CN114624197A - Method for detecting blood concentration of small molecule drug - Google Patents
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- CN114624197A CN114624197A CN202111454936.8A CN202111454936A CN114624197A CN 114624197 A CN114624197 A CN 114624197A CN 202111454936 A CN202111454936 A CN 202111454936A CN 114624197 A CN114624197 A CN 114624197A
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Abstract
The invention relates to the technical field of optical fiber biosensing, and discloses a method for detecting the blood concentration of a small molecule drug. The method for detecting the blood concentration of the small molecule drug comprises the steps of preparing an optical fiber probe, respectively obtaining a reference sample and a sample to be detected, competitively combining and amplifying signals of the reference sample, obtaining a standard curve of small molecule drug concentration-relative inhibition, competitively combining and amplifying signals of the sample to be detected, obtaining the small molecule drug concentration of the sample to be detected and the like. The invention establishes a rapid and high-sensitivity optical fiber biological sensing detection technology aiming at the blood concentration of small molecular drugs based on an optical fiber biological membrane interference technology, a direct competitive combination immunity method and a signal amplification strategy.
Description
Technical Field
The invention relates to the technical field of optical fiber biosensing, in particular to a method for detecting the blood concentration of a small molecular drug.
Background
Epilepsy is a chronic, recurrent and sudden brain dysfunction, and is a chronic central nervous system disease caused by sudden abnormal high-frequency discharge of cerebral neurons and diffusion to the periphery. Epilepsy can cause serious influence on the mind and body of a patient, and Carbamazepine (CBZ) is used as a common small molecule drug for treating epileptic focal seizures and grand seizures, the effective blood concentration range is narrow, the individual difference is large, the blood concentration is closely related to the curative effect, toxicity and adverse reactions of the blood concentration, and the blood concentration is difficult to control in a safe and effective range by empirical medication. In order to help reduce toxic and side effects and increase curative effects, the detection of the blood concentration of carbamazepine in patients is a clinically significant research work. The blood concentration detection can assist a doctor to find out a toxic reaction caused by excessive antiepileptic drugs in time in the application of epilepsy treatment, can provide scientific basis for judging the degree of poisoning and adjusting the drug administration scheme, shorten the time for exploring the drug administration dose, and formulate a drug administration scheme according with the characteristics of the patient with special pharmacokinetics.
At present, the common carbamazepine blood concentration detection method mainly comprises enzyme-linked immunosorbent assay and high performance liquid chromatography, and both have certain technical defects. The former can be used for high-throughput detection, but is greatly influenced by temperature and time, has poor repeatability and takes several hours; the latter has high sensitivity, can solve the problem of complicated sample analysis which cannot be realized by other analysis methods, but has excessively complicated blood sample pretreatment process and needs to be operated by professional staff.
Some abbreviations, English and key terms used in this application are defined as follows:
HRP, Horseradish Peroxidase and Horseradish Peroxidase;
DAB 3, 3' -diaminobenzidine tetrahydrochloride,3, 3-diaminobenzidine tetrahydrochloride.
Disclosure of Invention
The invention aims to provide a method for detecting the blood concentration of a small molecular drug, which realizes the rapid, high-sensitivity and full-automatic detection of the blood concentration of the small molecular drug by amplifying effective signals of a bio-layer interference technology.
In order to solve the above technical problems, a first aspect of the present invention provides a method for detecting a blood concentration of a small molecule drug, comprising:
(1) preparing an optical fiber probe: fixing the biotinylated specific micromolecular drug monoclonal antibody on the surface of a streptavidin optical fiber to obtain an optical fiber probe;
(2) respectively obtaining a reference sample and a sample to be detected:
adding small molecule drug standard substances with different concentrations into a blood reference substance to obtain a plurality of reference samples with different small molecule drug concentrations;
obtaining a blood sample of a person to be tested for the blood concentration of the small molecular drug to obtain a sample to be tested;
(3) competitive binding and signal amplification of the reference sample:
respectively mixing the plurality of reference samples with different small molecule drug concentrations with the small molecule drug-HRP conjugate in equal volume to obtain a mixed solution of the plurality of reference samples and the small molecule drug-HRP conjugate, and performing the following steps of competitive combination and signal amplification on the mixed solution of the plurality of reference samples and the small molecule drug-HRP conjugate:
competitive binding: contacting the fiber-optic probe with a mixed solution of the reference sample and the small molecule drug-HRP conjugate, wherein the small molecule drug and the small molecule drug-HRP conjugate in the reference sample are competitively combined with the specific small molecule drug monoclonal antibody on the fiber-optic probe;
signal amplification: then, the optical fiber probe is contacted with an HRP biological color developing agent, the HRP combined on the optical fiber probe is combined with the HRP biological color developing agent with high affinity, metal precipitates are generated, the spectral red shift of the surface of the optical fiber is brought, and the spectral shift of a reference sample is obtained;
(4) obtaining a standard curve of the concentration-relative inhibition (%) of the small molecule drug:
taking the concentration of the small-molecule drug in a reference sample as an X axis and the relative inhibition as a Y axis, and drawing a 'concentration-relative inhibition (%)' nonlinear standard curve; wherein, the calculation of the relative inhibition of the reference samples with different small molecule drug concentrations takes the spectral shift of the reference sample with zero small molecule drug concentration as a reference, and the formula is as follows:
relative inhibition (%) of a reference sample of a certain small molecule drug concentration
{ 1- (spectral shift of a reference sample of a certain small molecule drug concentration/spectral shift of a reference sample of zero small molecule drug concentration) } × 100%;
(5) competitive binding and signal amplification of the sample to be tested:
mixing the sample to be detected and the small molecule drug-HRP conjugate in equal volume to obtain a mixed solution of the sample to be detected and the small molecule drug-HRP conjugate, and performing the following steps of competitive combination and signal amplification on the mixed solution of the sample to be detected and the small molecule drug-HRP conjugate:
competitive binding: contacting the fiber probe with a mixed solution of the sample to be detected and the small molecule drug-HRP conjugate, wherein the small molecule drug and the small molecule drug-HRP conjugate in the sample to be detected are combined with the specific small molecule drug monoclonal antibody on the fiber probe in a competitive manner;
signal amplification: then, the optical fiber probe is contacted with an HRP biological color developing agent, the HRP combined on the optical fiber probe is combined with the HRP biological color developing agent with high affinity, metal precipitates are generated, the spectral red shift of the surface of the optical fiber is brought, and the spectral shift of a sample to be detected is obtained;
(6) obtaining the concentration of the small molecule drug of the sample to be detected:
calculating the relative inhibition (%) of the test sample:
relative inhibition (%) of the sample to be tested
{ 1- (spectral shift of sample to be tested/spectral shift of reference sample with zero small molecule drug concentration) } × 100%;
and obtaining the concentration of the small molecule drug in the sample to be detected based on the nonlinear standard curve of the small molecule drug concentration-relative inhibition (%) according to the relative inhibition (%) of the sample to be detected.
Preferably, in the detection method of the blood concentration of the small molecule drug provided by the invention, the HRP biological color developing agent is 3, 3-diaminobenzidine tetrahydrochloride.
Preferably, the method for detecting the blood concentration of the small molecule drug provided by the invention further comprises two elution steps. The first elution was: and after the preparation of the optical fiber probe, eluting the specific small molecule drug monoclonal antibody which is not fixed on the surface of the streptavidin chip. The second elution was: and in the competitive binding and signal amplification step of the reference sample and the competitive binding and signal amplification step of the sample to be detected, after the competitive binding, eluting the small molecule drug-HRP conjugate which is not bound with the specific small molecule drug monoclonal antibody.
Preferably, in the method for detecting the blood concentration of the small molecule drug provided by the invention, the blood control product is a whole blood matrix, serum or plasma of a person who does not use the small molecule drug; the blood sample of the person to be tested with the blood concentration of the small molecular drug is whole blood matrix, blood serum or blood plasma of the person to be tested with the blood concentration of the small molecular drug.
Preferably, in the step of obtaining the reference sample and the sample to be detected respectively, the reference sample and the sample to be detected are diluted by 15-25 times by using a high-salt sample dilution buffer solution; the high-salt sample dilution buffer solution is a 10mM pH 7.4 phosphate buffer solution containing 100-300 mM NaCl, 0.02% by mass of Tween-20 and 0.1% by mass of Bovine Serum Albumin (BSA).
Further preferably, in the step of obtaining the reference sample and the sample to be tested separately, a 20-fold dilution is performed using a high-salt sample dilution buffer, wherein the high-salt sample dilution buffer is a 10mM pH 7.4 phosphate buffer containing 274mM NaCl, 0.02% by mass of Tween-20, and 0.1% by mass of BSA.
Preferably, in the method for detecting the blood concentration of the small molecule drug provided by the invention, the small molecule drug-HRP conjugate is selected from a small molecule drug, a small molecule drug-bovine serum albumin complex or a small molecule drug-ovalbumin complex and an HRP conjugate.
Preferably, in the method for detecting the blood concentration of the small molecule drug provided by the invention, the small molecule drug is a small molecule drug requiring or potentially requiring blood concentration monitoring, such as carbamazepine, aminophylline, warfarin, quinidine, methotrexate, cyclosporine, vancomycin, digoxin, a small molecule antiviral drug (such as a small molecule anti-novel coronavirus drug) and the like.
Preferably, in the detection method of the blood concentration of the small molecule drug provided by the invention, the concentration range of the small molecule drug-HRP conjugate is 0.05-0.20 μ g/mL.
Preferably, in the method for detecting the blood concentration of the small molecule drug provided by the invention, the spectral shift of the sample to be detected and/or the spectral shift of the reference sample with different antibody concentrations are within a range of 10-25 nm.
Compared with the prior art, the invention establishes a rapid and high-sensitivity full-automatic optical fiber biological sensing technology aiming at small molecule drugs (such as carbamazepine and the like) based on an optical fiber biological membrane interference technology, a direct competitive combination immunity method and a signal amplification strategy. The method provided by the invention is not only suitable for serum samples, but also can directly and rapidly detect the micromolecule drugs in the whole blood. The innovative optical fiber sensing detection technology provided by the invention has the potential to be used for rapidly detecting other nervous micromolecular drugs and neurotransmitters on a whole blood sample, and provides a powerful bedside diagnostic tool for clinic.
Drawings
FIG. 1 is a schematic diagram of a method for detecting blood concentration of a small molecule drug according to the present invention; wherein 1-A is the condition when the small molecule drug is not present in the sample; 1-B is the case where a small molecule drug is present in the sample; wherein, 1: a streptavidin optical fiber; 2: biotinylation micromolecular drug monoclonal antibody; 3: a small molecule drug; 4: HRP-conjugated small molecule drugs; 5: HRP biological color developing agent;
FIG. 2 is a flow chart of the detection of blood concentration of small molecule drugs according to the present invention;
FIG. 3 is a non-linear calibration curve of "concentration versus relative inhibition (%)" for small molecule drugs established in an example of the present invention;
FIG. 4 is a graph comparing the results of the test of comparative example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below. However, it will be appreciated by those of ordinary skill in the art that numerous technical details are set forth in order to provide a better understanding of the present application in various embodiments of the present invention. However, the technical solution claimed in the present application can be implemented without these technical details and various changes and modifications based on the following embodiments.
The embodiment of the invention provides a method for detecting the blood concentration of a small molecule drug, which comprises the following steps:
(1) preparing an optical fiber probe: fixing the biotinylated specific micromolecular drug monoclonal antibody on the surface of a streptavidin optical fiber to obtain an optical fiber probe;
(2) respectively obtaining a reference sample and a sample to be detected: adding small molecule drug standard substances with different concentrations into a blood reference substance to obtain a plurality of reference samples with different small molecule drug concentrations; obtaining a blood sample of a person to be tested with the blood concentration of the small molecular drug to obtain a sample to be tested;
(3) competitive binding and signal amplification of the reference sample:
respectively mixing the plurality of reference samples with different small molecule drug concentrations with the small molecule drug-HRP conjugate in equal volume to obtain a mixed solution of the plurality of reference samples and the small molecule drug-HRP conjugate, and performing the following steps of competitive combination and signal amplification on the mixed solution of the plurality of reference samples and the small molecule drug-HRP conjugate:
competitive binding: contacting the fiber-optic probe with a mixed solution of the reference sample and the small molecule drug-HRP conjugate, wherein the small molecule drug and the small molecule drug-HRP conjugate in the reference sample are competitively combined with the specific small molecule drug monoclonal antibody on the fiber-optic probe;
signal amplification: then, the optical fiber probe is contacted with an HRP biological color developing agent, the HRP combined on the optical fiber probe is combined with the HRP biological color developing agent with high affinity, metal precipitates are generated, the spectral red shift of the surface of the optical fiber is brought, and the spectral shift of a reference sample is obtained;
(4) obtaining a standard curve of the concentration-relative inhibition (%) of the small molecule drug:
taking the concentration of the small-molecule drug in a reference sample as an X axis and the relative inhibition as a Y axis, and drawing a 'concentration-relative inhibition (%)' nonlinear standard curve; wherein, the calculation of the relative inhibition of the reference samples with different small molecule drug concentrations takes the spectral shift of the reference sample with zero small molecule drug concentration as a reference, and the formula is as follows:
relative inhibition (%) of a reference sample of a certain small molecule drug concentration
{ 1- (spectral shift of a reference sample of a certain small molecule drug concentration/spectral shift of a reference sample of zero small molecule drug concentration) } × 100%;
(5) detection and signal amplification of a sample to be detected:
mixing the sample to be detected and the small molecule drug-HRP conjugate in equal volume to obtain a mixed solution of the sample to be detected and the small molecule drug-HRP conjugate, and performing the following steps of competitive combination and signal amplification on the mixed solution of the sample to be detected and the small molecule drug-HRP conjugate:
competitive binding: contacting the fiber probe with a mixed solution of the sample to be detected and the small molecule drug-HRP conjugate, wherein the small molecule drug and the small molecule drug-HRP conjugate in the sample to be detected are combined with the specific small molecule drug monoclonal antibody on the fiber probe in a competitive manner;
signal amplification: then, the optical fiber probe is contacted with an HRP biological color developing agent, the HRP combined on the optical fiber probe is combined with the HRP biological color developing agent with high affinity, metal precipitates are generated, the spectral red shift of the surface of the optical fiber is brought, and the spectral shift of a sample to be detected is obtained;
(6) obtaining the concentration of the small molecule drug of the sample to be detected:
calculating the relative inhibition (%) of the test sample:
relative inhibition (%) of the sample to be tested
{ 1- (spectral shift of sample to be tested/spectral shift of reference sample with zero small molecule drug concentration) } × 100%;
and obtaining the concentration of the small molecule drug in the sample to be detected based on the nonlinear standard curve of the small molecule drug concentration-relative inhibition (%) according to the relative inhibition (%) of the sample to be detected.
According to the detection method of the blood concentration of the small molecule drug, firstly, a biotinylated specific small molecule drug monoclonal antibody is fixed to the surface of a streptavidin chip to obtain an optical fiber probe, and based on the optical fiber probe, a direct competitive binding method is adopted to specifically detect the small molecule drug. The direct competitive binding principle is that the micromolecule drug in a sample and the micromolecule drug-HRP conjugate are mixed in the same volume of 1:1, and the mixture directly competes with each other to bind the specific micromolecule drug monoclonal antibody fixed on the tip of the optical fiber. When the small molecule drug does not exist in the sample, the combined amount of the small molecule drug-HRP conjugate reaches the maximum; at this time, the HRP biological color developing agent with signal amplification effect will perform oxidation-reduction reaction with horseradish peroxidase (HRP), and then generate metal precipitate through oxidation-reduction reaction and polymerization reaction, and the metal precipitate will greatly enhance the signal intensity (shown in fig. 1-a). When a large amount of small molecule drug is present in the sample, the amount of bound small molecule drug-HRP conjugate is reduced to a minimum or zero, and then the signal amplification agent will not bind with HRP enzyme any more, no brown metal precipitate will be generated, and the signal intensity is zero (as shown in fig. 1-B).
Therefore, the embodiment of the application is based on the optical fiber biological layer interference technology, and realizes the rapid, high-sensitivity and automatic detection of the blood concentration of the small molecule drug by directly combining the immunity method and the signal amplification competitively. The bio-layer interference technique is a technique for detecting a sensor surface reaction by detecting a shift change of an interference spectrum. When a beam of visible light is emitted from the spectrometer, two reflection spectrums are formed on two interfaces of the optical film layer at the tail end of the optical fiber sensor, and an interference spectrum is formed, wherein the red shift size of the spectrum is in positive correlation with the concentration of the specific binding molecules at the tail end.
In the method for detecting the blood concentration of the small-molecule drug provided in some embodiments of the present application, the HRP biological color developing agent is 3, 3-diaminobenzidine tetrahydrochloride.
In some embodiments of the present application, two elution steps are also included. The first elution was: and after the preparation of the optical fiber probe, eluting the specific small molecule drug monoclonal antibody which is not fixed on the surface of the streptavidin chip. The second elution was: and in the competitive binding and signal amplification step of the reference sample and the competitive binding and signal amplification step of the sample to be detected, after the competitive binding, eluting the small molecule drug-HRP conjugate which is not bound with the specific small molecule drug monoclonal antibody.
In some embodiments of the present application, the blood control is a whole blood matrix, serum, or plasma of a subject without a small molecule drug; the blood sample of the person to be tested with the blood concentration of the small molecular drug is whole blood matrix, blood serum or blood plasma of the person to be tested with the blood concentration of the small molecular drug.
In some embodiments of the present disclosure, in the step of obtaining the reference sample and the sample to be tested, the reference sample and the sample to be tested are diluted by 15-25 times by using a high-salt sample dilution buffer; wherein the high-salt sample dilution buffer solution is a 10mM pH 7.4 phosphate buffer solution containing 100-300 mM NaCl, 0.02% by mass of Tween-20 and 0.1% by mass of BSA. Since the surface of the optical fiber is interfered by a large amount of cells, fat, protein and the like in a blood sample (serum, plasma, whole blood), the non-specific interference caused by the impurities such as the cells, the fat, the protein and the like in the whole blood sample can be reduced by diluting the whole blood sample by using the high-salt sample dilution buffer.
In some embodiments of the present application, in the step of obtaining the reference sample and the test sample separately, a 20-fold dilution is performed using a high-salt sample dilution buffer, wherein the high-salt sample dilution buffer is a 10mM pH 7.4 phosphate buffer containing 274mM NaCl, 0.02% by mass of Tween-20, and 0.1% by mass of BSA.
In some embodiments of the present application, the small molecule drug-HRP conjugate is a conjugate of a small molecule drug-bovine serum albumin complex and HRP.
Preferably, in some embodiments of the present application, the small molecule drug is carbamazepine, aminophylline, warfarin, quinidine, methotrexate, cyclosporine, vancomycin, digoxin, a small molecule antiviral drug (e.g., a small molecule anti-novel coronavirus drug) or other small molecule drugs that require or are potentially required for blood concentration monitoring.
In some embodiments of the present application, the concentration of the small molecule drug-HRP conjugate is 0.1. mu.g/mL.
In some embodiments of the present application, the spectral shift of the sample to be tested and/or the spectral shift of the reference sample with different antibody concentrations are in the range of 10-25 nm. The signal amplification agent DAB is used for amplifying the signal intensity, when the spectral shift of a sample to be detected and/or the spectral shift of a reference sample with different antibody concentrations are within the range of 10-25 nm, enough signal intensity is guaranteed, and fiber falling during system operation caused by the fact that the surface of the fiber tip is overloaded can be avoided.
Examples of detection
In this section, the detection of the blood concentration of the small molecule drug carbamazepine is taken as an example to specifically describe the detection process, and the detection of the blood concentration of other small molecule drugs can be carried out by referring to similar steps.
(1) Preparing an optical fiber probe:
and (3) fixing the biotinylated specific carbamazepine monoclonal antibody on the surface of the streptavidin optical fiber to obtain the optical fiber probe. Among them, specific Carbamazepine monoclonal antibodies are available from Bio-Rad Laboratories (Chinese: Berle Vital products Limited) as Mouse anti Carbamazepine antibody, clone CA1 (murine anti-Carbamazepine monoclonal antibody, clone CA 1). The biotinylation of a specific carbamazepine monoclonal antibody can be carried out by purchasing a commercial biotin kit (e.g., GENEMORE biotinylation kit from Jiangsu Bomada Life sciences, Ltd.), and referring to the instructions for the kit. Streptavidin fiber a fiber optic probe was prepared as described above from a Streptavidin (SA) biosensor available from Sartorius Stedim Biotech GmbH (chinese: sartoritei). The step of pre-functionalizing the fiber optic probe may be done in advance or may be done prior to the detection.
(2) 6 reference samples with different small molecule drug concentrations were prepared:
preparing a high-salt sample dilution buffer: the high-salt sample dilution buffer comprises 274mM NaCl, 0.02% by mass of Tween-20, and 0.1% by mass of BSA in a 10mM pH 7.4 phosphate buffer.
The carbamazepine standard was solubilized using high salt buffer to obtain 6 initial concentrations of carbamazepine (0, 1, 2, 4, 8 and 16 μ g/mL). Then, the above 6 initial concentrations were diluted 20-fold using healthy human whole blood (without carbamazepine administration) to obtain 6 reference samples (concentration range: 0, 50, 100, 200, 400 and 800ng/mL) with different concentrations of small molecule drug. For example: to obtain 200. mu.L of the reference sample, 10. mu.L of healthy whole blood + 10. mu.L of initial concentration + 190. mu.L of high salt buffer. Similarly, 6 carbamazepine reference samples based on healthy human serum, healthy human plasma matrix, can be obtained.
(3) Preparing a sample to be tested:
the method is used for taking blood from the veins of patients suffering from epilepsy and taking carbamazepine medicine, and a whole blood sample does not need any pretreatment and only needs to be diluted by 20 times of a high-salt sample buffer solution. Serum and plasma samples need to be pretreated by centrifugation, anticoagulation and the like, and then are diluted by 20 times by high-salt sample buffer solution.
(4) Preparing a small molecule drug-HRP conjugate:
the carbamazepine-HRP conjugate can be prepared by directly coupling carbamazepine and HRP (horse radish peroxidase) or indirectly coupling the carbamazepine and the HRP. The indirect coupling method comprises the following steps: the carbamazepine-bovine serum albumin complex is first prepared or purchased (e.g., available from U.S. gambo biotechnology, limited, wuhan), and then the carbamazepine-bovine serum albumin complex is coupled with HRP to prepare a carbamazepine-bovine serum albumin-HRP conjugate. The conjugation method may be referred to commercially available conjugation kits, for example: HRP Conjugation Kit-Lightning-
(ab102890) kit. Most preferably of the conjugatesThe concentrations used are preferably individually searched and selected according to the strength of the conjugate obtained. The carbamazepine conjugate in this assay example was obtained by indirect coupling, and the optimal concentration was chosen to be 0.1 μ g/mL to avoid poor signal enhancement at too low a concentration and significant background interference at too high a concentration.
(5) Mixing the small molecule drug-HRP conjugate with the reference sample in equal volume:
the carbamazepine-bovine serum albumin-HRP conjugate prepared above was taken at 100. mu.L, the reference sample (0ng/mL) prepared above at 20-fold dilution in healthy human whole blood matrix was taken at 100. mu.L, an equal volume was added to a 1.5mL tube, mixed briefly (less than 1 minute), and 200. mu.L of the mixed solution was tested for future use. Similarly, a mixture of the remaining 5 reference samples (50, 100, 200, 400 and 800ng/mL) was prepared and tested for future use.
(6) Mixing the small molecule drug-HRP conjugate with a sample to be detected in equal volume:
taking 100 μ L of the prepared carbamazepine-bovine serum albumin-HRP conjugate, taking 100 μ L of the prepared sample to be tested under 20-fold dilution, adding the sample to a 1.5mL test tube in an equal volume, mixing the sample for a short time (less than 1 minute), and testing 200 μ L of the mixed solution for later use (taking 2 samples to be tested in the example).
(7) Competitive binding and signal amplification of a mixture of a reference sample and a small molecule drug-HRP conjugate:
and (2) performing competitive combination and signal amplification steps on 6 carbamazepine reference samples with different concentrations prepared in the step (5) and a mixed solution of carbamazepine-HRP (namely 6 standard curve points) by using the optical fiber probe prepared in the step (1).
Competitive binding: contacting the fiber-optic probe with the mixed solution of the reference sample and the small-molecule drug-HRP conjugate, wherein the small-molecule drug and the small-molecule drug-HRP conjugate in the reference sample are competitively combined with the specific small-molecule drug monoclonal antibody on the fiber-optic probe;
signal amplification: and then contacting the fiber probe with an HRP biological color developing agent, and enabling the HRP bound on the fiber probe to be bound with the HRP biological color developing agent with high affinity, so as to generate metal precipitates and bring the spectral red shift of the surface of the optical fiber, thereby obtaining the spectral shift of the reference sample. The HRP biological color developing agent used in the detection example is 3, 3-diaminobenzidine tetrahydrochloride (DAB), and when the HRP biological color developing agent DBA is combined with HRP, brown metal precipitate is generated, so that the signal amplification effect is achieved.
Taking DAB as an example of a signal amplification reagent, the entire test can be completed in 465 seconds from the detection of the sample mixed solution, the cleaning and the signal amplification by using a pre-functionalized optical fiber, and the detection flow is shown in FIG. 2. Specifically, the preparation of the fiber-optic probe and the first elution can be done in advance, and thus, the test time of the sample includes 5min +45s +2 min-465 s (i.e., 7 minutes and 45 seconds). Each concentration corresponds to a different spectral shift, and the relative inhibition (%) is calculated as:
relative inhibition (%) of a reference sample of carbamazepine
{ 1- (spectral shift of a certain carbamazepine reference sample/spectral shift of a zero carbamazepine concentration reference sample) } × 100%;
in particular, the lowest detection line obtained according to the invention is 10ng/mL, the lowest quantification line is 50 ng/mL, and the highest detection line can reach 800ng/mL or higher. Therefore, for the convenience of quantitative determination, the following detection example shows that the setting of the quantitative range (0.5-800 ng/mL) corresponds to the concentration of carbamazepine in the whole blood sample of 1.0-16 μ g/mL, and meets the clinical quantitative determination of the diagnostic concentration of carbamazepine. Specific data include relative inhibition (%) data as shown in table 1 below.
TABLE 1
(8) Obtaining a standard curve of the concentration-relative inhibition (%) of the small molecule drug:
based on the data obtained above, the GraphPad Prism Software (version 9.02) from GraphPad Software, usa, was used to establish the carbamazepine "concentration-relative inhibitory property on a 20-fold diluted matrix of healthy human whole blood(%) "competitive specific binding Standard Curve (shown in FIG. 3, R)20.999; x-axis plotted against inhibition plotted against carbamazepine concentration data). The nonlinear standard curve is applied to data calculation of clinical samples, and 20-fold dilution is considered, so that the concentration range of the carbamazepine of real samples is 0-16 mu g/mL, and the quantitative concentration range is set as follows: 1-16 μ g/mL. Therefore, the concentration of carbamazepine (μ g/mL) in the sample to be tested is calculated by the formula:
X＝{Y*B*20/(A-Y)}/1000
{ (relative inhibitory 141.2)/(83.05-relative inhibitory) } 0.02 { (relative inhibitory 141.2) } b
Wherein: a represents maximum specific binding; b represents the equilibrium dissociation constant. 20 represents a 20-fold dilution of the sample and 1000 represents the conversion of concentration units from ng/mL to μ g/mL.
(9) Competitive binding and signal amplification are carried out on a mixed solution of a sample to be detected and a carbamazepine-HRP conjugate:
and carrying out competitive binding and signal amplification on the mixed solution of the sample to be detected by referring to the competitive binding and signal amplification steps of the mixed solution of the reference sample and the small molecule drug-HRP conjugate.
Competitive binding: contacting the fiber probe with a mixed solution of the sample to be detected and the small molecule drug-HRP conjugate, wherein the small molecule drug and the small molecule drug-HRP conjugate in the sample to be detected are combined with the specific small molecule drug monoclonal antibody on the fiber probe in a competitive manner;
signal amplification: and then contacting the optical fiber probe with an HRP biological color developing agent, combining the HRP combined on the optical fiber probe with the HRP biological color developing agent with high affinity, generating metal precipitates, bringing the spectral red shift of the surface of the optical fiber, and obtaining the spectral shift of the sample to be detected.
(10) Obtaining the concentration of the small molecule drug of the sample to be detected:
the following detection signal intensities (i.e., spectral shift data) were obtained for 2 samples to be measured, and the relative inhibitability (%) of the samples to be measured was calculated:
relative inhibition (%) of the sample to be tested
{ 1- (spectral shift of sample to be tested/spectral shift of reference sample with zero small molecule drug concentration) } × 100%;
and then obtaining the concentration of the small molecule drug in the sample to be detected based on the nonlinear standard curve of the concentration of the small molecule drug-relative inhibition (%). The calculation results are shown in table 2:
TABLE 2
Comparative example
Comparative example 1
The comparative example 1 is different from the detection example of the small molecule drug carbamazepine blood concentration only in that: no signal amplification step was performed, i.e. no HRP chromogenic reagent was used. The test results of comparative example 1 are shown in table 3.
TABLE 3
As can be seen from comparative example 1, in the case of no signal amplification, the detected spectral shift is very small (only between 0.04 and 0.1), and is close to the detection limit of the instrument, so that the detection method provided by the application cannot be effectively implemented.
Comparative example 2
The comparative example 2 is different from the detection example of the small molecule drug carbamazepine blood concentration only in that: comparative example 2A simulated test sample (also called quality control samples) was diluted 20-fold using a non-high salt sample dilution buffer (instead of a high salt sample dilution buffer), and the results of the test of comparative example 2 are shown in FIG. 4. from FIG. 4, it can be seen that at zero concentration of carbamazepine, the theoretical (i.e., in the absence of serum matrix) optical signal was around 15.5nm (see Table 1). when blood was diluted 20-fold using a non-high salt sample dilution buffer, the optical signal of the carbamazepine zero concentration sample increased to about 16.5-17.5 nm, the 1-2 nm signal difference resulted from the interference of serum components (including plasma proteins, fats and proteins, etc.) with the background signal of the fiber detection. however, when blood was diluted 20-fold using a high salt sample dilution buffer, the optical signal at zero concentration of carbamazepine was 15.6nm (see Table 1), maximally close to the theoretical value. It is known that high salt sample dilution buffers achieve two effects: on one hand, the background interference signal of the blood matrix to the optical fiber biosensing is minimized; on the other hand, the background interference signal of the blood matrix to the optical fiber biosensing is stabilized.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.
Claims (10)
1. A method for detecting the blood concentration of a small molecule drug is characterized by comprising the following steps:
(1) preparing an optical fiber probe: fixing a biotinylated specific micromolecular drug monoclonal antibody on the surface of a streptavidin optical fiber to obtain an optical fiber probe;
(2) respectively obtaining a reference sample and a sample to be detected:
adding small molecule drug standard substances with different concentrations into a blood reference substance to obtain a plurality of reference samples with different small molecule drug concentrations;
obtaining a blood sample of a person to be tested with the blood concentration of the small molecular drug to obtain a sample to be tested;
(3) competitive binding and signal amplification of the reference sample:
mixing the plurality of reference samples with different small molecule drug concentrations with the small molecule drug-horseradish peroxidase conjugate in equal volumes respectively to obtain a plurality of mixed liquor of the reference samples and the small molecule drug-horseradish peroxidase conjugate, and performing the following steps of competitive binding and signal amplification on the mixed liquor of the plurality of reference samples and the small molecule drug-horseradish peroxidase conjugate:
competitive binding: contacting the fiber-optic probe with a mixed solution of the reference sample and the small molecule drug-horseradish peroxidase conjugate, wherein the small molecule drug and the small molecule drug-horseradish peroxidase conjugate in the reference sample are competitively combined with the specific small molecule drug monoclonal antibody on the fiber-optic probe;
signal amplification: then contacting the optical fiber probe with a horseradish peroxidase biological color developing agent, and binding the horseradish peroxidase bound on the optical fiber probe with the horseradish peroxidase biological color developing agent at high affinity to generate metal precipitates, so as to bring the spectral red shift of the surface of the optical fiber and obtain the spectral shift of a reference sample;
(4) obtaining a standard curve of the concentration-relative inhibition (%) of the small molecule drug:
taking the concentration of the small-molecule drug in a reference sample as an X axis and the relative inhibition as a Y axis, and drawing a 'concentration-relative inhibition (%)' nonlinear standard curve; wherein, the calculation of the relative inhibition of the reference samples with different small molecule drug concentrations takes the spectral shift of the reference sample with zero small molecule drug concentration as a reference, and the formula is as follows:
relative inhibition (%) of a reference sample of a certain small molecule drug concentration
{ 1- (spectral shift of a reference sample of a certain small molecule drug concentration/spectral shift of a reference sample of zero small molecule drug concentration) } × 100%;
(5) competitive binding and signal amplification of the sample to be tested:
mixing the sample to be detected and the small molecule drug-horseradish peroxidase conjugate in equal volume to obtain a mixed solution of the sample to be detected and the small molecule drug-horseradish peroxidase conjugate, and performing the following competitive binding and signal amplification steps on the mixed solution of the sample to be detected and the small molecule drug-horseradish peroxidase conjugate:
competitive binding: contacting the optical fiber probe with the mixed liquid of the sample to be detected and the small molecule drug-horseradish peroxidase conjugate, wherein the small molecule drug and the small molecule drug-horseradish peroxidase conjugate in the sample to be detected are competitively combined with the specific small molecule drug monoclonal antibody on the optical fiber probe;
signal amplification: then contacting the optical fiber probe with a horseradish peroxidase biological color developing agent, and binding the horseradish peroxidase bound on the optical fiber probe with the horseradish peroxidase biological color developing agent at high affinity to generate metal precipitates, so as to bring the spectral red shift of the surface of the optical fiber and obtain the spectral shift of a sample to be detected;
(6) obtaining the concentration of the small molecule drug of the sample to be detected:
calculating the relative inhibition (%) of the test sample:
relative inhibition (%) of the sample to be tested
{ 1- (spectral shift of test sample/spectral shift of zero small molecule drug concentration reference sample) } × 100%;
and obtaining the concentration of the small molecule drug in the sample to be detected based on the nonlinear standard curve of the small molecule drug concentration-relative inhibition (%) according to the relative inhibition (%) of the sample to be detected.
2. The method for detecting the blood concentration of the small-molecule drug according to claim 1, wherein the horseradish peroxidase biological color-developing agent is 3, 3-diaminobenzidine tetrahydrochloride.
3. The method for detecting blood concentration of a small molecule drug according to claim 1, further comprising:
first elution: after the optical fiber probe is prepared, eluting the specific micromolecular drug monoclonal antibody which is not fixed on the surface of the streptavidin chip;
and (3) second elution: and in the competitive binding and signal amplification step of the reference sample and the competitive binding and signal amplification step of the sample to be detected, after the competitive binding, eluting the small molecule drug-horseradish peroxidase conjugate which is not bound with the specific small molecule drug monoclonal antibody.
4. The method for detecting the blood concentration of the small molecule drug according to claim 1, wherein the blood control is whole blood substrate, serum or plasma of a person who does not use the small molecule drug;
the blood sample of the person to be tested with the blood concentration of the small molecular drug is whole blood matrix, blood serum or blood plasma of the person to be tested with the blood concentration of the small molecular drug.
5. The method for detecting the blood concentration of the small molecule drug according to claim 1, wherein in the step of obtaining the reference sample and the test sample, respectively, the reference sample and the test sample are diluted 15-25 times by using a high-salt sample dilution buffer; wherein the high-salt sample dilution buffer solution is a 10mM pH 7.4 phosphate buffer solution containing 100-300 mM NaCl, 0.02% by mass of Tween-20 and 0.1% by mass of BSA.
6. The method for detecting the blood concentration of the small molecule drug according to claim 5, wherein the high-salt sample dilution buffer is used for respectively diluting a reference sample and a sample to be detected by 20 times; wherein the high-salt sample dilution buffer solution is a 10mM pH 7.4 phosphate buffer solution containing 274mM NaCl, 0.02% Tween-20 by mass and 0.1% bovine serum albumin by mass.
7. The method for detecting the blood concentration of a small molecule drug according to claim 1, wherein the small molecule drug-horseradish peroxidase conjugate is a small molecule drug, a small molecule drug-bovine serum albumin complex or a small molecule drug-ovalbumin complex and horseradish peroxidase conjugate.
8. The method for detecting the blood concentration of a small molecule drug according to claim 1, wherein the small molecule drug is selected from carbamazepine, aminophylline, warfarin, quinidine, methotrexate, cyclosporine, vancomycin, digoxin, or a small molecule antiviral drug.
9. The method for detecting the blood concentration of the small molecule drug according to claim 1, wherein the concentration of the small molecule drug-horseradish peroxidase conjugate is in a range of 0.05-0.20 μ g/mL.
10. The method for detecting the blood concentration of the small molecule drug according to claim 1, wherein the spectral shift of the sample to be detected and/or the spectral shift of the reference sample with different antibody concentrations are within a range of 10-25 nm.
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| WO2023098087A1 (en) * | 2021-12-01 | 2023-06-08 | 西湖大学 | Method for measuring plasma concentration of small molecule drug |
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| CN114624197B (en) | 2024-09-03 |
| WO2023098087A1 (en) | 2023-06-08 |
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