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A novel electrochemical cytosensing strategy was designed based on the specific recognition of integrin receptors on a cell surface to arginine-glycine-aspartic acid-serine (RGDS)-functionalized single-walled carbon nanotubes (SWNTs). The covalent conjugation of the RGDS tetrapeptide to SWNTs was proved with Raman and FT-IR spectra. The conjugated RGDS showed a predominant ability to capture cells on the electrode surface by the specific combination of RGD domains with integrin receptors. With the use of BGC-823 human gastric carcinoma cells (BGC cells) as a model, the cell surface mannosyl groups could specifically bind with horseradish peroxidase labeled concanavalin A, producing an electrochemical cytosensor. On the basis of the dual signal amplification of SWNTs and enzymatic catalysis, the cytosensor could respond down to 620 cells mL (-1) of BGC cells with a linear calibration range from 1.0 x 10 (3) to 1.0 x 10 (7) cells mL (-1), showing very high sensitivity. The dual signal amplification could be further used to evaluate the mannosyl groups on the cell surface, and the mannosyl groups on a single living intact BGC cell were detected to correspond to 5.3 x 10 (7) molecules of mannose. This strategy presents a promising platform for highly sensitive cytosensing and convenient evaluation of surface carbohydrates on living cells.

A cascade signal amplification strategy was proposed for detection of protein target at ultralow concentration by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized quantum dots (QDs), multiplex binding of the biotin-strepavidin system, and anodic stripping voltammetric detection. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of QDs, which presented per protein recognition event to numerous quantum dot tags for electrochemical readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human vascular endothelial growth factor as a model protein, the designed strategy could quantitatively detect protein down to 16 molecules in a 100 microL sample with a linear calibration range from 1 aM to 1 pM and was amenable to quantification of protein target in complex biological matrixes. The proposed cascade signal amplification strategy would become a powerful tool for proteomics research and clinical diagnostics.

The study of glycobiology has been seriously hampered due to lack of an ideal assay tool. This work proposes a robust carbohydrate monolayer platform to solve the problems of active site inaccessibility and lectin denaturation associated with protein arrays reported for detection of cell surface carbohydrates and develops a convenient method for monitoring cell surface carbohydrate sites of interest, with high sensitivity, acceptable rapidity, low cost, and excellent extensibility. It utilizes the competitive binding of solid-surface-confined and cell-surface-residing carbohydrates to quantum dot labeled carbohydrate recognition protein and subsequent voltammetric quantification of the metal signature. The mannan monolayer strategy exhibited sensitive response to K562 cells and possessed potential specificity due to the specific interaction between lectin and corresponding carbohydrate. By comparing the competitive binding of K562 cells with mannan in solutions, the average Con A binding capacity of a single K562 cell could be estimated to correspond to 6.9 pg or 2.3 x 10(10) mannose moieties. This strategy integrates the advantages of surface assembly, nanotechnology, bioconjugate techniques, and electrochemical detection and can be expanded for profiling cell surface carbohydrates and high-throughput multiple detection by simultaneously using more pairs of lectin and carbohydrate owing to the multiple coding capability of QDs, which provides an important protocol for the quantitative evaluation of cell surface carbohydrate sites.

Sugar and spice: The title method for the simultaneous multiplex analysis of intact cell‐surface glycans (see picture) shows excellent performance in sensitivity, stability, and practicality. The strategy can be used to analyze the dynamic variation of the cell‐surface glycome and to decipher cellular pathophysiological processes.

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A dual-functionalized nanoprobe was designed for highly sensitive and selective in situ evaluation of carbohydrates on living cells by integrating the specific carbohydrate recognition and enzymatic signal amplification of proteins on Au nanoparticles. A nanoscaffold of nanohorns functionalized with arginine-glycine-aspartic acid-serine tetrapeptide was also prepared on an electrode surface for cell capture and enhancing the electrical connectivity. Combined with the nanoprobe and peptide-nanohorns, a highly sensitive electrochemical strategy was developed for cytosensing, which showed a detection limit down to 15 cells, broad dynamic range, acceptable rapidity, and low cost. The proposed method was further used for monitoring of dynamic variation of carbohydrate expression on cancer cells in response to drugs, which obviated the destruction or labeling of cells and the covalent tagging of lectin and enzyme. The one-pot conjugation of three components was very convenient and could be extended for preparation of other lectin-functionalized nanoprobes. Further development of this technique would contribute considerably to meeting the challenges in comprehensive understanding of the glycomic codes.

A target-switched DNA nanotweezer is designed for AND logic gate operation and enzyme-free detection of microRNAs (miRNAs) by catalytic hairpin assembly (CHA) and proximity-dependent DNAzyme formation. The double crossover motif-based nanotweezer consists of an arched structure as the set strand for target inputs and two split G-rich DNAs at the termini of two arms for signal output. Upon a CHA, a small amount of binary target inputs can switch numerous open nanotweezers to a closed state, which leads to the formation of proximity-dependent DNAzyme in the presence of hemin to produce a highly sensitive biosensing system. The binary target inputs can be used for successful building of AND logic gate, which is validated by polyacrylamide gel electrophoresis, surface plasmon resonance and the biosensing signal. The developed biosensing system shows a linear response of the output chemiluminescence signal to input binary miRNAs with a detection limit of 30 fM. It can be used for miRNAs analysis in complex sample matrix. This system provides a simple and reusable platform for logic gate operation and enzyme-free, highly sensitive, and specific multianalysis of miRNAs.