Ultrasensitive multiplex SERS immunoassay based on porous Au-Ag alloy nanoparticle-amplified Raman signal probe and encoded photonic crystal beads.

An ultrasensitive multiplex surface-enhanced Raman scattering (SERS) immunoassay was developed using porous Au-Ag alloy nanoparticles (p-AuAg NPs) as Raman signal amplification probe coupling with encoded photonic crystal microsphere. p-AuAg NPs were synthesized and modified with the second antibody (Ab2) and Raman tag (mercaptobenzoic acid, MBA) to prepare a Raman signal-amplified probe. The high porosity of the p-AuAg NPs enables significant coupling of the localized surface plasmon resonance and thus abundant inherent hotspots for Raman signal enhancement. 3D-ordered silver nanoparticles-coated silica photonic crystal beads (Ag/SPCBs) were prepared as encoded SERS substrate for multiplex detection using their reflection peaks. The signal-amplified probe was used for multiplex detection of tumor markers carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP). The wide linear ranges of 10-7-103 ng/mL for CEA and 10-4-103 ng/mL for AFP with detection limits of 1.22 × 10-8 ng/mL and 2.47 × 10-5 ng/mL for CEA and AFP at a signal-to-noise ratio of 3 were obtained. The proposed multiplex SERS immunoassay method displays ultrahigh sensitivity, wide linear range, and excellent specificity, which can be successfully applied to measure clinical serum samples with satisfactory results. The research provides a novel SERS signal enhancement strategy for the multiplex bioassay.

Noncovalent functionalization of carbon nanotubes with lectin for label-free dynamic monitoring of cell-surface glycan expression.

A kind of concanavalin A functionalized multiwalled carbon nanotube (ConA-MWCNT) was constructed by noncovalent assembly of ConA on carboxylated MWCNT with poly(diallyldimethylammonium) as a linker. The novel nanomaterial was characterized with scanning electron microscopy and atomic force microscopy. It incorporated both the specific recognition ability of lectin for cell-surface mannosyl groups and the unique electronic and mechanical properties of MWCNT. An electrochemical label-free method for cytosensing was proposed by constructing a ConA-MWCNT interface on a glassy carbon electrode, which showed a linear response to K562 cells ranging from 1 × 10(4) to 1 × 10(7) cellsmL(-1). The ConA-MWCNT interface could be further used for monitoring of dynamic variation of glycan expression on K562 cells in response to drugs. A facile and high-throughput optical method for the analysis of dynamic glycan expression on living cells was also developed by constructing an array of ConA-MWCNT spots on a glass slide. This method showed acceptable rapidity and low cost. The noncovalent functionalization of MWCNTs with lectins could be potentially applied in cell biological studies based on cell-surface glycan expression.

Facile construction of ratiometric electrochemical immunosensor using hierarchical PtCoIr nanowires and porous SiO2@Ag nanoparticles for accurate detection of septicemia biomarker.

Procalcitonin (PCT) is a sensitive and specific biomarker for sepsis diagnosis. In this study, a novel ratio-typed electrochemical immunosensor was constructed for reliable and sensitive assay of PCT based on hierarchical PtCoIr nanowires/polyethylene polyamine-grafted-ferrocene (PtCoIr HNWs/PEPA-Fc) and porous SiO2@Ag nanoparticles-toluidine blue (porous SiO2@Ag NPs-TB). Importantly, the PtCoIr HNWs/PEPA-Fc was first modified on the sensing interface, which harvested stable and strong electrochemical signals for readout of Fc due to the enriched anchoring sites created by the PtCoIr HNWs. Meanwhile, porous SiO2@Ag NPs-TB behaved as the label to conjugate with secondary antibody (Ab2), which also provided another strong detection signals originated from TB confined in such porous structures. The resulting immunosensor displayed a measurable output of procalcitonin (PCT) in the dynamic scope of 0.001 ~ 100 ng mL-1 with a low limit of detection (LOD) of 0.46 pg mL-1 (S/N = 3). Moreover, we exploited this strategy for PCT assay in a diluted human serum sample with acceptable results, exhibiting promising applications in the clinical analysis.

In situ electrochemical imaging of membrane glycan expression on micropatterned adherent single cells.

A scanning electrochemical microscopic (SECM) method for in situ imaging of four types of membrane glycan motifs on single adherent cells was proposed using BGC-823 human gastric carcinoma (BGC) cells as the model. These adherent cells were first micropatterned in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the localized surface interaction, and the membrane glycans were then specifically recognized with corresponding lectins labeled with horseradish peroxidase (HRP). On the basis of the enzymatic oxidization of ferrocenylmethanol (FMA) by H(2)O(2) to yield FMA(+), the glycan expression level was detected by the reduction current of FMA(+) at the SECM tip. The cell-surface glycans could, thus, be in situ imaged by SECM at a single-cell level without peeling the cells from culture dish. Under the optimized conditions, four types of membrane glycan motifs showed statistically distinguishable expression levels. The SECM results for different glycan motifs on adherent single cells were consistent with those estimated by flow cytometric assay. This work provides a reliable approach for in situ evaluation of the characteristic glycopattern of single living cells and can be applied in cell biologic study based on cell surface carbohydrate expression.

Highly sensitive electrocatalytic biosensing of hypoxanthine based on functionalization of graphene sheets with water-soluble conducting graft copolymer.

A novel electrocatalytic biosensing platform was designed by the functionalization of reduced graphene oxide sheets (RGO) with conducting polypyrrole graft copolymer, poly(styrenesulfonic acid-g-pyrrole) (PSSA-g-PPY), via π-π noncovalent interaction. The resulting nanocomposite could well disperse in water for at least 2 months with a solubility of 3.0 mg mL(-1). The nanocomposite was characterized with atomic force microscopy, X-ray photoelectron spectroscopy, ultraviolet-visible absorption, contact angle measurement, and electrochemical impedance spectroscopy. Based on the advantageous functions of PSSA-g-PPY and RGO, the functional nanocomposite modified platinum electrode showed high electrocatalytic activity toward the oxidation of hydrogen peroxide and uric acid in neutral media. Further, a hypoxanthine biosensor was constructed by combining the modified electrode with the enzymatic reaction of xanthine oxidase. The biosensor exhibited a wide linear response ranging from 3.0×10(-8) to 2.8×10(-5) M with a high sensitivity of 673±4 µA M(-1) cm(-2). The detection limit of 10nM at a signal-to-noise ratio of 3 was one order of magnitude lower than that reported previously. The assay results of hypoxanthine in fish samples were in a good agreement with the reference values. The water-soluble conducting copolymer could serve as an efficient species for functionalization and solubilization of graphene sheets in biosensing and biocatalytic applications.