Colorimetric Detection of DNA by Modulation of Thrombin Activity on Gold Nanoparticles

Abstract

A colorimetric, non-cross-linking aggregation-based gold-nanoparticle (AuNP) probe has been developed for the detection of DNA and the analysis of single-nucleotide polymorphism (SNP). The probe acts by modulating the enzyme activity of thrombin relative to fibrinogen. A thrombin-binding aptamer with a 29-base-long oligonucleotide (TBA(29)) assembled on the nanoparticles (TBA(29)-AuNPs) through sandwich DNA hybridization was found to possess ultra-high anticoagulant potency. The enzyme inhibition of thrombin was determined by thrombin-induced aggregation of fibrinogen-functionalized 56 nm AuNPs (Fib-AuNPs). The potency of the inhibition of TBA(29)-AuNPs relative to thrombin--and thus the degree of aggregation of the Fib-AuNPs--is highly dependent on the concentration of perfectly matched DNA (DNA(pm)). Under optimal conditions [Tris-HCl (20 mM, pH 7.4), KCl (5 mM), MgCl(2) (1 mM), CaCl(2) (1 mM), NaCl (150 mM), thrombin (10 pM), and TBA(29)-AuNPs (20 pM)], the new TBA(29)-AuNP/Fib-AuNP probe shows linear sensitivity to DNA(pm) in the concentration range 20-500 pM with a correlation coefficient of 0.96. The limit of detection for DNA(pm) was experimentally determined to be 12 pM, based on a signal-to-noise ratio (S/N) of 3. The new probe was successfully applied to the analysis of an SNP that is responsible for sickle cell anemia. Relative to conventional molecular-beacon-based probes, the new probe offers the advantages of higher sensitivity and selectivity towards DNA and lower cost, showing its great potential for practical studies of SNPs.

Functional gold nanoparticles for sensing applications

“…Jian and Huang [131] reported a colorimetric detection of DNA sequences using a non-cross-linking approach. They found that the spatial distance between AuNP and thrombin (i.e., a serine protease involved in a multitude of biological processes) has a strong influence on the catalytic activity of thrombin.…”

Toward Non-Enzymatic Ultrasensitive Identification of Single Nucleotide Polymorphisms by Optical Methods

“…A second pool of Au-nps modified with thrombin substrate fibrinogen was not converted into aggregated fibrin-Au-nps due to binding of fully complementary DNA to the former Au-nps. This assay allowed detection of DNA by the naked eye at 12 pM concentration, although no information of the type of mismatch could be gained by this method [51]. Finally, a product of an SNP containing gene can also be applied for SNP sensing [52].…”

“…A colorimetric detection assay was reported using cross-linking [48] or non-cross-linking of Au-nps mediated by DNA aptamers [49]. Among other DNA aptamers, a 29-nt long thrombin-binding aptamer (TBA) has been well studied and already applied in SNP diagnostics [50,51]. Chen and co-workers used self-assembled bivalent thrombin-binding aptamers and fibrinogen-conjugated Au-nps to detect DNA, based on controlled thrombin activity ( Figure 4C) [50].…”

“…Using this assay, SNP could be detected at 20 pM concentration of target DNA with advantage of confirmed high selectivity. Alternatively, Au-nps modified with 29-nt long thrombin-binding aptamer inhibiting enzymatic activity of thrombin were applied in a sandwich hybridization assay [51]. A second pool of Au-nps modified with thrombin substrate fibrinogen was not converted into aggregated fibrin-Au-nps due to binding of fully complementary DNA to the former Au-nps.…”

“…As illustrated below, several non-enzymatic techniques provide advantages of improved repeatability, decreased cost and robustness. Based on the chemical nature of the utilized probe, these techniques can be divided into three groups: methods applying nano-and magnetic-particles , (1) aptamer-and protein-based assays [47][48][49][50][51][52], (2) and other techniques utilizing bright chemically modified nucleic acid analogues [53][54][55][56][57][58][59][60]…”