CN111308094A - Homogeneous method troponin chemiluminescence detection reagent and preparation method thereof - Google Patents
Homogeneous method troponin chemiluminescence detection reagent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a troponin chemiluminescence detection reagent by a homogeneous phase method and a preparation method thereof, and the detection reagent comprises the steps of adopting nano gold particles, adding a water-soluble photosensitizer into the nano gold particles to obtain a nano gold-photosensitizer compound, then coupling the nano gold-photosensitizer compound with a troponin I monoclonal antibody, and enabling the troponin I monoclonal antibody and the troponin I monoclonal antibody to generate electrostatic adsorption under the condition of isoelectric point environment through the charge property of the nano gold particles, so that the troponin I monoclonal antibody is coupled to the surfaces of the nano gold particles. The homogeneous phase troponin chemiluminescence detection reagent obtained by the preparation method of the invention improves the utilization rate of the photosensitizer, simplifies the production process, reduces the production cost, adopts the sandwich method detection principle to detect a detected object when in use, improves the effective light dose of irradiation, enables more photosensitizers to be activated and generate stronger photosensitization effect, enlarges the difference between the wavelength of exciting light and the emission wavelength, and improves the identification degree of detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a troponin chemiluminescence detection reagent by a homogeneous phase method and a preparation method thereof.
Background
Acute Myocardial Infarction (AMI) is a critical condition of the cardiovascular system, with high incidence, poor prognosis, and high mortality. For patients with myocardial infarction without typical chest pain and with insignificant changes of electrocardiogram, accurate diagnosis is difficult only by means of electrocardiogram, echocardiogram and cardiac nuclear magnetic resonance. Most of the traditional detection items are myocardial zymograms, but the defects of late rise of enzyme activity, poor specificity, short duration and the like exist. Cardiac troponin is the only contractile protein present in the myocardium and has high sensitivity and specificity for myocardial necrosis.
cTnI is one of three subunits (cTnI, cTnC and cTnT) of cardiac troponin (cTn), is a regulatory protein of muscle tissue contraction, is a polypeptide per se, is located on the thin muscle filament of contractile protein, and plays an important regulatory role in the processes of muscle contraction and relaxation. It has unique sequence in gene and molecular weight 24000. TnI from different tissue sources is tissue specific and not species specific. cTnI is present only in atrial and ventricular muscles, and only one type of TnI is present, and cTnI is the same in every stage of a fetus, a neonate, an adult, and the like. Thus, cTnI is an ideal cardiomyocyte-specific marker.
With intact myocardial cell membranes, cTnI cannot penetrate out of the cell membrane into the blood circulation. When myocardial cells are degenerated and necrotized by ischemia or hypoxia, cTnI is released into the blood through the damaged cell membrane. Since cTnI is of small molecular weight and continues to escape from denatured cells, AMI occurs early in the blood and persists for a considerable time after its onset. cTnI is a more specific marker of myocardial injury than other serozymes, and it is also very sensitive and can diagnose the 4h appearance of AMI in micro-infarcts and ischemia around cardiac surgery (e.g., calcified aortic stenosis and coronary bypass surgery). In contrast to CTnI, cTnC is not myocardial-specific and is not generally used for myocardial injury detection. Under normal conditions, both cTnI and cTnT can not penetrate cell membranes to enter blood, so that the cTnI and cTnT in the blood of healthy people are extremely low; such as myocardial cell damage, cTnI and cTnT enter the human intercellular matrix and blood. In diseases such as renal failure, pneumonia, and sepsis, the cTnT content in blood can also be increased, so its specificity is inferior to cTnI.
The cTnI content in normal human blood is very low, about 20.4 pg/ml. cTnI >20.4pg/ml, has diagnostic significance for myocardial injury. Minor myocardial damage in a patient several days before chest pain symptoms develop can be detected with cTnI. Its concentration begins to rise at 2.2-6.8 hours after AMI occurs, reaches a peak value of 195.9ng/ml at about 11.2 hours, lasts for a long time, and can fall to normal after 5-10 days. The cTnI is released rapidly after myocardial injury, has complete curve, obvious peak value and long window diagnosis period, and compared with the current index for diagnosing Myocardial Infarction (MI), the cTnI has the following advantages: the appearance time in blood is early, and the molecular weight is smaller than that of CK-MB, so the drug is easier to be released into blood after myocardial damage; the sensitivity is high; the specificity is high, the judgment of the result is not influenced by skeletal muscle damage, and the operation type of a patient without cardiac operation can be presumed to have no obvious influence on cTnI; the duration is long, so that the myocardial damage degree can be definitely diagnosed and judged by detecting cTnI (troponin I) in the early and later stages of myocardial damage.
For the troponin I detection method, the existing light-induced chemiluminescence method needs to adopt a water-insoluble photosensitizer (porphyrin, chlorophyll, phthalocyanine and derivatives thereof and the like), and the yield of singlet oxygen quantum of the unmodified photosensitizer is low, so that the detection performance is influenced; the derivatized photosensitizer is difficult to chemically synthesize and complex in process.
The photosensitizer used in the existing photoexcitation light system needs to be embedded with polystyrene microspheres in an organic phase, needs to be subjected to complicated purification modes such as centrifugation and ultrafiltration, needs to be recovered into a water phase matrix, is easy to generate loss in the embedding, purification and other treatment processes, and reduces the photosensitive effect.
The difference between the excitation wavelength and the detection wavelength of the existing light-excited chemiluminescence technology is not large, the interference is easy to occur, and the influence of the fluorescence background is large.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a troponin chemiluminescence detection reagent with a homogeneous method and a preparation method, wherein the detection precision, accuracy and sensitivity are enhanced, and the reagent is not easily interfered by exciting light.
In order to achieve the purpose, the invention designs a homogeneous method troponin chemiluminescence detection reagent which comprises a photosensitizer component, wherein the photosensitizer component is a coupling body of a nanogold-photosensitizer compound and a troponin I monoclonal antibody.
The invention provides a preparation method of a troponin chemiluminescence detection reagent by a homogeneous phase method, which comprises the steps of adopting nano-gold particles, carrying out nano-gold-photosensitizer compounding after obtaining the nano-gold particles, wherein the nano-gold-photosensitizer compounding comprises the steps of adding a water-soluble photosensitizer into the nano-gold particles, stirring overnight in a dark room at room temperature, then centrifuging to finally obtain a precipitated nano-gold-photosensitizer compound, and then re-dissolving the nano-gold-photosensitizer compound by using a buffer solution; coupling the re-dissolved nano gold-photosensitizer compound with troponin I monoclonal antibody, the coupling of the nanogold-photosensitizer and the troponin I monoclonal antibody is to add the troponin I monoclonal antibody into a re-dissolved nanogold-photosensitizer compound solution, adjust the reaction pH value to the isoelectric point (usually between plus or minus 0.5 of the isoelectric point value) of the troponin I monoclonal antibody, the troponin I monoclonal antibody and the gold nanoparticles are subjected to electrostatic adsorption under the condition of isoelectric point environment through the charge property of the gold nanoparticles, thereby coupling the troponin I monoclonal antibody to the surface of the nano-gold particles, removing the unbound troponin I monoclonal antibody in a centrifugal mode, then adding a redissolution buffer solution for redissolution to obtain a conjugate of the nanogold-photosensitizer compound and the troponin I monoclonal antibody suspended in the redissolution buffer solution.
The nanogold-photosensitizer compound provided by the invention has the advantages of higher photosensitizer utilization rate and higher detection sensitivity.
In order to obtain good composition of the nano gold particles and the photosensitizer and better coupling effect of the nano gold-photosensitizer compound and the troponin I monoclonal antibody, the molar ratio of the nano gold particles to the photosensitizer can be 2:1-10, the water-soluble photosensitizer can be a porphyrin derivative, a phthalocyanine derivative or a chlorophyll derivative, such as copper phthalocyanine-3, 4' -tetrasodium tetrasulfonate or sodium copper chlorophyllin, and the electrostatic adsorption time can be 1 hour to 2 hours.
In order to make the re-dissolving effect of the nanogold-photosensitizer compound in the buffer solution more remarkable, the buffer solution can be one of PBS, HEPES, borate, carbonate, Tris-HCl and citric acid; in order to make the effect of the conjugate of the nanogold-photosensitizer complex and troponin I on the reconstitution in a reconstitution buffer more remarkable, the reconstitution buffer can be one of PBS, HEPES, carbonate and Tris-HCl; in order to improve the stability of the nano-gold photosensitive particle suspension suspended in the re-dissolving buffer, a stabilizer, a surfactant, salt ions and preservative components in a determined proportion can be contained in the re-dissolving buffer, and the stabilizer can be BSA, Blockmaster, Tween and the like; the surfactant can be Triton, Tween, SDS, such as Tween-20, Triton-X100; the salt ion can be sodium chloride, potassium chloride and the like; the antiseptic component may be Proclin300, sodium azide, thimerosal, etc.
When the homogeneous phase troponin chemiluminescence detection reagent is used, commercially available luminescent microspheres are prepared into luminescent particles, the luminescent particles and the conjugate of the nanogold-photosensitizer compound and the troponin I monoclonal antibody provided by the invention form a kit, and a detection object is detected by adopting a sandwich method detection principle, so that the effective light dose of irradiation is increased in the detection process, more photosensitizers are activated and generate stronger photosensitization effect, the difference between the wavelength of excitation light and the wavelength of emission is increased, and the identification degree of detection is increased.
Drawings
FIG. 1 is a schematic diagram of a sandwich assay;
FIG. 2 is a calibration graph;
FIG. 3 is a linear range diagram of example 1;
FIG. 4 is a linear range diagram of example 2;
FIG. 5 is a graph of the linear range of example 3.
In the figure: exciting light 1, nano gold particles 2, a water-soluble photosensitizer 3, a troponin I monoclonal antibody 4, a detection object 5 and luminescent particles 6.
Detailed Description
The present invention will be further described with reference to the following examples and the corresponding test data charts.
Example 1:
the homogeneous phase troponin chemiluminescence detection reagent provided by this embodiment comprises a photosensitizer component, which is a conjugate of a nanogold-photosensitizer complex and a troponin I monoclonal antibody.
The preparation method of the homogeneous troponin chemiluminescence detection reagent provided by the embodiment comprises the steps of adopting gold nanoparticles, obtaining the gold nanoparticles, and then carrying out gold nanoparticle-photosensitizer compounding, wherein the gold nanoparticle-photosensitizer compounding is to add a water-soluble photosensitizer into the gold nanoparticles, stir the mixture in a dark room at room temperature overnight, then obtain a precipitated gold nanoparticle-photosensitizer compound through centrifugation, and then re-dissolve the gold nanoparticle-photosensitizer compound with a buffer solution; the re-dissolved nano gold-photosensitizer compound is coupled with a troponin I monoclonal antibody, the nano gold-photosensitizer compound and the troponin I monoclonal antibody are coupled, the troponin I monoclonal antibody is added into the re-dissolved nano gold-photosensitizer compound solution, the pH value of the reaction is adjusted to the isoelectric point of the troponin I monoclonal antibody, in the implementation process, the pH value of the isoelectric point is usually controlled to be between plus or minus 0.5 of the isoelectric point, the troponin I monoclonal antibody is subjected to electrostatic adsorption under the environment condition of the isoelectric point by the charge of the nano gold particles, so that the troponin I monoclonal antibody is coupled to the surfaces of the nano gold particles, the unbound troponin I monoclonal antibody is removed by a centrifugal mode, and then a re-dissolving buffer solution is added for re-dissolving to obtain the nano gold-photosensitizer compound and the troponin I monoclonal antibody which are suspended in the re-dissolving buffer solution A coupling body.
In this embodiment, the specific operation steps may be:
(1) preparation of gold nanoparticles
Heating 100ml of 0.01% chloroauric acid aqueous solution to boiling, accurately adding 0.7ml of l% trisodium citrate aqueous solution under stirring, enabling the golden chloroauric acid aqueous solution to turn into mauve within 2 minutes, continuously boiling for 15 minutes, cooling, and recovering the volume to the original volume with distilled water to obtain a 10-50 nanometer gold nanoparticle solution.
(2) Synthesis of nanogold-photosensitizer complexes
Adding copper phthalocyanine-3, 4 '-tetrasulfonic acid tetrasodium salt into the nano gold particle solution, wherein the molar ratio of nano gold to the copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt is 2: 1. The mixture was stirred overnight in a dark room at room temperature, centrifuged at 12000rpm to obtain the final complex precipitate, which was reconstituted with 50ml PB buffer.
(3) Coupling of nanogold-photosensitizer complex and troponin I monoclonal antibody
Adding 100 mu L of troponin I monoclonal antibody into the re-dissolved nano-gold-photosensitizer compound solution, adjusting the reaction pH to 8.0, coupling the troponin I monoclonal antibody to the surface of the nano-gold particles through electrostatic adsorption, removing unbound troponin I monoclonal antibody through centrifugation at 15000rpm for 15min after 2 hours, and adding 50ml of HEPES re-dissolving buffer solution for re-dissolving, wherein the re-dissolving buffer solution contains 0.5% of BSA, 0.1% of Tween-20, 0.9% of sodium chloride and 0.02% of proclin 300.
(4) Preparation of luminescent particles
The luminescent microspheres are purchased from Alpha Acceptor beads produced and sold by PE company, and the troponin I monoclonal antibody is coated in a covalent coupling mode. Adding 0.5-1mL of Buffer A into 1mL of luminescent microsphere, uniformly mixing, activating with 0.2-0.5mL of EDC for 10-30min, adding 0.3-1mL of antibody, carrying out shake reaction for 2-5 h, sealing for 5-30 min, adding Buffer B to the final volume of 50mL, and uniformly stirring to obtain the luminescent microparticle.
Wherein: buffer A: the pH value is 5-6, components containing MES, HEPES and the like provide certain buffering capacity, salt ions containing KCl, NaCl, CaCl2, MgCl2 and the like have the concentration of 1-10%, and certain ionic strength is provided to promote the activation reaction of the latex microspheres.
Buffer B: the pH value is 6-8, one of buffer solution containing MES, HEPES, PBS, Tris and the like provides the buffering capacity of the reaction, the concentration of salt ions containing one of KCl, NaCl, CaCl2, MgCl2 and the like is 1-10% (providing the ionic strength required by the antibody-microsphere coupling reaction), the concentration of one of substances containing stable substances such as Bovine Serum Albumin (BSA), cane sugar, trehalose, glycerol and the like is 0.1-0.5% (sealing the surfaces of microspheres which are not combined with the antibody and maintaining the stability of immune latex), and the preservative component can be 0.01-0.05% (inhibiting the propagation of bacteria and the like).
When the homogeneous method troponin chemiluminescence detection reagent provided in this embodiment is used, a kit is composed of a conjugate of a nanogold-photosensitizer complex suspended in a redissolving buffer solution and a troponin I monoclonal antibody and luminescent microparticles, and can detect a detected object according to the sandwich method detection principle shown in fig. 1, and the kit has the following properties by detection:
calibration curve:
the reagent is balanced to the ambient temperature before use, 20 mul of gradient calibrator is respectively added into calibration holes by adopting an instrument self-carried calibration program, and 30 mul of nanogold photosensitive particles and 30 mul of luminescent particles are sequentially added for calibration test.
Tables 1 to 1: detection result of calibration product
From the calibration curves obtained in Table 1-1 and FIG. 2, it can be seen that: the reaction degree is higher, and the detection range is wider.
Detection example 1 reagent linear range:
and (3) diluting the high-value serum sample with negative serum or physiological saline in a gradient manner, repeatedly measuring twice, and calculating the correlation between the theoretical concentration and the actually measured average value by using a least square method column equation.
Tables 1 to 2: table of the linear range of detection for the kit prepared by the method described in this example:
from tables 1-2, the linear range: the high-value serum sample is diluted by negative serum or normal saline in a gradient way, the measurement is repeated twice, the correlation between the theoretical concentration and the measured mean value is calculated by a least square method column equation, and the linear high value can be judged to reach 85.27. As shown in fig. 3, the linearity of the detection result obtained in this example is good, and the detection reagent has a good correlation (R2 ═ 0.9995).
The kit prepared by the method of the embodiment has the following detection precision:
precision is an important index for measuring the variation of the reagent between batches and is an important basis for evaluating the effectiveness of the products to be marketed, and usually comprises the precision between batches and the precision between batches.
The evaluation method of the precision in the batch comprises the following steps: using low (L) and high (H) value samples, independent analysis was performed on 2 batches of products, the measurement was repeated 10 times for each batch, the average value (x) and Standard Deviation (SD) of 10 measurements were calculated, and the Coefficient of Variation (CV) was calculated according to the formula CV ═ SD/x × 100%
The method for evaluating the batch precision comprises the following steps: using the low (L) and high (H) value samples, 3 batches of the product were analyzed independently, the measurement was repeated 10 times for each batch, the average value (x) and Standard Deviation (SD) of the 20 measurements were calculated, and the Coefficient of Variation (CV) was calculated according to the formula CV ═ SD/x × 100%.
The results of the intra-and inter-batch fine density tests were as follows:
tables 1 to 3: the kit prepared by the method of the embodiment has the following internal precision and batch-to-batch precision:
as can be seen from tables 1-3, the kits prepared by the methods described in this example all had batch precision<5％Inter-batch precision is equal<5％The kit prepared by the method of the embodiment has good repeatability and small random error in detection。
The detection accuracy of the kit prepared by the method of the embodiment is as follows:
the accuracy is the coincidence degree of the measured value and the actual value, and the detection error of the reaction reagent.
Carrying out three repeated detections by using an international reference substance NIST SRM2921 as a sample, and calculating the relative deviation B of the mean value X and the reference substance index value T, wherein the calculation formula is as follows: b ═ X-T)/T × 100%.
Tables 1 to 4: the detection accuracy of the kit prepared by the method of the embodiment
| Reference target value (ng/mL) | 31.2 |
| |
32.695 |
| |
32.765 |
| |
32.775 |
| Mean value of test | 32.745 |
| Relative deviation% | 4.95 |
The test result shows that the B is (32.745-31.2)/31.2 multiplied by 100 percent to 4.95 percent, and the relative deviation is less than 10 percent.
As can be seen from tables 1-4, the kit prepared by the method of this example has a small deviation of detection accuracy, the relative deviation is within 5%, which indicates that the measured value is close to the theoretical value, and the kit prepared by the method of this example has a small detection error.
Table 1-5 sensitivities:
the measurement of 0ng/mL of the calibration material was repeated 20 times, and the mean (X) and Standard Deviation (SD) D of the 20 times were calculated, and the analytical sensitivity calculation formula was: LOB is X +2SD, and the sensitivity can reach 0.009 ng/mL.
Example 2:
the preparation method of the homogeneous troponin chemiluminescence detection reagent provided by the embodiment comprises the following steps:
(1) the preparation of the gold nanoparticles was the same as in example 1;
(2) synthesis of nanogold-photosensitizer complexes
Adding copper phthalocyanine-3, 4 '-tetrasulfonic acid tetrasodium salt into the nano gold particle solution, wherein the molar ratio of the nano gold particles to the copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt is 2: 2. The mixture was stirred overnight in a dark room at room temperature, centrifuged at 12000rpm to obtain the final complex precipitate, which was reconstituted with 50ml HEPES.
(3) Coupling of Nanogold-photosensitizer complexes with troponin I
Adding 100 mu L of troponin I monoclonal antibody into the nanogold-photosensitizer complex, adjusting the reaction pH to 8.0, coupling the troponin I monoclonal antibody to the surface of the nanogold particles through electrostatic adsorption, removing unbound troponin I monoclonal antibody through centrifugation at 15000rpm for 15min after 2 hours, and adding 50ml of PB buffer solution for redissolution, wherein the redissolution buffer solution contains 0.5% of BSA, 0.1% of Tween20, 0.9% of sodium chloride and 0.02% of proclin300 in percentage by weight.
(4) The luminescent particles were prepared as in example 1, whereby a kit consisting of a conjugate of the nanogold-photosensitizer complex and troponin I monoclonal antibody and the luminescent particles in this example was obtained.
The kit obtained in this example, after detection, has the following results of the density test of the intra-batch and inter-batch samples:
table 2-1: the reagents prepared by the method of the invention have intra-batch precision and inter-batch precision
As can be seen from Table 2-1, the reagents prepared by the method of the present invention all have an intra-batch precision of less than 5% and an inter-batch precision of less than 5%, indicating that the reagent kit prepared by the method of the present invention has good repeatability and small random error when being used for detection.
Tables 2 to 2: detection accuracy of the kit obtained in this example
| Reference target value (ng/mL) | 31.2 |
| |
31.65 |
| |
31.7 |
| |
31.95 |
| Mean value of test | 31.767 |
| Relative deviation% | 1.82 |
As can be seen from table 2-2, the kit prepared by the method of this example has a small deviation of detection accuracy, the relative deviation is within 2%, which indicates that the measured value is close to the theoretical value, and the kit prepared by the method of this example has a small detection error.
The linear range of the kit of detection example 2 is shown in tables 2-3:
tables 2 to 3: table of the linear range of detection for the kit prepared by the method described in this example:
as can be seen from FIG. 4 and tables 2-3, the detection reagent prepared in example 2 can maintain good linearity and has good correlation (R)2＝0.9985)。
Example 3:
the preparation method of the homogeneous troponin chemiluminescence detection reagent provided by the embodiment comprises the following steps:
preparation of gold nanoparticles as in example 1, the gold nanoparticle-photosensitizer complex was synthesized by adding sodium copper chlorophyllin to a gold nanoparticle solution at a molar ratio of gold nanoparticles to sodium copper chlorophyllin of 2: 10. Stirring overnight in a dark room at room temperature, centrifuging at 12000rpm to obtain final compound precipitate, and re-dissolving with 50ml HEPES; the nano-gold-photosensitizer compound and the troponin I are coupled by adding 150 mu L of troponin I monoclonal antibody into the nano-gold-photosensitizer compound, adjusting the pH value of the reaction to 8.0, coupling the troponin I monoclonal antibody to the surface of a nano-gold particle through electrostatic adsorption, removing the unbound troponin I monoclonal antibody through a mode of centrifugation at 15000rpm for 15min after 2 hours, adding 50ml of HEPES buffer solution for redissolution, wherein the buffer solution contains 0.1% of Tween-20, 1.5% of sodium chloride and 0.02% of proclin 300.
The preparation of luminescent particles was the same as in example 1, whereby a kit consisting of a conjugate of the nanogold-photosensitizer complex and troponin I monoclonal antibody and luminescent particles in this example was obtained.
The results of the density test of the concentrates in batches and among batches are as follows:
table 3-1: the kit prepared by the method of this example has both batch-to-batch precision and batch-to-batch precision
As can be seen from Table 3-1, the kit prepared by the method of the present invention has an intra-batch precision of less than 5% and an inter-batch precision of less than 5%, which indicates that the kit prepared by the method of the present embodiment has good repeatability and small random error during detection.
The detection accuracy of the kit prepared by the method of the embodiment is as follows:
tables 3-2: detection accuracy of kit prepared by the method of the invention
| Reference target value (ng/mL) | 31.2 |
| |
32.03 |
| |
31.65 |
| |
32.59 |
| Mean value of test | 32.090 |
| Relative deviation% | 2.85 |
As can be seen from Table 3-2, the kit prepared by the method of this example has a small deviation of detection accuracy and a deviation of recovery rate within 3%, indicating that the measured value is close to the theoretical value and the detection error of the kit prepared by the method of this example is small.
The linear range of the kit for detecting the present embodiment is as follows:
tables 3 to 3: table of the linear range of detection for the kit prepared by the method described in this example:
as can be seen from FIG. 5 and tables 3-3, the detection kit prepared in this example 3 can maintain good linearity and has better correlation (R)2＝0.9986)。
Claims (10)
1. A homogeneous method troponin chemiluminescence detection reagent comprises a photosensitizer component, and is characterized in that the reagent is a coupling body of a nanogold-photosensitizer compound and a troponin I monoclonal antibody.
2. A homogeneous method troponin chemiluminescence detection reagent preparation method comprises adopting gold nanoparticles, and is characterized in that gold nanoparticles are obtained and then subjected to gold nanoparticle-photosensitizer compounding, wherein the gold nanoparticle-photosensitizer compounding is to add a water-soluble photosensitizer into the gold nanoparticles, stir the mixture at room temperature in a dark room overnight, then obtain a precipitated gold nanoparticle-photosensitizer compound through centrifugation, and then re-dissolve the gold nanoparticle-photosensitizer compound with a buffer solution; coupling the re-dissolved nano gold-photosensitizer compound with troponin I monoclonal antibody, the coupling of the nano-gold photosensitizer and the troponin I monoclonal antibody is realized by adding the troponin I monoclonal antibody into a re-dissolved nano-gold photosensitizer compound solution, adjusting the reaction pH value to the isoelectric point of the troponin I monoclonal antibody, the troponin I monoclonal antibody and the gold nanoparticles are subjected to electrostatic adsorption under the condition of isoelectric point environment through the charge property of the gold nanoparticles, thereby coupling the troponin I monoclonal antibody to the surface of the nano-gold particles, removing the unbound troponin I monoclonal antibody in a centrifugal mode, then adding a redissolution buffer solution for redissolution to obtain a conjugate of the nanogold-photosensitizer compound and the troponin I monoclonal antibody suspended in the redissolution buffer solution.
3. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 2, wherein the molar ratio of the gold nanoparticles to the photosensitizer is 2: 1-10.
4. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 2 or 3, wherein the water-soluble photosensitizer is a porphyrin derivative, a phthalocyanine derivative, or a chlorophyll derivative.
5. The method for preparing a reagent for the chemiluminescent detection of troponin according to claim 2 or 3 wherein the electrostatic adsorption is carried out for a period of time ranging from 1 hour to 2 hours.
6. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 4, wherein the electrostatic adsorption is performed for a period of 1 to 2 hours.
7. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 2 or 3, wherein the buffer is one of PBS, HEPES, borate, carbonate, Tris-HCl, and citric acid; the re-dissolving buffer solution is one of PBS, HEPES, carbonate and Tris-HCl.
8. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 4, wherein the buffer is one of PBS, HEPES, borate, carbonate, Tris-HCl and citric acid; the re-dissolving buffer solution is one of PBS, HEPES, carbonate and Tris-HCl.
9. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 5, wherein the buffer is one of PBS, HEPES, borate, carbonate, Tris-HCl, citric acid; the re-dissolving buffer solution is one of PBS, HEPES, carbonate and Tris-HCl.
10. The method for preparing the reagent for the homogeneous troponin chemiluminescence detection according to claim 6, wherein the buffer is one of PBS, HEPES, borate, carbonate, Tris-HCl, citric acid; the re-dissolving buffer solution is one of PBS, HEPES, carbonate and Tris-HCl.
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