CN106872425B - Modularized biological sample analysis chip - Google Patents
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- CN106872425B CN106872425B CN201710023509.1A CN201710023509A CN106872425B CN 106872425 B CN106872425 B CN 106872425B CN 201710023509 A CN201710023509 A CN 201710023509A CN 106872425 B CN106872425 B CN 106872425B
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention discloses a modularized biological sample analysis chip, and belongs to the technical field of biological detection. The modularized biological sample analysis chip comprises a substrate material for supporting, fixing and integrating an analysis module, wherein the analysis module is integrated on the substrate, and biomolecules such as protein, nucleic acid, polypeptide and the like or organic molecules can be coated on the surface of the analysis module to be used as capture molecules; the surface of the analysis chip further comprises at least one blocking component for avoiding nonspecific adsorption of proteins or other organisms and organic molecules, the hydrophilic substrate can be a substrate material which is hydrophilic or hydrophobic, and is modified into hydrophilic property through hydrophilic or blocking agent treatment, and the hydrophilic property and the blocking process can reduce nonspecific binding and interference. The modularized biological sample analysis chip solves the problems that the prior biological sample analysis chip has high requirement on a sample counter, low sample counting precision and non-independent reaction system.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a modularized biological sample analysis chip.
Background
In the 21 st century, the biochip technology has been developed at a high speed, and the detection of compounds, proteins, nucleic acids, cells or other biological components by using microarray biomolecule chips manufactured on materials such as silicon wafers, glass, gel or nylon membranes by automatic printing or light-guided chemical synthesis technology has been widely reported.
The preparation technology of the present biochip mainly couples or coats molecules such as protein and the like on a substrate for detection, patent 200710134477.9 discloses a covalently coupled coated chip, patent US20040009528a1 discloses a covalently coated chip with thiol groups, and these methods are directly coated on the substrate of the chip, and the coating amount of the antibody is generally configured into a solution by a spotting machine in the detection process, and spotting is carried out on the chip for coating, patent 01105795.5 discloses a method for preparing the biochip by using a high-speed spotting method, but the spotting method has extremely high requirements on the machine, even the best machine has the condition of failure or sample application failure, and the condition is difficult to detect, and an independent coupling reaction system is arranged between each droplet and the substrate, patent 200510013108.5 discloses a method for processing the substrate by using photolithography technology, the technology is improved to a certain extent by reserving an effective reaction area, but the problems of sample application precision and independent reaction system cannot be solved, and each sample point is a coating system theoretically, so that the application potential of the biochip in quantitative detection is limited in principle, and the biochip is a main application pain point at present.
Disclosure of Invention
The embodiment of the invention provides a modularized biological sample analysis chip. The invention changes the traditional sample application thought, invents a modularized high-precision analysis chip and aims to solve the problems that the existing biological sample analysis chip has high requirement on a sample application machine, low sample application precision and non-independent reaction system.
The embodiment of the invention is realized in such a way that the modularized biological sample analysis chip comprises a substrate material with a hydrophilic surface and an analysis module; the substrate material with the hydrophilic surface is used for supporting, fixing and integrating the analysis module; the analysis module is assembled on the substrate material; the surface of the analysis module is coated with capture molecules; the surface of the analysis module and the surface of the substrate material after the analysis module is assembled at least comprise a closed component, and the closed component is used for avoiding the nonspecific adsorption of proteins or other organisms and organic molecules; the analysis module is assembled on the substrate material after coating.
The hydrophilic substrate can be a substrate material which is modified by a hydrophilic or hydrophobic material and then becomes hydrophilic or becomes hydrophilic after being treated by a reagent or a blocking agent, and the non-specific binding and interference can be reduced by both the hydrophilic property and the blocking process.
The substrate material is one of metal material, glass material, silicon material or macromolecule plastic material.
The hydrophilic surface is a hydrophilic surface which is changed into hydrophilic property by the modification of a substrate material and a hydrophobic material or the treatment of a sealant.
The capture molecules are used for capturing the substance to be analyzed in the biological sample, and the capture molecules are selected from protein, glycoprotein, polypeptide, nucleic acid, organic small molecule, polysaccharide or antibody biological molecules.
The analysis module is selected from materials with the surface modified by carboxyl, amino, chloromethyl, epoxy alkyl, biotin, aldehyde group or sulfhydryl and the capability of coupling with biological molecules; a metal material having the ability to couple biomolecules; and one of polystyrene, nylon or cellulose acetate materials with the surface having biomolecule adsorption capacity.
The blocking component is biological or organic molecules with hydrophobic binding property, such as bovine serum albumin, tween, skimmed milk powder, casein, amino acid or xylitol.
The modular biological sample analysis chip is further assembled as a module on a larger substrate to form a new chip.
The modularized biological sample analysis chip also comprises a reference module for internal reference, wherein the reference module is directly marked with an object to be detected or an analogue of the object to be detected and is used for referencing the chip and a detection signal.
A biological sample analysis method using the modularized biological sample analysis chip comprises the following steps:
(1) coating the capture molecules on a material A with adsorption capacity or covalent coupling capacity or cutting the material into small modules for coating;
(2) cutting the cut material A or cutting the material A and then assembling the cut material A on a substrate material B, or assembling the material A on the substrate material B and cutting the material A to form an analysis chip C, wherein the analysis chip C can be further cut to generate a plurality of chips or a plurality of analysis chips C are further assembled on a new analysis chip formed on the substrate;
(3) the analysis chip C is sealed by a reagent containing a sealing component;
(4) contacting the analysis chip C with a molecular substance to be detected;
(5) cleaning the analysis chip C;
(6) the analysis chip C is contacted with a molecular reagent containing a label;
(7) if the labeled molecules in the step (6) are fluorescent molecules or quantum dots, exciting by a light source, and detecting by using a signal acquisition device; if the labeled molecules in the step (6) are enzymes or direct luminescent molecules, the analysis chip is further contacted with a reaction reagent to form luminescent signals, and the luminescent signals are detected by using a signal acquisition device.
The signal acquisition device may be selected from the group consisting of a CCD (charge coupled device), a PMT (photomultiplier tube), a PD (photoelectric cell), preferably a CCD.
A kit comprising
(1) The above-described modular biological sample analysis chip;
(2) a cleaning reagent;
(3) a reagent containing a labeled component capable of binding to the test substance.
(4) If the label in (3) is an enzyme or a direct light-emitting molecule, the kit further comprises a light-emitting reagent.
Compared with the prior art, the invention has the following advantages:
the invention changes the traditional sample application thought, invents a modularized high-precision analysis chip and aims to solve the problems that the existing biological sample analysis chip has high requirement on a sample application machine, low sample application precision and non-independent reaction system. The modularized biological sample analysis chip comprises a substrate material for supporting, fixing and integrating an analysis module, wherein the analysis module is integrated on the substrate, and biomolecules such as protein, nucleic acid, polypeptide and the like or organic molecules can be coated on the surface of the analysis module to be used as capture molecules; the surface of the analysis chip further comprises at least one blocking component for avoiding nonspecific adsorption of proteins or other organisms and organic molecules, the hydrophilic substrate can be a substrate material which is hydrophilic or hydrophobic, and is modified into hydrophilic property through hydrophilic or blocking agent treatment, and the hydrophilic property and the blocking process can reduce nonspecific binding and interference.
Drawings
Fig. 1 is an exemplary process of coating, cutting, assembling and sealing a modular biological sample analysis chip, wherein (a) the chip is coated and then cut and assembled, (b) the chip is cut and then coated and assembled, (c) the chip is cut during the assembly, and (d) the chip is cut and then assembled after the assembly.
Fig. 2 is a schematic diagram of a modular biological sample analysis chip.
FIG. 3 is a schematic view of the assembly of an analysis module with a substrate material; wherein FIG. (a) shows an analysis module assembled on a substrate having a recess; and (b) covering the surface of the adjustment analysis module by using the cover plate to be flush with the surface of the substrate.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The invention provides a modularized biological sample analysis chip, which comprises a substrate material with a hydrophilic surface and an analysis module; the substrate material with the hydrophilic surface is used for supporting, fixing and integrating the analysis module; the analysis module is assembled on the substrate material; the surface of the analysis module is coated with capture molecules; the surface of the analysis module and the surface of the substrate material after the analysis module is assembled at least comprise a closed component, and the closed component is used for avoiding the nonspecific adsorption of proteins or other organisms and organic molecules; the analysis module is assembled on the substrate material after coating.
The hydrophilic substrate can be a substrate material which is modified by a hydrophilic or hydrophobic material and then becomes hydrophilic or becomes hydrophilic after being treated by a reagent or a blocking agent, and the non-specific binding and interference can be reduced by both the hydrophilic property and the blocking process.
The substrate material is one of metal material, glass material, silicon material or macromolecule plastic material.
The hydrophilic surface is a hydrophilic surface which is changed into hydrophilic property by the modification of a substrate material and a hydrophobic material or the treatment of a sealant.
The capture molecules are used for capturing the substance to be analyzed in the biological sample, and the capture molecules are selected from protein, glycoprotein, polypeptide, nucleic acid, organic small molecule, polysaccharide or antibody biological molecules.
The analysis module is selected from materials with the surface modified by carboxyl, amino, chloromethyl, epoxy alkyl, biotin, aldehyde group or sulfhydryl and the capability of coupling with biological molecules; a metal material having the ability to couple biomolecules; and one of polystyrene, nylon or cellulose acetate materials with the surface having biomolecule adsorption capacity.
The blocking molecule is biological or organic molecule with hydrophobic binding property, such as bovine serum albumin, tween, skimmed milk powder, casein, amino acid or xylitol.
The modular biological sample analysis chip is further assembled as a module on a larger substrate to form a new chip.
The modularized biological sample analysis chip also comprises a reference module for internal reference, wherein the reference module is directly marked with an object to be detected or an analogue of the object to be detected and is used for referencing the chip and a detection signal.
A biological sample analysis method using the modularized biological sample analysis chip comprises the following steps:
(1) coating the capture molecules on a material A with adsorption capacity or covalent coupling capacity or cutting the material into small modules for coating;
(2) cutting the cut material A or cutting the material A and then assembling the cut material A on a substrate material B, or assembling the material A on the substrate material B and cutting the material A to form an analysis chip C, wherein the analysis chip C can be further cut to generate a plurality of chips or a plurality of analysis chips C are further assembled on a new analysis chip formed on the substrate;
(3) the analysis chip C is sealed by a reagent containing a sealing component;
(4) contacting the analysis chip C with a molecular substance to be detected;
(5) cleaning the analysis chip C;
(6) the analysis chip C is contacted with a molecular reagent containing a label;
(7) if the labeled molecules in the step (6) are fluorescent molecules or quantum dots, exciting by a light source, and detecting by using a signal acquisition device; if the labeled molecules in the step (6) are enzymes or direct luminescent molecules, the analysis chip is further contacted with a reaction reagent to form luminescent signals, and the luminescent signals are detected by using a signal acquisition device.
The signal acquisition device may be selected from the group consisting of a CCD (charge coupled device), a PMT (photomultiplier tube), a PD (photoelectric cell), preferably a CCD.
A kit comprising
(1) The above-described modular biological sample analysis chip;
(2) a cleaning reagent;
(3) a reagent containing a labeled component capable of binding to the test substance.
(4) If the label in (3) is an enzyme or a direct light-emitting molecule, the kit further comprises a light-emitting reagent.
A preparation method of a modularized biological sample analysis chip comprises the following specific steps:
firstly, coating the capture molecules on a material with adsorption capacity or covalent coupling capacity, wherein the material can be used in a relatively large area, cutting and assembling the material on a substrate material to form an analysis chip after coating, and further cutting the analysis chip to generate a plurality of chip modules or further assembling and assembling the chip modules on the substrate after cutting (including but not limited to the assembling process shown in (d) in fig. 1); or cutting before coating, and assembling on the substrate; or further cutting after the substrate is assembled after coating; including but not limited to the chip fabrication process shown in fig. 1 (a), (b), (c), (d).
As shown in FIG. 2, the modular chip of the present invention is assembled by a substrate and an analysis module, wherein the substrate includes but is not limited to metal, glass, nylon, silicon, polymer plastic, etc. with support material, a plurality of analysis modules can be assembled on the substrate, or a plurality of modules composed of analysis modules and substrate can be assembled as shown in FIG. 1 (d), the surface of the analysis module is coated with biomolecules, including but not limited to protein, antibody, polypeptide, amino acid, saccharide, enzyme, nucleic acid, etc. and organic molecules, the surface of the chip further includes a blocking molecule for blocking non-specific binding sites on the surface of the chip, the blocking molecule includes but not limited to BSA, amino acid, sugar, milk, casein, surfactant, polyethylene glycol, sugar alcohol, etc. with hydrophobic binding properties and organic molecules, the analytical modules of different chips are generated under the same reaction conditions, and the substrate can be made into a concave structure to place the chip thereon and keep the surface flat as shown in FIG. 3.
The method for analyzing a biological sample of the present invention comprises: (1) coating the capture molecules on a material A with adsorption capacity or covalent coupling capacity or cutting the material into small modules for coating; (2) cutting the cut material A or cutting the material A and then assembling the cut material A on a substrate material B, or assembling the material A on the substrate B for cutting to form an analysis chip C, wherein the analysis chip C can be further cut to generate a plurality of analysis chips, and the cut chip C can be further assembled on a new analysis chip formed on the substrate; (2) the analysis chip C is sealed by a reagent containing a sealing component; (3) contacting the analysis chip C with a molecular substance to be detected; (4) cleaning the analysis chip C; (5) the analysis chip C is contacted with a molecular reagent containing a label; (6) if the labeled molecules in the step 6 are fluorescent molecules or quantum dots, exciting by using a light source, and detecting by using a fluorescence detector; (7) if the labeled molecules in the step 6 are enzymes or direct luminescent molecules, the analysis chip is further contacted with a reaction reagent to form luminescent signals, and the luminescent signals are detected by a luminescent signal detector; the analysis chip can be sealed by assembling the substrate and the analysis module together after being sealed separately, or by assembling the analysis module and the substrate together, and the labeled molecules include but are not limited to fluorescent molecules, quantum dots, enzymes capable of catalyzing the substrate to emit light, and molecules capable of directly triggering the light emission through the reaction. In the detection process of the analysis signal, in the method of the invention, if the marker of the labeled molecule is a fluorescent molecule or a quantum dot, a light source is needed for excitation, and then a CCD (charge coupled device), PMT (photomultiplier tube) or PD (photoelectric tube) for signal acquisition is used for signal acquisition, if the labeled molecule is a molecule capable of triggering chemiluminescence, a luminous triggering reaction is firstly carried out, for example, horseradish peroxidase labeling needs to react with a luminol substrate reagent to trigger a chemiluminescence signal, and a chip substrate of the invention can also be integrated with an electrode circuit for detecting an electric signal generated by the reaction on the chip.
The chip of the invention can be used for preparing a kit, the kit comprises a chip, the chip comprises a substrate and an integrated analysis module, the analysis module is coated with capture molecules, the chip comprises blocking molecules which can effectively reduce nonspecific binding, the kit comprises a cleaning reagent, the cleaning reagent at least comprises a buffer salt and an antiseptic component which are used for cleaning the nonspecific binding molecules of the chip and luminous substrate molecules, the kit comprises a labeling reagent, labeling molecules in the reagent can be combined with the capture molecules, and the capture molecules, the capture molecules and the labeling molecules form a complex. If the labeling molecule is a fluorescent molecule or a quantum dot molecule, detection can be directly performed by optical excitation and CCD or PMT or PD (photoelectric cell). If the marker of the labeled molecule is an enzyme, such as horseradish peroxidase or alkaline phosphatase, the kit further comprises a substrate reagent, the substrate reagent contains a luminescent substrate, and if the marker of the labeled molecule is a direct luminescent molecule, such as acridinium ester, acridinium sulfonamide, acridinium amide or analogues thereof, the substrate reagent contains a reaction reagent which reacts with the direct luminescent molecule to form a luminescent signal, and the signal detection is carried out by CCD or PMT.
The application example is as follows:
reagent 1:
Tris10mL0.01M/L；HCL 0.0003N/L；
adjusting pH to 8.5, filtering with 0.22 μm filter
Reagent 2:
imidazole 2.72 g/L; tween 0.2 mL/l; 8.76g/L of NaCL; pH to 8.5,0.22 μm, and filtering with a filter
Reagent 3:
0.58g/L Na2HPO4,0.05g/L KCl,0.05g/L K3PO4,2.0g/L NaCl, 10g/L BSA, TWEEN4 mL/L; filtering with a 0.22 μm filter at pH 7.4
Reagent 4:
Mab PCT 16B5 Cat.#4PC47 hytest 1mg/mL
sodium azide 0.1%
And (5) reagent:
Mab PCT 42 Cat.#4C10 hytest 2mg/mL
sodium azide 0.1%
Reagent 6:
PCT recombinant protein Cat. #8PC 550 ng/mL
Sodium azide 0.1%
And (7) reagent:
Mab CRP C2 Cat.#4C28 hytest 1mg/mL
sodium azide 0.1%
Reagent 8:
Mab CRP C6 Cat.#4C28 hytest 2mg/mL
sodium azide 0.1%
Reagent 9
CRP antigen Cat. #8C72 hystest 20ug/mL
Sodium azide 0.1%
Reagent 10
HNO30.15M；H2O20.7％，Triton-100 0.25％；NaOH 0.125M；
Reagent 11
9- (4-chlorophenyl-thiobenzoyl oxymethylene) -10-methyl-9, 10-acridan-disodium salt 100mg/L, N, N dimethylacridine nitrate, 1.0mg/L sodium dodecyl sulfate, 0.8g/L sodium sulfite, 5mg/L TWEEN-20, 0.31g/L0.26mol/L of Tris buffer system pH 9.10.
Application example 1
Washing and drying a polystyrene plastic plate with the size of 100mm multiplied by 1mm by using a reagent 1, placing the polystyrene plastic plate in a plate, diluting the protein concentration of the reagent 4 to 20ug/mL, adding the polystyrene plastic plate into the plate, immersing the polystyrene plastic plate in the solution, and standing the solution at room temperature for 2 hours and 4 ℃ overnight; the plates were washed with reagents and air dried under clean conditions. Precisely cutting a small block (PCT analysis module) of 5mm multiplied by 5mm by laser;
washing and drying a polystyrene plastic plate with the size of 100mm multiplied by 1mm by using a reagent 1, placing the polystyrene plastic plate in a plate, diluting the protein concentration of the reagent 7 to 15ug/mL, adding the polystyrene plastic plate into the plate, immersing the polystyrene plastic plate in the solution, and standing the solution at room temperature for 2 hours and 4 ℃ overnight; the plates were washed with reagents and air dried under clean conditions. Precisely cutting a small block (CRP analysis module) with the size of 5mm multiplied by 5mm by laser;
cutting the glass plate into small blocks of 1cm multiplied by 3cm multiplied by 2mm as a substrate; adhering a PCT analysis module and a CRP analysis module on each substrate to form an analysis chip;
placing the analysis chip in a plate, injecting a reagent 3 into the plate, sealing, and taking out the analysis chip at room temperature for 3 hours;
adding reagent 580ul into 600ul reagent 1, then adding 150ul of 0.5mmol/l acridinium ester, mixing, keeping out of the sun for 20 minutes at room temperature, adding lysine and 180ul reagent 1, and standing for 30 minutes to obtain the acridinium ester label. The PCT-labeling reagent is used for labeling a cell,
adding 870ul of reagent into 600ul of reagent 1, then adding 150ul of 0.5mmol/l acridinium ester, mixing, keeping the mixture at room temperature in a dark place for 20 minutes, adding lysine and 180ul of reagent 1, and standing for 30 minutes to obtain the acridinium ester label. A CRP-labeling agent;
the PCT-labeling reagent and the CRP labeling reagent were mixed according to a 1:1 proportion to obtain a PCT-CRP labeling reagent;
reagent 6 was diluted to PCT concentration and reagent 9 was diluted to configuration:
standard solution 1: PCT 0.1ng/mL CRP 0.5 ug/mL; standard solution 2: PCT 0.5ng/mL CRP 1 ug/mL; standard solution 3: PCT 1ng/mL CRP 2 ug/mL; standard solution 4: PCT 10ng/mL CRP 10 ug/mL; standard solution 5: PCT 30ng/mL CRP 20 ug/mL;
the test procedure was as follows, with the assay chip immersed in the standard solution (as a sample), incubated for 5 minutes at room temperature, washed twice with reagent 2, then immersed in the PCT-CRP labeling reagent for incubation for 5 minutes, washed twice with washing reagent 2, and placed in reagent 10 while the CCD detects the luminescent signal.
After a standard luminescence signal curve is established through standard reagent detection, a reagent 6 and a reagent 9 are added into serum, and the standard luminescence signal curve is obtained through weighing method configuration, wherein a sample 1: CRP 2ug/MLPCT 0.5ng/mL, sample 2: CRP 5ug/MLPCT5ng/mL, sample 3: CRP 10ug/MLPCT 30ng/mL, the above procedure was repeated to test the samples with the analytical chip, and the test results are shown in Table 1:
TABLE 1
| CRP(ug/ml) | Configured concentration | Chip 1 | Chip 2 | Chip 3 | Chip 4 | Chip 5 | Chip 6 | Chip 7 | Chip 8 | CV | Mean value |
| Sample 1 | 2 | 2.10 | 2.10 | 2.04 | 2.10 | 2.20 | 1.90 | 2.20 | 2.20 | 4.86％ | 2.11 |
| Sample 2 | 5 | 5.15 | 5.15 | 5.15 | 5.15 | 4.85 | 4.85 | 4.85 | 5.15 | 3.08％ | 5.04 |
| Sample 3 | 10 | 10.80 | 10.40 | 9.40 | 9.60 | 9.60 | 9.80 | 9.90 | 10.40 | 4.92％ | 9.99 |
| PCT(ng/ml) | Configured concentration | Chip 1 | Chip 2 | Chip 3 | Chip 4 | Chip 5 | Chip 6 | Chip 7 | Chip 8 | CV | Mean value |
| Sample 1 | 0.5 | 0.51 | 0.48 | 0.52 | 0.52 | 0.53 | 0.56 | 0.53 | 0.48 | 5.45％ | 0.51 |
| Sample 2 | 5 | 5.15 | 5.15 | 5.15 | 5.00 | 5.15 | 4.90 | 5.15 | 5.80 | 5.16％ | 5.18 |
| Sample 3 | 30 | 30.90 | 29.10 | 29.10 | 30.90 | 29.10 | 29.10 | 29.10 | 29.10 | 2.82％ | 29.55 |
Application example 2
Washing an epoxy alkyl modified chip (Telechemistry national CAT: SME225 × 76mm) by using a reagent 1, placing the chip in a plate, diluting a reagent 4 protein concentration to 20ug/mL, adding the diluted reagent into the plate, immersing a polystyrene plate in the diluted reagent, and standing the polystyrene plate at room temperature for 2 hours and at 4 ℃ overnight; the plates were washed with reagents and air dried under clean conditions. Precisely cutting a small block (PCT analysis module) of 5mm multiplied by 5mm by laser;
washing an epoxy alkyl modified chip (Telechemistry national CAT: SME225 × 76mm) by using a reagent 1, placing the chip in a plate, diluting the concentration of a reagent 7 protein to 15ug/mL, adding the diluted solution into the plate, immersing a polystyrene plate in the diluted solution, and standing the solution at room temperature for 2 hours and at 4 ℃ overnight; the plates were washed with reagents and air dried under clean conditions. Precisely cutting a small block (CRP analysis module) with the size of 5mm multiplied by 5mm by laser;
cutting the glass plate into small blocks of 1cm multiplied by 3cm multiplied by 2mm as a substrate; adhering a PCT analysis module and a CRP analysis module on each substrate to form an analysis chip;
taking 5, 300uL of reagent, adding 14uL of Sulfo-NHS-LC-Biotinylation Kit (THERMO), incubating at 37 ℃ for 39 minutes, adding into an ultrafiltration tube, centrifuging at 12000Xg for 10 minutes, mixing by a pipette, centrifuging for 10 minutes, adding 0.3mL of labeling buffer solution, and centrifuging at 6000Xg for 10 minutes. The solution in the centrifuge tube was collected, supplemented with labeling buffer to 0.5mL, and 0.4mL Streptavidin-labeled alkaline phosphatase (THERMO AP Streptavidin 434322) was added and incubated for 30 minutes at room temperature. The cross-linked material was purified by Sephadex g200 to obtain PCT-labeled reagent.
Taking 8, 500uL of reagent, adding 14uL of Sulfo-NHS-LC-Biotinylation Kit (THERMO), incubating for 39 minutes at 37 ℃, adding into an ultrafiltration tube, centrifuging for 10 minutes at 12000Xg, uniformly mixing by a pipette, centrifuging for 10 minutes, adding 0.3mL of labeling buffer solution, and centrifuging for 10 minutes at 6000 Xg. The solution in the centrifuge tube was collected, supplemented with labeling buffer to 0.5mL, and 0.6mL Streptavidin-labeled alkaline phosphatase (THERMO AP Streptavidin 434322) was added and incubated for 30 minutes at room temperature. The cross-linked material was purified by Sephadex g200 to obtain the CRP-labeling reagent.
Mixing a PCT labeling reagent and a CRP labeling reagent in a ratio of 1: 1;
placing the analysis chip in a plate, injecting a reagent 3 into the plate, sealing, and taking out the analysis chip at room temperature for 3 hours;
reagent 6 was diluted to PCT concentration and reagent 9 was diluted to configuration:
standard solution 1: CRP 0.5 ug/mL; standard solution 2: PCT 0.5ng/mL CRP 1 ug/mL; standard solution 3: PCT 1ng/mL CRP 2 ug/mL; standard solution 4: PCT 10ng/mL CRP 10 ug/mL; standard solution 5: PCT 30ng/mLCRP 20 ug/mL;
the test procedure was as follows, with the assay chip immersed in the standard solution (as a sample), incubated for 5 minutes at room temperature, washed twice with reagent 2, then immersed in the PCT-CRP labeling reagent for incubation for 5 minutes, washed twice with washing reagent 2, and placed in reagent 11 while the CCD detects the luminescent signal.
After a standard luminescence signal curve is established through standard reagent detection, a reagent 6 and a reagent 9 are added into serum, and the standard luminescence signal curve is obtained through weighing method configuration, wherein a sample 1: CRP 2ug/MLPCT 0.5ng/mL, sample 2: CRP 5ug/mLPCT5ng/mL, sample 3: CRP 10ug/MLPCT 30ng/mL, the above procedure was repeated to test the samples with the analytical chip, and the test results are shown in Table 2:
TABLE 2
| CRP(ug/ml) | Configured concentration | Chip 1 | Chip 2 | Chip 3 | Chip 4 | Chip 5 | Chip 6 | Chip 7 | Chip 8 | CV | Mean value |
| Sample 1 | 2 | 1.92 | 1.92 | 1.92 | 2.08 | 2.08 | 2.08 | 2.08 | 1.92 | 4.28％ | 2.00 |
| Sample 2 | 5 | 4.85 | 5.15 | 4.85 | 5.15 | 4.85 | 4.85 | 4.85 | 5.15 | 3.13％ | 4.96 |
| Sample 3 | 10 | 10.25 | 9.75 | 9.75 | 9.75 | 9.75 | 10.25 | 9.75 | 9.75 | 2.34％ | 9.88 |
| PCT(ng/ml) | Configured concentration | Chip 1 | Chip 2 | Chip 3 | Chip 4 | Chip 5 | Chip 6 | Chip 7 | Chip 8 | CV | Mean value |
| Sample 1 | 0.5 | 0.53 | 0.48 | 0.48 | 0.53 | 0.53 | 0.48 | 0.53 | 0.48 | 5.35％ | 0.50 |
| Sample 2 | 5 | 5.15 | 4.85 | 5.15 | 5.15 | 5.15 | 5.15 | 5.15 | 4.85 | 2.74％ | 5.08 |
| Sample 3 | 30 | 30.81 | 30.81 | 30.81 | 30.81 | 30.81 | 29.19 | 29.19 | 29.19 | 2.78％ | 30.20 |
Application example 3
Washing an epoxy alkyl modified chip (Telechemistry national CAT: SME225 × 76mm) by using a reagent 1, placing the chip in a plate, diluting a reagent 4 protein concentration to 20ug/mL, adding the diluted reagent into the plate, immersing a polystyrene plate in the diluted reagent, and standing the polystyrene plate at room temperature for 2 hours and at 4 ℃ overnight; the plates were washed with reagents and air dried under clean conditions. Precisely cutting a small block (PCT analysis module) of 5mm multiplied by 5mm by laser;
washing an epoxy alkyl modified chip (Telechemistry national CAT: SME225 × 76mm) by using a reagent 1, placing the chip in a plate, diluting the concentration of a reagent 7 protein to 10ug/mL, adding the diluted solution into the plate, immersing a polystyrene plate in the diluted solution, and standing the solution at room temperature for 2 hours and at 4 ℃ overnight; the plates were washed with reagents and air dried under clean conditions. Precisely cutting a small block (CRP analysis module) with the size of 5mm multiplied by 5mm by laser;
cutting the glass plate into small blocks of 1cm multiplied by 3cm multiplied by 2mm as a substrate; adhering a PCT analysis module and a CRP analysis module on each substrate to form an analysis chip;
placing the analysis chip in a plate, injecting a reagent 3 into the plate, sealing, and taking out the analysis chip at room temperature for 3 hours;
washing fluorescent latex microspheres with carboxyl groups with the particle size of 100nm and the excitation wavelength of 480nm by using a 50mM MES buffer solution with the pH value of 6.0, wherein the dilution concentration is 0.01mg/mL, taking 2mL, adding 200uL EDC (5mg/mL), uniformly mixing, adding 200uL sulfo-NHS (2mg/ML), carrying out reaction activation reaction for 0.5 hour, adding 80uL reagent 5, reacting for 2 hours at room temperature, adding 500uL ethylenediamine with the concentration of 0.01g/mL, reacting for 1 hour, washing by using a buffer solution for centrifugation, adding 3 reagent for washing twice, adding 3 and 10mL buffer solution reagent, and obtaining the PCT-microsphere labeling reagent.
Washing fluorescent latex microspheres with carboxyl groups with the particle size of 100nm and the excitation wavelength of 480nm by using a 50mM MES buffer solution with the pH value of 6.0, wherein the dilution concentration is 0.01mg/mL, taking 2mL, adding 200uL EDC (5mg/mL), uniformly mixing, adding 200uL sulfo-NHS (2mg/ML), carrying out reaction activation reaction for 0.5 hour, adding 60uL reagent for reaction for 2 hours at room temperature, adding 500uL ethylenediamine with the concentration of 0.01g/mL, carrying out reaction for 1 hour, washing by using a buffer solution for centrifugation, adding 3 reagent for washing twice, adding 3 and 10mL buffer solution reagent, and obtaining the CRP microsphere labeling reagent.
Mixing the PCT-microsphere labeling reagent and the CRP microsphere labeling reagent to obtain a PCT-CRP microsphere labeling reagent;
diluting a reagent 6 to a PCT concentration, and diluting a reagent 9 for preparation;
standard solution 1: CRP 0.5 ug/mL; standard solution 2: PCT 0.5ng/mL CRP 1 ug/mL; standard solution 3: PCT 1ng/mL CRP 2 ug/mL; standard solution 4: PCT 10ng/mL CRP 10 ug/mL; standard solution 5: PCT 30ng/mLCRP 20 ug/mL;
the assay procedure was as follows, with the assay chip immersed in standard solution (as sample), incubated for 5 minutes at room temperature, the chip washed twice with reagent 2, further immersed in PCT-CRP labeling reagent for incubation for 5 minutes, washed twice with washing reagent 2, excited with laser, while detecting the fluorescent signal with PMT.
After a standard luminescence signal curve is established through standard reagent detection, a reagent 6 and a reagent 9 are added into serum, and the standard luminescence signal curve is obtained through weighing method configuration, wherein a sample 1: CRP 2ug/MLPCT 0.5ng/mL, sample 2: CRP 5ug/MLPCT5ng/mL, sample 3: CRP 10ug/MLPCT 30ng/mL, the above procedure was repeated to test the samples with the analytical chip, and the test results are shown in Table 3:
TABLE 3
| CRP(ug/ml) | Configured concentration | Chip 1 | Chip 2 | Chip 3 | Chip 4 | Chip 5 | Chip 6 | Chip 7 | Chip 8 | CV | Mean value |
| Sample 1 | 2 | 1.86 | 2.10 | 2.10 | 2.14 | 1.90 | 1.86 | 1.86 | 1.86 | 6.55％ | 1.96 |
| Sample 2 | 5 | 5.30 | 5.30 | 5.20 | 5.30 | 5.10 | 4.70 | 5.30 | 5.10 | 4.70 | 5.16 |
| Sample 3 | 10 | 9.40 | 9.40 | 10.60 | 10.60 | 9.40 | 10.50 | 10.50 | 9.40 | 6.17％ | 9.98 |
| PCT(ng/ml) | Configured concentration | Chip 1 | Chip 2 | Chip 3 | Chip 4 | Chip 5 | Chip 6 | Chip 7 | Chip 8 | CV | Mean value |
| Sample 1 | 0.5 | 0.46 | 0.46 | 0.46 | 0.49 | 0.47 | 0.54 | 0.46 | 0.46 | 5.95％ | 0.48 |
| Sample 2 | 5 | 4.65 | 4.65 | 4.65 | 5.35 | 4.65 | 5.35 | 4.65 | 4.65 | 6.72％ | 4.83 |
| Sample 3 | 30 | 32.10 | 27.90 | 27.90 | 27.90 | 27.90 | 32.10 | 32.10 | 27.90 | 7.37％ | 29.48 |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A modular biological sample analysis chip, comprising: a substrate material comprising a hydrophilic surface, an analysis module; the substrate material with the hydrophilic surface is used for supporting, fixing and integrating the analysis module; the analysis module is assembled on the substrate material; the surfaces of the analysis modules are coated with capture molecules, wherein the surfaces of the analysis modules of different analysis types are coated with the capture molecules of different types; the surface of the analysis module and the surface of the substrate material after the analysis module is assembled at least comprise a closed component, and the closed component is used for avoiding the nonspecific adsorption of proteins or other organisms and organic molecules; the analysis module is assembled on a substrate material after coating is finished so as to obtain the modularized biological sample analysis chip; the modular biological sample analysis chip is further diced to produce a plurality of chips or a new analysis chip formed by further assembling a plurality of analysis chips on a substrate.
2. The modular biological sample analysis chip of claim 1, wherein: the substrate material is one of metal material, glass material, silicon material or macromolecule plastic material.
3. The modular biological sample analysis chip of claim 1, wherein: the hydrophilic surface is a hydrophilic surface which is changed into hydrophilic property by the modification of a substrate material and a hydrophobic material or the treatment of a sealant.
4. The modular biological sample analysis chip of claim 1, wherein: the capture molecules are used for capturing the substance to be analyzed in the biological sample, and the capture molecules are selected from protein, glycoprotein, polypeptide, nucleic acid, organic small molecule, polysaccharide or antibody biological molecules.
5. The modular biological sample analysis chip of claim 1, wherein: the analysis module is selected from materials with the surface modified by carboxyl, amino, chloromethyl, epoxy alkyl, biotin, aldehyde group or sulfhydryl and the capability of coupling with biological molecules; a metal material having the ability to couple biomolecules; and one of polystyrene, nylon or cellulose acetate materials with the surface having biomolecule adsorption capacity.
6. The modular biological sample analysis chip of claim 1, wherein: the blocking component is biological or organic molecules with hydrophobic binding property, such as bovine serum albumin, tween, skimmed milk powder, casein, amino acid or xylitol.
7. The modular biological sample analysis chip of claim 1 further assembled as a module on a larger substrate to form a new chip.
8. The modular biological sample analysis chip of claim 1, wherein: the reference module is directly marked with an object to be detected or an analogue of the object to be detected and is used for referencing the chip and the detection signal.
9. A biological sample analysis method using the modularized biological sample analysis chip of claim 1, wherein: the method comprises the following steps:
(1) coating the capture molecules on a material A with adsorption capacity or covalent coupling capacity or cutting the material into small modules for coating; wherein, different kinds of capture molecules are coated on the surfaces of the materials A of different analysis kinds;
(2) cutting the cut material A or cutting the material A and then assembling the cut material A on a substrate material B, or assembling the material A on the substrate material B and cutting the material A to form an analysis chip C, wherein the analysis chip C can be further cut to generate a plurality of chips or a plurality of analysis chips C are further assembled on a new analysis chip formed on the substrate;
(3) the analysis chip C is sealed by a reagent containing a sealing component;
(4) contacting the analysis chip C with a molecular substance to be detected;
(5) cleaning the analysis chip C;
(6) the analysis chip C is contacted with a reagent containing the labeled molecules;
(7) if the labeled molecules in the step (6) are fluorescent molecules or quantum dots, exciting by a light source, and detecting by using a signal acquisition device; if the labeled molecules in the step (6) are enzymes or direct luminescent molecules, the analysis chip is further contacted with a reaction reagent to form luminescent signals, and the luminescent signals are detected by using a signal acquisition device.
10. A kit, characterized in that: comprises that
(1) The assay chip of claim 1;
(2) a cleaning reagent;
(3) a reagent containing a labeled component capable of binding to the test substance.
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