•A photoelectrochemical biosensor based on the surface defect recognition and multiple signal amplification of metal-organic frameworks has been used for protein kinase activity analysis.•[Ru(bpy)3]2+ loaded UiO-66 probes were linked to the phosphorylated kemptide modified electrode through the chelation between the Zr4+ defects on the surface of UiO-66 and the phosphate groups in kemptide.•The biosensor was applied for quantitative kinase inhibitor evaluation and PKA activities detection.A turn-on photoelectrochemical (PEC) biosensor based on the surface defect recognition and multiple signal amplification of metal-organic frameworks (MOFs) was proposed for highly sensitive protein kinase activity analysis and inhibitor evaluation. In this strategy, based on the phosphorylation reaction in the presence of protein kinase A (PKA), the Zr-based metal-organic frameworks (UiO-66) accommodated with [Ru(bpy)3]2+ photoactive dyes in the pores were linked to the phosphorylated kemptide modified TiO2/ITO electrode through the chelation between the Zr4+ defects on the surface of UiO-66 and the phosphate groups in kemptide. Under visible light irradiation, the excited electrons from [Ru(bpy)3]2+ adsorbed in the pores of UiO-66 injected into the TiO2 conduction band to generate photocurrent, which could be utilized for protein kinase activities detection. The large surface area and high porosities of UiO-66 facilitated a large number of [Ru(bpy)3]2+ that increased the photocurrent significantly, and afforded a highly sensitive PEC analysis of kinase activity. The detection limit of the as-proposed PEC biosensor was 0.0049 U mL−1 (S/N!=!3). The biosensor was also applied for quantitative kinase inhibitor evaluation and PKA activities detection in MCF-7 cell lysates. The developed visible-light PEC biosensor provides a simple detection procedure and a cost-effective manner for PKA activity assays, and shows great potential in clinical diagnosis and drug discoveries.Download high-res image (251KB)Download full-size image

A large amount of proteins are post-translationally modified with a sialic acid terminal oligosaccharide, and sialylation directly affects the function of glycoproteins and adjusts relevant biological processes. Herein, we developed a method for imaging analysis of protein-specific sialylation on the cell surface via silver nanoparticle (AgNPs) plasmonic enhanced Förster resonance energy transfer (FRET). In this strategy, the target monosaccharide was labelled with the FRET acceptor of Cy5 via bioorthogonal chemistry. In addition, aptamer linked AgNPs were combined with the Cy3 fluorophore by DNA hybridization as the FRET donor probe, which could be conjugated to the target glycoprotein based on specific aptamer-protein recognition. The Cy5 fluorescence signal was obtained under the Cy3 excitation wavelength via FRET. Moreover, the FRET fluorescence signal was obviously enhanced owing to the plasmonic effect of AgNPs at an appropriate distance to Cy3 on the cell surface. Hence, the protein-specific sialic acids were detected with high contrast. The results showed that the AgNP plasmonic enhanced FRET method was not only superior to the bare FRET method but also can be used to evaluate the expression of sialoglycoproteins in different cell types under pharmacological treatments. The AgNP plasmonic enhanced FRET method provides a valuable tool in the research of glycan metabolism biological processes, the active site of glycoproteins and drug screening.

A deep understanding of distinct functional differences of various defects in semiconductor materials is conducive to effectively control and rationally tune defect-induced functionalities. However, such research goals remain a substantial challenge due to great difficulties in identifying the defect types and distinguishing their own roles, especially when various defects coexist in bulk or nanoscale material. Hereby, we subtly selected a molecular-type semiconductor material as structural mode composed of supertetrahedral chalcogenide Cd–In–S nanoclusters (NCs) with intrinsic vacancy point defect at the core site and antisite point defects at the surface of supertetrahedron and successfully established the correlation of those point defects with their own electrochemiluminescence (ECL) behaviors. The multichannel ECL properties were recorded, and the corresponding reaction mechanisms were also proposed. The predominant radiation recombination path of ECL emission peak at 585 nm was significantly distinguished from asymmetrically broad PL emission with a peak at 490 nm. In addition, the ECL performance of the coreless supertetrahedral chalcogenide nanocluster can be modulated by atomically precise doping of monomanganese ion at the core vacant site. A relatively high ECL efficiency of 2.1% was also gained. Actually, this is the first investigation of ECL behavior of semiconductor materials based on supertetrahedral chalcogenide nanocluster in aqueous solution. Current research may open up a new avenue to probe the roles of various different defects with defined composition and position in the NC. The versatile and bright ECL properties of Cd–In–S NC combined with tunable ECL potential and ECL peak suggest that the new kind of NC-based ECL material may hold great promising for its potential applications in electrochemical analysis, sensing, and imaging.

A novel visible-light photoelectrochemical (PEC) biosensor based on localized surface plasmon resonance (LSPR) enhancement and dye sensitization was fabricated for highly sensitive analysis of protein kinase activity with ultralow background. In this strategy, DNA conjugated gold nanoparticles (DNA@AuNPs) were assembled on the phosphorylated kemptide modified TiO2/ITO electrode through the chelation between Zr4+ ions and phosphate groups, then followed by the intercalation of [Ru(bpy)3]2+ into DNA grooves. The adsorbed [Ru(bpy)3]2+ can harvest visible light to produce excited electrons that inject into the TiO2 conduction band to form photocurrent under visible light irradiation. In addition, the photocurrent efficiency was further improved by the LSPR of AuNPs under the irradiation of visible light. Moreover, because of the excellent conductivity and large surface area of AuNPs that facilitate electron-transfer and accommodate large number of [Ru(bpy)3]2+, the photocurrent was significantly amplified, affording an extremely sensitive PEC analysis of kinase activity with ultralow background signals. The detection limit of as-proposed PEC biosensor was 0.005 U mL–1 (S/N = 3). The biosensor also showed excellent performances for quantitative kinase inhibitor screening and PKA activities detection in MCF-7 cell lysates under forskolin and ellagic acid stimulation. The developed dye-sensitization and LSPR enhancement visible-light PEC biosensor shows great potential in protein kinases-related clinical diagnosis and drug discovery.

In this manuscript, a sandwich electrogenerated chemiluminescence (ECL) biosensor for galactosyltransferase (Gal T) activity analysis based on a graphitic carbon nitride (g-C3N4) nanosheet interface and polystyrene microsphere-enhanced responses is described. The carboxylated g-C3N4 nanosheet was firstly coated on the GC electrode surface, and then the bovine serum albumin conjugated with N-acetylglucosamine (GlcNAc-BSA) was further modified on the g-C3N4 modified GC electrode. In the presence of Gal T and UDP-Gal as a coreactor, galactose was conjugated to GlcNAc-BSA, and the ECL signal of the GlcNAc-BSA conjugated g-C3N4 nanosheet modified electrode decreased. After the adsorption of Artocarpus integrifolia lectin (AIA) conjugated polystyrene microsphere (PSM) nanoprobes through the specific interaction between galactose and AIA, the ECL signal decreased significantly due to the poor conductivity of the nanoprobes, which inhibited the electron transfer on the electrode interface, leading to high sensitivity toward Gal T activity analysis. The detection limit of 7 × 10−5 U mL−1 was obtained. The proposed ECL sensor has been applied to Gal T activity analysis in serum samples as well as Gal T activity expression in different cell lines, and may be a novel tool to for glycosyltransferase activity evaluation and inhibition in clinic diagnostics. This strategy can also be extended to detect and monitor other glycosyltransferases.

A novel reusable and dual-potential responsive electrogenerated chemiluminescence (ECL) biosensor was fabricated for synchronous detection of cancer cells and their surface N-glycan. In this strategy, a cancer cell recognized aptamer hybridized with a capture DNA was immobilized on electrochemically reduced MoS2 nanosheets, and Ru(phen)32+ as ECL probes was intercalated into the grooves of the double-strand DNA. In the presence of target cells, the capture DNA and Ru(phen)32+ were released from the electrode interface owing to the specific interaction between cancer cells and the aptamer. Meanwhile, concanavalin A (Con A), a mannose binding protein, and a conjugated gold nanoparticle modified graphite-C3N4 (Con A@Au-C3N4) was used as a negative ECL nanoprobe and applied for the cell surface N-glycan evaluation owing to the excellent ECL properties of g-C3N4 at negative potential. The cytosensing and cell surface N-glycan evaluation could be simultaneously realized with high sensitivity and excellent selectivity based on the ratio of ECL intensity between the negative potential and positive potential (ΔECLn/ΔECLp), avoiding the traditional routing cell counting procedures. Moreover, the aptamer modified electrode can be regenerated in the presence of capture DNA solutions for cyclic utilization. As a proof-of-concept, the ECL cytosensor showed excellent performances for the analysis of the MCF-7 cancer cell and its surface N-glycan evaluation in human serum samples. The reusable and dual potential response ECL biosensor endows a feasibility tool for clinical diagnosis and drug screening especially in complex biological systems.

Highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) were prepared by a simple and green route employing sodium alginate as a carbon source and tryptophan as both a nitrogen source and a functional monomer. The as-synthesized N-CNPs exhibited excellent water solubility and biocompatibility with a fluorescence quantum yield of 47.9%. The fluorescence of the N-CNPs was intensively suppressed by the addition of ascorbic acid (AA). The mechanism of the fluorescence suppression of the N-CNPs was investigated, and the synergistic action of the inner filter effect (IFE) and the static quenching effect (SQE) contributed to the intensive fluorescence suppression, which was different from those reported for the traditional redox-based fluorescent probes. Owing to the spatial effect and hydrogen bond between the AA and the groups on the N-CNP surface, excellent sensitivity and selectivity for AA detecting was obtained in a wide linear relationship from 0.2 μM to 150 μM. The detection limit was as low as 50 nM (signal-to-noise ratio of 3). The proposed sensing systems also represented excellent sensitivity and selectivity for AA analysis in human biological fluids, providing a valuable platform for AA sensing in clinic diagnostic and drug screening.

Graphene has attracted an enormous amount of interest because of its excellent properties. The relatively versatile possibilities in the doping of graphene sheets afford them with tuneable electronic properties, and provide a simple but efficient approach in tuning their catalytic activity at an atomic level. Recently, a lot of efforts have been carried out to further tailor the electronic and catalytic properties of graphene by doping, realizing the precise structure control and superior characteristics of graphene, which also broadens their applications in nanoelectronics and optoelectronics. In this review, the synthesis methods developed during the last decade for the fabrication of doped graphene with excellent properties are summarized. Moreover, as a rising and brilliant research direction, the bioanalysis applications based on doped graphene also were demonstrated.

FePt bimetallic nanoparticles were formed on reduced graphene oxide(rGO) with the help of double-stranded DNA(dsDNA) via a simple and universal route to obtain a FePt/DNA-rGO composite. The FePt nanoparticles with an average size of about 5 nm were well dispersed on rGO. FePt/DNA-rGO modified glassy carbon electrode(GCE) exhibited excellent electrocatalytic activity for the oxidation of dopamine(DA) with a detection limit of 100 nmol/L(S/N = 3). In addition, the FePt/DNA-rGO based electrochemical sensor showed an excellent selectivity for DA in the presence of ascorbic acid(AA), uric acid(UA) and other interference reagents. The as-prepared electrochemical biosensor shows great promise in the application of clinical diagnostics.

A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode interface, affording fast and highly sensitive ECL cytosensing and cell surface glycan evaluation. Combining the multivalent aptamer interface and ALP nanoprobes, the ECL cytosensor showed a detection limit of 38 CCRF-CEM cells per mL in human serum samples, broad dynamic range and excellent selectivity. In addition, the proposed biosensor provided a valuable insight into dynamic profiling of the expression of different glycans on cell surfaces, based on the carbohydrates recognized by lectins applied to the nanoprobes. This biosensor exhibits great promise in clinical diagnosis and drug screening.

We demonstrate a multivalent recognition and highly selective aptamer signal amplification strategy for electrochemical cytosensing and dynamic cell surface N-glycan expression evaluation by the combination of concanavalin A (Con A), a mannose binding protein, as a model, conjugated poly(amidoamine) dendrimer on a chemically reduced graphene oxide (rGO–DEN) interface, and aptamer- and horseradish peroxidase-modified gold nanoparticles (HRP–aptamer–AuNPs) as nanoprobes. In this strategy, the rGO–DEN can not only enhance the electron transfer ability but also provide a multivalent recognition interface for the conjugation of Con A that avoids the weak carbohydrate–protein interaction and dramatically improves the cell capture efficiency and the sensitivity of the biosensor for cell surface glycan. The high-affinity aptamer- and HRP-modified gold nanoparticles provide an ultrasensitive electrochemical probe with excellent specificity. As proof-of-concept, the detection of CCRF-CEM cell (human acute lymphoblastic leukemia) and its surface N-glycan was developed. It has demonstrated that the as-designed biosensor can be used for highly sensitive and selective cell detection and dynamic evaluation of cell surface N-glycan expression. A detection limit as low as 10 cells mL–1 was obtained with excellent selectivity. Moreover, this strategy was also successfully applied for N-glycan expression inhibitor screening. These results imply that this biosensor has potential in clinical diagnostic and drug screening applications and endows a feasibility tool for insight into the N-glycan function in biological processes and related diseases.

A highly sensitive electrochemical biosensor was built for the detection of kinase activity based on the DNA induced gold nanoparticles (AuNPs) polymeric network block signal amplification. In this strategy, the DNA1 conjugated AuNPs were integrated with the phosphorylated peptide by Zr4+ and assembled into DNA-AuNPs polymeric network block by the hybridization of cDNA with each side sequences of DNA1 and joint DNA2. The kinase activity was determined by the amperometric responses of [Ru(NH3)6]3+ absorbed on the network block by electrostatic interaction. Due to its excellent electroactivity and high accommodation of the DNA-AuNPs polymeric network block for [Ru(NH3)6]3+, the current signal was significantly amplified, affording a highly sensitive electrochemical analysis of kinase activity. The as-proposed biosensor presents a low detection limit of 0.03 U mL–1 for protein kinase A (PKA) activity, wide linear range (from 0.03 to 40 U mL–1), and excellent stability even in cell lysates and serum samples. This biosensor can also be applied for quantitative kinase inhibitor screening. Finally, the PKA activities from BE4S-2B, A549, and MCF-7 cell lysates were further analyzed, which provided a valuable strategy in developing a high-throughput assay of in vitro kinase activity and inhibitor screening for clinic diagnostics and therapeutics.

•Nanomaterials make sensitive, selective detection of carbohydrates possible.•Recent advances in nanomaterials for construction of carbohydrate biosensors.•These nanomaterials also include upconverting NPs, composite nanomaterials and nanopores.•Carbohydrate biosensors in profiling carbohydrate-lectin interaction.•Carbohydrate biosensors in cancer and pathogen detection, glycosylation analysis.Nanomaterials have received much attention for their fascinating catalytic activity, large surface area, and excellent photonic and electronic features. These characteristic properties have been used to improve the sensitivity and the specificity of biosensors. This article reviews nanomaterials, including metal nanoparticles (NPs), carbon materials, quantum dots, magnetic NPs and silicon NPs, and evaluates their different functions when applied to the construction of carbohydrate biosensors. Furthermore, we discuss specialized nanomaterials, e.g., photo-to-electron conversion nanomaterials, upconverting NPs, composite nanomaterials and nanopores. Finally, we present applications of nanomaterial-based carbohydrate biosensors, including profiling of carbohydrate-lectin interaction, cancer-cell and pathogen detection, and glycosylation analysis.

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate–lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.

In this work, a novel 1,4-bis (4- aminophenylethynyl)benzene (OPE-NH2, a symmetric linear conjugated oligo(phenylene ethynylene)s derive) and chemically-reduced graphene oxide (rGO) nanocomposite (OPE-NH2/rGO) was synthesized by a simple self-assembly method. The OPE-NH2/rGO nanocomposite was stable and water soluble. The formation of OPE-NH2/rGO nanocomposite was ascribed to the π–π stacking interaction between the conjugated structure of OPE-NH2 and rGO as well as the electrostatic force between the amino group of OPE-NH2 and the carboxyl group on rGO, which was characterized by FT-IR, UV–vis spectra and fluorescence spectra. The OPE-NH2/rGO nanocomposite exhibited significantly improved electrocatalytic activity to the oxidization of dopamine (DA) than that of rGO or OPE-NH2. The electrochemical performances of OPE-NH2/rGO were dependent on the OPE-NH2 contents, and OPE-NH2 content of 5 wt% exhibited the highest activity. Compared with that of rGO, the nanocomposite presented superior high sensitivity with detection limit of 5 nM, excellent selectivity, wide linear range (0.01–60 μM) and good stability on the determination of DA. The practical application of the developed OPE-NH2/rGO nanocomposite modified electrode was successfully demonstrated for DA determination in human serum samples.Graphical abstractHighlights► A novel oligo (phenylene ethynylene)s/graphene nanocomposite (OPE-NH2/rGO) was synthesized. ► The OPE-NH2/rGO modified electrode exhibited improved electroactivity to dopamine oxidation. ► The sensor showed good performances for DA detection and can be used for human serum samples.

In this work, uniform and stable multi-walled carbon nanotubes (MWCT) and chemically reduced graphene (GR) composite electrode interface was fabricated by using layer-by-layer assembly method. The performances of these GR–MWCT assembled electrode interfaces were studied by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). It was demonstrated that the assembled composite film significantly improved the interfacial electron transfer rate compared with that of GR or MWCT modified electrode. Based on the GR–MWCT assembled interface, a sandwich-type electrochemical immunosensor was constructed using human IgG as a model target. In this assay, human IgG was fixed as the target antigen, the HRP-conjugated IgG as the probing antibody and hydroquinone as the electron mediator. The detection limit of the immunosensor was 0.2 ng mL−1 (signal-to-noise ratio of 3). A good linear relationship between the current signals and the concentrations of Human IgG was achieved from 1 ng mL−1 to 500 ng mL−1. Moreover, this electrochemical immunosensor exhibited excellent selectivity, stability and reproducibility, and can be used to accurately detect IgG concentration in human serum samples. The results suggest that the electrochemical immunosensor based on GR–MWCT assembled composite will be promising in the point-of-care diagnostics application of clinical screening of multiple diseases.Highlights► We constructed a uniform and highly porous GR–MWCT composite electrode interface. ► The GR–MWCT modified GC electrodes exhibited significant improvement on electron transfer rate, mass transfer. ► We fabricated a sandwich-type electrochemical immunosensor based on the GR–MWCT electrode interface. ► The immunosensor shows excellent selectivity, stability and reproducibility with the detection limit of 0.2 ng mL−1.

In this work, we report a cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca. 0.05 V vs Ag/AgCl) with a strong light emission on the graphene-modified glass carbon electrode. The resulted graphene-modified electrode offers an excellent platform for high-performance biosensing applications. On the basis of the cathodic ECL signal of luminol on the graphene-modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab2). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Ab1) due to it large surface area. The gold nanorods were not only used as carriers of secondary antibody (Ab2) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low-potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from 10 pg mL–1 to 8 ng mL–1. The detection limit of PSA was 8 pg mL–1 (signal-to-noise ratio of 3). Moreover, the as-proposed low-potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene-based ECL immunosensor accurately detected PSA concentration in 10 human serum samples from patients demonstrated by excellent correlations with standard chemiluminescence immunoassay. The results suggest that the as-proposed graphene ECL immunosensor will be promising in the point-of-care diagnostics application of clinical screening of cancer biomarkers.

A novel photoelectrochemical cell (PEC) based on graphene/P3OT (poly(3-octyl-thiophene)) nanocomposites was developed for photovoltaic solar energy conversion. Based on the noncovalent functionalization with pyrenebutyrate (PB), solution processable graphene was achieved and used to prepare the graphene/P3OT nanocomposites for PEC application. It has been shown that the doping of the graphene in P3OT film significantly improved the photocurrent as well as the photovoltaic conversion efficiency of the PEC cells by over 10 folds. The highest on–off ratio of photocurrent from the graphene/P3OT nanocomposites reached about 100. Moreover, the performances of the photoelectrochemical cells were largely dependent on the graphene content and morphology of the graphene/P3OT nanocomposites, and the highest photovoltaic conversion efficiency was obtained at a graphene content of 5 wt.% in the nanocomposites. The solution-processed graphene/P3OT nanocomposites PEC provides a general platform for next generation solar energy conversion, photoconductivity and photodetectors.

A novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticles as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after the phosphorylation process in the presence of adenosine 59-[c-thio] triphosphate (ATP-s) cosubstrate. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of 0.07 U mL−1, wide linear range (from 0.07 to 32 U mL−1), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC50 of ellagic acid, a PKA inhibitor, was estimated, which was in agreement with those characterized with the conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor, but not PKA inhibitor, shows its excellent performance in kinase inhibitor screening. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.