Ying-Lin Zhou

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.

Synthesis of orientedly bioconjugated core/shell Fe3O4@Au magnetic nanoparticles for cell separation

Orientedly bioconjugated core/shell Fe(3)O(4)@Au magnetic nanoparticles were synthesized for cell separation. The Fe(3)O(4)@Au magnetic nanoparticles were synthesized by reducing HAuCl(4) on the surfaces of Fe(3)O(4) nanoparticles, which were further characterized in detail by TEM, XRD and UV-vis spectra. Anti-CD3 monoclonal antibody was orientedly bioconjugated to the surface of Fe(3)O(4)@Au nanoparticles through affinity binding between the Fc portion of the antibody and protein A that covalently immobilized on the nanoparticles. The oriented immobilization method was performed to compare its efficiency for cell separation with the non-oriented one, in which the antibody was directly immobilized onto the carboxylated nanoparticle surface. Results showed that the orientedly bioconjugated Fe(3)O(4)@Au MNPs successfully pulled down CD3(+) T cells from the whole splenocytes with high efficiency of up to 98.4%, showing a more effective cell-capture nanostructure than that obtained by non-oriented strategy. This developed strategy for the synthesis and oriented bioconjugation of Fe(3)O(4)@Au MNPs provides an efficient tool for cell separation, and may be further applied to various fields of bioanalytical chemistry for diagnosis, affinity extraction and biosensor.

Increased release of immunoreactive CCK-8 by electroacupuncture and enhancement of electroacupuncture analgesia by CCK-B antagonist in rat spinal cord

Cholecystokinin octapeptide (CCK-8) in CNS has been shown to function as a neuropeptide with potent anti-opioid activity. It hinders opioid analgesia and facilitates opioid tolerance. The present study showed that electroacupuncture (EA) stimulation produced a marked increase of the CCK-8 immunoreactivity (ir) in the perfusate of the rat spinal cord. The increase of CCK-8-ir was most marked in response to EA of 100 Hz and 15 Hz, and less marked in response to EA of 2 Hz. Since CCK-8 has been shown to possess potent anti-opioid activity at the spinal level, blockade of the spinal CCK effect would be expected to potentiate EA-induced analgesia which is known to be opioid-mediated. Intrathecal (i.t.) administration of CCK-B antagonist L-365,260 per se did not affect tail flick latency (TFL) to any significant extent, yet it potentiated EA induced analgesia in a dose- and frequency-dependent manner. The potentiation was most marked at a dose range of 2.5-5.0 ng (i.t.) and at a frequency rank order of 100 Hz > 15 Hz > 2 Hz. The results suggest that an increased release of CCK-8 following EA may limit the effect of opioid peptides, and that the CCK-B receptor mediates the anti-opioid effect of CCK-8 in rat spinal cord.

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.

The fabrication of nanoelectrodes based on a single carbon nanotube.

Nanoelectrodes are fabricated from individual carbon nanotubes (CNTs) connected to tungsten probes or carbon fibers. The whole electrode was covered by an insulating HfO(2) layer except for a section of conducting CNT at the apex. The fabrication process includes mounting individual CNTs to the conductive support through nanomanipulation, coating the whole probe by a dielectric layer using atomic layer deposition and removing the dielectric layer at the apex through a nanomanipulation process. Differential pulse voltammetry and cyclic voltammetry measurements show the CNT nanoelectrode has similar electrochemical behavior to the widely used carbon fiber probe, but has a much smaller active electrode area and can provide much higher spatial resolution and signal-to-noise ratio.

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.

Cocaine detection by structure‐switch aptamer‐based capillary zone electrophoresis

Aptamers, which are nucleic acid oligonucleotides that can bind targets with high affinity and specificity, have been widely applied as affinity probes in capillary electrophoresis (CE). Due to relative weak interaction between aptamers and small molecules, the application of aptamer‐based CE is still limited in certain compounds. A new strategy that is based on the aptamer structure‐switch concept was designed for small molecule detection by a novel CE method. A carboxyfluorescein (fluorescein amidite, FAM) label DNA aptamer was first incubated with partial complementary strand (CS), and then the free aptamer and the aptamer‐CS duplex were well separated and determined by metal cation mediated CE/laser‐induced fluorescence. When the target was introduced into the incubated sample, the hybridized form was destabilized, resulting in the changes of the fluorescence intensities of the free aptamer and the aptamer‐CS duplex. The length of CS was investigated and 12 mer CS showed the best sensitivity for the detection of cocaine. The presented CE‐LIF method, which combines the separation power of CE with the specificity of interactions occurring between target, aptamer, and CS, could be a universal detection strategy for other aptamer‐specified small molecules.

The coating of smart pH-responsive polyelectrolyte brushes in capillary and its application in CE.

A novel pH‐responsive coating technique was developed and applied to CE successfully in this paper. The coating was formed by bonding mixed opposite charge poly(acrylic acid) and poly(2‐vinylpyridine) randomly onto the inner wall of a silica capillary. The coating processes were first characterized by ellipsometry and atomic force microscopy at macroscale and microscale, respectively. Measurements of EOF were implemented to confirm the coating. Direction and velocity of EOF became controllable from negative to positive, showing a perfect sigmoidal curve as the coating net charges alternated by the pH of BGE. The control of the EOF makes it possible to analyze different kinds of small molecules, peptides, and proteins successfully in the same capillary. Results showed that the stability and reproducibility for separations of fluoroquinolone standards were satisfactory for more than a hundred separations. A series of basic and acidic protein standards were separated with admirable efficiency and minimal adsorption using both polarities. The separation of tryptic BSA digest showed that the prepared capillary has immense potential in analyzing a single sample with both acidic and basic separations, which achieved the expectation in proteomics study by CE‐MS.