| Therapeutic drug monitoring(TDM)is an important means to understand and control the concentration of drugs in the body fluids of patients in clinical therapeutics.It can provide individual information of drugs used by patients for clinical therapeutics,and is an important part of precision medicine system.The analysis of TDM data can provide patients with the optimal therapeutic schedule,which can not only reduce the damage of adverse drug reactions,but also reduce unnecessary medical expenditure for patients from the economic aspect.TDM is mainly applied to drugs with narrow therapeutic window or concentrationdependent adverse drug reactions,such as immunosuppressants,antiepileptics,antibiotics,antipsychotics,etc.At present,the common methods of TDM include chromatography and immunoassay.The chromatography technology is mature,but the sample processing is complicated and the turnover time is long.Immunoassay has the advantages of simple operation and rapid detection,but the results are easily affected by endogenous substances.According to the current investigations of TDM in hospitals and laboratories,most hospitals are limited by cost,time,technician and other factors,resulting in the role of TDM in clinical therapeutics is not as useful as expected.It indicates that the existing detection methods cannot fully meet the needs of TDM in clinical routine detection.Therefore,it is urgent to establish a new detection method which can quickly,simply and accurately quantitatively analyze drug concentration in clinical samples.Surface plasmon resonance(SPR)biosensor is a kind of biosensor based on physical optics technology to detect the interaction between two molecules.It has the characteristics of non-label,real-time monitoring,high specificity and high sensitivity.It has been used in food analysis,clinical diagnosis,environmental monitoring,pathogenic microorganism detection,new drug development and other fields.In the field of concentration detection,SPR biosensors have been used to detect pesticide residues and illegal drugs in food and water pollutants.However,few existing quantitative detection studies have focused on the quantitative detection of drugs,metabolites and biomarkers in complex matrices such as body fluids.This study mainly focused on the quantitative analysis method of SPR biosensor for antibacterial drugs.Based on the research of quantitative analysis methods of samples in vitro,the quantitative detection of animal blood samples,milk samples,and clinical human blood samples has been realized step by step.In addition,the exploration of new aptamer recognition element provides a scientific basis for the selection and use of SPR biosensor recognition elements.Firstly,the quantitative detection of chloramphenicol based on antibody-SPR biosensor was studied.Chloramphenicol,whose structure,function and mechanism of action were clearly studied,was used as the model drug for the analysis method,and the feasibility of SPR biosensor for quantitative analysis was confirmed.Firstly,a SPR biosensor with chloramphenicol antibody was constructed and the performance of the biosensor was detected.The affinity between biosensor and chloramphenicol was strong.The chip had good activity,binds specifically to chloramphenicol,and remains stable during the experiment.Secondly,the method was verified from the aspect of the detection range,limit of detection(LOD),precision and accuracy.The detection range was 0.1-50 ng/m L and the LOD was 0.099±0.023 ng/m L.The intra-day and inter-day precision of medium-and highconcentration samples were lower than 15%,and the accuracy was 98% to 122%.The influence of methanol solvent and serum matrix in blood samples and the recovery rate of sample pretreatment were further studied.The response value was positively correlated with the concentration of methanol.The matrix effect of medium-and high-concentration samples were less than 10%,and the extraction recovery was less than 20%.Finally,the quantitative determination of chloramphenicol in rat blood samples was realized.This study systematically evaluated the feasibility of SPR biosensors for quantitative detection and demonstrated that SPR biosensors can be applied to quantitative analysis of blood samples.Secondly,the quantitative detection of chloramphenicol based on aptamer-SPR biosensor was studied.The SPR biosensor using chloramphenicol aptamer as the recognition element was explored from the aspects of fixing aptamer and chip construction strategy,selection of suitable running buffer and sample diluent,and suitable sample detection conditions.Firstly,after comparing different chip construction strategies,it was found that the method of fixing streptavidin on the surface of CM5 chip and then fixing biotin labeled aptamer could achieve the optimal effect.Secondly,after comparing different buffer combinations,the best buffer condition for chloramphenicol aptamer was confirmed.After comparing the influence of different dissociation time on the binding of samples,it was found that 60 s was the most suitable dissociation condition.Finally,the aptamer-SPR biosensor was used to detect chloramphenicol in milk samples.This study confirms that aptamers,as new biometric components,have the potential to be used for quantitative analysis,but their application conditions are more stringent and complex.To develop this type of SPR biosensor,further studies on the mechanism and conditions of binding of aptamer to target molecules are needed.Finally,the quantitative detection of polymyxin B(PMB)based on SPR biosensor was studied.Using polymyxin B as the detection object,SPR biosensor with polymyxin B antibody as the recognition element was established to detect clinical blood samples,and further confirmed the feasibility of SPR biosensor for clinical TDM.Firstly,the SPR biosensor based on PMB antibody was constructed and its performance was tested.The affinity between the chip and PMB was strong and the activity of the chip was good.Only PMB could respond and had a much higher response value than other drugs which could be administrated in combination,indicating strong specificity between the chip and PMB.The samples were stable during the testing.Secondly,the SPR curve signal conversion method was optimized to reduce the background signal interference of endogenous substances in the sample.Thirdly,the pretreatment method of serum samples was studied.The influence of different dilution ratio of sample on the test results and the influence of thermal denaturation on the test results were investigated.Then a simple and convenient blood sample pretreatment method suitable for SPR biosensor was proposed.Furthermore,the detection range,LOD,precision and accuracy of SPR method were investigated,confirming that the chip could be used for the quantitative detection of PMB in serum.Finally,the SPR method was used for the detection of clinical serum samples,and the results were compared with the TDM reports from the hospital using LC-MS/MS method,which showed that the results of the two detection methods were significantly correlated.This study showed that the TDM of PMB in clinical blood samples applied by SPR biosensor was in good agreement with the classical method.Interestingly,the sample pretreatment method was simple,which could improve the efficiency of TDM in clinical work.In summary,this research established the quantitative analysis method of some antibacterial drugs based on SPR biosensor,systematically evaluated the methodological index of this method,and scientifically explored the practical application of this method.The feasibility of SPR biosensor for quantitative detection of antibiotics was demonstrated by three different examples.This study takes the quantitative detection of antibacterial drugs in in vitro samples,animal samples,clinical samples and food samples as examples,illustrates the principle and method of SPR biosensor for concentration determination,and expands the application field of surface plasmon resonance technology.Meanwhile,more SPR biosensors can be developed for the quantitative analysis of drugs in vivo by using different recognition elements.Moreover,due to it is sensitive,accuracy,simple and rapid,SPR biosensor can meet the diversified needs of clinical TDM,and has the potential to become a new TDM technology. |
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