We present a simple and efficient colorimetric assay strategy for ultrasensitive visual detection of human α-thrombin, which is essentially based on the formation of the DNA1-thrombin-DNA2 sandwich complex-bridged gold nanoparticle (Au NP) oligomers. Unlike the traditional colorimetric sensing strategies which induced the nanoparticle aggregates with uncontrolled aggregate size. In this work, the DNA1with rich G bases was firstly conjugated on the surfaces of Au NPs fixed on the hexadecyl trimethylammonium bromide (CTAB)-coated glass slide, and thrombin was captured by the DNA1. Then, the other DNA2 with rich G bases interacted with the former DNA1-thrombin complex and formed a DNA1-thrombin-DNA2 sandwich complex. The subsequently added Au NPs can be bound to the Au NP-DNA1-thrombin-DNA2 via Au-S bond to trigger the formation of Au NP oligomers, an apparent color change of the single Au NPs from green to yellow and red was observed under dark field microscopy. By measuring the intensity change of the yellow and red Au NPs, the concentration of target thrombin could be accurately quantified. As a proof of concept experiment, the formation of Au NP oligomers resulted in significantly improved sensitivity (10 fM of limit of detection and 20 fM of limit of quantity) and wider linear dynamic range of thrombin detection (20 fM-20 nM), the relative standard deviation (RSD) was less than 5.73% (n = 5). In addition, in order to validate the potential application in clinical diagnosis, the content of thrombin in a human serum samples was also quantified.
Keywords: Colorimetric; DNA1-thrombin-DNA2 sandwich; Dark-field microscopy; Gold nanoparticle oligomers; Thrombin.
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