Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano‑biological events.
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http://dx.doi.org/10.3390/microarrays4020228 | DOI Listing |
Sci Rep
January 2025
Johnson & Johnson, Therapeutics Discovery, Spring House, PA, USA.
Solution-based affinity assays are used for the selection and characterization of proteins that could be developed into therapeutic molecules. However, these assays have limitations for cell-surface proteins as in most cases their purification requires detergent solubilization and are unlikely to assume conformations in solution that resemble their native states in cell membranes. This report describes a novel electrochemiluminescence-based method, called MSD-CAT, for the affinity analysis of antibodies binding to cell-surface receptors.
View Article and Find Full Text PDFAnal Chem
January 2025
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
Ligand binding to membrane proteins initiates numerous therapeutic processes. Surface plasmon resonance (SPR), a popular method for analyzing molecular interactions, has emerged as a promising tool for in situ determination of membrane protein binding kinetics owing to its label-free detection, high surface sensitivity, and resistance to intracellular interference. However, the excitation of SPR relies on noble metal films, typically gold, which are biologically incompatible and can cause fluorescence quenching.
View Article and Find Full Text PDFAnal Chem
January 2025
School of Pharmaceutical Sciences, Guizhou University, Guiyang 550025, China.
5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are crucial epigenetic modifications in eukaryotic genomic DNA that regulate gene expression and are associated with the occurrence of various cancers. Here, we combined bisulfite conversion with 4-acetamido-2,2,6,6-tetramethyl-1-oxopiperridinium tetrafluoroborate (ACTBF, TCI) oxidation to develop a label-free and sequence-independent isothermal amplification (BTIA) assay for a genome-wide 5mC and 5hmC analysis. The BTIA strategy can distinguish 5mC and 5hmC signatures from other bases with high sensitivity and good specificity, avoiding sophisticated chemical modifications and expensive protein labeling.
View Article and Find Full Text PDFJ Proteome Res
January 2025
Discovery Research, AbbVie, Inc., 1 North Waukegan Rd., North Chicago, Illinois 60064, United States.
Affinity capture (AC) combined with mass spectrometry (MS)-based proteomics is highly utilized throughout the drug discovery pipeline to determine small-molecule target selectivity and engagement. However, the tedious sample preparation steps and time-consuming MS acquisition process have limited its use in a high-throughput format. Here, we report an automated workflow employing biotinylated probes and streptavidin magnetic beads for small-molecule target enrichment in the 96-well plate format, ending with direct sampling from EvoSep Solid Phase Extraction tips for liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis.
View Article and Find Full Text PDFThe polymerase gamma (POLG) gene mutation is associated with mitochondria and metabolism disorders, resulting in heterogeneous responses to immunological activation and posing challenges for mitochondrial disease therapy. Optical metabolic imaging captures the autofluorescent signal of two coenzymes, NADH and FAD, and offers a label-free approach to detect cellular metabolic phenotypes, track mitochondria morphology, and quantify metabolic heterogeneity. In this study, fluorescence lifetime imaging (FLIM) of NAD(P)H and FAD revealed that POLG mutator macrophages exhibit a decreased NAD(P)H lifetime, and optical redox ratio compared to the wild-type macrophages, indicating an increased dependence on glycolysis.
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