| Aptamers have been widely used in the field of biological analysis due to their outstanding advantages of good chemical and thermodynamic stability,easy synthesis and low cost.Nowadays,multifarious aptamer-based methods for detection of a variety of disease biomarkers have been developed,such as colorimetry and fluorescence.In this paper,new analytical methods and sensors were explored for one cardiac biomarker(cardiac troponin I,c Tn I),on the basis of aptamers,to overcome the limitation of classic immunoassays,and reproducible and sensitive determination of c Tn I was realized.Specific research contents are outlined as follows.1.An enzyme-linked hybrid sandwich assay(ELHSA)was constructed to detect c Tn I.A unique "antibody-target-signal aptamer" structure was designed compare to traditional "antibody-antigen" immunoassay.The optimal antibody-aptamer combination and amount ratio between streptavidin-horseradish peroxidase conjugate and biotin-modified aptamer were obtained through systematical study.And one of the most beneficial interaction modes between c Tn I and the two recognition elements was revealed,that is,the binding of c Tn I protein to the aptamer was well recognized by antibodies.In addition to being a blocker,bovine serum albumin(BSA)can also be introduced into signal probe solution as an enhancer with high sensitivity and reproducibility.BSA-assisted ELHSA can detect c Tn I as low as0.24 ng/m L.2.ELHSA-based aptasensing methods including colorimetry and fluorescence,were designed and compared for c Tn I detection.The optimal type and concentration of antibody were obtained,and it was disclosed that the recognition site of antibody had an important impact on the recognition efficiency of c Tn I.It was demonstrated that the best recognition site of c Tn I was 41 aa～50 aa,when the aptamer(Apt2)was used in ELHSA.The detection limit of optimized colorimetric method for c Tn I is 48 pg/m L,which is an order of magnitude lower than before.In addition,the enzyme in aforementioned streptavidin-horseradish peroxidase conjugate was substituted with streptavidin-alkaline phosphatase conjugate(SALP),resulting in a fluorescent aptasensing method.On the basis of previous works,the amount ratio between S-ALP and biotin-modified aptamer were optimized and a detection limit of 3.47 ng/m L was obtained.3.A lateral flow assay-based test strip was developed for colorimetric detection of c Tn I,based on aforementioned hybrid recognition by antibody and aptamer.Streptavidin-modified gold nanoparticles(Au NPs)were utilized as the signal indicator,instead of streptavidinenzyme conjugate,and critical factors including the type and concentration of antibody on the test line,and the concentration of aptamer in detection solution,were optimized.It was demonstrated that aptamer concentration had an important impact on the recognition efficiency of target c Tn I.If the concentration of aptamer was too low,Au NPs would be very easy to aggregate;high steric hindrance on Au NPs would be produced by utilization of too much aptamer,lead to low recognition ability of aptamer toward c Tn I,and meanwhile affect the hybridization between aptamer and its complementary strand on the quality control line.The detection limit of lateral flow assay-based test strip for c Tn I is 6.54 ng/m L,and good selectivity for c Tn I was also observed. |
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