Dual Quinone Tagging for MALDI-TOF Mass Spectrometric Quantitation of Cysteine-Containing Peptide

A dual quinone tagging strategy is designed for quantitation of cysteine-containing peptide (CCP) with MALDI-TOF mass spectrometry. The quinone compounds can rapidly and specifically bind to the thiol group of cysteine residues by a Michael addition reaction, which is used to identify both CCP and the number of cysteine residues in CCP through the direct observation of untagged and tagged products. After reduced with DL-dithiothreitol, the intramolecular disulfide bond can also be identified. Using benzoquinone (BQ) and methyl-<i>p</i>-benzoquinone (MBQ) as dual tags and a peptide with an amino acid sequence of SSDQFRPDDCT (C-pep1) as a model target, respectively, the quantitation strategy is performed through the intensity ratio of MBQ-tagged C-pep1 to BQ-tagged C-pep1 as the internal standard. The logarithm value of the intensity ratio is proportional to C-pep1 concentration in a range from 5.0 to 5000 nM. The limit of detection is as low as 2.0 nM. The proposed methodology provides a novel tool for rapid characterization, identification, and quantitation of biomolecules containing thiol reactive sites and has a promising application in the large-scale detection and analysis of cysteine-containing biomolecules

Host–Guest Interaction of Adamantine with a β‑Cyclodextrin-Functionalized AuPd Bimetallic Nanoprobe for Ultrasensitive Electrochemical Immunoassay of Small Molecules

A modular labeling strategy was presented for electrochemical immunoassay via supramolecular host–guest interaction between β-cyclodextrin (β-CD) and adamantine (ADA). An ADA-labeled antibody (ADA–Ab) was synthesized via amidation, and the number of ADA moieties loaded on a single antibody was calculated to be ∼7. The β-CD-functionalized gold–palladium bimetallic nanoparticles (AuPd–CD) were synthesized in aqueous solution via metal-S chemistry and characterized with transmission electron microscopy and X-ray photoelectron spectra. After the ADA–Ab was bound to the antigen-modified electrode surface with a competitive immunoreaction, AuPd–CD as a signal tag was immobilized onto the immunosensor by a host–guest interaction, leading to a large loading of AuPd nanoparticles. The highly efficient electrocatalysis by AuPd nanoparticles for NaBH₄ oxidation produced an ultrasensitive response to chloramphenicol as a model of a small molecule antigen. The immunoassay method showed a wide linear range from 50 pg/mL to 50 μg/mL and a detection limit of 4.6 pg/mL. The specific recognition of antigen by antibody resulted in good selectivity for the proposed method. The host–guest interaction strategy provided a universal labeling approach for the ultrasensitive detection of small molecule targets

Perylene Diimide and Luminol as Potential-Resolved Electrochemiluminescence Nanoprobes for Dual Targets Immunoassay at Low Potential

In the field of clinical diagnosis, it is important to construct a potential-resolved multiplex electrochemiluminescence (ECL) biosensor for decreasing the false-positive rate and improving the diagnostic accuracy. However, the shortage of low-potential cathodic luminophores between −1 and 0 V (vs Ag/AgCl) severely limited the development of the biosensor. Herein, we synthesized a novel luminophore N,N-bis-(3-dimethyl aminopropyl)-3,4,9,10-perylene tetracarboxylic acid diimide (PDI), which gave dual emissions at −0.25/–0.26 V with K2S2O8 as a co-reactant in aqueous solution. The ECL was assigned to excited J-type PDI dimers. Then, PDI and luminol were used as luminophores to respectively combine with graphite oxide and gold nanoparticles and form potential-resolved ECL nanoprobes. Also, this potential-resolved ECL nanoprobes were respectively functionalized by secondary antibodies (Ab2) to construct a low-potential sandwiched ECL immunosensor for tumor markers carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) simultaneous determination during linear scanning potential range from −0.6 to 0.6 V. The prepared multiplex immunosensor exhibited sensitive ECL response for CEA at −0.6 V due to PDI and that for AFP at 0.6 V due to luminol, and both linear semilogarithmical ranges were from 0.1 pg to 1 ng mL–1. In addition, PDI with dual ECL peaks showed enticing prospect of built-in self-calibration for a precise quantitative and bioimaging analysis