Sensitive Method for Determination of Protein and Cell Concentrations Based on Competitive Adsorption to Nanoparticles and Time-Resolved Luminescence Resonance Energy Transfer between Labeled Proteins

Abstract

A sensitive mix-and-measure method for the determination of protein and cell concentrations was developed. It is based on the competitive adsorption between the analyte and donor- and acceptor-labeled proteins to carboxylate-modified polystyrene nanoparticles. A high time-resolved luminescence resonance energy transfer (TR-LRET) signal is detected in the absence of the analyte due to the close proximity of the nanoparticle-adsorbed labeled proteins. The increased concentration of the analyte decreases the adsorption of the labeled proteins, leading to the loss of proximity and thus a decrease in the TR-LRET. The detection limit of the assay was 2.6 ng of proteins, which is higher than that of the most sensitive commercial methods. The method was also applied to cell counting, and 200 eukaryotic cells were measured in a microtiter well under the optimized conditions. The average coefficient of variation for both developed assays was approximately 10%, and the protein-to-protein variability for 11 different proteins was no more than 20%. The developed method requires no labeled particles, making the concept optimally applicable to varying targets as the material of the particle may be selected according to the application.

Protein Quantification Using Resonance Energy Transfer between Donor Nanoparticles and Acceptor Quantum Dots

“…These results indicate that only the pH of the sample must be adjusted to be applicable for various research interests. The adsorption of analyte BSA in the developed assay was not studied as a function of pH, as the most sensitive detection has been observed earlier at low pH in the nonspecific particle-based assays for protein quantification. − Thus, 3.0 mM citrate, pH 5.0, was selected as an assay buffer, providing the highest signal-to-background ratio.…”

“…BSA was chosen to demonstrate the assay performance, as it is widely used as a calibration biomolecule. Different proteins were quantified using the nonspecific nanoparticle-based assays ,,, based on the competitive protein adsorption as presented earlier by our group. Similar EuNPs and assay conditions were used in the TR-LRET assay .…”

“…Previously, we have reported competitive adsorption-based methods for the quantification of proteins using luminescent donor or quencher nanoparticles and labeled proteins. − The increase in the concentration of the analyte protein led to the decrease in RET between the acceptor-labeled protein and the donor-labeled nanoparticles or to the enhanced fluorescence of the labeled protein separated from the quencher nanoparticles. Here, we have developed a two-particle system relying on RET between polystyrene nanoparticles loaded with Eu 3+ chelates (EuNPs) and streptavidin-coated CdSe/ZnS (QD655-strep) or CdSeTe/ZnS (QD705-strep) acceptor QDs for the first time (Figure ).…”

Lanthanide-Functionalized Nanoparticles as MRI and Luminescent Probes for Sensing and/or Imaging Applications

“…Overall, luminescent lanthanide(III) ions have been predominantly incorporated as dopants in inorganic NPs, being usually made from rare-earth oxides, phosphates, or fluorides, and used as linear and/or nonlinear optical probes for biological applications. − Polymeric lanthanide(III)-based NPs, consisting of lanthanide(III) complexes embedded in polystyrene NPs, have been developed by Härmä and collaborators and successfully applied for nucleic acid diagnostics as well as for heterogeneous and homogeneous bioaffinity assays. − More recently, the same authors developed a range of NP assays for the determination of protein and cell concentrations. − Each of these assays was based on time-resolved luminescence resonance energy transfer (TR-LRET), a mechanism that took place from the europium(III)-based polystyrene NPs to diverse acceptors, such as labeled proteins or protein-coated quantum dots.…”

Ultrasensitive Protein Concentration Detection on a Micro/Nanofluidic Enrichment Chip Using Fluorescence Quenching

“…Recently, Pihlasalo et al. proposed highly sensitive methods for protein quantification based on competition adsorption of proteins to particles. − A low-end detection limit of 7.0 μg/mL for bovine serum albumin (BSA) was achieved . These methods are significant for advancing the progress in protein quantitative analyses.…”