Label-Free Electrochemical Immunoassay for C-Reactive Protein

Abstract

C-reactive protein (CRP) is one of the most expressed proteins in blood during acute phase inflammation, and its minute level increase has also been recognized for the clinical diagnosis of cardio vascular diseases. Unfortunately, the available commercial immunoassays are labour intensive, require large sample volumes, and have practical limitations, such as low stability and high production costs. Hence, we have developed a simple, cost effective, and label-free electrochemical immunoassay for the measurement of CRP in a drop of serum sample using an immunosensor strip made up of a screen printed carbon electrode (SPE) modified with anti-CRP functionalized gold nanoparticles (AuNPs). The measurement relies on the decrease of the oxidation current of the redox indicator Fe3+/Fe2+, resulting from the immunoreaction between CRP and anti-CRP. Under optimal conditions, the present immunoassay measures CRP in a linear range from 0.4–200 nM (0.047–23.6 µg mL−1), with a detection limit of 0.15 nM (17 ng mL−1, S/N = 3) and sensitivity of 90.7 nA nM−1, in addition to a good reproducibility and storage stability. The analytical applicability of the presented immunoassay is verified by CRP measurements in human blood serum samples. This work provides the basis for a low-priced, safe, and easy-to-use point-of-care immunosensor assay to measure CRP at clinically relevant concentrations.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

“…The two systems share the use of ligands markedly smaller than IgG and that extend roughly 3 nm away from the transducing surface. Since IgG has been already successfully used in CRP diagnostics, for instance, as a reagent in lateral flow, fluorescence immunochromatography and different sandwich ELISA assays [ 33 , 34 , 35 , 36 , 37 ] with sensitivity until 1 ng/mL −1 , it can be expected that nanobodies might also substitute the original immunoreagents in these diagnostic methods since their small dimensions enable a higher ligand density, a condition that reduces background signal and favors sensitivity. Nanobodies are particularly stable, simple to engineer and functionalize and (as other recombinant small ligands) can be produced inexpensively and already fused to different tags suited to specific downstream applications that speed up the coating steps and, consequently, significantly reduce the analysis costs and, therefore, make diagnostics more accessible with respect to the use of IgG monoclonal antibodies.…”

Recent Advances in CRP Biosensor Based on Electrical, Electrochemical and Optical Methods

“…CRP is synthesized in the liver and circulates in the body via plasma. Under normal conditions, the plasma concentration of CRP is generally less than 2.0 mg/L [ 18 ]; excess CRP concentrations above 2.0 mg/L can therefore be used to suspect pathologies such as cardiovascular disease, infection, and inflammation [ 19 ].…”

A Surface Acoustic Wave Sensor with a Microfluidic Channel for Detecting C-Reactive Protein

“…However, while these methods reported sufficient sensitivity, they were time consuming, expensive, and involved complicated detection procedures. To overcome these limitations, various biosensors based on electrochemical and optical methods [12,[15][16][17][18][19][20][21][22] for CRP detection analysis have been reported. However, there is a need to develop a simple and highly efficient CRP detection technique for routine clinical testing.…”