A Novel Metabolite as a Hapten to Prepare Monoclonal Antibodies for Rapid Screening of Quinoxaline Drug Residues.

Quinoxalines (Qx) are chemically synthesized antibacterial drugs with strong antibacterial and growth-promoting effects. Qx is heavily abused by farmers, resulting in large residues in animal-derived foods, which pose a serious threat to human health. Desoxyquinoxalines (DQx), which have the highest residue levels, have been identified as the major toxicant and have become a new generation of residue markers. In this study, we prepared monoclonal antibodies (mAb) based on a new generation metabolite (desoxymequindox, DMEQ) and establish an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the rapid determination of Qx residues in food. The mAb exhibited high sensitivity with half maximal inhibitory concentration (IC50) and a linear range of 2.84 µg/L and 0.8-12.8 µg/L, respectively. Additionally, the cross-reactivity (CR) of the mAb showed that it recognized multiple DQx to varying levels. The limits of detection (LOD), limits of quantification (LOQ), and recoveries for the ic-ELISA assay of pork, swine liver, swine kidney, chicken, and chicken liver were 0.48-0.58 µg/kg, 0.61-0.90 µg/kg, and 73.7-107.8%, respectively, and the coefficients of variation (CV) were less than 11%. The results of the ic-ELISA showed a good correlation with LC-MS/MS in animal-derived foods. This suggests that this analytical method can be used for the rapid screening of QX residues.

Plasmonic coupling of dual gold nanoprobes for SERS imaging of sialic acids on living cells.

This work reports a benzoic group functionalized gold nanoflower as a bridge probe for both recognition of target sialic acids and assembly of poly(N-acetylneuraminic acid) modified gold nanoparticles, which leads to plasmonic coupling of two kinds of gold nanoprobes in a single-core-multi-satellite nanostructure to produce a sensitive surface-enhanced Raman scattering (SERS) signal for the imaging of sialic acids on living cells.

Liberation of Protein-Specific Glycosylation Information for Glycan Analysis by Exonuclease III-Aided Recycling Hybridization.

A strategy for information liberation of protein-specific glycosylation is designed via an exonuclease III-aided recycling "hybridization and cleavage" process with glycan and protein probes, which achieves homogeneous quantification of cell surface glycan. The protein probe contains matching and spacer DNA sequences and an aptamer specific to target protein. The glycan probe contains a complementary sequence modified with neighboring fluorescein and quencher, a spacer sequence, and a dibenzocyclooctyne-amine end to bind azide-tagged glycan. Upon sequential binding to their targets, the complementary sequences of two probes approach enough for their hybridization, which leads to the cleavage of hybridized glycan probe by exonuclease III and followed recycling "hybridization and cleavage" process of protein probe with other adjacent glycan probes to release the labeled fluorescein for obtaining the information on protein-specific glycosylation. This protocol has been used to in situ quantify EpCAM-specific sialic acid on MCF-7 cell surface and monitor its variation during drug treatment. This work demonstrates a powerful quantification tool for research of glycosylation.

Protein-specific Raman imaging of glycosylation on single cells with zone-controllable SERS effect.

A zone-controllable SERS effect is presented for Raman imaging of protein-specific glycosylation on a cell surface using two types of newly designed nanoprobes. The signal probe, prepared using a Raman signal molecule and dibenzocyclooctyne-amine to functionalize a 10 nm Au nanoparticle, exhibits a negligible SERS effect and can recognize and link the azide-tagged glycan via a click reaction. The substrate probe, an aptamer modified 30 or 40 nm Au nanoparticles, can specifically recognize the target protein to create an efficient SERS zone on the target protein. By controlling the size of the substrate probe to match the expression zone of the protein-specific glycan, an efficient SERS signal can be generated. This method has been successfully used for in situ imaging of sialic acids on the target protein EpCAM on an MCF-7 cell surface and for the monitoring of the expression variation of protein-specific glycosylation during drug treatment. The concept of zone control can also be used to measure the distance between glycoproteins on a cell surface. This protocol shows promise in uncovering glycosylation-related biological processes.

Micro-competition system for Raman quantification of multiple glycans on intact cell surface.

A micro-competition system is designed for simultaneous quantification of multiple glycans on intact cell surfaces, by integrating two-surface-one-molecule competition with surface enhanced Raman scattering (SERS). The micro-competition is achieved among multiple-polysaccharide-coated gold nanostars functionalized silica bubbles, target cells and gold nanoprobes at a micron scale. The gold nanoprobes are prepared by coating distinct Raman molecules and lectins on gold nanoparticles for signal resolution and glycan recognition, respectively. The silica bubble surface serves as an artificial glycan surface and a SERS substrate. Upon the competitive recognition of lectin to the corresponding glycan, the gold nanoprobes can be specifically captured by the bubbles and cells in a homogeneous system, and the amounts of different gold nanoprobes on bubbles are simultaneously detected by SERS to reflect the corresponding glycan amounts on the cell surface. This micro-competition system with multiple quantification capability provides a powerful tool for investigation of the complex glycan-related biological processes.