Studies Of Quantitative Detection Technique Based On CdSe/ZnS Fluorescent Quantum Dots And Lateral Flow Immunoassay

Lateral flow immunoassay?LFIA?technology is one of the most important and widely applied detection technologies in the field of point-of-care test?POCT?.It has been considered to be a very promising and cost-effective POCT technology in developing countries because of its various advantages,such as rapid detection,low cost,and easy operation et al.Colloidal gold and organic fluorescent dyes are commonly used as traditional labels for LFIA.However,the detection results are limited to be only qualitative or semi-quantitative.Quantum dots?QDs?,as a novel semiconductor nanomaterial,possess unique optical properties,such as wide excitation and narrow emission,adjustable emission spectra,good photochemical stability,high fluorescence intensity,long life time,etc.It has been considered as an excellent fluorescent label materials being widely used for immunofluorescence diagnosis,biosening,cell labelling,vivo imaging,photodynamic therapy.A combination of QDs and LFIA?QD-based LFIA?can reach a rapid,sensitive and quantitative detection of bacteria,viral,protein and antigen.This combination is of great significance in POCT field,which could promote a wider application of QDs materials in the biomedical field.In the present study,the fluorescent quantum dots?CdSe/ZnS,635 nm?modified by amphiphilic polymers and silica coating?QDs@PMAH and QDs@SiO₂?are used as labels.The conditions of preparation of QD fluorescence probe and the composition of QD-based LFIA system are optimized.The principle of double-antibody sandwich method and competitive method are adopted.The developed QD-based LFIA realize a rapid and quantitative detection of C-reactive protein?CRP?,procalcitonin?PCT?and aflatoxin B1?AFB1?,respectively.The QD-based LFIA for simultaneous detection of CRP and PCT has also been developed.The main research contents are as follows:1.Quantitative detection of C-reactive protein by QD-based LFIA.The QDs@PMAH are used as labels,and the coupling conditions between QDs and CRP antibodies are optimized.The QD-based LFIA system for CRP quantitative detection has been constructed employing the double-antibody sandwich method.The related factors affecting the performance of LFIA have been explored,such as sample conjugate pad,nitrocellulose membrane,amounts of probe and dilution,etc.Using the optimized detection system,the developed quantitative QD-based LFIA has a good linear regression in the range of 0.5-1000 ng/mL for CRP.The limit of detection?LOD?is 0.30 ng/mL.Compared to other POCT methods for CRP,the present method has advantages of high sensitivity,short detection time?only 3 min?and wide detection range.At the same time,the intra-assay and inter-assay coefficient of variation?CV?are all less than 15%,and the recoveries are within 95%-110%,indicating that this QD-based LFIA method are reliable,accurate and stable.Clinical samples have been used for correlation analysis between the QD-based LFIA and the gold standard method?Roche immunoturbidimetry?.The results turn out to show that they have a good concordance for all 135samples.2.Quantitative detection of PCT by QD-based LFIA employing QDs@PMAH and QDs@SiO₂.Two kinds of QDs,QDs@PMAH and QDs@SiO₂,as labels have been introduced into the QDs-based LFIA for procalcitonin quantitative detection.By optimizing the LFIA system and employing the double-antibody sandwich method,the developed quantitative LFIA for PCT has high sensitivity and requires only 12 min of detection time.Both kinds of QDs-based LFIA deliver good linear regression in the range of 0.1-100 ng/mL for intial PCT concentrations in serum sample.The LOD is 0.03 ng/mL for QDs@PMAH and 0.01 ng/mL for QDs@SiO₂,respectively.The sensitivity based on QDs@SiO₂ is higher?10 folds?than that of the commercially available POCT methods,such as colloidal gold.At the mean time,compared to the LFIA employing QDs@PMAH,the amount of probes used in the detection system based on QDs@SiO₂ is relatively less.However,the sensitibity has been higher and the stability has been greatly improved?CV<10%?.Clinical PCT serum samples have been detected by applying this QD-based LFIA.Compared with the commonly used gold standard method?VIADS enzyme-linked immunofluorescence method?in the hospital,our results show good agreement for total 140 samples.3.Quantitative detection for AFB1 by QD-based LFIA employing QDs@SiO₂.Using the more stable QDs@SiO₂ as labels,a competitive QD-based LFIA method has been developed to quantitative detection for aflatoxin B1 in foods.The detection system exhibits a good linear regression in the range of 2.5-100 ng/mL for intial AFB1 concentration in the corn sample with a median inhibitory concentration?IC50?of 0.294ng/mL for diluted sample.The LOD for AFB1 is 0.796 ng/mL and the theoretical LOD is calculated to be0.057 ng/g because of 14-fold dilution for the sample detection.Its sensitivity is 2.5 times that of the domestic industry standard colloidal gold rapid quantitative detection method.Moreover,this QD-based LFIA has good stability and high accuracy,and the recoveries are within 80%-120%.In addition,the sample extraction method is rather simple with only one step extraction and the detection time only needs 15 min.Therefore,it is very suitable for rapid and quantitative detection of aflatoxin B1 in food,feed industry or primary inspection department.4.We have tried to develop a quantitative detection method for CRP and PCT simultaneously using QDs@SiO₂ as labels.In this QD-based LFIA system,the principle of detection for PCT with double-antibody sandwich assays and detection for CRP with competitive assays have been adopted to realize simultaneous detection.Our results show that the simultaneous detection of two inflammatory markers could be achieved with the same QD labels.The detection range and the LOD are comparable to that of separate detection.However,the stability of LFIA for CRP are not good enough to be further improved.Above results show that the fluorescent QDs,as excellent labels,could be used for the quantitative detection of target analytes in LFIA,which has the advantages of quantitative,sensitive and rapid detection.In particular,the detection system based QDs@SiO₂ as label materials showes the characteristics of less immunological reagents,higher sensitivity,and better stability.As a promising label materials,the QD-based LFIA will make a powerful tool for lateral flow assay,and we believe that it can be easily applied for economic,sensitive,quantitative,and rapid detection of common diagnostics tests in the future.It could also promote POCT technology for the development of hierarchical medical system in China.