| N terminal brain natriuretic peptide?NT-proBNP?and cardiac troponin I?cTnI?are ideal markers for the prediction and diagnosis of cardiovascular disease.While they usually were detected by the traditional immunological detection technology with the shortcomings of tedious,long detection time and bulky instrument,it can not achieve point-of-care testing?POCT?.At present,lateral flow immunoassay?LFIA?has been widely applied to quantitative analysis and detection of various biomolecules because of its advantages of fast detection,convenient portability,low cost and simple operation.However,there is still the problem of non-specific adsorption and low sensitivity in lateral flow immunoassay?LFIA?,which limits its application and research in high-sensitivity biochemical markers,such as myocardial indicators,tumor markers.In the recent years,owing to the long fluorescence lifetime,good biocompatibility and strong anti-bleaching,quantum dots?QDs?have been widely used in the field of biomedical detection.Therefore,we introduce quantum dots to LFIA detection system and combine the advantages of quantum dots and LFIA to overcome the shortcomings of LFIA for the fast quantitative detection of the N terminal brain natriuretic peptide?NT-proBNP?and cardiac troponin I?cTnI?in the sera sample.The main content of this paper is as follows:?1?Quantitative and rapid detection of N terminal brain natriuretic peptide?NT-proBNP?by QDs-based lateral flow immunoassay?LFIA?.In this chapter,aqueous CdSe/ZnS@PMAH@SiO2?75 nm?,simply written as QDs@SiO2,was selected as the best labeling material for LFIA detection.Afterwards,the experimental conditions for the covalent coupling of aqueous QDs with antibody were optimized,and the fluorescent probes were prepared via an amide bond formed between active carboxyl groups and the amino groups from the antibodies with the activation of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride?EDC·HCl?and N-hydroxysulfosuccinimide?Sulfo-NHS?.After coupling,the fluorescence peak of quantum dots had no significant change,fluorescence recovery rate was 80.4%,the hydrodynamic size increased,but the uniformity of particle size distribution reduced and the Zeta potential was-36.1 mV.The above results showed that the fluorescence probes were prepared successfully,and the fluorescence characteristics of the probe and its stability in the aqueous solution were still good.Next,the experimental conditions of QD-LFIA detection system were optimized and determined.Based on the best experimental conditions,the quantitative standard curve for NT-proBNP detection was obtianed with Y=3.596×10-4X-3.57×10-3?r=0.9992?,which shows the linear relationship is good within the range of50-15000 pg/mL.Then the detection performance of the QD-LFIA system was evaluated:the intra-assay CV and inter-assay CV of this system was less than 15%,limit of detection?LOD?was 10.58 pg/mL.The established QD-LFIA offers a lower LOD and a shorter detection time than traditional colloidal gold LFIA.Finally,we compared the concentrations of 63 clinical serum NT-proBNP samples detected by QD-LFIA and the established standard method Roche diagnostic system,and the results showed that the established QD-LFIA had a good accuracy with the good linear correlation coefficient?r=0.9607?.The assay satisfies the need of sensitivity,rapid,accurate,so it has great application value in the development of in vitro diagnostic POCT reagents.?2?Quantitative and rapid detection of cardiac troponin I?cTnI?by QDs-based lateral flow immunoassay?LFIA?.First of all,fluorescence probe?QDs@SiO2@mAb?was prepared.Then,aqueous quantum dots?QDs@SiO2?and the probe?QDs@SiO2@mAb?were characterized by fluorescence spectroscopy,dynamic light scattering and agarose gel electrophoretic,which can be proved the successful coupling of aqueous QDs with antibody.Then,we detected the serum samples from 5 patients with cTnI gradient concentration using QD-LFIA,whose result showed that the corresponding T/C value increases with the increase of cTnI concentration in the sera sample,thus proving that the conjugated antibody still had a good activity.After that,we choosed the clinical serum samples to optimize the experimental conditions of LFIA system.Based on the optimized experimental conditions,we established the standard curve by detecting 12 concentration gradient cTnI calibrations,and the final quantitative standard curve was adjusted with a reference comparison curve.The quantitative linear equation is Y=0.0228X+1.2805×10-4?r=0.9995?,which shows the linear relationship is good within the cTnI concentration of 0.8-200 ng/mL.Next,the detection performance of the QD-LFIA system was evaluated:the intra-assay CV and inter-assay CV of this system was less than 10%,limit of detection?LOD?was5.63×10-3 ng/mL.The established QD-LFIA offers igher sensitivity,wider range of quantitative detection and shorter detection time than traditional colloidal gold LFIA.Finally,we compared the concentrations of118 clinical serum cTnI samples detected by QD-LFIA and the established standard method siemens direct chemiluminescence?ADVIA Centaur?,and the results showed that the established QD-LFIA has a good accuracy with the good linear correlation coefficient?r=0.9829?.The assay satisfies the need of sensitivity,rapid,accurate,so it has immense application value in the development of in vitro diagnostic POCT reagents. |
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