CN109187450B - Biomolecule concentration detection method based on quantum dots - Google Patents
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Abstract
The invention discloses a biomolecule concentration detection method based on quantum dots, which comprises the following steps: coupling a quantum dot with an antibody of a target biomolecule to be detected, mixing and culturing a quantum dot-antibody stock solution and a solution to be detected containing the target biomolecule to combine the quantum dot-antibody with the target biomolecule, passing the mixed solution through a microfluidic channel, and controlling the flow of the mixed solution to enable one quantum dot-antibody or one quantum dot-antibody-biomolecule to pass through the microfluidic channel; measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or the quantum dot-antibody-biomolecule passing through the microfluidic channel, and distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule; and calculating the concentration of the target biomolecule in the solution to be detected. The method can detect the biomolecule with fM concentration, and the detection speed is greatly improved.
Description
Technical Field
The invention relates to a biomolecule concentration detection method, in particular to a biomolecule concentration detection method based on quantum dots.
Background
The high-speed development of the society greatly improves the living standard of people, and puts forward new requirements for living sanitation, disease prediction and the like, however, the existing detection means for the biological molecules is difficult to realize rapid identification and measurement, so that the practical application is difficult to break through in aspects such as bacteria monitoring in drinking water, bacterial drug resistance investigation, identification and quantification of cancer biomarkers and the like. Methods used in the prior art for identifying and quantifying low concentrations of biomolecules include:
liquid chromatography (Liquid chromatography) and mass spectrometry (LC-MS): LC physically separates components in the liquid solution; MS provides structural characteristics of each component by measuring the mass-to-charge ratio of each component. Enzyme-linked immunosorbent assay (ELISA): antibody-based immunoassays, such as ELISA, can be used to detect the presence of an antigen in a sample. To quantify the antigen in solution, the antigen is first immobilized on a solid surface (polystyrene microtiter plate) by direct adsorption to the surface or using "capture antibodies" that have been coated on the surface. Specific primary antibodies linked to enzymes are then added to bind the immobilized antigen. Finally, a substrate for the enzyme is added, the color of which changes upon reaction with the enzyme.
Western blotting: detection and quantification of proteins in lysed cells and tissues is usually performed by western blotting, which mainly comprises the steps of sample preparation, gel/transfer/blocking, primary antibody binding and signal detection.
Flow cytometry, Fluorescence In Situ Hybridization (FISH), Polymerase Chain Reaction (PCR): the identification of microorganisms in water is generally carried out by molecular techniques such as flow cytometry, magnetic separation techniques, Fluorescence In Situ Hybridization (FISH), Polymerase Chain Reaction (PCR) and microarray.
The quantum dot (abbreviated as QD) can be produced industrially, the quantum dot can be coupled with an antibody (abbreviated as Ab) to form a quantum dot-antibody (QD-Ab) coupling body through surface modification, and the quantum dot is coupled with a biomolecule to be detected in a targeted manner through the Ab on the QD-Ab coupling body. The quantum dots emit fluorescence under the irradiation of external exciting light so as to calibrate the targeted biomolecules. In the prior art, a QD-Ab-biomolecule and a QD-Ab coupling body are distinguished by detecting the change of fluorescence intensity or fluorescence wavelength of quantum dots, so that the aim of detecting a target biomolecule is fulfilled. However, in theory, the change in fluorescence intensity and wavelength of the QD-Ab before and after coupling to the biomolecule is small. And in practical optical measurement applications, quantitative measurement of small changes in fluorescence intensity and fluorescence wavelength is difficult to achieve. Meanwhile, the synthesis of quantum dots is influenced by many experimental conditions, and even the quantum dots grown in the same batch are difficult to find the quantum dots with the same fluorescence intensity and wavelength, so the quantum dots for biomolecule detection have the nonuniformity of fluorescence intensity and wavelength, which brings insurmountable difficulty for the realization of an instrument for detecting biomolecules by using the change of the fluorescence intensity and wavelength of the quantum dots. Therefore, the instrument for quantum dot biological detection based on the method is proposed from about 2000 years, but the marketization of the instrument is not realized at present, which explains that QD-Ab-biomolecule is difficult to carry out quantitative recognition by measuring fluorescence intensity and wavelength change after quantum dots are coupled with the biomolecule from another aspect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a biomolecule concentration detection method based on quantum dots, which solves the problem of QD-Ab-biomolecule and QD-Ab identification and realizes the rapid quantitative identification of low-concentration biomolecules.
The technical scheme of the invention is as follows: a biomolecule concentration detection method based on quantum dots comprises the following steps:
s1, coupling the quantum dot with an antibody of a target biomolecule to be detected to form a quantum dot-antibody stock solution;
s2, mixing and culturing the quantum dot-antibody stock solution and the solution to be detected containing the target biomolecule to combine the quantum dot-antibody and the target biomolecule to obtain a mixed solution containing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s3, enabling the mixed solution obtained in the step 2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or quantum dot-antibody-biomolecule passing through the microfluidic channel, and distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s5, counting the flow time of the mixed solution when the quantum dot-antibody-biomolecule number is in the effective concentration identification number, obtaining the volume of the mixed solution according to the flow time and the flow speed of the mixed solution, and calculating the concentration of the target biomolecule in the solution to be detected according to the quantum dot-antibody-biomolecule number and the volume of the mixed solution.
Further, the step S4 of distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule is to calculate the pulse width probability density of the fluorescence pulse according to the photon pulse sequence generated by the continuous light excitation of the quantum dot-antibody and the photon pulse sequence generated by the excitation of the quantum dot-antibody-biomolecule.
Further, the step S4 of distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule is to calculate a fluorescence pulse correlation spectrum according to the photon pulse sequence generated by the continuous light excitation of the quantum dot-antibody and the photon pulse sequence generated by the excitation of the quantum dot-antibody-biomolecule.
Further, the step S4 of distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule is to calculate the pulse interval probability density of the fluorescence pulse according to the photon pulse sequence generated by the continuous light excitation of the quantum dot-antibody and the photon pulse sequence generated by the excitation of the quantum dot-antibody-biomolecule.
Further, the step of calculating the width probability density of the fluorescence pulse for distinguishing is to perform ms-level sampling on the photon pulse sequence and calculate the pulse width probability density of the fluorescence pulse with the pulse width of more than 1ms for distinguishing.
Further, the calculating the fluorescence pulse correlation spectrum is to perform 10 the photon pulse sequence-7s-level sampling and calculating correlation by the following formula
Wherein g isA(τ) is the correlation value, IA(t) is the time-resolved fluorescence spectrum intensity, τ is the lag time,<>tis averaged over time with a lag time of 10-7～100s。
Further, the step of calculating the pulse interval probability density of the fluorescence pulse for distinguishing is to perform ns-level sampling on the photon pulse sequence and calculate the pulse interval probability density with the pulse interval of 1-100 ns for distinguishing.
Further, the photon pulse sequence generated according to the continuous light excitation quantum dot-antibody and the photon pulse sequence generated according to the excitation quantum dot-antibody-biomolecule are the photon pulse sequence generated according to the continuous light excitation quantum dot-antibody within a time span and the photon pulse sequence generated according to the excitation quantum dot-antibody within a time span, and the time span of the pulse sequences is 1 ms-1 s.
Preferably, the coupling of the quantum dot and the antibody of the target biomolecule to be detected is to directly couple the antibody and the quantum dot by using ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, wherein the surface of the quantum dot is activated by carboxyl, the surface of the antibody is activated by amino, and the quantum dot and the antibody are coupled to form an amide bond.
Preferably, the coupling of the quantum dot to the antibody of the target biomolecule to be detected is performed by covalent reaction of amino and thiol using succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate coupling reaction to couple the thiol-activated quantum dot to the amino-activated antibody.
Further, the method comprises the following steps:
s1, coupling the multiple quantum dots with antibodies of multiple target biomolecules to be detected respectively, so that the same quantum dot is coupled with the same antibody to form multiple quantum dot-antibody stock solutions;
s2, mixing and culturing various quantum dot-antibody stock solutions and a solution to be detected containing various target biomolecules to combine the quantum dot-antibody with the target biomolecules to obtain a mixed solution containing various quantum dot-antibodies and quantum dot-antibody-biomolecules;
s3, enabling the mixed solution obtained in the step 2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or quantum dot-antibody-biomolecule passing through the microfluidic channel, and distinguishing different quantum dot-antibodies and different quantum dot-antibody-biomolecules;
s5, counting the flow time of the mixed solution when the number of the single quantum dots-antibody-biomolecules reaches the effective identification number of the concentrations, obtaining the volume of the mixed solution according to the flow time of the mixed solution and the flow speed of the mixed solution, and calculating the concentration of the target biomolecule corresponding to the quantum dots-antibody-biomolecules in the solution to be detected according to the number of the quantum dots-antibody-biomolecules and the volume of the mixed solution.
The technical scheme provided by the invention has the advantages that: the quantum dot-antibody and the quantum dot-antibody-biomolecule are distinguished by the time-resolved single photon characteristic spectrum, so that the identification error caused by the individual difference of the quantum dots in the distinguishing mode of the fluorescence intensity and the fluorescence wavelength can be avoided, and the problem that the distinguishing is difficult due to the difference of the fluorescence intensity and the fluorescence wavelength which are too small is also avoided. The quantum dot-antibody and the quantum dot-antibody-biomolecule can be distinguished and distinguished in a very short time by distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule by the time-resolved single photon characteristic spectrum, so that the method is suitable for measuring trace biomolecule concentration liquid, the biomolecule with fM concentration can be detected in hours, and the detection speed is greatly improved.
Drawings
FIG. 1 is a flow chart of a biomolecule concentration detection method based on quantum dots.
Fig. 2 is a photon pulse sequence diagram of continuously photoexcited quantum dot-antibody and quantum dot-antibody-biomolecule.
Fig. 3 is a pulse width probability density plot of photon pulse sequences for quantum dot-antibody and quantum dot-antibody-biomolecule.
Fig. 4 is a correlation spectrum of photon pulse sequences of quantum dot-antibody and quantum dot-antibody-biomolecule.
Fig. 5 is a pulse width interval probability density plot of photon pulse sequences for quantum dot-antibody and quantum dot-antibody-biomolecule.
Fig. 6 is a correlation spectrum of photon pulse sequences at a ratio of green quantum dot-antibody-biomolecule to red quantum dot-antibody-biomolecule of 7.5: 2.5.
Fig. 7 is a correlation spectrum of photon pulse sequences at a green quantum dot-antibody-biomolecule to red quantum dot-antibody-biomolecule ratio of 2.5: 7.5.
Detailed Description
The present invention is further described in the following examples, which are intended to be illustrative only and not to be limiting as to the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalent modifications thereof which would occur to persons skilled in the art upon reading the present specification and which are intended to be within the scope of the present invention as defined in the appended claims.
Please refer to fig. 1, example 1
The method for detecting the concentration of the biomolecule based on the quantum dot comprises the following steps:
s1, coupling the water-soluble quantum dot QD surface modification (carboxyl and sulfydryl) with an antibody of a target biomolecule to be detected through different cross-linking agents:
EDC (ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) is utilized to directly couple an antibody and a quantum dot, wherein the surface of the quantum dot is activated by carboxyl, the surface of the antibody is activated by amino, and the antibody and the quantum dot form an amido bond after being coupled to form a quantum dot-antibody stock solution.
S2, stirring the quantum dot-antibody stock solution and the solution to be detected containing the target biomolecule for 20 minutes, and performing mixed culture to combine the quantum dot-antibody and the target biomolecule to obtain a mixed solution containing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s3, enabling the mixed solution obtained in the step 2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or the quantum dot-antibody-biomolecule passing through the microfluidic channel, and according to the photon pulse sequence generated by the continuous light excitation quantum dot-antibody in the ms level and the photon pulse sequence generated by the excitation quantum dot-antibody-biomolecule in the ms level, as shown in figure 2, the sampling time length is 5.2 ms. Sampling is performed in ms level, and the pulse width probability density of the fluorescent pulse with the pulse width of more than 1ms is calculated, so that the result shown in fig. 3 is obtained, and the quantum dot-antibody or quantum dot-antibody-biomolecule is distinguished by the difference of the pulse widths corresponding to the same width probability density.
S5, counting the flow time of the mixed solution when the quantum dot-antibody-biomolecule number is counted to the effective identification number of the concentration, wherein the effective identification number of the concentration is generally 600 times, obtaining the volume of the mixed solution according to the flow time of the mixed solution and the flow rate of the mixed solution, and calculating the concentration of the target biomolecule in the solution to be detected by dividing the counted quantum dot-antibody-biomolecule number by the volume of the mixed solution.
Example 2
The method for detecting the concentration of the biomolecule based on the quantum dot comprises the following steps:
s1, coupling the water-soluble quantum dot QD surface modification (carboxyl and sulfydryl) with an antibody of a target biomolecule to be detected through different cross-linking agents:
and (2) coupling the sulfydryl activated quantum dot with the amino activated antibody by using SMCC (succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate European Union reaction, and performing covalent reaction of amino and sulfydryl to couple the antibody and the quantum dot to form a quantum dot-antibody stock solution.
S2, stirring the quantum dot-antibody stock solution and the solution to be detected containing the target biomolecule for 20 minutes, and performing mixed culture to combine the quantum dot-antibody and the target biomolecule to obtain a mixed solution containing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s3, enabling the mixed solution obtained in the step 2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or quantum dot-antibody-biomolecule passing through the microfluidic channel, and exciting the quantum dot-antibody at 10 deg.C according to continuous light-7Photon pulse sequence generated in s-level and excited quantum dot-antibody-biomolecule 10-7sequence of s-order generated photon pulses, proceed 10-7s-level sampling and calculating correlation by the following formula
Wherein g isA(τ) is the correlation value, IA(t) is the time-resolved fluorescence spectrum intensity, τ is the lag time,<>tis averaged over time with a lag time of 10-7～100s, the results shown in fig. 4 were obtained, and the quantum dot-antibody or quantum dot-antibody-biomolecule was distinguished by the difference between the correlation peak and the half-peak width.
S5, counting the flow time of the mixed solution when the quantum dot-antibody-biomolecule number is counted to the effective identification number of the concentration, wherein the effective identification number of the concentration is generally 600 times, obtaining the volume of the mixed solution according to the flow time of the mixed solution and the flow rate of the mixed solution, and calculating the concentration of the target biomolecule in the solution to be detected by dividing the counted quantum dot-antibody-biomolecule number by the volume of the mixed solution.
Example 3
The method for detecting the concentration of the biomolecule based on the quantum dot comprises the following steps:
s1, coupling the water-soluble quantum dot QD surface modification (carboxyl and sulfydryl) with an antibody of a target biomolecule to be detected through different cross-linking agents:
and (2) coupling the sulfydryl activated quantum dot with the amino activated antibody by using SMCC (succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate European Union reaction, and performing covalent reaction of amino and sulfydryl to couple the antibody and the quantum dot to form a quantum dot-antibody stock solution.
S2, stirring the quantum dot-antibody stock solution and the solution to be detected containing the target biomolecule for 20 minutes, and performing mixed culture to combine the quantum dot-antibody and the target biomolecule to obtain a mixed solution containing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s3, enabling the mixed solution obtained in the step 2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or quantum dot-antibody-biomolecule passing through the microfluidic channel, and exciting the photon pulse sequence generated by the quantum dot-antibody in ns level and the photon pulse sequence generated by the quantum dot-antibody-biomolecule in ns level according to continuous light. Sampling is carried out at 0.1ns level, pulse interval probability density with pulse interval of 1-100 ns is calculated, results shown in figure 5 are obtained, and quantum dot-antibody or quantum dot-antibody-biomolecule is distinguished according to different half peak widths of pulse interval probability density spectrums.
S5, counting the flow time of the mixed solution when the quantum dot-antibody-biomolecule number is counted to the effective identification number of the concentration, wherein the effective identification number of the concentration is generally 600 times, obtaining the volume of the mixed solution according to the flow time of the mixed solution and the flow rate of the mixed solution, and calculating the concentration of the target biomolecule in the solution to be detected by dividing the counted quantum dot-antibody-biomolecule number by the volume of the mixed solution.
Troponin I is a key cardiac and skeletal muscle protein for clinical diagnosis of heart disease, and for this example, 5-10 ng/l troponin I (ca24 kDa) is detected, and this level is indicative of an increased probability of myocardial injury. 1pM of QD antibody was added to each 1nL of blood sample, followed by incubation for 20 minutes, and the 1nL and 1000nL of the mixture were measured, respectively, as follows:
by adopting the method, various biomolecules can be measured simultaneously, different quantum dots (red and green quantum dots in the embodiment) are adopted to be coupled with antibodies of different biomolecules, a quantum dot-antibody stock solution and a solution to be measured containing target biomolecules are stirred for 20 minutes, mixed culture is carried out to combine the quantum dot-antibody with the target biomolecules, and a mixed solution containing the quantum dot-antibody and the quantum dot-antibody-biomolecules is obtained; controlling the flow rate of the mixed solution and enabling one quantum dot-antibody or quantum dot-antibody-biomolecule to pass through the microfluidic channel; measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or the quantum dot-antibody-biomolecule passing through the microfluidic channel, and calculating the correlation spectrum of the photon pulse sequence according to the photon pulse sequence generated by continuous light excitation of the quantum dot-antibody and the photon pulse sequence generated by excitation of the quantum dot-antibody-biomolecule. The correlation spectra of the photon pulse sequences of two quantum dots-antibody-biomolecules when red quantum dots and green quantum dots are mixed in different ratios (7.5:2.5 and 2.5:7.5) are shown in fig. 6 and 7, the green quantum dots are small and light, the diffusion coefficient is large, and the lag time in the correlation spectra of the emitted photon pulse sequences is shorter than that of the red quantum dots. The types and the quantities of the biomolecules corresponding to different quantum dots can be distinguished by comparing the photon lag time correlation diagrams.
Claims (7)
1. A biomolecule concentration detection method based on quantum dots is characterized by comprising the following steps:
s1, coupling the quantum dot with an antibody of a target biomolecule to be detected to form a quantum dot-antibody stock solution;
s2, mixing and culturing the quantum dot-antibody stock solution and the solution to be detected containing the target biomolecule to combine the quantum dot-antibody and the target biomolecule to obtain a mixed solution containing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s3, enabling the mixed solution obtained in the step S2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or the quantum dot-antibody-biomolecule passing through the microfluidic channel, and calculating the pulse width probability density of the fluorescence pulse according to the photon pulse sequence generated by continuous light excitation of the quantum dot-antibody and the photon pulse sequence generated by excitation of the quantum dot-antibody-biomolecule; or calculating a fluorescence pulse correlation spectrum for distinguishing according to a photon pulse sequence generated by the continuous light excitation quantum dot-antibody and a photon pulse sequence generated by the excitation quantum dot-antibody-biomolecule; or calculating the pulse interval probability density of the fluorescence pulse according to the photon pulse sequence generated by exciting the quantum dot-antibody by continuous light and the photon pulse sequence generated by exciting the quantum dot-antibody-biomolecule, and distinguishing the quantum dot-antibody and the quantum dot-antibody-biomolecule;
s5, counting the flow time of the mixed solution when the quantum dot-antibody-biomolecule number is in the effective concentration identification number, obtaining the volume of the mixed solution according to the flow time and the flow speed of the mixed solution, and calculating the concentration of the target biomolecule in the solution to be detected according to the quantum dot-antibody-biomolecule number and the volume of the mixed solution.
2. The method of claim 1, wherein the step of calculating the width probability density of the fluorescence pulse for discrimination is performed by sampling the photon pulse sequence in ms-order and calculating the pulse width probability density of the fluorescence pulse having a pulse width of 1ms or more.
3. The method of claim 1, wherein the step of differentiating the calculated fluorescence pulse correlation spectrum is a step of performing 10 on the photon pulse sequence-7s-level sampling and calculating correlation by the following formula
Wherein g isA(τ) is the correlation value, IA(t) is the time-resolved fluorescence spectrum intensity, τ is the lag time,<>tis averaged over time with a lag time of 10-7～100s。
4. The method for detecting the concentration of the biomolecule based on the quantum dots, according to claim 1, wherein the step of calculating the pulse interval probability density of the fluorescence pulse for distinguishing is to sample the photon pulse sequence in 0.1ns order and calculate the pulse interval probability density with the pulse interval of 1-100 ns for distinguishing.
5. The method of any one of claim 1, wherein the photon pulse sequence generated from the sequential photoexcited quantum dot-antibody and the photon pulse sequence generated from the excited quantum dot-antibody-biomolecule are a photon pulse sequence generated from the sequential photoexcited quantum dot-antibody within a time span and a photon pulse sequence generated from the excited quantum dot-antibody-biomolecule within a time span, and the time span of the pulse sequences is 1ms to 1 s.
6. The method for detecting the concentration of biomolecules based on quantum dots according to claim 1, wherein the coupling of the quantum dots with the antibody of target biomolecules to be detected is directly coupling the antibody and the quantum dots by using ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, wherein the surface of the quantum dots is activated by carboxyl groups, the surface of the antibody is activated by amino groups, and the quantum dots are coupled with the antibody to form amide bonds; or the covalent reaction of amino and sulfhydryl is carried out by using succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate coupling reaction to couple the sulfhydryl activated quantum dot with the amino activated antibody.
7. A biomolecule concentration detection method based on quantum dots is characterized by comprising the following steps:
s1, coupling the multiple quantum dots with antibodies of multiple target biomolecules to be detected respectively, so that the same quantum dot is coupled with the same antibody to form multiple quantum dot-antibody stock solutions;
s2, mixing and culturing various quantum dot-antibody stock solutions and a solution to be detected containing various target biomolecules to combine the quantum dot-antibody with the target biomolecules to obtain a mixed solution containing various quantum dot-antibodies and quantum dot-antibody-biomolecules;
s3, enabling the mixed solution obtained in the step S2 to pass through a microfluidic channel, controlling the flow rate of the mixed solution, and enabling a quantum dot-antibody or a quantum dot-antibody-biomolecule to pass through the microfluidic channel;
s4, measuring the time-resolved single photon characteristic spectrum of the quantum dot-antibody or the quantum dot-antibody-biomolecule passing through the microfluidic channel, and calculating the pulse width probability density of the fluorescence pulse according to the photon pulse sequence generated by continuous light excitation of the quantum dot-antibody and the photon pulse sequence generated by excitation of the quantum dot-antibody-biomolecule; or calculating a fluorescence pulse correlation spectrum for distinguishing according to a photon pulse sequence generated by the continuous light excitation quantum dot-antibody and a photon pulse sequence generated by the excitation quantum dot-antibody-biomolecule; or according to the photon pulse sequence generated by exciting the quantum dot-antibody by continuous light and the photon pulse sequence generated by exciting the quantum dot-antibody-biomolecule, calculating the pulse interval probability density of the fluorescence pulse, and distinguishing different quantum dot-antibodies and different quantum dot-antibody-biomolecules;
s5, counting the flow time of the mixed solution when the number of the single quantum dots-antibody-biomolecules reaches the effective identification number of the concentrations, obtaining the volume of the mixed solution according to the flow time of the mixed solution and the flow speed of the mixed solution, and calculating the concentration of the target biomolecule corresponding to the quantum dots-antibody-biomolecules in the solution to be detected according to the number of the quantum dots-antibody-biomolecules and the volume of the mixed solution.
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