Jianping Lei

A glucose biosensor based on direct electrochemistry of glucose oxidase immobilized on nitrogen-doped carbon nanotubes

A novel biosensor for glucose was prepared by immobilizing glucose oxidase (GOx) on nitrogen-doped carbon nanotubes (CNx-MWNTs) modified electrode. The CNx-MWNTs membrane showed an excellent electrocatalytic activity toward the reduction of O(2) due to its diatomic side-on adsorption on CNx-MWNTs. The nitrogen doping accelerated the electron transfer from electrode surface to the immobilized GOx, leading to the direct electrochemistry of GOx. The biofunctional surface showed good biocompatibility, excellent electron-conductive network and large surface-to-volume ratio, which were characterized by scanning electron microscopy, contact angle and electrochemical impedance technique. The direct electron transfer of immobilized GOx led to stable amperometric biosensing for glucose with a linear range from 0.02 to 1.02 mM and a detection limit of 0.01 mM (S/N=3). These results indicated that CNx-MWNTs are good candidate material for construction of the third-generation enzyme biosensors based on the direct electrochemistry of immobilized enzymes.

Carbon nanohorn sensitized electrochemical immunosensor for rapid detection of microcystin-LR.

A sensitive electrochemical immunosensor was proposed by functionalizing single-walled carbon nanohorns (SWNHs) with analyte for microcystin-LR (MC-LR) detection. The functionalization of SWNHs was performed by covalently binding MC-LR to the abundant carboxylic groups on the cone-shaped tips of SWNHs in the presence of linkage reagents and characterized with Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and a transmission electron micrograph. Compared with single-walled carbon nanotubes, SWNHs as immobilization matrixes showed a better sensitizing effect. Using home-prepared horseradish peroxidase-labeled MC-LR antibody for the competitive immunoassay, under optimal conditions, the immunosensor exhibited a wide linear response to MC-LR ranging from 0.05 to 20 microg/L with a detection limit of 0.03 microg/L at a signal-to-noise of 3. This method showed good accuracy, acceptable precision, and reproducibility. The assay results of MC-LR in polluted water were in a good agreement with the reference values. The proposed strategy provided a biocompatible immobilization and sensitized recognition platform for analytes as small antigens and possessed promising application in food and environmental monitoring.

Low-Potential Photoelectrochemical Biosensing Using Porphyrin-Functionalized TiO2 Nanoparticles

A novel photoelectrochemical biosensing platform for the detection of biomolecules at relatively low applied potentials was constructed using porphyrin-functionalized TiO₂ nanoparticles. The functional TiO₂ nanoparticles were prepared by dentate binding of TiO₂ with sulfonic groups of water-soluble [meso-tetrakis(4-sulfonatophenyl)porphyrin] iron(III) monochloride (FeTPPS) and characterized by transmission electron microscopy; contact angle measurement; and Raman, X-ray photoelectron, and ultraviolet-visible absorption spectroscopies. The functional nanoparticles showed good dispersion in water and on indium tin oxide (ITO) surface. The resulting FeTPPS-TiO₂-modified ITO electrode showed a photocurrent response at +0.2 V to a light excitation at 380 nm, which could be further sensitized through an oxidation process of biomolecules by the hole-injected FeTPPS. Using glutathione as a model, a methodology for sensitive photoelectrochemical biosensing at low potential was thus developed. Under optimal conditions, the proposed photoelectrochemical method could detect glutathione ranging from 0.05 to 2.4 mmol L⁻¹ with a detection limit of 0.03 mmol L⁻¹ at a signal-to-noise ratio of 3. The photoelectrochemical biosensor had an excellent specificity against anticancer drugs and could be successfully applied to the detection of reduced glutathione in gluthion injection, showing a promising application in photoelectrochemical biosensing.

Electrochemical synthesis of reduced graphene sheet-AuPd alloy nanoparticle composites for enzymatic biosensing

A simple, fast, green and controllable approach was developed for electrochemical synthesis of a novel nanocomposite of electrochemically reduced graphene oxide (ERGO) and gold-palladium (1:1) bimetallic nanoparticles (AuPdNPs), without the aid of any reducing reagent. The electrochemical reduction efficiently removed oxygen-containing groups in ERGO, which was then modified with homogeneously dispersed AuPdNPs in a good size distribution. ERGO-AuPdNPs nanocomposite showed excellent biocompatibility, enhanced electron transfer kinetics and large electroactive surface area, and were highly sensitive and stable towards oxygen reduction. A biosensor was constructed by immobilizing glucose oxidase as a model enzyme on the nanocomposites for glucose detection through oxygen consumption during the enzymatic reaction. The biosensor had a detection limit of 6.9μM, a linear range up to 3.5mM and a sensitivity of 266.6μAmM(-1)cm(-2). It exhibited acceptable reproducibility and good accuracy with negligible interferences from common oxidizable interfering species. These characteristics make ERGO-AuPdNPs nanocomposite highly suitable for oxidase-based biosensing.

Fluorescence Quenching of Carbon Nitride Nanosheet through Its Interaction with DNA for Versatile Fluorescence Sensing

This work investigates the interaction of carbon nitride nanosheet (CNNS), a recently developed two-dimensional nanomaterial, with DNA and its fluorescence quenching mechanism on fluorophore labeled single-stranded DNA probes. The static quenching through the photoinduced electron transfer (PET) from the excited fluorophore to the conductive band of CNNS is identified. Utilizing the affinity change of CNNS to DNA probes upon their recognition to targets and the PET-based fluorescence quenching effect, a universal sensing strategy is proposed for design of several homogeneous fluorescence detection methods with short assay time and high sensitivity. This strategy is versatile and can be combined with different amplification tools for quick fluorescence sensing of DNA and extensive DNA related analytes such as metal cations, small molecules, and proteins. As examples, two simple fluorescence detection methods for DNA and Hg(2+), one facile detection method coupled with Exo III-mediated target recycling for sensitive DNA analysis, and a ratiometric fluorescence protocol for DNA detection are proposed. This work provides an avenue for understanding the interaction between two-dimensional nanomaterials and biomolecules and designing novel sensing strategies for extending the applications of nanomaterials in bioanalysis.

Effective cell capture with tetrapeptide-functionalized carbon nanotubes and dual signal amplification for cytosensing and evaluation of cell surface carbohydrate.

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.

Characterization, Direct Electrochemistry, and Amperometric Biosensing of Graphene by Noncovalent Functionalization with Picket‐Fence Porphyrin

Reduced graphene oxide (RGO) was prepared and functionalized with picket-fence porphyrin, 5,10,15,20-tetrakis [αααα-2-trismethylammoniomethylphenyl] porphyrin iron(III) pentachloride (FeTMAPP), through π-π interactions. The resulting nanocomposite was characterized by atomic force microscopy (AFM); transmission electron microscopy (TEM); contact angle measurements; and fluorescence, Raman, and UV/Vis absorption spectroscopy. On account of the introduction of positively charged FeTMAPP, the functionalized RGO showed good dispersion in aqueous solution. The RGO could greatly accelerate the electron transfer of FeTMAPP to produce a well-defined redox couple of Fe(III)/Fe(II) at -0.291 and -0.314 V. Due to the synergic effect between RGO and the porphyrin, the nanocomposite showed excellent electrocatalytic activity toward the reduction of chlorite, thus leading to highly sensitive amperometric biosensing at low applied potential. The biosensor for chlorite showed a linear range from 5.0×10(-8) to 1.2×10(-4) mol L(-1) with a detection limit of 2.4×10(-8) mol L(-1) at a signal-to-noise ratio of 3. The picket-fence porphyrin could serve as an efficient species to functionalize graphene for electronic and optical applications.

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

MicroRNAs (miRNAs) are a class of short, endogenous, noncoding regulatory RNAs (approximately 18–25 nucleotides), encoded in the genomes of plants, animals, and viruses. They partially complement the 3’ untranslated region of target mRNAs, causing mRNA cleavage or inhibiting protein synthesis within the Dicer/Argonaute complex. MiRNAs play key regulatory roles in a diverse range of biological processes, including cell development, differentiation, metabolism, and apoptosis. In particular, distinct miRNA expression patterns are associated with various cancer phenotypes. Therefore, miRNAs are also an emerging class of useful diagnostic and prognostic markers. However, miRNA analysis is challenging owing to the unique characteristics of miRNAs, such as their small size, sequence similarity among family members, low abundance, susceptibility to degradation, and the technical impediments in delivering miRNAs across the plasma membrane of cells. An improved delivery strategy is thus needed to successfully monitor intracellular miRNA levels in situ. Generally, transmission of oligonucleotides for gene therapy falls into two broad categories, viral vectors or nonviral carriers. Viral vectors exhibit high transfection efficiency but also some fundamental problems, such as immunogenicity and toxicity. These problems limit their broad application. Currently, the nonviral vectors, such as liposomes, cationic polymers, dendrimers, polypeptides, and nanomaterials have attracted significant interest, owing to their good biocompatibility and potential for large-scale production, in contrast to viral vectors. However, many of these carrier systems do not inhibit genes in a cell-specific manner and cannot be used to monitor their intracellular delivery or therapeutic response. An excellent nonviral vector should efficiently facilitate cell-specific gene-probe (a single-stranded oligonucleotide designed to recognize a single target-nucleotide sequence) uptake and gene-probe endosomal escape for intracellular delivery. Fluorescent nanoparticles are highly attractive materials for gene-probe delivery because of their unique properties, including uniform size, superior imaging characteristics, and facile surface modification. The rational functionalization of these nanomaterials with target-specific moieties, such as antibodies, aptamers, and other molecules, to recognize receptors on the cell surface has led to versatile theragnostic nanosystems. These multifunctional nanosystems not only allow efficient delivery of gene probes to target cells with fewer side effects, but also allow the simultaneous monitoring of delivery and the therapeutic response of the targeted genes, using the advanced optical properties of the nanosystem. However, these nanosystems have not been used for in situ detection of intracellular miRNAs yet, because the detection strategy has not been optimized. In this study we design a novel multifunctional SnO2 nanoprobe (mf-SnO2), which contains a cell-targeting moity, as well as a conjugated gene probe to specifically recognize the target sequence, thus providing a detection strategy or inhibitor. Also, visualization of the delivery and intracellular response is possible through fluorescence of the SnO2. As shown in Scheme 1, cell-specific delivery is achieved by functionalizing SnO2 nanoparticles (SnO2NPs) with folic acid (FA), which targets cancer cells; a gene probe, in this case a molecular beacon (MB) to detect target miRNAs, is conjugated by a disulfide linkage, which is sensitive to pH values. Cleavage of the disulfide linkage between the gene probe and the nanoparticle enhances the efficiency of intracellular delivery. Using miRNA-21 in HeLa cells as a model, a method for in situ detection of intracellular miRNA by the multifunctional nanoprobe is reported. To verify the practicality of this approach, another nanoprobe was also designed, by substituting the MB with an anti-miR of miRNA-21, to down-regulate the expression of a target miRNA. The proposed method, with a MB as the recognition probe, can be used subsequently to monitor the change in miRNA levels from negligible cytotoxicity, and to monitor the ability of the multifunctional nanoprobe to [*] Dr. H. Dong, Prof. J. Lei, Prof. H. Ju, Z. Zhu State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210093 (P.R. China) E-mail: hxju@nju.edu.cn

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification with self-produced coreactant from oxygen reduction.

A highly sensitive competitive immunosensor based on the electrochemiluminescence (ECL) of quantum dots (QDs) was proposed by coupling with an enzymatic amplification. The fabrication process of the immunosensor was traced with atomic force microscopic images and electrochemical impedance spectra. The strong cathodic ECL emission of the immobilized QDs could be detected at a relatively low emission potential. The reduction of dissolved oxygen during the cathodic process provided a self-produced coreactant, H(2)O(2), for the ECL emission. Using human IgG (HIgG) as a model protein, upon the immuno-recognition of the immobilized HIgG to its antibody labeled simply with horseradish peroxidase, the ECL intensity decreased due to the steric hindrance of the proteins to electron transfer. The decrease could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant, leading to a wide calibration range of 0.05 ng mL(-1) approximately 5 microg mL(-1) and a low limit of detection for the competitive immunoassay of HIgG. This immunosensor showed good stability and fabrication reproducibility. The immunoassays of practical samples showed acceptable results. This facile immunosensing strategy opened a new avenue for detection of proteins and application of QDs in ECL biosensing.

Electrochemiluminescent Quenching of Quantum Dots for Ultrasensitive Immunoassay through Oxygen Reduction Catalyzed by Nitrogen-Doped Graphene-Supported Hemin

A hemin functionalized graphene sheet was prepared via the noncovalent assembly of hemin on nitrogen-doped graphene. The graphene sheet could act as an oxygen reduction catalyst to produce sensitive electrochemiluminescent (ECL) quenching of quantum dots (QDs) due to the annihilation of dissolved oxygen, the ECL coreactant, by its electrocatalytic reduction. With the use of the catalyst with high loading of hemin as a signal tag of the secondary antibody, a novel ultrasensitive immunoassay method for biomarker detection was proposed. In an air-saturated pH 8.0 buffer, the immunosensor constructed by a stepwise immobilization of bidentate-chelated CdTe QDs and capture antibody showed an intensive cathodic ECL irradiation, which could be scavenged upon the formation of the catalyst-bound sandwich immunocomplex. With the use of the carcinoembryonic antigen as a model analyte, the immunoassay method showed a linear range from 0.1 pg mL(-1) to 10 ng mL(-1) and a detection limit of 24 fg mL(-1). The immunosensor exhibited good stability, acceptable fabrication reproducibility, and practicability. The electrocatalytic reduction-based ECL quenching strategy provided a powerful avenue for the design of the ultrasensitive detection method, showing great promise for clinical application.