High-sensitivity single-particle colorimetric detection heparin, method and application

Abstract

Establishment and application of a method for detecting heparin by a single-particle level high-sensitivity colorimetric method are as follows: firstly, respectively marking quantum dots with different emission wavelengths by antithrombin III and protamine, and forming a sandwich compound through affinity interaction between the thrombin III and the heparin and electrostatic interaction between the heparin and the protamine; and then judging the formation of a compound through primary spectral stripes of the two quantum dots, and establishing a new heparin detection method on the basis of correlation between the formation quantity of the two quantum dot compounds and the heparin concentration. The linear range of heparin detection is 0.001-0.02 U / mL, and the detection limit is 5.4 * 10 <-6 > U / mL. Meanwhile, the method is used for carrying out heparin labeling recovery detection on the diluted serum, and the recovery rate is 92-106%. The method has the advantages of excellent detection performance, high sensitivity and intuitive and rapid detection result, and provides a new method choice for clinical heparin detection.

Description

Technical Field

[0001] The present invention relates to the establishment and application of a quantum dot single particle imaging analysis technology, belonging to the field of chemical detection technology, and specifically to the establishment and application of a quantum dot single particle imaging analysis technology. Background Art

[0002] Heparin is a linear mucopolysaccharide that is highly acetylated and sulfated. It is one of the most electronegative substances in nature. The pentasaccharide sequence of heparin can react with antithrombin III in a 1:1 ratio and is widely used as an anticoagulant in the treatment of cardiovascular diseases and hemodialysis

[95] . However, studies have shown that heparin drugs can inhibit the number of platelets. Long-term use of heparin anticoagulants can lead to problems such as spontaneous bleeding, inhibition of platelet count, and even a series of complications such as intracranial hemorrhage. Under normal circumstances, the heparin content in the blood during cardiovascular surgery is 2-8U / ml (17-67μM), and the heparin content in the blood after surgery and during long-term care is 0.2-1.2U / ml (1.7-10μM). Therefore, at this stage, the accurate quantification of heparin concentration and activity in the blood is still of great significance for disease prevention and clinical treatment.

[0003] The traditional method of detecting heparin is called the coagulation time method, including the activated thrombin time method (ACT) and the activated partial thromboplastin time method (aPPT), but this type of method can only identify heparin but cannot accurately quantify heparin. In recent years, a series of sensor methods for accurately quantifying heparin have been established, such as using heparin to restore the quenched fluorescence system or induce the quenching of the fluorescence system, or using heparin to induce the aggregation or dissociation of fluorescent substances and gold and silver nanoparticles, thereby generating signal enhancement or weakening. Such methods generally require the preparation of signal probes and require complex sample processing to achieve accurate determination of the heparin content in blood samples.

[0004] The single particle detection technology based on quantum dots can detect and image samples at the single molecule level, and has the advantages of high selectivity, high sensitivity, high signal-to-noise ratio, low consumption and rapid detection. A grating is added in front of the detector (such as EMCCD), and the bleaching order of the primary spectrum is used to identify the size of quantum dot aggregates. By converting the fluorescence signal of quantum dots into spectral signals, the detection method is greatly simplified, making the detection of quantum dot complexes extremely convenient. This method can achieve specific recognition and detection of heparin at the single molecule level. However, there are still some problems: the self-aggregation of quantum dots brings very strong background noise and low signal-to-noise ratio; excessive heparin causes the aggregates to be too large, and quantum dot aggregates cannot be identified; the blue shift of the quantum dot spectrum is not obvious, and data processing is extremely troublesome; single signal identification of heparin is not accurate enough, etc. Therefore, it is very necessary to develop a simple and accurate heparin quantification method in biological samples. Summary of the invention

[0005] Based on this, the present invention provides a highly sensitive single particle colorimetric detection of heparin, a method and an application thereof, so as to solve the problems of complicated detection process and low sensitivity in the existing heparin detection method in the prior art.

[0006] To achieve the above object, the present invention provides a highly sensitive single particle colorimetric detection of heparin, a method and an application thereof, the innovation of which is that it comprises the following steps:

[0007] Step S1: QD545 labeling of antithrombin III

[0008] After activating 1.2-1.3 μL of QD545 with a concentration of 8 μmol by 8-11 μL of EDC with a concentration of 13 mg / mL for 14-15 min, 478.75 μL of 10 mmol of boric acid buffer solution with a pH value of 7.4 and 9-10 μL of 100 IU of thrombin III were added to form a mixed solution, the mixed solution was allowed to stand at 4°C-5°C for 10-12 hours, and then ultrafiltered with a 100 kDa ultrafiltration tube at a speed of 14000 r / min for 5 minutes, and finally the mixed solution was washed three times with 9-10 ml of boric acid buffer solution with a pH value of 8.3 for standby use;

[0009] Step S2: QD655 labeling of protamine

[0010] After activating 1.2-1.3 μL of QD545 with a concentration of 8 μmol by 8-11 μL of QD545 with a concentration of 13 mg / mL for 14-15 min, 478.75 μL of 10 mmol boric acid buffer solution with a pH value of 7.4 and 5-7 μL of 0.2 mg / mL protamine were added to form a mixed solution, the mixed solution was allowed to stand for 1-2 hours in an environment of 23° C.-25° C., and then ultrafiltered for 5 minutes using a 100 kDa ultrafiltration tube at a speed of 14000 r / min, and finally the mixed solution was washed three times with 10 ml of boric acid buffer solution with a pH value of 8.3 for standby use;

[0011] Step S3: Aggregation of QD545-labeled antithrombin III, heparin, and QD655-labeled protamine

[0012] Take 20 nanometers 9-10 μL of the mixed solution in step S1, add a 0.5-0.6U / mL heparin sodium standard solution, add 107.5 μL of boric acid buffer and let it stand for half an hour, then add 20 nanometers 9-10 μL of the mixed solution in step S2 to form an agglomeration solution, wherein the total agglomeration volume is 200 μL, let the agglomeration solution stand for 1 hour for standby use, and two quantum dots are formed in the agglomeration solution, namely QD655 quantum dots and QD545 quantum dots;

[0013] Step S4: The agglomerate solution in step 3 is imaged in an electron coupling device and an EMCCD spectrum imaging with a grating added to analyze the quantum dot dimer; the quantum dot image of the agglomerate solution in step S3 is taken, and the quantum dot QD655 is used as a benchmark, combined with the ratio of QD545 quantum dots as data, the quantum dot dimer is calculated, and then the quantum dot dimer is judged by the primary stripes of the quantum dot agglomerates, and a standard curve is drawn according to the heparin sodium concentration and the ratio of the formed quantum dot dimer.

[0014] Preferably, the EDC in step S1 and step S2 is prepared with cold secondary water and is ready for use.

[0015] Preferably, in step S3, the amount of the mixed solution in step S1 is 10 μL, wherein the heparin sodium standard solution is added with a concentration of 0.5 U / mL, and other heparin sodium standard solutions of different concentrations may also be added, and the amount of the mixed solution in step S2 is 10 μL.

[0016] Preferably, in step S1, 1.25 μL of QD545 is used for activation with 10 μL of LEDC for 15 min, 10 μL of thrombin III is used, and 9-10 mm of boric acid buffer solution with a pH value of 8.3 is used, wherein the mixed solution in step S1 is allowed to stand at 4°C for 12 hours.

[0017] Preferably, in step S2, 1.25 μL of QD545 is used for activation with 10 μL of LEDC for 15 min, 6 μL of protamine is used, and 10 ml of boric acid buffer solution with a pH value of 8.3 is used, wherein the mixed solution in step S1 is allowed to stand at 4°C for 12 hours.

[0018] Preferably, in step S3, 10 mm of boric acid buffer is added, and when the reaction temperature is 25° C., after the QD655 quantum dots and QD545 quantum dots are mixed for 1 hour, the aggregation ratio of the QD655 quantum dots and QD545 quantum dots is 1:1.

[0019] Preferably, in step S4, the quantum dot imaging is collected using a color electron coupling device, and then Image J is used to determine the agglomeration of the quantum dots.

[0020] Preferably, the amount of the boric acid buffer solution with a pH value of 8.3 is 10 mm.

[0021] The invention discloses a highly sensitive single particle colorimetric detection method for heparin.

[0022] A highly sensitive single particle colorimetric detection of heparin is used for application in heparin detection. A highly sensitive single particle colorimetric detection of heparin is used for application in heparin detection.

[0023] The advantages of the present invention are:

[0024] The present invention modifies antithrombin III and protamine respectively by dual-color quantum dots, the specific binding of antithrombin and heparin, and the electrostatic binding of heparin and protamine to form a sandwich structure, and detects heparin sensitively and specifically at the single molecule level by counting methods. Compared with previous methods, this detection system overcomes the influence of quantum dot agglomeration, specifically identifies heparin with ATIII, and the dual-color sandwich structure can achieve co-localization and specific identification. At the same time, the single molecule counting method is used to overcome the errors caused by the use of signals such as fluorescence intensity in the past. Therefore, this experiment realizes the detection of heparin activity while reducing the detection limit of heparin. It has certain demonstration significance for the construction of related sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1A is a schematic diagram of the technical principle of single-particle colorimetric detection of heparin in Example 2 of the present invention;

[0027] Figure 1B This is the aggregation of quantum dots without adding heparin in Example 2 of the present invention;

[0028] Figure 1C This is the aggregation of quantum dots in the presence of heparin in Example 2 of the present invention;

[0029] Figure 2 This is a trend diagram of the agglomeration of two quantum dots at different ratios in Example 2 of the present invention.

[0030] Figure 3 This is the effect of different pH values ​​on the aggregation degree of the two quantum dots in Example 2 of the present invention.

[0031] Figure 4 This is the effect of different reaction temperatures on the degree of agglomeration of the two quantum dots in Example 2 of the present invention.

[0032] Figure 5 This is the effect of different reaction times on the aggregation degree of the two quantum dots in Example 2 of the present invention.

[0033] Figure 6 This is a light spot spectral imaging diagram corresponding to the agglomeration formed by two quantum dots after adding heparin in Example 2 of the present invention;

[0034] Figure 7 The effect of different biomolecules on the aggregation degree of quantum dots in Example 2 of the present invention;

[0035] Figure 8 This is a graph showing the trend of the aggregation degree of two-color quantum dots when different concentrations of heparin are added in Example 2 of the present invention;

[0036] Fig. 9 This is a linear relationship diagram between the aggregation degree of two-color quantum dots and the heparin concentration in Example 2 of the present invention.

[0037] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0038] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0039] The technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0040] A highly sensitive single particle colorimetric method for detecting heparin of the present invention comprises the following steps:

[0041] Step S1: QD545 labeling of antithrombin III

[0042] After activating 1.2-1.3 μL of QD545 with a concentration of 8 μmol by 8-11 μL of EDC with a concentration of 13 mg / mL for 14-15 min, 478.75 μL of 10 mmol of boric acid buffer solution with a pH value of 7.4 and 9-10 μL of 100 IU of thrombin III were added to form a mixed solution, the mixed solution was allowed to stand at 4°C-5°C for 10-12 hours, and then ultrafiltered with a 100 kDa ultrafiltration tube at a speed of 14000 r / min for 5 minutes, and finally the mixed solution was washed three times with 9-10 ml of boric acid buffer solution with a pH value of 8.3 for standby use;

[0043] Step S2: QD655 labeling of protamine

[0044] After activating 1.2-1.3 μL of QD545 with a concentration of 8 μmol by 8-11 μL of QD545 with a concentration of 13 mg / mL for 14-15 min, 478.75 μL of 10 mmol boric acid buffer solution with a pH value of 7.4 and 5-7 μL of 0.2 mg / mL protamine were added to form a mixed solution, the mixed solution was allowed to stand for 1-2 hours in an environment of 23° C.-25° C., and then ultrafiltered for 5 minutes using a 100 kDa ultrafiltration tube at a speed of 14000 r / min, and finally the mixed solution was washed three times with 10 ml of boric acid buffer solution with a pH value of 8.3 for standby use;

[0045] Step S3: Aggregation of QD545-labeled antithrombin III, heparin, and QD655-labeled protamine

[0046] Take 20 nanometers 9-10 μL of the mixed solution in step S1, add a 0.5U / mL heparin sodium standard solution, add 107.5 μL of boric acid buffer and let it stand for half an hour, then add 20 nanometers 9-10 μL of the mixed solution in step S2 to form an agglomeration solution, wherein the total agglomeration volume is 200 μL, let the agglomeration solution stand for 1 hour for standby use, and two quantum dots are formed in the agglomeration solution, namely QD655 quantum dots and QD545 quantum dots;

[0047] Step S4: The agglomerate solution in step 3 is imaged in an electron coupling device and an EMCCD spectrum imaging with a grating added to analyze the quantum dot dimer; the quantum dot image of the agglomerate solution in step S3 is taken, and the quantum dot QD655 is used as a benchmark, combined with the ratio of QD545 quantum dots as data, the quantum dot dimer is calculated, and then the quantum dot dimer is judged by the primary stripes of the quantum dot agglomerates, and a standard curve is drawn according to the heparin sodium concentration and the ratio of the formed quantum dot dimer.

[0048] In the above steps S1 and S2, the EDC is cold secondary water and is ready for use.

[0049] In the above step S3, the amount of the mixed solution in step S1 is 10 μL, wherein a heparin sodium standard solution with a concentration of 0.5 U / mL can also be added. The amount of the mixed solution in step S2 is 10 μL.

[0050] In the above step S1, the amount of QD545 is 1.25 μL and the amount of LEDC is 10 μL for activation for 15 minutes, the amount of thrombin III is 10 μL, and the amount of boric acid buffer solution with a pH value of 8.3 is 9-10 mm, wherein the mixed solution in step S1 is allowed to stand at 4°C for 12 hours.

[0051] In the above step S2, 1.25 μL of QD545 and 10 μL of LEDC were used for activation for 15 min, 6 μL of protamine was used, and 10 ml of boric acid buffer solution with a pH value of 8.3 was used, wherein the mixed solution in step S1 was allowed to stand at 4°C for 12 hours.

[0052] In the above step S3, 10 mM boric acid buffer was added. When the reaction temperature was 25°C, after the QD655 quantum dots and QD545 quantum dots were mixed for 1 hour, the aggregation ratio of QD655 quantum dots and QD545 quantum dots was 1:1. The solution in step S3 was taken and analyzed for quantum dot dimers using Nikon microscope combined with color electron coupled device (CCD) imaging and EMCCD spectral imaging with grating.

[0053] In the above step S4, the quantum dot imaging is collected by a color electronic coupling device, and then Image J is used to judge the aggregation of quantum dots. Step S4 Aggregate formation conditions: add 0.5U / mL heparin to 10μL QD545-AT-Ⅲ with a volume of 20nM, add an appropriate amount of boric acid buffer solution, pre-react for half an hour, add 10μL QD655-Protamine (20nM), the final volume of the solution is 200μL, and the reaction is terminated after 1 hour of reaction. Without adding other ions and 10mM boric acid buffer solution, the aggregation ratio of the two colors of quantum dots reaches the maximum. At 25°C, the aggregation ratio of the two colors of quantum dots reaches the highest aggregation ratio. The aggregation ratio reaches the maximum after the two colors of quantum dots are mixed for 1 hour. The aggregation ratio reaches the maximum when the ratio of the two colors of quantum dots is 1:1. Quantum dot imaging is collected by a color electronic coupling device (CCD), and then Image J is used to judge the aggregation of quantum dots. In the experiment, the aggregation degree is based on the relatively stable quantum dot QD655, and the quantum dots are used as effective data to calculate the aggregation ratio. The correctness of the calculation method of aggregation is then judged by the quantum dot color primary stripes.

[0054] The amount of the borate buffer solution with a pH value of 8.3 used in the above step is 10 mM.

[0055] The invention discloses a highly sensitive single particle colorimetric detection method for heparin.

[0056] A highly sensitive single particle colorimetric detection of heparin is used for application in heparin detection. A highly sensitive single particle colorimetric detection of heparin is used for application in heparin detection.

[0057] The invention discloses a method for detecting heparin based on high-sensitivity single particle colorimetry.

[0058] The invention discloses an application of detecting heparin based on high-sensitivity single particle colorimetric method, and an application of detecting heparin based on high-sensitivity single particle colorimetric method is applied in heparin detection.

[0059] Embodiment 1:

[0060] First, under the optimal experimental conditions, 10 μM human serum albumin, glutathione, uric acid, lysine, tryptophan, cysteine, ascorbic acid and leucine were added to two quantum dot solution systems of different colors, and the changes in the degree of quantum dot aggregation were observed.

[0061] The results showed that these biomacromolecules and small molecules had little interference with the experiment. The main reason was the specific binding of antithrombin III to heparin and the electrostatic binding of heparin to protamine. Therefore, this technical method has extremely high selectivity for the detection of heparin.

[0062] Embodiment 2:

[0063] Recovery rates of serum samples at different dilution factors.

[0064] In order to investigate the application of this technology in the detection of complex serum samples, we calculated the recovery rate of heparin spiked serum samples at different dilution multiples. Serum solution was taken and diluted to 50 times, 100 times, 500 times, 1000 times, and 2000 times with boric acid buffer solution (10mM pH 8.3). 107.5μL of serum dilution was added with 6.25μL of 20nM QD545-ATIII solution and 5μL of 0.5 U / mL sodium heparin aqueous solution, mixed well, reacted at 25℃ for 30 minutes, and then 6.25μL of 20 nM QD655-protamine was added, mixed well, reacted for 1 hour, diluted five times, prepared, and observed the aggregation under a fluorescence microscope.

[0065] Table 1 lists the aggregation degree and recovery rate of dual-color quantum dots after the serum was diluted at different times and 0.01U / mL heparin was added as standard. As the dilution factor increases, the aggregation ratio of quantum dots increases, and the recovery rate also improves accordingly. When diluted to 1000 times and 2000 times, almost the same recovery is obtained. Therefore, it can be considered that when the serum is diluted 1000 times, the background interference is almost non-existent. Therefore, our method can also be used for the detection of serum samples.

[0066] Table 1 Spiked recovery of serum samples at different dilution factors

[0067]

[0068] (2) Recovery rates of different serum samples at 1000-fold dilution

[0069] Through the recovery of serum sample dilution, we believe that interference is almost non-existent after human serum samples are diluted 1000 times, so we selected three concentrations of heparin in the detection linear range for spike recovery. The serum solutions of three people were diluted 1000 times with 10mM pH 8.3 boric acid buffer solution, and different concentrations of heparin were added respectively. After the serum of multiple people was tested, as shown in Table 2, the experimental batches under several spiked concentrations all obtained a recovery rate of 92% to 106%, indicating that there was no interference in the experiment. When the spiked sample is multiplied by 1000 times, the initial heparin concentration is between 0.2 and 8U / mL. Therefore, this method has potential practical application value for the detection of heparin.

[0070] Table 2 Spiked recovery of different serum samples diluted 1000 times

[0071] It should be understood that the above description is for illustration and not for limitation.

[0072]

[0073] in Figure 6 This is the spectral imaging diagram of the light spot corresponding to the agglomeration formed by the two quantum dots after adding heparin. The light spot is diffracted into zero-order light spot and first-order stripes under the action of the grating, and the corresponding scale is 4μm. Figure 1A , Figure 1B and Figure 1C The scale bar is 10 μm.

[0074] It should be noted that in the description of this specification, the terms are only used for descriptive purposes and to distinguish similar objects. There is no order of precedence between the two, and they cannot be understood as indicating or implying relative importance. In addition, in the description of this specification, unless otherwise specified, the meaning of "multiple" is two or more.

[0075] Any numerical value quoted herein includes all values ​​of the lower and upper values ​​with one unit increment from the lower limit to the upper limit, and there is at least two units between any lower value and any higher value. These are just examples intended to be clearly expressed, and it can be considered that all possible combinations of numerical values ​​listed between the lowest value and the highest value are clearly stated in this specification in a similar manner.

[0076] It should be understood that the above description is for illustration and not for limitation. Many embodiments and many applications beyond the examples provided will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of the present invention should not be determined with reference to the above description, but rather with reference to the appended claims and the full range of equivalents to which such claims are entitled. For the purpose of comprehensiveness, all articles and references, including disclosures of patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein in the preceding claims is not intended to be a waiver of the subject matter, nor should it be considered that the inventors did not consider the subject matter to be part of the disclosed inventive subject matter.

Claims

1. A highly sensitive single particle colorimetric method for detecting heparin, characterized in that: The highly sensitive single particle colorimetric method for detecting heparin comprises the following steps: Step S1: QD545 labeling of antithrombin III After activating 1.2-1.3 μL of QD545 with a concentration of 8 μmol by 8-11 μL of EDC with a concentration of 13 mg / mL for 14-15 min, 478.75 μL of 10 mmol of boric acid buffer solution with a pH value of 7.4 and 9-10 μL of 100 IU of thrombin III were added to form a mixed solution, the mixed solution was allowed to stand at 4°C-5°C for 10-12 hours, and then ultrafiltered with a 100 kDa ultrafiltration tube at a speed of 14000 r / min for 5 minutes, and finally the mixed solution was washed three times with 9-10 ml of boric acid buffer solution with a pH value of 8.3 for standby use; Step S2: QD655 labeling of protamine After activating 1.2-1.3 μL of QD545 with a concentration of 8 μmol by 8-11 μL of QD545 with a concentration of 13 mg / mL for 14-15 min, 478.75 μL of 10 mmol boric acid buffer solution with a pH value of 7.4 and 5-7 μL of 0.2 mg / mL protamine were added to form a mixed solution, the mixed solution was allowed to stand for 1-2 hours in an environment of 23° C.-25° C., and then ultrafiltered for 5 minutes using a 100 kDa ultrafiltration tube at a speed of 14000 r / min, and finally the mixed solution was washed three times with 10 ml of boric acid buffer solution with a pH value of 8.3 for standby use; Step S3: QD545 labeled antithrombin III, heparin and QD655 labeled protamine form agglomerates. Take 20 nanometers 9-10 μL of the mixed solution in step S1, add 0.5-0.6U / mL heparin sodium standard solution, add 107.5 μL boric acid buffer and let stand for half an hour, then add 20 nanometers 9-10 μL of the mixed solution in step S2 to form agglomeration solution, wherein the total agglomeration volume is 200 μL, let the agglomeration solution stand for 1 hour for standby use, and two quantum dots are formed in the agglomeration solution, namely QD655 quantum dots and QD545 quantum dots; Step S4: The agglomerate solution in step 3 is imaged in an electron coupling device and an EMCCD spectrum imaging with a grating added to analyze the quantum dot dimer; the quantum dot image of the agglomerate solution in step S3 is taken, and the quantum dot QD655 is used as a benchmark, combined with the ratio of QD545 quantum dots as data, the quantum dot dimer is calculated, and then the quantum dot dimer is judged by the primary stripes of the quantum dot agglomerates, and a standard curve is drawn according to the heparin sodium concentration and the ratio of the formed quantum dot dimer.

2. A highly sensitive single particle colorimetric method for detecting heparin according to claim 1, characterized in that: In the step S1 and the step S2, the EDC is prepared as cold secondary water and is ready for use.

3. A highly sensitive single particle colorimetric method for detecting heparin according to claim 1, characterized in that: In step S3, the amount of the mixed solution in step S1 is 10 μL, wherein the heparin sodium standard solution is added with a concentration of 0.5 U / mL, and the amount of the mixed solution in step S2 is 10 μL.

4. A highly sensitive single particle colorimetric method for detecting heparin as claimed in claim 1, characterized in that: In the step S1, 1.25 μL of QD545 is used for activation with 10 μL of LEDC for 15 min, 10 μL of thrombin III is used, and 9-10 mm of boric acid buffer solution with a pH value of 8.3 is used. The mixed solution in step S1 is allowed to stand at 4° C. for 12 hours.

5. A highly sensitive single particle colorimetric method for detecting heparin as claimed in claim 1, characterized in that: In step S2, 1.25 μL of QD545 and 10 μL of LEDC are used for activation for 15 min, 6 μL of protamine is used, and 10 ml of boric acid buffer solution with a pH value of 8.3 is used, wherein the mixed solution in step S1 is allowed to stand at 4° C. for 12 hours.

6. A highly sensitive single particle colorimetric method for detecting heparin as claimed in claim 1, characterized in that: In the step S3, 10 mM boric acid buffer was added. When the reaction temperature was 25° C., after the QD655 quantum dots and QD545 quantum dots were mixed for 1 hour, the aggregation ratio of the QD655 quantum dots and QD545 quantum dots was 1:

1.

7. A highly sensitive single particle colorimetric method for detecting heparin according to claim 1, characterized in that: In step S4, the quantum dot imaging is collected using a color electron coupling device, and then Image J is used to determine the aggregation of the quantum dots.

8. A highly sensitive single particle colorimetric method for detecting heparin as claimed in claim 4, characterized in that: The amount of the boric acid buffer solution with a pH value of 8.3 is 10 mM.

9. A highly sensitive single particle colorimetric detection of heparin, using a highly sensitive single particle colorimetric detection of heparin method according to any one of claims 1 to 8, characterized in that: The highly sensitive single particle colorimetric detection of heparin is prepared by a highly sensitive single particle colorimetric detection of heparin method.

10. An application of a highly sensitive single particle colorimetric detection of heparin, using the highly sensitive single particle colorimetric detection of heparin as claimed in claim 9, characterized in that: A highly sensitive single particle colorimetric heparin detection method is used in heparin detection.

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