Electrochemical Sandwich Assays for Biomarkers Incorporating Aptamers, Antibodies and Nanomaterials for Detection of Specific Protein Biomarkers

The development of sensitive and selective assays for protein biomarkers and other biological analytes is important for advancing the fields of clinical diagnostics and bioanalytical chemistry. The potential advantages of using aptamers in electrochemical sandwich assays are being increasingly recognized. These assays may include an aptamer as both capture and detection agent or a combination of an aptamer with a different partner such as an antibody, a lectin or a nanomaterial. The second binding partner in the sandwich structure is typically conjugated to a redox marker, a catalyst or an enzyme that can be used to generate the signal needed for electrochemical detection. Nanoparticles and other nanostructures can be used as the carriers for multiple molecules of the detection partner and thereby increase the signal. Nanostructured surfaces can be used to increase surface area and improve electron transfer. Sensitive electrochemical methods including impedance, differential and square-wave voltammetry and chronocoulometry have been used for electrochemical signal read-out. Impressive results have been achieved using electrochemical sandwich assays in terms of limit of detection and linear range for a growing range of analytes. The recent progress for this type of assay for proteins and other biomarkers is the subject of this review.

Biotin interference in several electrochemiluminescence immunoassays (eclia)

– Background: The aim of this study is to define the interference of biotin in several endocrine, tumor marker, and vitamin assays performed by an electrochemiluminescence method, trying to determinate the critical level that causes biotin interference. – Material and methods: Working biotin solutions were prepared in phosphate-buffered saline (PBS) at different concentrations (10000, 7500, 5000, 2500, 1250, 625, and 312.5 ng/mL), which were spiked on the samples to obtain final concentrations ten-fold lower. Each serum biotin dilution was tested in triplicate, using at least two levels of analytes. Determinations of several endocrine, vitamins, tumor and bone markers were carried-out with eletrochemilumenescent immunoassays on the cobas e801 and cobas e411. Comparison between the results obtained by analyzing the biotin-spiked samples and the reference PBS-spiked samples was performed using Microsoft Excel. The relative bias with the interfering-free specimen was calculated for each biotin concentration. Interference was considered significant when the relative bias exceeded 10%. Glick´s interferographs were performed plotting the percentage of change vs. biotin concentration. – Results: Analyte concentrations were spuriously decreased in 12 sandwich immunoassays and falsely increased in 11 competitive immunoassays. However thyrotropin and CA 15.3 antigen were not significantly affected. – Conclusions: Except CA 15.3 and TSH, the methods tested were susceptible to biotin interference. Falsely low values occurred in sandwich assays and high bias in competitive assays. Clinicians and laboratorians should be aware of the medical importance of biotin interference as a cause of misdiagnosis and incorrect treatment.

Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins

Analytical performance of an alternative binding protein (rcSso7d) and antibodies are compared in cellulose-based, full sandwich assays.

Machine Learning Approach to Enhance the Performance of MNP-Labeled Lateral Flow Immunoassay

The use of magnetic nanoparticle (MNP)-labeled immunochromatography test strips (ICTSs) is very important for point-of-care testing (POCT). However, common diagnostic methods cannot accurately analyze the weak magnetic signal from ICTSs, limiting the applications of POCT. In this study, an ultrasensitive multiplex biosensor was designed to overcome the limitations of capturing and normalization of the weak magnetic signal from MNPs on ICTSs. A machine learning model for sandwich assays was constructed and used to classify weakly positive and negative samples, which significantly enhanced the specificity and sensitivity. The potential clinical application was evaluated by detecting 50 human chorionic gonadotropin (HCG) samples and 59 myocardial infarction serum samples. The quantitative range for HCG was 1–1000 mIU mL−1 and the ideal detection limit was 0.014 mIU mL−1, which was well below the clinical threshold. Quantitative detection results of multiplex cardiac markers showed good linear correlations with standard values. The proposed multiplex assay can be readily adapted for identifying other biomolecules and also be used in other applications such as environmental monitoring, food analysis, and national security.

Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase

The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with β-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.