CN113433329A - PCT/IL-6 duplex detection kit based on quantum dot fluorescent microspheres and preparation method thereof - Google Patents
PCT/IL-6 duplex detection kit based on quantum dot fluorescent microspheres and preparation method thereof Download PDFInfo
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Abstract
A PCT/IL-6 duplex detection kit based on quantum dot fluorescent microspheres and a preparation method thereof are characterized in that: the kit comprises a reaction area, a first detection site, a second detection site and a third detection site, wherein an antibody-labeled quantum dot fluorescent microsphere is arranged in the reaction area, the first detection site is coated with an IL-6 capture antibody, the second detection site is coated with a PCT capture antibody, and the third detection site is coated with an anti-chicken IgY capture antibody. The method combines the quantum dot fluorescent microspheres with good stability and high sensitivity with the micro-fluidic chip for instant detection, thereby realizing the technical effect of rapidly and accurately carrying out combined detection on the PCT and the IL-6 markers simultaneously.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a PCT/IL-6 dual detection kit based on quantum dot fluorescent microspheres and a preparation method thereof.
Background
Procalcitonin (PCT) is a precursor of calcitonin with no hormonal activity, is a glycoprotein consisting of 116 amino acids and has a molecular weight of approximately 13000. The in vivo generation speed of the PCT is high, and the half-life period of the PCT is usually 20-24 h. Typically, healthy adults have serum PCT levels below 10 pg/mL. When the serum PCT concentration is 0.1-0.5 ng/mL, the human body is generally considered to have bacterial infection, the concentration reaches about 2.0 ng/mL to prompt the occurrence of systemic inflammatory response syndrome, the patient is considered to have sepsis when the concentration ranges from 1 ng/mL to 10 ng/mL, and the concentration ranges from 10 ng/mL to 100 ng/mL indicates the possibility of septic shock. In addition, studies have shown that PCT exhibits high specificity and sensitivity in distinguishing between bacterial and non-bacterial infections. Generally, after 3-4 hours of bacterial infection, the PCT level in the patient begins to rise, reaches a peak value within 12-48 hours, and reaches a maximum value after 24 hours. Compared with inflammatory factors such as c-reactive protein (CRP), white blood cell count (WBC), Tumor Necrosis Factor (TNF) -alpha, interleukin-6 (IL-6) and the like, PCT has higher specificity and sensitivity, and can be used for early diagnosis, judgment of disease severity, prognosis and evaluation of anti-infection curative effect of severe bacterial infection. The clinical common important tool for judging sepsis, which is the diagnostic value of systemic and local infection, can guide the reasonable use of antibiotics.
Interleukin 6 (IL-6), a multifunctional cytokine consisting of 184 amino acids produced by fibroblasts, monocytes, macrophages, T lymphocytes, B lymphocytes, epithelial cells, keratinocytes and various neoplastic cells, has received increasing attention as the most promising biomarker for early identification of sepsis. IL-6 is closely related to bacterial infection, can be rapidly increased after infection, can regulate immune response, and can be used for assisting early diagnosis of acute infection and reflecting infection degree. However, inflammatory or autoimmune diseases may affect the concentration of IL-6 in human serum. IL-6 concentrations in ICU patients are reported to be generally >3000 pg/ml, whereas IL-6 levels in healthy adults are reported to be about 6.4 pg/ml, with a sensitivity of about 85% and specificity of about 62%. After pathogen infection, the concentration of IL-6 rose to a peak at 3 h and dropped to the baseline after 8 h. In addition, IL-6 showed kinetics to monitor the efficacy of antibiotic therapy. IL-6 is present in very low concentrations in the blood (in the picogram range), and the technical threshold for accurate and stable measurements is very high. Therefore, manufacturers of IL-6 currently have a fair number of fingers. The quantum dots are also called semiconductor nanocrystals, are approximately spherical, and can receive exciting light to generate fluorescence. As a novel semiconductor nano material, the quantum dot has unique advantages compared with a common fluorescent marker, such as wide and continuous excitation spectrum, narrow and adjustable emission spectrum, high fluorescence intensity, long luminescence time, good photochemical stability, easy surface modification and the like. These advantages make quantum dots the optimal choice for a new generation of fluorescent-labeled probes, and have been applied and developed to some extent in the field of biomarker detection. However, the size of the quantum dot is small, the fluorescence intensity of a single quantum dot is limited, and when the quantum dot is used for biological labeling, the fluorescence stability of the quantum dot is easily damaged when the quantum dot is exposed to a complex physiological environment. The quantum dots are encapsulated in the polymer microspheres to form the quantum dot fluorescent microspheres, so that the fluorescence intensity of a single microsphere can be improved, and the quantum dot fluorescent microspheres are easy to modify and convenient to combine with biological macromolecules. In addition, the quantum dots are loaded in the microspheres, so that the bleaching resistance, the thermal stability, the chemical environment stability and the like of the quantum dots can be improved.
Microfluidic technology refers to the science and technology involved in systems that process or manipulate tiny fluids using microchannels (tens to hundreds of microns in size), and is an emerging interdiscipline that involves chemical, fluid physics, microelectronics, new materials, biology, and biomedical engineering. The micro-fluidic chip is a main platform for realizing the micro-fluidic technology. Compared with the traditional in-vitro diagnosis product, the system can integrate a series of basic operation units such as sample preparation, reaction, separation, detection and the like related in the fields of chemistry, biology and the like on a micron-sized chip, control the flow of fluid with extremely small volume in a tiny space, and easily meet the requirements of life science on low-dose, more-efficient, highly-sensitive and rapid separation and analysis of biological samples. At present, more and more people try to combine quantum dots and microfluidic technology to realize quantitative analysis of biomarkers and make important progress.
The consensus expert consensus on clinical significance interpretation of biomarkers related to infection, officially released in 4 months in 2017, indicates that none of the biomarkers is absolutely sensitive and specific, and diseases cannot be diagnosed by the change of a certain biomarker. The time difference between abnormal levels of PCT and IL-6 may lead to misdiagnosis of infectious diseases, if the clinician detects only one biomarker from a patient sample. Therefore, it is necessary to detect PCT and IL-6 simultaneously, which can not only distinguish bacterial infection from non-bacterial infection, but also guide antibiotic treatment and improve clinical treatment effect, and the joint inspection of two items can put two items on a reagent card for detection at the same time, and the increase of cost for one charge item, which is lower than the cost accumulation of two items, can increase the profit margin of hospitals, and is adapted to the current appeal of the strict control cost of hospital clinical laboratory, so that the combination has greater market potential.
The PCT detection method mainly comprises a colloidal gold method, a radioimmunoassay method, a transmission immunoturbidimetry method and a double-antibody sandwich immunochemiluminescence method. The radioimmunoassay has high sensitivity reaching picogram level, but cannot distinguish the related peptides of free type and combined type PCT and CT genes, and simultaneously, because of containing radioactive elements, the radioimmunoassay is easy to cause pollution, is long in time consumption, and is greatly limited in clinical and laboratory research. The principle of the transmission immunoturbidimetry is that the turbidity of a system is increased by using a PCT antigen-antibody reaction, and the turbidity is increased in proportion to the concentration of a PCT sample. The most widely used methods in clinical practice are the colloidal gold method and the ELISA method. The colloidal gold method is simple and convenient, but the colloidal gold method can only distinguish the approximate concentration of the PCT by comparing the human subjectivity with a colorimetric card, belongs to semi-quantitative method, has strong subjectivity, cannot further confirm the specific concentration of the PCT, and influences the means and dosage for later treatment. The ELISA chemiluminescence method has strong specificity and high sensitivity, but the method needs more precise operation of professionals and detection of larger instruments, and all processes can be finished under the operation of the professionals, so that the portability of PCT detection is hindered. The most widely used method for rapid detection of IL-6 is the gold lateral flow test, which is simple to operate, fast in response speed and low in cost. The low sensitivity and qualitative analysis of this method limits its further application to the detection of trace targets in complex samples. Other methods with high sensitivity are mainly chemiluminescence immunoassay, fluorescence immunoassay, enzyme-linked immunosorbent assay and electrochemical immunoassay. Although these methods have been used to measure PCT and IL-6 levels in human serum, most methods use cumbersome instruments and are unable to detect both biomarkers simultaneously, which can lead to misdiagnosis and considerable treatment delays.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the PCT/IL-6 dual detection kit based on the quantum dot fluorescent microspheres, which combines the quantum dot fluorescent microspheres with good stability and high sensitivity with a micro-fluidic chip for instant detection and simultaneously carries out rapid and accurate combined detection on two markers of PCT and IL-6 based on the dual characteristics of quantum dot fluorescence and micro-fluidic technology.
In order to solve the technical problems, the application provides a PCT/IL-6 dual detection kit based on quantum dot fluorescent microspheres, the kit is provided with a reaction region, a first detection site, a second detection site and a third detection site, the reaction region is provided with antibody-labeled quantum dot fluorescent microspheres, the first detection site is coated with an IL-6 capture antibody, the second detection site is coated with a PCT capture antibody, and the third detection site is coated with an anti-chicken IgY capture antibody.
Further, the kit specifically comprises an upper shell, a lower shell and a microfluidic chip area positioned on the lower shell; the microfluidic chip area comprises a sample receiving area, a sample storage area, a reaction area, a time valve, a detection channel and a waste liquid area, wherein the sample receiving area, the sample storage area, the reaction area, the time valve, the detection channel and the waste liquid area are sequentially connected along the flow direction of a medium; the detection channel is sequentially provided with a first detection site, a second detection site and a third detection site along the medium flowing direction.
By adopting the structure, the antibody-labeled quantum dot fluorescent microspheres, the IL-6 antibody, and the capture antibodies corresponding to the PCT antibody and the chicken IgY antibody are simultaneously arranged on the microfluidic chip area, so that the rapid and accurate combined detection of PCT and IL-6 can be realized simultaneously, and the effect of combining the quantum dot fluorescent microspheres with good stability and high sensitivity with the microfluidic chip for immediate detection is realized; the product can be used for multiple departments and multiple scenes after being produced in batches, has important clinical value, and can provide data support for the development of subsequent different products.
The application also provides a preparation method of the kit for carrying out PCT/IL-6 duplex detection by using the quantum dot fluorescent microsphere labeled antibody, which comprises the following steps: the method comprises the following steps:
(1) quantum dot fluorescent microsphere labeling (antibody labeling quantum dot fluorescent microsphere): firstly, activating quantum dot fluorescent microspheres (QBC 620, Shanghai Kun Dai Biotechnology Co., Ltd.), then adding a PCT antibody or an IL-6 antibody or a chicken IgY antibody into an activated quantum dot fluorescent microsphere solution, ultrasonically mixing uniformly for marking, centrifugally cleaning after reaction is finished, adding a confining liquid for confining, centrifugally cleaning the confined fluorescent microspheres to obtain antibody-labeled quantum dot fluorescent microspheres, and then storing by using a microsphere storage solution;
(2) assembling the microfluidic chip: after the surface of the microfluidic chip is subjected to hydrophilic treatment, drying the microfluidic chip in an oven at 37 ℃, and loading a capture antibody at a corresponding position of a detection channel; sealing the surface of the chip, adding the quantum dot fluorescent microsphere solution prepared in the step (1) into a reaction zone, drying in a 37 ℃ oven, and performing ultrasonic bonding to complete combination after the sample application of the chip is completed;
(3) preparation of a standard substance:
diluting the antigen concentrated solution by using a standard substance diluent to obtain a standard substance antigen mother solution for later use; the standard substance diluent is 10% BSA solution, and is preserved at 2-8 deg.C; the standard substance comprises a standard substance A and a standard substance B, wherein the standard substance A is PCT antigen mother liquor diluted to 100 ng/mL and stored at the temperature of-20 ℃, and the standard substance B is IL-6 antigen mother liquor diluted to 1000 pg/mL and stored at the temperature of-20 ℃;
obtaining the quantum dot fluorescent microsphere-based PCT/IL-6 duplex detection kit.
Preferably, the activation treatment in step (1) is: and (3) placing the quantum dot fluorescent microspheres in 0.25 mg/mL EDC-NHS, and activating for 10-20 min, wherein the 0.25 mg/mL EDC-NHS is the final concentration of the EDC-NHS in the buffer solution.
Preferably, the blocking solution in step (1) is 0.1% BSA (0.1% bovine serum albumin,% by mass), and the blocking solution of 0.1% BSA refers to the final concentration of BSA in the buffer.
Preferably, in the step (1), the final concentration of the PCT antibody in the activated quantum dot fluorescent microsphere solution is 0.3 mg/mL (final concentration in the quantum dot fluorescent microsphere solution, the same applies below), the final concentration of the IL-6 antibody in the activated quantum dot fluorescent microsphere solution is 0.2 mg/mL, the final concentration of the chicken IgY antibody in the activated quantum dot fluorescent microsphere solution is 0.2 mg/mL, and the activated quantum dot fluorescent microsphere solution is an activated quantum dot fluorescent microsphere solution containing a buffer solution.
Further preferably, the buffers used in the activation, labeling and blocking processes in step (1) above (i.e. the buffers mentioned in each of the above processes) are all 50mM HEPES ((4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) pH =7.4, and the stock solution of microspheres in step (1) contains 10 mg/mL BSA, 10 mg/mL trehalose; the addition of trehalose and BSA can improve the dispersion degree of the antibody labeled microspheres and prevent the microspheres from aggregating to form precipitates to influence the use effect in the long-time standing process.
Preferably, the capture antibody (stationary phase antibody) in step (2) (the stationary phase antibody is a counterpart capture antibody (i.e. PCT capture antibody, IL-6 capture antibody, anti-chicken IgY capture antibody) corresponding to the "PCT antibody or IL-6 antibody or chicken IgY antibody" mentioned in step (1), and the PCT capture antibody or IL-6 capture antibody or anti-chicken IgY capture antibody is 0.3-0.5 μ L, 1.5 mg/mL for forming a "labeled antibody-antigen-capture antibody" double-antibody sandwich structure at the corresponding position of the detection channel, respectively, so as to achieve the purpose of specifically binding to the corresponding antigen in the sample); (ii) a The chip surface was blocked with 0.1% BSA; the total mixed microsphere spot size in the reaction zone was 0.66 mg/mL, 3.3. mu.L.
Furthermore, the total mixed microsphere sample amount of the reaction area is 0.66 mg/mL, and 3.3 muL is the concentration and the using amount formed by mixing 0.272 mg/mL labeled PCT microspheres, 0.272 mg/mL labeled IL-6 microspheres and 0.136mg/mL labeled chicken IgY microspheres respectively.
Preferably, the standard dilution of 10% BSA in step (3) is the final concentration of BSA in a standard dilution buffer of 50mM BB buffer (50 mM Tris-HCl, 5mM KCl, 100mM NaCl, 1mM MgCl) at pH =8.52）。
Compared with the prior art, the quantum dot fluorescent microsphere-based PCT/IL-6 duplex detection kit has the following advantages:
1. the invention is based on dual effects of quantum dot fluorescence and micro-fluidic for the first time, and realizes the rapid and accurate combined detection of the specific biomarker PCT/IL-6 by combining the quantum dot fluorescent microspheres with good stability and high sensitivity with the micro-fluidic chip for instant detection.
2. The technical scheme of the invention can realize high-sensitivity, quick and accurate combined detection of the PCT and IL-6 biomarkers. The product can be used for multiple departments and multiple scenes after being produced in batches, has important clinical value, and can provide data support for the development of subsequent different products.
3. The application adopts an immune double-antibody sandwich method to realize PCT/IL-6 combined detection; firstly, specifically combining PCT and IL-6 antigens contained in a sample in a receiving area with PCT and IL-6 marked by quantum dot fluorescent microspheres arranged in a reaction area to form a quantum dot fluorescent microsphere marked antibody-antigen compound; then, the quantum dot fluorescent microsphere labeled antibody-antigen composite enters the detection channel through the time valve to control the flow rate and is combined with corresponding capture antibodies (IL-6 capture antibody and PCT capture antibody) loaded on the stationary phase of the detection channel to form a sandwich structure of quantum dot fluorescent microsphere labeled antibody-antigen-capture antibody, so that the quantum dot fluorescent microsphere labeled antibody-antigen-capture antibody composite is fixed at the corresponding position of the detection zone; then, ultraviolet light on a single-channel fluorescence immunoassay instrument is adopted to excite the fixed fluorescent microspheres, so that fluorescent signals are emitted and collected to realize PCT/IL-6 duplex detection.
4. According to the application, based on dual effects of quantum dot fluorescence and microfluidics, and a microfluidic chip with a specific structure is combined, so that the final kit achieves the purpose of high-sensitivity, rapid and accurate combined detection of two biomarkers of PCT and IL-6.
Drawings
Fig. 1 is a schematic structural view of a microfluidic chip region of the present application.
As shown in the attached drawings: a. the kit comprises a lower shell, 1, a sample receiving area, 2, a sample storage area, 3, a reaction area, 4, a time valve, 5, a detection channel, 5.1, a first detection site, 5.2, a second detection site, 5.3, a third detection site and 6, a waste liquid area.
FIG. 2. Duplex kit PCT Standard test curves.
FIG. 3 Duplex kit PCT clinical sample calibration curves.
FIG. 4 shows a standard curve for IL-6 detection in the duplex kit.
FIG. 5 Duplex kit IL-6 clinical sample calibration curves.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. The content of each component% in the application is the content of mass percent without special description.
The% of each component in the present application is the mass percentage concentration in the solution in which it is present, without specific description.
As shown in the attached drawing 1, the application provides a PCT/IL-6 duplex detection kit based on quantum dot fluorescent microspheres, which comprises an upper shell (the upper shell is just a shell structure of an industry conventional kit, and is matched with a lower shell in the attached drawing in a corresponding way, and the drawing is not shown), a lower shell a and a microfluidic chip area positioned on the lower shell; the microfluidic chip area comprises a sample receiving area 1, a sample storage area 2, a reaction area 3, a time valve 4, a detection channel 5 and a waste liquid area 6, wherein the sample receiving area 1, the sample storage area 2, the reaction area 3, the time valve 4, the detection channel 5 and the waste liquid area 6 are sequentially connected along the medium flowing direction; the detection device is characterized in that an antibody-labeled quantum dot fluorescent microsphere is arranged in the reaction region 3, a first detection site 5.1, a second detection site 5.2 and a third detection site 5.3 are sequentially arranged on the detection channel 5 along the medium flow direction, an IL-6 capture antibody is coated on the first detection site 5.1, a PCT capture antibody is coated on the second detection site 5.2, and an anti-chicken IgY capture antibody is coated on the third detection site 5.3.
The specific structure of the detection kit is consistent with that disclosed in CN201720773638.8, wherein the main difference points are that quantum dot fluorescent microspheres marked by antibodies are arranged in a reaction zone, and three antibody-capturing sites corresponding to the antibodies are arranged on a detection channel; the specific antibody arrangement mode of synchronously and jointly detecting procalcitonin and IL-6 of the micro-fluidic chip based on the quantum dot fluorescent microspheres and the specific structure is combined with the kit structure of CN201720773638.8 for the first time, so that the final kit completely meets the technical effects of realizing high sensitivity, rapidness and accuracy joint detection of two biomarkers of PCT and IL-6.
Specific preparation examples of the kit of the present application are described below, and the raw materials mentioned in the examples of the present application can be purchased on the market without specific description.
Examples
(1) Labeling a PCT antibody by using quantum dot fluorescent microspheres: activating quantum dot fluorescent microspheres (QBC 620, Shanghai Kun Dai Biotechnology limited) in 0.25 mg/mL EDC-NHS reagent for 10-20 min, adding 0.3 mg/mL PCT antibody, performing shaking table reaction for 2 h, ultrasonically mixing uniformly, performing centrifugal cleaning after the reaction is finished, adding 0.1% BSA (bovine serum albumin) as a confining liquid for reaction for 2 h, performing centrifugal cleaning, and collecting a microsphere storage solution;
(2) marking an IL-6 antibody by using quantum dot fluorescent microspheres: activating quantum dot fluorescent microspheres in 0.25 mg/mL EDC-NHS for 10-20 min, adding 0.2 mg/mL IL-6 antibody, reacting for 2 h in a shaker, ultrasonically mixing uniformly, after the reaction is finished, centrifugally cleaning, adding confining liquid 0.1% BSA for reacting for 2 h, centrifugally cleaning, and collecting microsphere storage liquid;
(3) marking chicken IgY antibody with quantum dot fluorescent microspheres: activating quantum dot fluorescent microspheres in 0.25 mg/mL EDC-NHS for 10-20 min, adding 0.2 mg/mL IL-6 chicken IgY antibody, reacting for 2 h in a shaking table, ultrasonically mixing uniformly, after the reaction is finished, centrifugally cleaning, adding confining liquid 0.1% BSA for reacting for 2 h, centrifugally cleaning, and collecting microsphere storage liquid;
(4) assembling the microfluidic chip: after hydrophilic treatment is carried out on the micro-fluidic chip by using air plasma, the micro-fluidic chip is dried, and 0.3-0.5 muL of stationary phase antibody (namely capture antibody, wherein the capture antibody is 0.3-0.5 muL, 1.5 mg/mL of PCT capture antibody or IL-6 capture antibody or anti-chicken IgY capture antibody; also can be described as 0.3-0.5 muL, 1.5 mg/mL of PCT antibody or IL-6 antibody or anti-chicken IgY) is loaded in sequence; sealing the surface of the chip by using 0.1% BSA, adding the fluorescent microsphere mixed solution obtained in the steps (1), (2) and (3) into a reaction area (the three antibodies are added after being mixed, the mixing ratio of the three antibodies is 2: 2: 1), wherein the sample amount of the total mixed microspheres in the reaction area is 0.66 mg/mL (0.272 mg/mL labeled PCT microspheres +0.272 mg/mL labeled IL-6 microspheres +0.136mg/mL labeled chicken IgY microspheres), and 3.3 muL; after the sample application of the chips is finished, the ultrasonic waves are used for finishing the combination;
(5) preparation of a standard substance: diluting the antigen concentrated solution by using a standard substance diluent to obtain a standard substance antigen mother solution for later use; the standard dilution was 10% BSA solution (i.e., 10% BSA final concentration in standard dilution buffer), the standard dilution buffer was pH = 8.550 mM BB, and was stored at 2-8 ℃; the standard substance comprises a standard substance A and a standard substance B, wherein the standard substance A is PCT antigen mother liquor diluted to 100 ng/mL and stored at-20 ℃; the standard substance B is IL-6 antigen mother liquor diluted to 1500 pg/mL and stored at the temperature of minus 20 ℃;
(6) and (3) standard curve drawing detection performance evaluation: taking a standard substance antigen mother solution, diluting the standard substance antigen mother solution by using a standard substance diluent according to a certain dilution ratio, or obtaining PCT and IL-6 standard substance antigen diluents with a certain gradient concentration, establishing a standard curve according to the relation between the concentrations of the antigen diluents with different concentrations and corresponding signal intensities, evaluating the linear range, the detection capability, the stability and other performance indexes of the kit, and comparing the performance indexes with clinical samples; specifically, fig. 2 is a dual kit PCT detection standard curve, fig. 3 is a dual kit PCT standard calibration curve, fig. 4 is a dual kit IL-6 detection standard curve, and fig. 5 is a dual kit IL-6 clinical sample calibration curve.
TABLE 1 detection of PCT/IL-6 Signal intensity and stability by the duplex detection kit
(7) Analysis of results
The kit has the advantages that the lowest detection concentration of PCT is 0.05 ng/ml, the detection range is 0.05-100 ng/ml (r 2= 0.99), the relative standard deviation is 5.5-14.55%, and the detection of clinical samples r2= 0.93. The minimum concentration of IL-6 detection is 3 pg/ml, the detection range is 3-1500 pg/ml (r 2= 0.99), the relative standard deviation is 6.1-17%, and the clinical sample detection r2= 0.93.
Therefore, the above examples of the present application demonstrate that the present invention can realize the high-sensitivity, rapid and accurate combined detection of two biomarkers of PCT and IL-6.
Claims (10)
1. A PCT/IL-6 duplex detection kit based on quantum dot fluorescent microspheres is characterized in that: the kit comprises a reaction area, a first detection site, a second detection site and a third detection site, wherein an antibody-labeled quantum dot fluorescent microsphere is arranged in the reaction area, the first detection site is coated with an IL-6 capture antibody, the second detection site is coated with a PCT capture antibody, and the third detection site is coated with an anti-chicken IgY capture antibody.
2. The quantum dot fluorescent microsphere-based PCT/IL-6 duplex detection kit of claim 1, wherein: the kit comprises an upper shell, a lower shell and a microfluidic chip area positioned on the lower shell; the microfluidic chip area comprises a sample receiving area, a sample storage area, a reaction area, a time valve, a detection channel and a waste liquid area, wherein the sample receiving area, the sample storage area, the reaction area, the time valve, the detection channel and the waste liquid area are sequentially connected along the flow direction of a medium; the detection channel is sequentially provided with a first detection site, a second detection site and a third detection site along the medium flowing direction.
3. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres as claimed in claim 1, which is characterized in that: the method comprises the following steps:
(1) marking the quantum dot fluorescent microspheres: firstly, activating quantum dot fluorescent microspheres, then adding a PCT antibody or an IL-6 antibody or a chicken IgY antibody into an activated quantum dot fluorescent microsphere solution, ultrasonically mixing uniformly for marking, centrifugally cleaning after reaction is finished, adding a sealing liquid for sealing, centrifugally cleaning the sealed fluorescent microspheres to obtain antibody-marked quantum dot fluorescent microspheres, and then storing by using a microsphere storage liquid;
(2) assembling the microfluidic chip: after the surface of the microfluidic chip is subjected to hydrophilic treatment, drying the microfluidic chip in an oven at 37 ℃, and loading a capture antibody at a corresponding position of a channel; sealing the surface of the chip, adding the quantum dot fluorescent microsphere solution prepared in the step (1) into a reaction zone, drying in a 37 ℃ oven, and performing ultrasonic bonding to complete combination after the sample application of the chip is completed;
(3) preparation of a standard substance:
diluting the antigen concentrated solution by using a standard substance diluent to obtain a standard substance antigen mother solution for later use; the standard substance diluent is 10% BSA solution, and is preserved at 2-8 deg.C; the standard substance comprises a standard substance A and a standard substance B, wherein the standard substance A is PCT antigen mother liquor diluted to 100 ng/mL and stored at the temperature of-20 ℃, and the standard substance B is IL-6 antigen mother liquor diluted to 1000 pg/mL and stored at the temperature of-20 ℃;
obtaining the quantum dot fluorescent microsphere-based PCT/IL-6 duplex detection kit.
4. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres as claimed in claim 3, which is characterized in that: the activation treatment of the quantum dot fluorescent microspheres in the step (1) comprises the following steps: and (3) placing the quantum dot fluorescent microspheres in 0.25 mg/mL EDC-NHS, and activating for 10-20 min, wherein the 0.25 mg/mL EDC-NHS is the final concentration of the EDC-NHS in the buffer solution.
5. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres as claimed in claim 3, which is characterized in that: the blocking solution in the step (1) is BSA with the mass percent of 0.1%, and the 0.1% BSA refers to the final concentration of the BSA in the buffer solution.
6. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres as claimed in claim 3, which is characterized in that: in the step (1), the final concentration of the PCT antibody in the activated quantum dot fluorescent microsphere solution is 0.3 mg/mL, the final concentration of the IL-6 antibody in the activated quantum dot fluorescent microsphere solution is 0.2 mg/mL, the final concentration of the chicken IgY antibody in the activated quantum dot fluorescent microsphere solution is 0.2 mg/mL, and the activated quantum dot fluorescent microsphere solution is an activated quantum dot fluorescent microsphere solution containing a buffer solution.
7. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres according to any one of claims 4 to 6, which is characterized in that: the buffers used in the activation, labeling and blocking processes in step (1) are all 50mM HEPES with pH = 7.4; the microsphere stock solution in step (1) contained 10 mg/mL BSA, 10 mg/mL trehalose.
8. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres as claimed in claim 3, which is characterized in that: the capture antibody in the step (2) is 0.3-0.5 muL and 1.5 mg/mL of PCT capture antibody or IL-6 capture antibody or anti-chicken IgY capture antibody; the chip surface was blocked with 0.1% BSA; the total mixed microsphere spot size in the reaction zone was 0.66 mg/mL, 3.3. mu.L.
9. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres according to claim 8, which is characterized in that: the total mixed microsphere sample amount of the reaction area is 0.66 mg/mL, and 3.3 muL refers to the concentration and the use amount formed by mixing 0.272 mg/mL marked PCT microspheres, 0.272 mg/mL marked IL-6 microspheres and 0.136mg/mL marked chicken IgY microspheres respectively.
10. The preparation method of the PCT/IL-6 duplex detection kit based on the quantum dot fluorescent microspheres as claimed in claim 3, which is characterized in that: the standard dilution of step (3) is a 10% BSA solution, which means that BSA is added to a final concentration of a standard dilution buffer, which is a BB buffer of pH =8.5 and 50 mM.
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