Ultrasensitive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching

Abstract

A new easy-to-use method for quantification of proteins in solution has been developed. It is based on adsorption competition of the sample protein and fluorescently labeled bovine serum albumin (BSA) onto gold particles. The protein concentration is determined by observing the magnitude of fluorescence altered by quenching the fluorescence on the gold particles in a homogeneous assay format. Under optimal low pH conditions, the assay allowed the determination of picogram quantities (7.0 microg/L) of proteins with an average variation of 4.5% in a 10 min assay. The assay sensitivity was more than 10-fold improved from those of the commonly used most sensitive commercial methods. In addition, the particle sensor provides a simple and rapid assay format without requirements for hazardous test compounds and elevated temperature. Eleven different proteins were tested with the constructed sensor exhibiting a protein-to-protein variability less than 15% allowing protein concentration measurements without the need for recalibration of different proteins.

DNA template-synthesized silver nanoparticles: A new platform for high-performance fluorescent biosensing of biothiols

“…Through fluorescence resonance energy transfer (FRET) process or photo-induced electron transfer (PET) process, most fluorescent dyes could be quenched by metal nanoparticles [6][7][8][9][10][11][12][13]. When gold nanoparticles (GNPs) were employed as metal nanoquenchers to establish fluorescent platforms for biosensing and received great success during last decades, however, little attention was paid to such applications of AgNPs.…”

“…In recent years, to develop sensitive, rapid and cost-effective biosensors, metal nanoparticles were employed as super nanoquenchers instead of traditional organic dyes in design of fluorescent sensors [6][7][8][9][10][11][12][13]. For the much higher quenching efficiency, metal nanoparticle-based fluorescent sensors were successfully developed with advantages of lower background and better sensitivity.…”

“…An alternative way is to non-cross link them by weak interactions as electrostatic force and π-π stacking interaction (Scheme 1(b)). Sensors for label-free fluorescent detections are developed under this strategy with advantages of simplicity and fast responding [8][9][10][11][12][13]. However, the relatively weak interactions make the non-cross linked combinations unstable in complex conditions, which increase the background and limit their applications in real samples.…”

An Indirect Immunoassay for Detecting Antigen Based on Fluorescence Resonance Energy Transfer

“…Pihlasalo et al reported a new and highly sensitive method to detect protein concentrations which relying on protein adsorption and quenching of fluorescently labeled protein. This method is based on the competitive assay principle with noncovalent adsorption of sample protein and dipyrrylmethene-BF2 530 labeled BSA on nanosized gold particles in a homogeneous assay format [10]. Mayilo et al reported the homogeneous sandwich immunoassay with GNPs as fluorescence quenchers.…”

High Sensitivity Luminescence Nanoparticle Assay for the Detection of Protein Aggregation

“…The developed assay is based on the nonspecific adsorption of proteins and protein aggregates to the solid surface, which, in contrast to the commercial assays, is not highly dependent on the surface characteristics of proteins or protein aggregates as also shown earlier. 24,25 The ThT assay possesses high background fluorescence from the ThT dye and short dynamic range for the aggregate sizes. 28,29 Wide size range from approximately 10 to 1 000 nm of aggregated protein was detectable with the TR-LRET nanoparticle assay suggesting that the method may be more widely applicable to follow aggregation, reactions, and decomposition of various substances in biochemical laboratories and in quality assessment of protein products in industry.…”