Nonamplification Sandwich Assay Platform for Sensitive Nucleic Acid Detection Based on AuNPs Enumeration with the Dark-Field Microscope

Abstract

A simple and efficient assay platform with high sensitivity, convenient implementation, and moderate cost in reagents and instrumentation is most appropriate for routine applications. On the basis of the gold nanoparticle (AuNP) enumeration signal readout mode established in our laboratory, we have developed a nonamplification sandwich assay for nucleic acid detection with the 3 fM limit of detection for a sequence related to Alzheimer's disease. This AuNP counting based method takes advantages of the distinctive and strong localized surface plasmon resonance light scattering with the dark-field microscope and magnetic separation. It is shown that the presence of 20 nM random DNA sequence or calf thymus DNA with a mass up to 10(6)-fold of the targets do not significantly interfere with the assay signal. The spike recoveries of Hela cell lysate sample at 109.3% for 20 pM target and 110.5% for 100 pM target indicate the potential of this proposed method in practical sample applications. This nonamplification sandwich assay platform in principle is applicable to other assays such as the immunoassay and thus would be expected to find a breadth of applications that can make the best use of the simplicity and sensitivity.

Multiplexed Detection of Attomoles of Nucleic Acids Using Fluorescent Nanoparticle Counting Platform

“…Specifically, criteria boundaries were ( C * > 10, 40° < h° < 72°) for red, ( C * > 10, 130° < h ° < 195°) for green, and ( C * > 10, 200° < h ° < 290°) for blue FNPs. The image processing software for the automatic counting of FNPs was developed in C# programming language based on our previous automatic counting method for gold nanoparticle recognition. ,, Compared with the manual counting, the automatic counting yielded greater than a 96% recognition rate at different concentration levels (Table S5), demonstrating the good performance of the automatic enumeration method.…”

Carbon Nanodot–Based Fluorescent Method for Virus DNA Analysis with Isothermal Strand Displacement Amplification

“…However, those traditional methods are time‐consuming, laborious, less sensitive, which limit their clinical applications. Recently, some researchers tried to develop novel types of biosensor for virus DNA analysis, such as nanoparticle‐assembled photonic crystal arrays, electrochemical assays, dark field sensors using plasmonic nanoparticles, piezoelectric plate sensors and fluorescence assays . To improve the sensitivity, some amplification strategies have been used like polymerase chain reaction (PCR), ligation‐based amplification, rolling circle amplification (RCA), loop mediated isothermal amplification (LAMP), entropy‐driven catalysis, and hybridization chain reaction (HCR) .…”

Individual Plasmonic Nanoprobes for Biosensing and Bioimaging: Recent Advances and Perspectives

“…The level of target single‐stranded DNA (ssDNA) is highly correlated with the number of target‐bound AuNPs, thereby allowing to easily quantify the amount of target DNA by counting the number of target‐bound AuNPs under DFM. [ 144 ] Compared with traditional DNA detection systems using fluorescence intensity measurements, the sensitivity of this method is about 60‐fold higher, underscoring the promising potential of PNPs for the detection and quantification of gene expression. [ 145 ] Furthermore, the introduction of functionalized PNPs causes a pseudo‐increase in the mass of the analyte and results in the significant signal amplification, allowing the applications of single particle counting in SPR technique.…”