mentioned single - channel and dual - channel detection chip cards according to needs to obtain multiple detection channels to meet the synchronous detection requirements of samples with different concentrations.

[0036] (3) In the dual - channel chip card provided in the preferred embodiment of the present invention, a boss protruding relative to the bottom surface of the second tank is provided on the side of the waste liquid outlet hole of the chip card facing the second outlet hole, and a semi - annular bulge is formed on the side wall of the second tank relative to the position of the boss, so that the second tank forms a U - shaped fluid channel. Such a special design can well avoid the sample solution diffusing to the surface of the reference chip and contaminating the reference chip when coating the first molecule, such as a fixed protein molecule, on the detection chip for the first step, ensuring the normal function of the reference chip during the dual - channel detection process.

[0037] (4) The nano - plasma resonance desktop molecular interaction analysis system based on the above - mentioned chip card proposed by the present invention includes an optical path unit, a liquid path unit, a control unit, and the chip card proposed by the present invention. Only by connecting each component in an orderly manner according to a specific connection method and following the established analysis steps, injecting samples through a six - way valve quantitative loop successively, and carrying out the fixation of the first molecule and the interaction analysis of the first and second molecules under pump pressure, the kinetic process of the interaction between the first molecule and the second molecule can be detected in real - time, accurately, and completely. Moreover, the whole system has a simple structure, is easy to build, portable, and has a low cost.

[0038] (5) In a preferred embodiment of the present invention, for an analysis system using a dual - channel chip card, the detection accuracy is further improved by optimizing the sample injection method. For example, by transporting the ligand solution for fixation through the central fluid transfer hole of the dual - channel chip card to fix the ligand molecules on the detection chip and discharging the fixed waste liquid from the left - most fluid transfer hole; then transporting the second - molecule sample solution from the right - most fluid transfer hole and discharging the waste liquid from the left - most fluid transfer hole, it is possible to completely avoid the contamination of the reference chip by the residual ligand solution during the first - step fixation of ligand molecules, thereby improving the accuracy of the detection results.

[0039] (6) The present invention is designed based on a chip card with a specific structure, and an analysis system including an optical path unit, a liquid path unit, and a control unit is built. The light emitted by the light source is transmitted through an optical fiber to the surface of the chip in the chip card, where surface plasmon resonance occurs at the chip. The obtained feedback signal is transmitted into the detector through the optical fiber. After the detector collects the signal, it is processed and then transmitted to the computer, which analyzes, processes, and displays the collected signal. Compared with the method of using an enzyme-linked immunosorbent assay (ELISA) reader and an SPR sensor with a nano-columnar array to measure the molecular binding amount, it can obtain complete molecular interaction information in real time, including the complete kinetic binding and dissociation detection and simulation processes of equilibrium, binding, dissociation, and regeneration, and the results are reliable, accurate, and highly sensitive. Description of the Drawings

[0040] Figure 1 It is the front view of a single-channel chip card in some embodiments of the present invention;

[0041] Figure 2 is Figure 1 the side view of the single-channel chip card in

[0042] Figure 3 It is the front view of a double-channel chip card in some embodiments of the present invention;

[0043] Figure 4 is Figure 3 the side view of the double-channel chip card in

[0044] Figure 5 It is the front view of a double-channel chip card in some other embodiments of the present invention;

[0045] Figure 6 is Figure 5 the side view of the double-channel chip card in

[0046] Figure 7 It is a nano-plasmonic resonance desktop molecular interaction analysis system based on a single-channel chip card in some embodiments of the present invention;

[0047] Figure 8 It is a nano-plasmonic resonance desktop molecular interaction analysis system based on a double-channel chip card in some embodiments of the present invention;

[0048] Figure 9 It is a nano-plasmonic resonance desktop molecular interaction analysis system based on a double-channel chip card in some other embodiments of the present invention;

[0049] Figure 10 It is the full-spectrum diagram of narrow-spectrum detection of different sucrose concentrations in Example 1;

[0050] Figure 11 It is the full-spectrum diagram of wide-spectrum detection of different sucrose concentrations in Example 1;

[0051] Figure 12 For Example 1, the contrast between the difference in light intensity between 660 nm and 600 nm of the narrow spectrum and the difference in light intensity between 620 nm and 610 nm of the wide spectrum;

[0052] Figure 13 For Example 2, detecting the kinetic interaction between Protein A and IgG, and the typical atlas of the detection process;

[0053] Figure 14 For Example 2, detecting the kinetic interaction between Protein A and IgG, and the kinetic binding and dissociation detection and simulation processes of the two;

[0054] Figure 15 For Example 2, the detection linearity R of IgG using a Protein A chip 2 Fitting result;

[0055] Figure 16 For Example 3, detecting the kinetic interaction between FcRn and IgG, and the kinetic binding and dissociation simulation process of the two;

[0056] In all the drawings, the same reference numerals are used to represent the same elements or structures, where:

[0057] 1 - Card body; 2 - First groove; 3 - Second groove; 4 - First fluid transfer hole; 5 - Detection chip; 6 - Second fluid transfer hole; 7 - Third fluid transfer hole; 8 - Reference chip; 9 - Boss; 10 - Semi-circular bulge; 11 - Buffer storage device; 12 - Quantification component; 13 - First multi-channel valve; 14 - Fluid flow power device; 15 - Waste liquid collection device; 16 - Second multi-channel valve; 17 - Light source; 18 - Optical fiber; 19 - Detector; 20 - Computer; 21 - Optical fiber probe; 22 - Light wave. Detailed implementation manners

[0058] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0059] Regarding the technical defects of the method for measuring the molecular binding amount using an enzyme-linked immunosorbent assay (ELISA) reader and a surface plasmon resonance (SPR) sensor with a nano-columnar array, such as the inability to detect the starting point, the need to separately detect binding and dissociation, and the inability to directly obtain the kinetic curve pattern. The present invention proposes to use a detector such as a spectrometer and an SPR sensor with a nano-columnar array (hereinafter abbreviated as NanoSPR) to measure the molecular binding amount. However, in the specific experimental process, how to connect the spectrometer to the NanoSPR sensor for real-time detection, how to fix the chip in the flow channel, and how to ensure no liquid leakage during fluid flow, and how to avoid the diffusion and contamination of the reference chip by the protein-containing solution during the process of fixing protein molecules in dual-channel detection, etc., are all technical problems that need to be overcome. For example, the commercial OpenSPR sensor uses a glass chip to accelerate the PDMS pressing and sealing method, which has poor stability and is also prone to liquid leakage.

[0060] In view of the above defects, the present invention proposes a chip card for molecular interaction analysis and a nano-plasmonic resonance desktop molecular interaction analysis system based on the chip card. As Figure 1 and Figure 2 shown, in some embodiments, a chip card for molecular interaction analysis includes a card body 1, a first groove 2 opened on the surface of the card body 1, and a second groove 3 opened at the bottom of the first groove 2; the first groove 2 can be of various shapes, including but not limited to a rectangular groove. The second groove 3 is actually a channel for the sample solution to flow, and can be opened as a groove of various shapes according to needs, including but not limited to a long rectangular body.

[0061] A fluid first transmission hole 4, a detection chip 5, and a fluid second transmission hole 6 are axially arranged in the second groove 3; both the fluid first transmission hole 4 and the fluid second transmission hole 6 are through holes passing through the card body 1, and the peripheral surfaces of the holes of the fluid first transmission hole 4 and the fluid second transmission hole 6 are flush with the bottom surface of the second groove 3.

[0062] A transparent cover body (not shown in the figure) is embedded in the first groove 2, and the transparent cover body covers the surface of the second groove 3, making the second groove 3 a closed groove to prevent fluid from flowing out. In some embodiments, the transparent cover body is glass, a PET film, or an acrylic plate; the surface of the transparent cover body facing the second groove itself has hydrophilicity or is made to have hydrophilicity after hydrophilic treatment so that the surface facing the second groove has hydrophilicity. The hydrophilic transparent cover body can promote fluid diffusion to ensure that after the sample solution enters the second groove 3 from the fluid first transmission hole 4, it can quickly spread evenly in the second groove 3, ensure uniform coverage of the solution on the surface of the detection chip 5, and ensure uniform interaction of substances on the chip surface.

[0063] In some embodiments, the size of the transparent cover body embedded in the first groove body 2 is the same as the size of the grooving area of the first groove body and larger than the grooving area of the second groove body, covering the surface of the second groove body 3, so that the second groove body 3 becomes a closed groove body, which can better prevent the fluid from flowing out. Preferably, the thickness of the transparent cover body is the same as the grooving depth of the first groove body, so that the overall surface of the chip card is a flat surface.

[0064] In order to ensure the smooth progress of the molecular interaction analysis reaction, in some preferred embodiments, the thickness of the card body is 5 mm - 15 mm, the grooving depth of the first groove body is 1 mm - 5 mm, the grooving depth of the second groove body is 0.5 mm - 1.5 mm, and the width of the detection chip (the width in the direction perpendicular to the axial direction) is 35 mm - 45 mm.

[0065] In some other embodiments, as Figure 3 and Figure 4 shown, a third fluid transfer hole 7 is further provided on the extension line of the detection chip 5 and the second fluid transfer hole 6 in the second groove body 3. A reference chip 8 is further provided between the third fluid transfer hole 7 and the second fluid transfer hole. The third fluid transfer hole 7 is a through hole passing through the card body 1, and the outer peripheral surface of the hole of the third fluid transfer hole 7 is flush with the bottom surface of the second groove body 3. The setting of the reference chip 8 is used to provide reference data for the molecular interaction analysis detection or deduct background noise from the curve to reduce the non-specific adsorption of the chip. In the present invention, the chip card with both a detection chip and a reference chip is called a dual-channel detection chip card, and the chip card with only a detection chip is called a single-channel chip card.

[0066] In order to avoid resistance when the sample solution flows through the detection chip 5 and the reference chip 8 after entering the second groove body from the first fluid transfer hole 4, in some embodiments, a first chip groove and a second chip groove (not shown in the figure) are provided in the second groove body. The first chip groove is used to accommodate and fix the detection chip 5, and the second chip groove is used to accommodate and fix the reference chip 8, so that the surfaces of the detection chip 5 and the reference chip 8 are flush or substantially flush with the bottom surface of the second groove body 3.

[0067] Furthermore, since dual-channel detection is employed, i.e., when a detection chip and a reference chip are simultaneously provided in the chip card of the present invention, in the first step of fixing the first molecule, such as the ligand molecule, only the detection chip needs to fix the ligand molecule, and the reference chip does not need to fix the ligand molecule. In order to prevent the sample solution from diffusing onto the reference chip, in some preferred embodiments, a boss 9 protruding relative to the bottom surface of the second trough body 3 is provided on the side of the second fluid transmission hole 6 facing the third fluid transmission hole 7, and a semi-annular bulge 10 is formed on the side wall of the second trough body 3 relative to the position of the boss 9, so that the second trough body 3 forms an "X"-shaped fluid channel. In this way, when the solution used to fix the ligand molecule for the detection chip 5 enters from the first fluid transmission hole 4, gradually flows through the detection chip 5 and then to the second fluid transmission hole 6, due to the blocking effect of the boss 9, experiments have shown that it can effectively prevent the sample solution from further diffusing to the side of the reference chip 8, thereby causing the reference chip 8 to be contaminated.

[0068] Figure 2 、 Figure 4 and Figure 6 They are Figure 1 、 Figure 3 and Figure 5 The side views of the three chip cards illustrate that the first fluid transmission hole 4, the second fluid transmission hole 6 and the third fluid transmission hole 7 are all protruding relative to the bottom of the card body 1 to facilitate the connection of liquid pipelines.

[0069] The present invention also provides a molecular interaction analysis system based on the chip card, comprising an optical path unit, a liquid path unit, the chip card and a control unit; Figure 7 、 Figure 8 and Figure 9 As shown, where:

[0070] The optical path unit is used to provide the chip card with the illumination required for molecular interaction analysis, and includes a light source 17, an optical fiber 18 and a detector 19. The present invention uses a nanoplasmon resonance chip to test molecular interactions. The light source used can be any light source that can provide light waves, including but not limited to a tungsten lamp light source or an LED lamp; the optical fiber can be a reflective fiber or a transmissive fiber; the detector is used to detect molecular interactions on the chip surface, including but not limited to a spectrometer or a photoelectric detector (such as a PD detector).

[0071] The liquid path unit is used to provide the chip card with the fluid path required for molecular interaction analysis, and includes a fluid flow power device, a liquid pipeline and a waste liquid collection device; the sample is transmitted to the liquid pipeline by the fluid flow power device, flows through the chip on the chip card, and under the light provided by the optical path unit, molecular interaction analysis and detection are realized, and the waste liquid flows into the waste liquid collection device for collection; the chip on the chip card is a plasma resonance chip sensor with a nanocolumnar array.

[0072] The control unit is a computer 20. The light source 17 is connected to the optical fiber 18, and the optical fiber 18 is connected to the detector 19, and the detector 19 is connected to the computer 20. The computer 20 controls the detector 19 through a data transmission line. The light emitted by the light source 17 is transmitted through the optical fiber 18 (the light wave 22 emitted through the optical fiber probe 21) to the surface of the chip in the chip card, where surface plasmon resonance occurs at the chip. The obtained feedback signal is transmitted into the detector 19 through the optical fiber 17. After the detector 19 collects the signal and processes it, the signal is transmitted to the computer 20, and the computer analyzes and processes the collected signal and displays it.

[0073] The chip card design provided by the present invention can not only meet the requirements of single-channel molecular interaction analysis, but also meet the requirements of dual-channel molecular interaction analysis. Compared with single-channel molecular interaction analysis, a reference chip is added to the chip card in dual-channel molecular interaction analysis. By setting the reference chip, the background noise of the reference chip can be deducted on the basis of the detection chip to reduce the non-specific adsorption of the chip.

[0074] In some embodiments, such as Figure 7 described, a single-channel chip card is adopted. The structural schematic diagram of the chip card is as shown in Figure 1 and Figure 2 shown. In the second groove body of the chip card, a first fluid transmission hole 4, a detection chip 5, and a second fluid transmission hole 6 are axially arranged; wherein the first fluid transmission hole 4 is a sample inlet hole, the second fluid transmission hole 6 is a waste liquid outlet hole. A buffer solution storage device 11, a quantitative component 12, a first multi-channel valve 13, the chip card, a fluid flow power device 14, and a waste liquid collection device 15 are sequentially arranged on the fluid passage; wherein the first multi-channel valve 13 is connected to the sample inlet hole (the first fluid transmission hole 4), and the waste liquid outlet hole (the second fluid transmission hole 6) is connected to the fluid flow power device 14.

[0075] In other embodiments, a reference chip is added to the chip card, which is called a dual-channel chip card by the present invention. Taking the dual-channel chip card shown in Figure 3 , Figure 4 , Figure 5 and Figure 6 as an example, in an analysis system including the chip card, in the second groove body of the chip card, a first fluid transmission hole 4, a detection chip 5, a second fluid transmission hole 6, a reference chip 8, and a third fluid transmission hole 7 are axially arranged; the first fluid transmission hole 4 is a sample inlet hole, the second fluid transmission hole 6 is a waste liquid outlet hole, and the third fluid transmission hole 7 is a second outlet hole, as shown in Figure 8As shown, a buffer storage device 11, a metering component 12, a first multi-channel valve 13, the chip card, a second multi-channel valve 16, a fluid flow power device 14, and a waste liquid collection device 15 are sequentially arranged on the fluid passage; wherein the first multi-channel valve 13 is connected to the sample inlet hole, and the waste liquid outlet hole and the second outlet hole are both connected to the second multi-channel valve 16. During use, by switching the opening direction of the second multi-channel valve, the power provided by the fluid flow power device 14 is used to make the waste liquid flow out from the waste liquid outlet hole or the second outlet hole.

[0076] With the above-mentioned molecular interaction analysis system based on a dual-channel chip card, the single-channel or dual-channel molecular interaction analysis requirements can be realized according to needs by selecting and setting the waste liquid outlet channel.

[0077] When using the analysis system of the present invention for molecular interaction, the process is similar to the general process of using a NanoSPR chip for molecular interaction analysis. Generally, the chip is first carboxylated, then the carboxylated chip is activated with an activation solution, and then the first molecule (such as a ligand) is immobilized on the activated carboxylated chip, and then the second molecule is made to interact with the first molecule under light conditions, and the analysis result is obtained with the help of this analysis system.

[0078] In some embodiments, the NanoSPR chip is immersed in 50 mM 3-MPA at 4°C overnight to carboxylate the chip surface, and then the carboxylated chip is stored in PBS buffer at 4°C for later use. The carboxylated chip is pasted on the chip card and assembled into the detection system for detection. An activation solution EDC / NHS (400 mM / 100 mM) mixture is injected into the metering component through the sample inlet of the first multi-channel valve, and the opening direction of the first multi-channel valve is switched to make the mixture flow in from the first fluid transfer hole and flow through the detection chip and the reference chip to activate the carboxyl group of the chip, and then flow out from the third fluid transfer hole to the waste liquid collection device at a flow rate of 10 μL / min.

[0079] Specifically, using the Figure 7 single-channel chip card shown to build an analysis system, the method for analyzing the interaction between the first molecule and the second molecule includes the following steps:

[0080] (1) Inject the activation solution for activating the carboxylated chip into the quantitative component 12 through the injection port of the first multi-channel valve 13. Switch the opening direction of the first multi-channel valve 13 so that the activation solution follows the buffer solution and enters the chip card through the first fluid transfer hole 4 and flows through the detection chip 5. The detection chip 5 is a carboxylated detection chip to activate the carboxylated detection chip. Use the power provided by the fluid flow power device 14 to make the waste liquid generated during the activation process flow out from the waste liquid outlet hole, that is, the second fluid transfer hole, to the waste liquid collection device 15, and obtain the activated carboxylated detection chip;

[0081] (2) Inject the first molecular sample into the quantitative component 12 through the sample injection port of the first multi-channel valve 13. Switch the opening direction of the first multi-channel valve 13 so that the first molecule follows the buffer solution and enters the chip card through the first fluid transfer hole 4 and flows through the activated carboxylated detection chip to fix the first molecule on the detection chip 5. Use the power provided by the fluid flow power device 14 to make the waste liquid generated during the fixation process flow out from the second fluid transfer hole 6 to the waste liquid collection device 15, and obtain the detection chip with the first molecule fixed;

[0082] (3) Inject the second molecular sample into the quantitative component 12 through the sample injection port of the first multi-channel valve 13. Switch the opening direction of the first multi-channel valve 13 so that the second molecule follows the buffer solution and enters the chip card through the first fluid transfer hole 4 and flows through the detection chip with the first molecule fixed, so that the second molecule interacts with the first molecule fixed on the detection chip. Use the power provided by the fluid flow power device 14 to make the detection waste liquid flow out from the second fluid transfer hole to the waste liquid collection device.

[0083] In some embodiments, between the above steps (2) and (3), there is also a step:

[0084] (2.5) Inject the blocking solution for blocking the chip into the quantitative component 12 through the sample injection port of the first multi-channel valve 13. Switch the opening direction of the first multi-channel valve 13 so that the blocking solution follows the buffer solution and enters the chip card through the first fluid transfer hole 4 and flows through the detection chip with the first molecule fixed to block the detection chip. Use the power provided by the fluid flow power device 14 to make the blocking waste liquid flow out from the second fluid transfer hole to the waste liquid collection device.

[0085] Use the above dual-channel chip card to build an analysis system as shown in Figure 8 for the interaction analysis method of the first molecule and the second molecule, including the following steps:

[0086] (1) Inject the activation solution for activating the carboxylated chip into the quantitative component 12 through the injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, so that the activation solution enters the chip card with the buffer solution from the first fluid transfer hole 4 and flows through the carboxylated detection chip 5 and the carboxylated reference chip 8 in sequence, to activate the carboxylated detection chip and the carboxylated reference chip; switch the opening direction of the second multi-channel valve 16, and use the power provided by the fluid flow power device 14 to make the waste liquid generated during the activation process flow out from the third fluid transfer hole 7 to the waste liquid collection device 15, to obtain the activated carboxylated detection chip and the activated carboxylated reference chip;

[0087] (2) Inject the first molecular sample into the quantitative component 12 through the injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, so that the first molecule enters the chip card with the buffer solution from the first fluid transfer hole 4 and flows through the activated carboxylated detection chip, to fix the first molecule on the detection chip; use the power provided by the fluid flow power device 14 to make the waste liquid generated during the fixation process flow out from the second fluid transfer hole 6 to the waste liquid collection device 15;

[0088] (3) Inject the second molecular sample into the quantitative component 12 through the injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, so that the second molecule enters the chip card with the buffer solution from the first fluid transfer hole 4 and flows through the detection chip 5 fixed with the first molecule and the activated carboxylated reference chip 8 in sequence, to make the second molecule interact with the first molecule fixed on the detection chip; switch the opening direction of the second multi-channel valve 16, and use the power provided by the fluid flow power device 14 to make the detection waste liquid flow out from the third fluid transfer hole 7 to the waste liquid collection device.

[0089] In some embodiments, between the above step (2) and step (3), there is also a step:

[0090] (2.5) Inject the sealing solution for sealing the chip into the quantitative component 12 through the injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, so that the sealing solution enters the chip card with the buffer solution from the first fluid transfer hole 4 and flows through the detection chip 5 fixed with the first molecule and the activated carboxylated reference chip 8 in sequence, to seal the detection chip and the reference chip; switch the opening direction of the second multi-channel valve 16, and use the power provided by the fluid flow power device 14 to make the sealing waste liquid flow out from the third fluid transfer hole 7 to the waste liquid collection device.

[0091] In order to avoid the contamination of the reference chip by the residual ligand solution during the immobilization of the ligand molecule as much as possible, in a more preferred embodiment, the Figure 5 and Figure 6 shown dual-channel chip card is still taken as an example. The corresponding analysis system requires the ligand solution to be transported from the second fluid transfer hole and the waste liquid to be discharged from the first fluid transfer hole. Specifically, in the second groove of the chip card, a first fluid transfer hole, a detection chip, a second fluid transfer hole, a reference chip, and a third fluid transfer hole are axially arranged; the second fluid transfer hole is the first sample inlet hole, the third fluid transfer hole is the second sample inlet hole, and the first fluid transfer hole is the waste liquid outlet hole. As Figure 9 shown, a buffer storage device 11, a quantification component 12, a first multi-channel valve 13, a second multi-channel valve 16, the chip card, a fluid flow power device 15, and a waste liquid collection device 15 are sequentially arranged on the fluid passage; wherein the first multi-channel valve 13 is connected to the second multi-channel valve 16, and the second multi-channel valve 16 is connected to the first sample inlet hole and the second sample inlet hole. During use, by switching the opening direction of the second multi-channel valve 16, the sample is made to enter from the first sample inlet hole or the second sample inlet hole by the power provided by the fluid flow power device 14.

[0092] When performing molecular interaction analysis using the analysis system as Figure 9 shown, the following steps are included:

[0093] (1) Inject the activation solution for activating the carboxylated chip into the quantification component 12 through the injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, and make the activation solution enter the chip card from the third fluid transfer hole 7 along with the buffer and flow through the carboxylated reference chip 8 and the carboxylated detection chip 5 in sequence to activate the carboxylated reference chip and the carboxylated detection chip; switch the opening direction of the second multi-channel valve 16, and make the activation waste liquid flow out from the first fluid transfer hole 4 to the waste liquid collection device by the power provided by the fluid flow power device 15 to obtain the activated carboxylated detection chip and the activated carboxylated reference chip;

[0094] (2) Inject the first molecular sample into the quantification component 12 through the injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, and make the first molecule enter the chip card from the second fluid transfer hole 6 along with the buffer and flow through the activated carboxylated detection chip to fix the first molecule on the detection chip; make the waste liquid generated during the fixing process flow out from the first fluid transfer hole 4 to the waste liquid collection device 15 by the power provided by the fluid flow power device 14;

[0095] (3) Inject the second molecular sample into the quantitative component 12 through the sample injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, so that the second molecule flows through the activated carboxylated reference chip and the detection chip with the first molecule immobilized thereon in sequence from the third fluid transfer hole 7 along with the buffer solution; under the illumination provided by the optical path unit, make the second molecule interact with the first molecule immobilized on the detection chip; use the power provided by the fluid flow power device 14 to make the detection waste liquid flow out from the first fluid transfer hole 4 to the waste liquid collection device 15.

[0096] In some embodiments, between the above steps (2) and (3), there is also a step:

[0097] (2.5) Inject the blocking solution for blocking the chip into the quantitative component 12 through the sample injection port of the first multi-channel valve 13, switch the opening direction of the first multi-channel valve 13, so that the blocking solution flows through the activated carboxylated reference chip and the detection chip with the first molecule immobilized thereon in sequence from the third fluid transfer hole 7 along with the buffer solution to block the reference chip and the detection chip; use the power provided by the fluid flow power device 14 to make the blocking waste liquid flow out from the first fluid transfer hole 4 to the waste liquid collection device 15.

[0098] In some embodiments of the present invention, the buffer solution storage device 11 is a buffer solution bottle for containing buffer solution; the quantitative component 12 can be various components for quantitatively injecting samples, including but not limited to a quantitative loop; the first multi-channel valve 13 can regulate the fluid flow path by switching the multi-channel openings, and can be a three-way valve, a six-way valve, etc.; the fluid flow power device 14 is used to provide power for fluid flow, including but not limited to a peristaltic pump, an injection pump or a high-pressure pump, etc.; the fluid is pumped by alternately squeezing and releasing the elastic delivery hose of the pump, and the flow rate of the fluid is controlled by controlling the appropriate alternate squeezing and releasing speed during the pumping process. The waste liquid collection device 15 is used to collect waste liquid.

[0099] In some embodiments, after immobilizing the first molecule on the detection chip in the analysis system of the present invention, before introducing the second molecule for detection, the detection chip and the reference chip are also blocked to reduce the non-specific adsorption of the chip. In some embodiments, the blocking solution for blocking the chip is a PBS solution of casein.

[0100] According to the design concept of the chip card and the analysis system of the present invention, a single-channel, dual-channel or any other multi-channel chip card can be set as needed. For example, multiple second grooves can be opened on the chip card, the structure of the second groove can be replicated, and the liquid flow path can be configured to simultaneously realize the analysis of molecular interactions at multiple different concentrations.

[0101] The detection chip and the reference chip adopted in the present invention are nano-columnar array plasmon resonance (Nano-SPR) chip sensors. The chip sensors can be fabricated by replication molding technology. Among them, a nano-columnar array pattern is fabricated on a quartz substrate by laser interference lithography. Then, an ultraviolet-curable polymer (such as NOA-61) is evenly dispersed on the mold, and the top is supported by polyethylene terephthalate; it is cured by ultraviolet light, and then the polyethylene terephthalate substrate together with the periodic nanopore pattern is peeled off from the mold; a titanium adhesion layer, a silver layer, and a gold layer are formed by electron beam evaporation deposition to realize the formation of plasmonic components. In the embodiments of the present invention, the specific chip structure and preparation can refer to the patent document CN111781370A. In the following embodiments, the thicknesses of the titanium adhesion layer, the gold layer, and the titanium dioxide resonator layer in the detection chip and the reference chip are 16nm, 220nm, and 180nm respectively, and the curing time is 3 minutes.

[0102] The chip card, the nano-plasmon resonance desktop molecular interaction analysis system and the analysis method based on the chip card provided by the present invention can be used for real-time monitoring of kinetic biomolecular interactions, including but not limited to the analysis of interactions between proteins and proteins, proteins and nucleic acids, proteins and small molecules, nucleic acids and small molecules, nucleic acids and nucleic acids, etc. In the following embodiments of the present invention, the feasibility of the chip card and the nano-plasmon resonance molecular interaction analysis system based on the chip card proposed by the present invention is verified by detecting the affinity between Protein A and IgG and performing a linear detection on IgG.

[0103] Example 1

[0104] Comparing the narrow spectrum and the wide spectrum with different concentrations of sucrose

[0105] 1. Experimental procedure

[0106] Sucrose is diluted with ultrapure water into different concentrations (0-60%) and injected into the detection system as shown in Figure 7 where the schematic diagram of the chip card structure is shown in Figure 1 and Figure 2 shown. The buffer solution adopts a peristaltic pump power device, switches the opening direction of the first multi-channel valve, that is, the six-way valve, and uses the pump pressure to make different concentration sucrose solutions enter the chip card with the buffer solution from the first fluid transmission hole, flow through the chip, and the waste liquid flows out from the second fluid transmission hole to the waste liquid bottle; and is detected with a narrow spectrum (wavelength range 400nm - 800nm) and a wide spectrum (wavelength range 200nm - 1100nm) respectively.

[0107] 2. Detection results

[0108] Figure 10The full-spectrum diagram for detecting different sucrose concentrations with a narrow spectrum shows that the sensitivity of this chip is very high. Figure 11 The full-spectrum diagram for detecting different sucrose concentrations with a wide spectrum shows that the sensitivity of this chip is very high. Figure 12 The comparison is made between the difference in light intensity at 660 nm and 600 nm in the narrow spectrum and the difference in light intensity at 620 nm and 610 nm in the wide spectrum. The result shows that the narrow-spectrum effect is better. Therefore, a narrow-spectrum detector is selected for detection.

[0109] Example 2

[0110] The equilibrium affinity constant K of Protein A and immunoglobulin G (IgG) D

[0111] Experimental materials:

[0112] Mouse anti-human IgG: Beijing Solarbio Science & Technology Co., Ltd.

[0113] Protein A: Beijing Sino Biological Inc.

[0114] The NanoSPR one component includes a tungsten light source (Hangzhou Saiman Technology Co., Ltd., 220609W132), a spectrometer (Guangzhou Changhui Electronic Technology Co., Ltd., CHS-3000), an optical fiber (Guangzhou Changhui Electronic Technology Co., Ltd., CH-IR-05M), a six-port valve, a three-port valve, a plastic tube, a nano-plasma chip, and a peristaltic pump.

[0115] Adopt the chip card as shown in Figure 5 and Figure 6 The thickness of the card body is 8 mm, the depth of the first groove is 2 mm, the depth of the second groove is 1 mm, the width of the detection chip (the width in the direction perpendicular to the axial direction) is 40 mm, the transparent cover is a hydrophilic acrylic plate with the same thickness as the depth of the first groove and the same size as the internal cross-sectional size of the first groove, which is embedded in the first groove and covers the surface of the second groove to seal the second groove. A convex platform 9 protruding relative to the bottom surface of the second groove 3 is provided on the side of the fluid second transmission hole 6 facing the fluid third transmission hole 7, and a semi-circular bulge 10 is formed on the side wall of the second groove 3 relative to the position of the convex platform 9, so that a U-shaped fluid channel is formed in the second groove 3. When the solution for fixing ligand molecules to the detection chip 5 enters from the fluid first transmission hole 4 and gradually flows through the detection chip 5 and then to the fluid second transmission hole 6, due to the blocking effect of the convex platform 9, experiments prove that it can well block the further diffusion of the sample solution to the side of the reference chip 8, resulting in the contamination of the reference chip 8.

[0116] Use this chip card to build as shown in Figure 8The analysis system shown is used to test the equilibrium affinity constant of Protein A and immunoglobulin G (IgG), and the specific steps are as follows:

[0117] 1. Experimental procedure

[0118] (1) Immerse the NanoSPR chip in 50 mM 3-MPA overnight at 4 °C to carboxylate the chip surface. Then store the carboxylated chip in PBS at 4 °C for subsequent molecular interaction studies. Cut the chip into 1 cm × 0.3 cm and paste it with double-sided tape into Figure 5 and Figure 6 the first and second chip slots in the detection chip area of the chip card shown. Then seal the sensor surface with an acrylic plate. Inject the EDC / NHS (400 mM / 100 mM) mixture into the quantitative loop through the six-port valve. Switch the three-way valve to allow the mixture to enter through the first fluid transfer hole, flow through the detection chip and the reference chip in sequence, activate the carboxyl groups on the chip, and let the activated waste liquid flow out through the third fluid transfer hole into the waste collection bottle. Then inject 100 μL of 20 μg / mL Protein A to enter through the first fluid transfer hole and flow through the detection chip, and flow out through the second fluid transfer hole into the waste collection bottle to fix the ligand Protein A. Then inject 100 μL of 1% casein PBS solution into the quantitative loop, switch the three-way valve to allow the mixture to enter through the first fluid transfer hole, flow through the detection chip and the reference chip, and let the waste liquid flow out through the third fluid transfer hole to block the detection chip and the reference chip and reduce the non-specific adsorption of the chip. Finally, obtain the Protein A chip and store it in a 4 °C refrigerator for later use.

[0119] (2) Use the analysis system shown in Figure 8 for detection. Dilute IgG to 5 different concentrations (0, 1, 3.5, 7, 14, 28, 56 nM) with PBST buffer. Perform reflectance spectroscopy detection on the chip at two wavelengths of 600 nm and 660 nm, with a balanced baseline of 30 s. Inject different concentrations of IgG (0 - 56 nM) from low to high concentrations into the detection system respectively. Switch the direction of the three-way valve to allow the sample to enter through the first fluid transfer hole, flow through the detection chip and the reference chip respectively, and flow out through the third fluid transfer hole into the waste collection bottle. The detection time for each group is 10 min, and the flow rate is 68 μL / min. The regeneration condition between each group is to inject 100 μL of glycine solution (pH = 1.5) into the detection system, switch the direction of the three-way valve, and according to the same flow path setting, let the sample flow through the reference chip and the test chip respectively, with a flow rate of 150 μL / min. After the baseline is stable, set the flow rate to 68 μL / min, and then perform the next group of concentration detections.

[0120] (3) The original data was fitted using Origin 8.0 software to obtain the association rate constant K on and the dissociation rate constant K off The fitting equations are respectively

[0121] and

[0122] Then, according to the formula the equilibrium affinity constant K D of the analyte to be measured was fitted; where [B] is the concentration of the protein to be measured, and a is the asymptote.

[0123] 2. Detection Results

[0124] In this experiment, Protein A was directly immobilized on the chip surface, and the affinity was detected by orientedly binding to the Fc region of IgG. The kinetic interaction between Protein A and IgG was detected by a reflective optical fiber in this experiment, and a typical graph of the detection process is as Figure 13 shown, including equilibrium, binding, dissociation, and regeneration. And the kinetic binding and dissociation detection and simulation processes of the two are as Figure 14 shown, the K D value is 0.25 nM, and this experimental result is consistent with the previous literature reports (Yang D, Singh A, Wu H, et al. Comparison of biosensor platforms in the evaluation of high affinity antibody - antigen binding kinetics[J]. Analytical biochemistry, 2016, 508:78 - 96.). It shows that the nanoplasmonic resonance spectrometer of the present invention can be applied to the Protein A universal chip, and the results are reliable, accurate, and usable. Figure 15 For the linear detection of IgG using the Protein A chip, its R 2 is 0.99, indicating good linearity.

[0125] Example 3

[0126] Test the equilibrium affinity constant K D

[0127] Experimental Materials:

[0128] Mouse anti - human IgG: Beijing Solarbio Science & Technology Co., Ltd.;

[0129] FcRn Beijing Proteintech Group Co., Ltd.; The NanoSPR one component includes a tungsten light source (Hangzhou Saiman Technology Co., Ltd., 220609W132), a spectrometer (Guangzhou Changhui Electronic Technology Co., Ltd., CHS-3000), an optical fiber (Guangzhou Changhui Electronic Technology Co., Ltd., CH-IR-05M), a six-port valve, a three-port valve, a plastic tube, a nano-plasma chip, and a peristaltic pump.

[0130] The chip card used is the same as that in Example 2. Using this chip card, an analysis system as shown in Figure 9 is built. The specific analysis process includes the following steps:

[0131] 1. Experimental process

[0132] (1) Immerse the NanoSPR chip in 50 mM 3-MPA at 4 °C overnight to carboxylate the chip surface. Then store the carboxylated chip in PBS at 4 °C for subsequent molecular interaction studies or paste the chip on the detection chip area of the chip card cut to 1 cm × 0.3 cm and pasted with double-sided tape to Figure 6 as shown. Then seal the sensor surface with an acrylic board. Inject the EDC / NHS (400 mM / 100 mM) mixture into the six-port valve metering loop. Switch the three-port valve to allow the mixture to enter from the third fluid transfer hole and flow through the reference chip and the detection chip in sequence to activate the carboxyl groups on the chip. The activated waste liquid flows out from the first fluid transfer hole into the waste liquid collection bottle. Then inject 100 μL of 20 μg / mL FcRn into the second fluid transfer hole to flow through the detection chip for the immobilization of the ligand FcRn. The immobilization waste liquid flows out from the first fluid transfer hole. Then inject 100 μL of 1% casein PBS solution into the metering loop. Switch the three-port valve to allow the mixture to enter from the third fluid transfer hole and flow through the reference chip and the detection chip in sequence. The waste liquid flows out from the first fluid transfer hole to block the detection chip and the reference chip and reduce the non-specific adsorption of the chip. Finally, obtain the Protein A chip and store it in a 4 °C refrigerator for later use.

[0133] (2) Use as shown in Figure 9The analysis system shown was used for detection. IgG was diluted to different concentrations (0, 47, 94, 188, 376, 752 nM) with PBST buffer. The chip was detected for reflection spectrum using dual wavelengths of 600 nm and 660 nm, and the baseline was balanced for 30 s. IgG at different concentrations (0 - 752 nM) was injected into the detection system from low concentration to high concentration. The direction of the three-way valve was switched to allow the sample to enter from the third fluid transfer hole, and then it flowed through the reference chip and the test chip in sequence. The detection time for each group was 10 min, and the flow rate was 68 μL / min. The regeneration condition between each group was to inject 100 μL of PBS solution (pH = 8) into the detection system, switch the direction of the three-way valve to allow the sample to flow through the reference chip and the test chip respectively, with a flow rate of 150 μL / min. After the baseline was stable, the flow rate was set to 68 μL / min, and then the next group of concentration detection was carried out.

[0134] (3) The origin 8.0 software was used to fit the original data to obtain the fitting equations for the affinity constant (Ka) and dissociation constant (Kd), which were respectively

[0135]

[0136] Then, according to the formula the equilibrium affinity constant K of the protein to be detected was obtained by fitting D the fitting equation;

[0137] wherein, K on is the association rate constant, K off is the dissociation rate constant; [B] is the concentration of the protein to be detected; a is the asymptote.

[0138] 2. Detection results

[0139] The kinetic binding process between FcRn and IgG is as Figure 16 shown, the K D value is 68.4 nM, which is consistent with the result given by the merchant. It shows that the nano - plasmon resonance spectrometer of the present invention can be applied to the rapid binding and dissociation detection similar to FcRn and Herceptin, and the results are reliable, accurate and usable.

[0140] It is easy for those skilled in the art to understand that the above - mentioned is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A chip card for molecular interaction analysis, characterized in that, It includes a card body, a first groove formed on the surface of the card body, and a second groove formed at the bottom of the first groove; A fluid first transmission hole, a detection chip, and a fluid second transmission hole are axially arranged in the second groove; both the fluid first transmission hole and the fluid second transmission hole are through holes passing through the card body, and the hole surfaces of the fluid first transmission hole and the fluid second transmission hole are flush with the bottom surface of the second groove; A transparent cover body is embedded in the first groove, and the transparent cover body covers the surface of the second groove to make the second groove a closed groove; A fluid third transmission hole is further arranged on the extension line of the detection chip and the fluid second transmission hole, a reference chip is arranged between the fluid third transmission hole and the fluid second transmission hole, the fluid third transmission hole is a through hole passing through the card body, and the hole surface of the fluid third transmission hole is flush with the bottom surface of the second groove; A convex platform protruding relative to the bottom surface of the second groove is arranged on one side of the fluid second transmission hole facing the fluid third transmission hole, and a semi-circular bulge is formed on the side wall of the second groove relative to the position of the convex platform, so that a U-shaped fluid channel is formed in the second groove; When the solution for fixing ligand molecules to the detection chip enters from the fluid first transmission hole and gradually flows through the detection chip and then to the fluid second transmission hole, due to the blocking effect of the convex platform, it can block the further diffusion of the sample solution to the side of the reference chip, resulting in the contamination of the reference chip.

2. A molecular interaction analysis system based on the chip card as described in claim 1, characterized in that, It includes an optical path unit, a liquid path unit, the chip card as claimed in claim 1, and a control unit; wherein: The optical path unit is used to provide the light required for molecular interaction analysis for the chip card, and it includes a light source, an optical fiber, and a detector; The liquid path unit is used to provide a fluid path required for molecular interaction analysis for the chip card. A fluid flow power device, a liquid pipeline, and a waste liquid collection device are arranged on the fluid path; the sample is transported to the liquid pipeline through the fluid flow power device, flows through the chip on the chip card, and realizes the detection of molecular interaction analysis under the light provided by the optical path unit, and the waste liquid flows into the waste liquid collection device for collection; the chip on the chip card is a nano-columnar array plasmon resonance chip sensor; The control unit is a computer, which controls the detector and the optical fiber through a data transmission line. The light emitted by the light source enters the chip in the chip card through the optical fiber, and plasmon resonance occurs at the chip. The obtained feedback signal is collected by the detector and then transmitted to the computer, and the computer analyzes and processes the collected signal.

3. The molecular interaction analysis system according to claim 2, wherein In the chip card, a fluid first transfer hole, a detection chip, a fluid second transfer hole, a reference chip, and a fluid third transfer hole are axially arranged in the second slot; the fluid second transfer hole is the first sample inlet hole, the fluid third transfer hole is the second sample inlet hole, and the fluid first transfer hole is the waste liquid outlet hole. A buffer solution storage device, a quantification component, a first multi-channel valve, a second multi-channel valve, the chip card, a fluid flow power device, and a waste liquid collection device are sequentially arranged on the fluid path; wherein the first multi-channel valve is connected to the waste liquid outlet hole, and both the first sample inlet hole and the second sample inlet hole are connected to the second multi-channel valve; during use, by switching the opening direction of the second multi-channel valve, the sample is made to enter from the first sample inlet hole or the second sample inlet hole by the power provided by the fluid flow power device.

4. A method for performing molecular interaction analysis using the analysis system according to claim 3, characterized in that, It includes the following steps: (1) Inject the activation solution for activating the carboxylated chip into the quantification component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the activation solution enters the chip card from the fluid third transfer hole along with the buffer solution and sequentially flows through the carboxylated reference chip and the carboxylated detection chip to activate the carboxylated reference chip and the carboxylated detection chip; switch the opening direction of the second multi-channel valve, and use the power provided by the fluid flow power device to make the activation waste liquid flow out from the fluid first transfer hole to the waste liquid collection device to obtain the activated carboxylated detection chip and the activated carboxylated reference chip; (2) Inject the first molecular sample into the quantification component through the injection port of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the first molecule enters the chip card from the fluid second transfer hole along with the buffer solution and flows through the activated carboxylated detection chip to fix the first molecule on the detection chip; use the power provided by the fluid flow power device to make the waste liquid generated during the fixing process flow out from the fluid first transfer hole to the waste liquid collection device; (3) Inject the second molecular sample into the quantification component through the sample inlet of the first multi-channel valve, switch the opening direction of the first multi-channel valve, so that the second molecule enters the chip card from the fluid third transfer hole along with the buffer solution and sequentially flows through the activated carboxylated reference chip and the detection chip fixed with the first molecule; under the illumination provided by the optical path unit, make the second molecule interact with the first molecule fixed on the detection chip; use the power provided by the fluid flow power device to make the detection waste liquid flow out from the fluid first transfer hole to the waste liquid collection device.

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