De-Wen Zhang

Amperometric hydrogen peroxide biosensor based on the immobilization of heme proteins on gold nanoparticles–bacteria cellulose nanofibers nanocomposite

A novel matrix, gold nanoparticles-bacterial cellulose nanofibers (Au-BC) nanocomposite was developed for enzyme immobilization and biosensor fabrication due to its unique properties such as satisfying biocompatibility, good conductivity and extensive surface area, which were inherited from both gold nanoparticles (AuNPs) and bacterial cellulose nanofibers (BC). Heme proteins such as horseradish peroxidase (HRP), hemoglobin (Hb) and myoglobin (Mb) were successfully immobilized on the surface of Au-BC nanocomposite modified glassy carbon electrode (GCE). The immobilized heme proteins showed electrocatalytic activities to the reduction of H(2)O(2) in the presence of the mediator hydroquinone (HQ), which might be due to the fact that heme proteins retained the near-native secondary structures in the Au-BC nanocomposite which was proved by UV-vis and IR spectra. The response of the developed biosensor to H(2)O(2) was related to the amount of AuNPs in Au-BC nanocomposite, indicating that the AuNPs in BC network played an important role in the biosensor performance. Under the optimum conditions, the biosensor based on HRP exhibited a fast amperometric response (within 1s) to H(2)O(2), a good linear response over a wide range of concentration from 0.3 μM to 1.00 mM, and a low detection limit of 0.1 μM based on S/N=3. The high performance of the biosensor made Au-BC nanocomposite superior to other materials as immobilization matrix.

Novel Homogeneous Label-Free Electrochemical Aptasensor Based on Functional DNA Hairpin for Target Detection

We first developed a label-free and immobilization-free homogeneous electrochemical aptasensor, which combined a smart functional DNA hairpin and a designed miniaturized electrochemical device. Cocaine was chosen as a model target. The anticocaine aptamer and peroxidase-mimicking DNAzyme were integrated into one single-stranded DNA hairpin. Both aptamer and G-quadruplex were elaborately blocked by the stem region. The conformation switching induced by the affinity interaction between aptamer and cocaine released G-quadruplex part and turned on DNAzyme activity. The designed electrochemical device, constructed by a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode, was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level. The aptasensor realized the quantification of cocaine ranging from 1 to 500 μM with high specificity. The clever combination of the functional DNA hairpin and the novel device achieved an absolutely label-free electrochemical aptasensor, which showed excellent performance like low cost, easy operation, rapid detection, and high repeatability.

G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection

A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay-Sachs genetic disorder mutant.

Methylene blue as a G-quadruplex binding probe for label-free homogeneous electrochemical biosensing.

Herein, G-quadruplex sequence was found to significantly decrease the diffusion current of methylene blue (MB) in homogeneous solution for the first time. Electrochemical methods combined with circular dichroism spectroscopy and UV-vis spectroscopy were utilized to systematically explore the interaction between MB and an artificial G-quadruplex sequence, EAD2. The interaction of MB and EAD2 (the binding constant, K ≈ 1.3 × 10(6) M(-1)) was stronger than that of MB and double-stranded DNA (dsDNA) (K ≈ 2.2 × 10(5) M(-1)), and the binding stoichiometry (n) of EAD2/MB complex was calculated to be 1.0 according to the electrochemical titration curve combined with Scatchard analysis. MB was proved to stabilize the G-quadruplex structure of EAD2 and showed a competitive binding to G-quadruplex in the presence of hemin. EAD2 might mainly interact with MB, a positive ligand of G-quadruplex, through the end-stacking with π-system of the guanine quartet, which was quite different from the binding mechanism of dsDNA with MB by intercalation. A novel signal read-out mode based on the strong affinity between G-quadruplex and MB coupling with aptamer/G-quadruplex hairpin structure was successfully implemented in cocaine detection with high specificity. G-quadruplex/MB complex will function as a promising electrochemical indicator for constructing homogeneous label-free electrochemical biosensors, especially in the field of simple, rapid, and noninvasive biochemical assays.

Micropipet Tip-Based Miniaturized Electrochemical Device Combined with Ultramicroelectrode and Its Application in Immobilization-Free Enzyme Biosensor

In this study, a simple miniaturized microliter electrochemical device was constructed using a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode. The novel electrochemical device set the electrochemical reaction in a micropipet tip containing an ultramicroelectrode. We investigated the feasibility of the designed electrochemical device by cyclic voltammetric measurements of redox probe. Its application in an immobilization-free enzyme electrochemical biosensor was also evaluated. Horseradish peroxidase and glucose oxidase were selected to test sensor feasibility. Our results showed that the micropipet tip-based electrochemical device could detect low substrate or enzyme concentration or enzymatic reaction rate. The electrochemical device was applied to analyze the glucose content in human blood samples. With the advantages of low cost, easy operation, rapid detection and high reproducibility, this design provides a new approach in immobilization-free enzyme biosensor construction. Integrated with an ultramicroelectrode, our micropipet tip-based electrochemical device could replace most normal electrodes and electrochemical cells in common laboratories for electroanalysis.

Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis

Aptamers are nucleic acid oligonucleotides, which can recognize targets with high affinity and specificity. Fluorescently labeled aptamers have been used as affinity probes in CE for interaction analysis. In this study, a method of labeling aptamers chemiluminescently with isoluminol isothiocyanate (ILITC) through covalent bonds was proposed and realized. The ILITC‐labeled aptamers were characterized by HPLC‐MS and purified by HPLC. After desalination, the ILITC‐labeled aptamers were employed as the affinity probe for interaction analysis in CE coupled with chemiluminescence detection (CE‐CL) by interface of end column reaction mode, the apparatus of which was home‐designed and setup. CE‐CL experiment conditions, including buffer pH, concentrations of horseradish peroxidase and H2O2, were optimized first. The system of thrombin and its 29‐mer aptamer was chosen as the model. Binding parameters, namely the dissociation constant (Kd) and the binding site number (n), were calculated. The Kd obtained was 124.0±6.9 nM in agreement with the reported values. Thus, interaction analysis method based on chemiluminescently labeled aptamers as the affinity probe in CE‐CL has been established. This method can be widely applied due to the ease and universality of the labeling method, simplicity of CE‐CL apparatus and combination with aptamers for a wide range of targets.

Soft coordination supramolecular polymers: novel materials for dual electro-catalysis

We report in this paper a dual catalytic effect of water soluble soft coordination supramolecular polymers (SCSPs) based on Fe3+ and a bis-ligand. Upon assembling the Fe(III)-SCSPs onto electrodes via layer-by-layer technique, the electroactive films may lower the half-wave potential for all the tested molecules and enhance the current for neutral or oppositely charged electroactive species. The decrease of the half-wave potential was attributed to the mediation of the Fe(III)-SCSPs in electron transferring, whereas the enhancement of current is a consequence of accumulation of electrons in the films. We verified that this dual catalytic effect is general to neutral and cationic redox active molecules, and can be used to detect hydrogen peroxide owing to the significant enlargement of catalytic current. Our results proved that the water soluble soft coordination supramolecular polymers containing metal centers are indeed a novel class of advanced catalytic materials, which may open a new vista on the design and study of materials of this class.

Biological imaging using light-addressable potentiometric sensors and scanning photo-induced impedance microscopy

Light-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) use photocurrent measurements at electrolyte–insulator–semiconductor substrates for spatio-temporal imaging of electrical potentials and impedance. The techniques have been used for the interrogation of sensor arrays and the imaging of biological systems. Sensor applications range from the detection of different types of ions and the label-free detection of charged molecules such as DNA and proteins to enzyme-based biosensors. Imaging applications include the temporal imaging of extracellular potentials and dynamic concentration changes in microfluidic channels and the lateral imaging of cell surface charges and cell metabolism. This paper will investigate the current state of the art of the measurement technology with a focus on spatial and temporal resolution and review the biological applications, these techniques have been used for. An outlook on future developments in the field will be given.

Study of the electrical connection mechanism of sheathless interface for capillary electrophoresis-electrospray ionization-mass spectrometry.

With the combination of high separation ability of capillary electrophoresis (CE) and strong identification ability of mass spectrometry (MS), CE/MS is becoming a powerful tool for polar and ionic analytes analysis. Different interfaces have been developed to enhance the sensitivity and reliability since the first introduction of CE/MS in 1987. A sheathless porous interface based on a new ions transferring electric connection technique was reported to be with high sensitivity and reliability. In this work, a series of optical and electrochemical experiments were designed to study the electric connection process. The results indicated that closing CE electrical circuit and applying MS spray voltage were achieved by the small ions transferring through the interface porous wall. The new electric connection method significantly enhanced the sensitivity, resolution and stability of the CE/MS analysis. The interface was applied in CE/MS detection of morphine and 6-monoacetylmorphine in urine sample and showed an equal sensitivity to LC/MS. With the significant improvement of sensitivity and stability, the CE/MS with the new interface showed strong potential for the determination of low abundance analytes.

LAPS and SPIM Imaging Using ITO-Coated Glass as the Substrate Material

Light-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) use photocurrent measurements for spatiotemporal imaging of ion concentrations, electrical potentials, and impedance. In this work, ITO-coated glass was confirmed to produce photocurrents at anodic potentials with 405 nm diode laser illumination. Therefore, it was developed as a low cost and robust substrate material for LAPS and SPIM imaging compared to traditional expensive ultrathin Si substrates. ITO showed good ac photocurrent and pH response without surface modification and insulator. Local photocurrents were produced by scanning a focused laser beam across the sample, which proved the light addressability of ITO-coated glass. With a high-impedance PMMA dot deposited onto the ITO as a model system, a lateral resolution of about 2.3 μm was achieved.