CN112683820A - Detection method for latex immunoturbidimetry enhanced by plasma optical sensing chip - Google Patents
Detection method for latex immunoturbidimetry enhanced by plasma optical sensing chip Download PDFInfo
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Abstract
A detection method for latex immunoturbidimetry enhanced by a plasma optical sensing chip comprises the following steps: adding a first reagent and a sample to be detected into a porous microporous plate, and uniformly mixing the first reagent and the sample to be detected, wherein the porous microporous plate is provided with a nano optical sensor chip; adding a second reagent into the uniformly mixed solution in the porous microporous plate, and mixing the solution in the porous microporous plate for a first predetermined period of time at a predetermined temperature; measuring the initial absorbance value of a reaction system in the porous microporous plate in a preset wavelength range by using an enzyme-labeling instrument; after the solution in the porous microporous plate is continuously incubated for a second time period at a preset temperature, the final absorbance value of the reaction system in the porous microporous plate is measured in a preset wavelength range by using an enzyme-labeling instrument; calculating the absorbance value variation according to the initial absorbance value and the final absorbance value; and comparing the calculated change of the absorbance value with a standard curve representing the relationship between the known quantity of the standard sample to be detected and the change of the absorbance value to obtain the content of the sample to be detected.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a detection method of enhanced latex immunoturbidimetry by a plasma optical sensing chip.
Background
In the fields of biology and chemistry, it is often necessary to determine the content of substances such as C-reactive protein (CRP), immunoglobulins (IgG/IgA/IgM), apolipoproteins, ferritin, fibrinogen, cancer embryo antigens, complement C4\ D-dimers.
However, the existing content measurement method is high in cost, insufficient in accuracy, or complicated in measurement operation.
Thus, it is desirable to provide a low-cost solution that is easy to operate and has high accuracy.
Disclosure of Invention
The invention aims to solve the technical problem of providing a detection method of enhanced latex immunoturbidimetry by using a plasma optical sensor chip, which is simple to operate, high in precision and low in cost, aiming at the defects in the prior art.
According to the invention, the detection method of the enhanced latex immunoturbidimetry of the plasma optical sensing chip is provided, which comprises the following steps:
adding a first reagent and a sample to be detected into a porous microporous plate, and uniformly mixing the first reagent and the sample to be detected, wherein the porous microporous plate is provided with a nano optical sensor chip;
adding a second reagent into the uniformly mixed solution in the porous microporous plate, and mixing the solution in the porous microporous plate for a first predetermined period of time at a predetermined temperature;
measuring the initial absorbance value of a reaction system in the porous microporous plate in a preset wavelength range by using an enzyme-labeling instrument;
after the solution in the porous microporous plate is continuously incubated for a second time period at a preset temperature, the final absorbance value of the reaction system in the porous microporous plate is measured in a preset wavelength range by using an enzyme-labeling instrument;
calculating the absorbance value variation according to the initial absorbance value and the final absorbance value;
and comparing the calculated change of the absorbance value with a standard curve representing the relationship between the known quantity of the standard sample to be detected and the change of the absorbance value to obtain the content of the sample to be detected.
Preferably, the predetermined temperature is 37 degrees celsius, the first predetermined period of time is one minute, and the second period of time is five minutes.
Preferably, the predetermined wavelength range is at 590nm and 610nm wavelengths; the formula for calculating the absorbance value variation according to the initial absorbance value and the final absorbance value is that delta OD (OD 2)610-OD1610)-(OD2590-OD1590) Where OD1 represents the initial absorbance value, OD2 represents the final absorbance value, and the subscripts represent the wavelength used to measure the absorbance value.
Preferably, the first reagent comprises: 10-200mM of reaction buffer solution, 0.5-5% (w/v) of reaction aggregation promoter, 0.01-1% (w/v) of preservative, 0.05-2% (w/v) of surfactant and protective agent.
Preferably, the second reagent comprises: reaction buffer solution, preservation solution and protein or antibody modified nano-microspheres, wherein the microspheres comprise one or more of polystyrene microspheres, colored latex microspheres, fluorescent microspheres and magnetic microspheres; the microsphere has a particle size of 20nm-3 μm, and the reaction buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, MES buffer solution, HEPES buffer solution, glycine buffer solution, acetic acid buffer solution and DIPSO buffer solution.
According to the invention, the invention also provides a detection method of the enhanced latex immunoturbidimetry of the plasma optical sensing chip, which comprises the following steps:
uniformly mixing a first reagent and a sample to be detected, and adding the mixture into a porous microporous plate, wherein a nano optical sensor chip is arranged in the porous microporous plate;
adding the second reagent into the uniformly mixed solution in the porous microporous plate, and immediately measuring the absorbance change rate curve of the reaction system in the porous microporous plate within a preset wavelength range by using an enzyme-labeling instrument;
and comparing the obtained absorbance change rate curve with a standard curve representing the relation between the known quantity of the sample to be detected and the absorbance change rate curve, and obtaining the content of the sample to be detected.
Preferably, the first reagent comprises: 10-200mM of reaction buffer solution, 0.5-5% (w/v) of reaction aggregation promoter, 0.01-1% (w/v) of preservative, 0.05-2% (w/v) of surfactant and protective agent.
Preferably, the second reagent comprises: reaction buffer solution, preservation solution and protein or antibody modified nano-microspheres, wherein the microspheres comprise one or more of polystyrene microspheres, colored latex microspheres, fluorescent microspheres and magnetic microspheres; the microsphere has a particle size of 20nm-3 μm, and the reaction buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, MES buffer solution, HEPES buffer solution, glycine buffer solution, acetic acid buffer solution and DIPSO buffer solution.
Preferably, the predetermined wavelength range is 560-600 nm.
Preferably, the predetermined wavelength ranges are 590nm and 610nm wavelengths.
Drawings
A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:
FIG. 1 is a schematic diagram illustrating the principle of the detection method of latex immunoturbidimetry enhanced by the plasma optical sensing chip according to the preferred embodiment of the invention.
FIG. 2 is a schematic diagram illustrating the principle of the detection method of latex immunoturbidimetry enhanced by the plasma optical sensing chip according to the preferred embodiment of the invention.
It is to be noted, however, that the appended drawings illustrate rather than limit the invention. It is noted that the drawings representing structures may not be drawn to scale. Also, in the drawings, the same or similar elements are denoted by the same or similar reference numerals.
Detailed Description
In order that the present disclosure may be more clearly and readily understood, reference will now be made in detail to the present disclosure as illustrated in the accompanying drawings.
A method for detecting the enhanced latex immunoturbidimetry of a plasma optical sensing chip adopts a porous microporous plate carrying a novel nano optical sensor chip, wherein a reagent for measuring the enhanced latex immunoturbidimetry of the plasma optical sensing chip comprises a first reagent R1 and a second reagent R2. The detection method of the plasma optical sensing chip enhanced latex immunoturbidimetry can be used for detecting various proteins, antibodies, antigens, small molecules and the like. The detection method of the plasma optical sensing chip enhanced latex immunoturbidimetry amplifies optical signals by utilizing the action of nano plasma optical resonance, so that the detection method is more sensitive and stable, the repeatability can be improved, and the minimum detection limit can be reduced to pg/ml; and the linear range can be improved by changing the prescription ratio of the first reagent R1 and the second reagent R2. The method for enhancing latex immunoturbidimetry by using the plasma optical sensing chip has the advantages of high sensitivity, wide detection line, high detection speed, simple operation, good stability, cyclic utilization, low production cost, capability of measuring on a common microplate reader and the like.
< first embodiment >
Fig. 1 and 2 schematically show a principle schematic diagram of a detection method of latex immunoturbidimetry enhanced by a plasma optical sensing chip according to a preferred embodiment of the invention.
Referring to fig. 1 and 2, the method for detecting latex immunoturbidimetry enhanced by a plasma optical sensing chip according to the first preferred embodiment of the invention comprises:
the first step is as follows: adding a first reagent R1 and a sample to be detected into a porous microporous plate, and uniformly mixing, wherein a nano optical sensor chip is arranged in the porous microporous plate;
the second step is as follows: adding a second reagent R2 into the uniformly mixed solution in the porous microplate, and mixing the solution in the porous microplate for a first predetermined period of time at a predetermined temperature; for example, the solution in the multi-well microplate may be mixed at 37 degrees Celsius for one minute to allow the solution in the multi-well microplate to mix uniformly.
The third step: measuring the initial absorbance value OD1 of a reaction system in the porous microporous plate in a preset wavelength range by using a microplate reader;
the fourth step: after continuing to incubate the solution in the multiwell microplate for a second period of time at the predetermined temperature (e.g., five minutes of mixing at 37 ℃), the final absorbance value OD2 of the reaction system in the multiwell microplate is determined with a microplate reader over a predetermined wavelength range;
the fifth step: calculating the absorbance value variation delta OD according to the initial absorbance value OD1 and the final absorbance value OD 2;
wherein, for example, the predetermined wavelength range is 560-600nm, and preferably the predetermined wavelength ranges are 590nm and 610 nm. Further preferably, Δ OD ═ (OD 2)610-OD1610)-(OD2590-OD1590) Where the subscript indicates the wavelength used to measure the photometric values.
A sixth step: and comparing the calculated change of the absorbance value with a standard curve representing the relationship between the known quantity of the standard sample to be detected and the change of the absorbance value to obtain the content of the sample to be detected.
< second embodiment >
Fig. 1 and 2 schematically show a principle schematic diagram of a detection method of latex immunoturbidimetry enhanced by a plasma optical sensing chip according to a second preferred embodiment of the invention.
Referring to fig. 1 and 2, the method for detecting latex immunoturbidimetry enhanced by a plasma optical sensing chip according to the preferred embodiment of the invention comprises the following steps:
the first step is as follows: uniformly mixing a first reagent R1 with a sample to be detected, and adding the mixture into a porous microporous plate, wherein a nano optical sensor chip is arranged in the porous microporous plate;
the second step is as follows: adding a second reagent R2 into the uniformly mixed solution in the porous microporous plate, and immediately measuring the absorbance change rate curve of the reaction system in the porous microporous plate within a preset wavelength range by using an enzyme-labeling instrument;
the third step: and comparing the obtained absorbance change rate curve with a standard curve representing the relation between the known quantity of the sample to be detected and the absorbance change rate curve, and obtaining the content of the sample to be detected.
< reagent example >
Preferably, the first reagent R1 is formulated as follows: 10-200mM of reaction buffer solution (one or more of PBS, Tris-HCl buffer solution, MES buffer solution, HEPES buffer solution, glycine buffer solution, acetic acid buffer solution and DIPSO buffer solution), 0.5-5% (w/v) of reaction aggregation promoter (one or more of PEG5000, PEG6000, PEG8000 and PEG 12000), 0.01-1% (w/v) of preservative (Proclin-300, gentamicin or NaN3), 0.05-2% (w/v) of surfactant (one or more of Tween20, TritonX-100 and Tergitol NP 9), 0.05-5% of other protective agents such as bovine serum albumin, sucrose and trehalose, and the pH is 7.0-9.0.
Preferably, the raw material formula of the second reagent R2 is as follows: protein or antibody modified nano-microspheres, wherein the microspheres comprise polystyrene microspheres, colored latex microspheres, fluorescent microspheres, magnetic microspheres and the like; the particle size range of the microspheres is 20nm-3 μm, preferably 50nm-300 nm; the preservation solution is 10-200mM of buffer solution, 0.1-0.5% (w/v) of preservative, 0.1-2% (w/v) of surfactant and 0.05-5% of other protective agents. Wherein the reaction buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, MES buffer solution, HEPES buffer solution, glycine buffer solution, acetic acid buffer solution and DIPSO buffer solution; the antiseptic is Proclin-300, gentamicin or NaN 3; the surfactant is one or more of Tween20, TritonX-100 and Tergitol NP 9. Other protective agents are bovine serum albumin, sucrose, trehalose, polyethylene glycol, and the like.
The detection method of the latex immunoturbidimetry enhanced by the plasma optical sensor chip can be used for quantitative real-time detection of various proteins, nucleic acids, antigen antibodies, microbial particles, bacteria, viruses and the like, and particularly relates to the detection of C-reactive protein, apolipoprotein E, immunoglobulin, apolipoprotein, ferritin, fibrinogen, oncofetal antigen, complement C4, D-dimer, N-terminal pro-brain natriuretic peptide, troponin, myoglobin, cardiac fatty acid binding protein, phospholipase A2, various viral particles (spherical viruses, baculovirus, brick-shaped viruses, filarial viruses, coronavirus and the like), bacteria (bacilli, cocci, spirochetes and the like), and other microorganisms.
It should be noted that the terms "first", "second", "third", and the like in the description are used for distinguishing various components, elements, steps, and the like in the description, and are not used for indicating a logical relationship or a sequential relationship between the various components, elements, steps, and the like, unless otherwise specified.
It is to be understood that while the present invention has been described in conjunction with the preferred embodiments thereof, it is not intended to limit the invention to those embodiments. It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. A detection method for latex immunoturbidimetry enhanced by a plasma optical sensing chip is characterized by comprising the following steps:
adding a first reagent and a sample to be detected into a porous microporous plate, and uniformly mixing the first reagent and the sample to be detected, wherein the porous microporous plate is provided with a nano optical sensor chip;
adding a second reagent into the uniformly mixed solution in the porous microporous plate, and mixing the solution in the porous microporous plate for a first predetermined period of time at a predetermined temperature;
measuring the initial absorbance value of a reaction system in the porous microporous plate in a preset wavelength range by using an enzyme-labeling instrument;
after the solution in the porous microporous plate is continuously incubated for a second time period at a preset temperature, the final absorbance value of the reaction system in the porous microporous plate is measured in a preset wavelength range by using an enzyme-labeling instrument;
calculating the absorbance value variation according to the initial absorbance value and the final absorbance value;
and comparing the calculated change of the absorbance value with a standard curve representing the relationship between the known quantity of the standard sample to be detected and the change of the absorbance value to obtain the content of the sample to be detected.
2. The method for detecting immunoturbidimetry of latex enhanced by a plasma optical sensor chip according to claim 1, wherein the predetermined temperature is 37 degrees celsius, the first predetermined time period is one minute, and the second time period is five minutes.
3. The method for detecting latex immunoturbidimetry enhanced by a plasma optical sensor chip according to claim 1 or 2, wherein the predetermined wavelength ranges are 590nm and 610 nm; the formula for calculating the absorbance value variation according to the initial absorbance value and the final absorbance value is that delta OD (OD 2)610-OD1610)-(OD2590-OD1590) Where OD1 represents the initial absorbance value, OD2 represents the final absorbance value, and the subscripts represent the wavelength used to measure the absorbance value.
4. The method for detecting the latex immunoturbidimetry enhanced by the plasma optical sensing chip according to claim 1 or 2, wherein the first reagent comprises: 10-200mM of reaction buffer solution, 0.5-5% (w/v) of reaction aggregation promoter, 0.01-1% (w/v) of preservative, 0.05-2% (w/v) of surfactant and protective agent.
5. The method for detecting latex immunoturbidimetry enhanced by the plasma optical sensing chip according to claim 1 or 2, wherein the second reagent comprises: reaction buffer solution, preservation solution and protein or antibody modified nano-microspheres, wherein the microspheres comprise one or more of polystyrene microspheres, colored latex microspheres, fluorescent microspheres and magnetic microspheres; the microsphere has a particle size of 20nm-3 μm, and the reaction buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, MES buffer solution, HEPES buffer solution, glycine buffer solution, acetic acid buffer solution and DIPSO buffer solution.
6. A detection method for latex immunoturbidimetry enhanced by a plasma optical sensing chip is characterized by comprising the following steps:
uniformly mixing a first reagent and a sample to be detected, and adding the mixture into a porous microporous plate, wherein a nano optical sensor chip is arranged in the porous microporous plate;
adding the second reagent into the uniformly mixed solution in the porous microporous plate, and immediately measuring the absorbance change rate curve of the reaction system in the porous microporous plate within a preset wavelength range by using an enzyme-labeling instrument;
and comparing the obtained absorbance change rate curve with a standard curve representing the relation between the known quantity of the sample to be detected and the absorbance change rate curve, and obtaining the content of the sample to be detected.
7. The method for detecting latex immunoturbidimetry enhanced by the plasma optical sensor chip according to claim 6, wherein the first reagent comprises: 10-200mM of reaction buffer solution, 0.5-5% (w/v) of reaction aggregation promoter, 0.01-1% (w/v) of preservative, 0.05-2% (w/v) of surfactant and protective agent.
8. The method for detecting latex immunoturbidimetry enhanced by the plasma optical sensor chip according to claim 6 or 7, wherein the second reagent comprises: reaction buffer solution, preservation solution and protein or antibody modified nano-microspheres, wherein the microspheres comprise one or more of polystyrene microspheres, colored latex microspheres, fluorescent microspheres and magnetic microspheres; the microsphere has a particle size of 20nm-3 μm, and the reaction buffer solution is one or more of PBS buffer solution, Tris-HCl buffer solution, MES buffer solution, HEPES buffer solution, glycine buffer solution, acetic acid buffer solution and DIPSO buffer solution.
9. The method for detecting latex immunoturbidimetry enhanced by the plasma optical sensor chip as claimed in claim 6 or 7, wherein the predetermined wavelength range is 560-600 nm.
10. The method for detecting latex immunoturbidimetry enhanced by a plasma optical sensor chip according to claim 6 or 7, wherein the predetermined wavelength ranges are 590nm and 610 nm.
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