Sensitive Luminometric Method for Protein Quantification in Bacterial Cell Lysate Based on Particle Adsorption and Dissociation of Chelated Europium

Abstract

A sensitive and rapid assay for the quantification of proteins, based on sample protein adsorption to Eu(3+)-chelate-labeled nanoparticles, was developed. The lanthanide ion of the surface-conjugated Eu(3+) chelate is dissociated at a low pH, decreasing the luminescence signal. The increased concentration of the sample protein prevents dissociation of the chelate, leading to a high luminescence signal due to the nanoparticle-bound protein. The assay sensitivity for the quantification of proteins was 130 pg for bovine serum albumin (BSA), which is an improvement of nearly 100-fold from the most sensitive commercial methods. The average coefficient of variation for the assay of BSA was 8%. The protein-to-protein variability was sufficiently low; the signal values varied within a 28% coefficient of variation for nine different proteins. The developed method is relatively insensitive to the presence of contaminants, such as nonionic detergents commonly found in biological samples. The existing methods tested for the total protein quantification failed to measure protein concentration in the presence of bacterial cell lysate. The developed method quantified protein also in samples containing insoluble cell components reducing the need for additional centrifugal assay steps and making the concept highly attractive for routine laboratory work.

Method for Estimation of Protein Isoelectric Point

“…The performance of the developed assay was examined in the presence of four contaminants commonly found in biological samples. Due to the high tolerance shown for the corresponding protein quantification assay based on the same detection principle, we expected the developed method to have a good tolerance for contaminants . Detergent Triton X-100, NaCl salt, organic solvent ethanol, and polymer PEG 3000 were tested for their highest tolerated concentration.…”

“…Amino-modified polystyrene particles, 240 nm in diameter, were labeled with 9-dentate Eu 3+ chelate, {2,2′,2″,2‴-{[4′-(4‴-isothiocyanatophenyl)-2,2′:6,6″-terpyridine-6,6″-diyl]bis(methylenenitrilo)}tetrakis(acetato)}europium, as described previously. 18 Before labeling, the particles were washed by centrifugation (19000g for 5 min) several times. The 9-dentate (20 nmol) Eu 3+ chelate was added to 250 μL of a 30 mM carbonate buffer (pH 9.9) containing 0.11 pmol of particles.…”

“…Previously, we have constructed and reported a mix-and-measure fluorescence quenching, time-resolved luminescence resonance energy transfer (TR-LRET), and dissociation-based methods for the quantification of proteins using the adsorption of an analyte to nanoparticles. − These microtiter-well assays are extremely sensitive with detection limits of subnanogram quantities of the protein. Here, we developed a sensitive and fast method for the estimation of the protein isoelectric point.…”

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

“…Standard luminescence plate readers found in most laboratories are utilized, making the methods amenable for the end-users. As labeled particles are not required in the current method, the concept may be applied to match particles which may have more specificity to the adsorbing target analytes in contrast to the methods developed earlier by us. − , Moreover, the study on the effects of the particle surface properties, counting of particles, or estimation of the size of the particles can be potential applications for the method.…”

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.…”