Wenhua Gao

A novel electrochemiluminescence ethanol biosensor based on tris(2,2′-bipyridine) ruthenium (II) and alcohol dehydrogenase immobilized in graphene/bovine serum albumin composite film

We developed a novel electrochemiluminescence (ECL) ethanol biosensor based on Ru(bpy)(3)(2+) and alcohol dehydrogenase (ADH) immobilized by graphene/bovine serum albumin composite film. The graphene film was directly formed on a glassy carbon electrode surface via an in situ reduction of graphene oxide (GO) and Ru(bpy)(3)(2+) was immobilized during its formation. The graphene film acted as both a decorating agent for immobilization of Ru(bpy)(3)(2+) and a matrix to immobilize bovine serum albumin (BSA), meanwhile BSA not only acted as a reductant to reduce GO, but also provided a friendly environment for ADH immobilization. Furthermore, ADH was separated from Ru(bpy)(3)(2+) by the electron-conductive graphene/BSA composite film to retain its enzymatic activity. The experimental results indicated that the biosensor had excellent electrochemical activity, ECL response to ethanol and stability. Such a design of Ru(bpy)(3)(2+)-graphene/BSA film to modify electrode holds a great promise as a new biocompatible platform for the development of enzyme-based ECL biosensors.

Application of single drop liquid-liquid-liquid microextraction for the determination of fluoroquinolones in human urine by capillary electrophoresis.

A simple and novel method of single drop liquid-liquid-liquid microextraction (SD-LLLME) coupled with capillary electrophoresis (CE) for the determination of six fluoroquinolones (FQs) was developed. The method was eventually applied to extraction and preconcentration of FQs in human urine samples. Good linear relationships were obtained for all analytes in a range of 40-1000 μg L⁻¹ with the correlation coefficients from 0.9913 to 0.9995. The limit of detections (LODs) varied from 7.4 to 31.5 μg L⁻¹ at a signal-to-noise (S/N) of 3. The recoveries at two spiking levels were 81.8-104.9% with relative standard deviations <8.3%.

The interaction of 2-mercaptobenzimidazole with human serum albumin as determined by spectroscopy, atomic force microscopy and molecular modeling

The interaction of 2-mercaptobenzimidazole (MBI) with human serum albumin (HSA) was studied in vitro by equilibrium dialysis under normal physiological conditions. This study used fluorescence, ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared (FT-IR), circular dichroism (CD) and Raman spectroscopy, atomic force microscopy (AFM) and molecular modeling techniques. Association constants, the number of binding sites and basic thermodynamic parameters were used to investigate the quenching mechanism. Based on the fluorescence resonance energy transfer, the distance between the HSA and MBI was 2.495 nm. The ΔG(0), ΔH(0), and ΔS(0) values across temperature indicated that the hydrophobic interaction was the predominant binding Force. The UV, FT-IR, CD and Raman spectra confirmed that the HSA secondary structure was altered in the presence of MBI. In addition, the molecular modeling showed that the MBI-HSA complex was stabilized by hydrophobic forces, which resulted from amino acid residues. The AFM results revealed that the individual HSA molecule dimensions were larger after interaction with MBI. Overall, this study suggested a method for characterizing the weak intermolecular interaction. In addition, this method is potentially useful for elucidating the toxigenicity of MBI when it is combined with the biomolecular function effect, transmembrane transport, toxicological testing and other experiments.

Directly suspended droplet microextraction combined with single drop back-extraction as a new approach for sample preparation compatible with capillary electrophoresis.

A simple and novel method of directly suspended droplet microextraction (DSDME) combined with single drop back-extraction prior to capillary electrophoresis (CE) measurement is developed. In this technique, DSDME was firstly carried out under the maximum stirring rate for a desired time. Then, an aqueous droplet as back-extractive phase suspended at the needle tip was immersed in droplet of organic phase for back-extracted. After extraction, the aqueous droplet was transferred into a suitable vial and injected into CE for analysis. Three alkaloids were selected as model compounds for developing and evaluating the method performance. Under the optimum conditions, the enrichment factors ranged from 231 to 524. The relative standard deviations for five replicates were in the range of 4.8-8.1%. The calibration graph was linear in the range of 20-1000 ng mL(-1) yielding correlation coefficients higher than 0.9983. The limit of detections varied from 8.1 to 14.1 ng mL(-1). Human urine samples were spiked with three alkaloids standard to assess the matrix effects and satisfactory results were obtained. The advantages of this method are simplicity of operation, rapid detection, low cost, high enrichment factor and little solvent consumption.

Selective extraction of alkaloids in human urine by on-line single drop microextraction coupled with sweeping micellar electrokinetic chromatography

A novel method of on-line single drop microextraction (SDME) coupled with sweeping micellar electrokinetic chromatography (MEKC) for the selective extraction and dual preconcentration of alkaloids was developed. In this technique, analytes of three alkaloids were firstly extracted from 4.0 mL basic aqueous sample solution (donor phase, 500 mM NaOH) into a layer of n-octanol at temperature 30 °C with the stirring rate of 1150 rpm, then back-extracted into the acidified aqueous acceptor (acceptor phase, 50 mM H₃PO₄) suspended at the tip of a capillary at 650 rpm. Then, the aqueous acceptor was introduced into capillary by hydrodynamic injection with a height difference of 15 cm between the inlet and outlet of capillary for 300 s, and analyzed directly by on-line sweeping MEKC. With the selective SDME, we were able to extract three alkaloids without any interfering components in human urine samples. Under the optimum conditions, the proposed method achieved limits of detections (LOD) of between 0.2 ng mL⁻¹ and 1.5 ng mL⁻¹ with 1583-3556-fold increases in detection sensitivity for three analytes, which indicated that it was a promising method for analysis of alkaloids in human urine.

Preparation of cyano‐functionalized multiwalled carbon nanotubes as solid‐phase extraction sorbent for preconcentration of phenolic compounds in environmental water

In the present work, we showed a novel method to synthesize cyano-functionalized multiwalled carbon nanotubes (MWCNTs-CN) and utilize it as a solid-phase extraction sorbent for preconcentration of phenolic compounds in environmental water samples. MWCNTs-CN was synthesized through surface functionalization of multiwalled carbon nanotubes (MWCNTs). The functional groups on the surface of modified MWCNTs were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis and scanning electron microscopy. The analytical procedure was based on a conventional solid-phase extraction step for which 100 mg of MWCNTs-CN were packed in a 3 mL polypropylene cartridge. Analytes were thus isolated and preconcentrated from the pretreated samples and subsequently detected on high-performance liquid chromatography-ultraviolet detection. The results showed the proposed method exhibited good sensitivity and precision for the extraction and elution of analytes. The limit of detections (S/N = 3) of the method were 0.45, 0.09, 0.08, and 3.00 ng mL(-1) for p-chlorophenol, 1-naphthol, 2-naphthol, and 2,4-dichlorophenol, respectively. The mean relative recoveries (n = 3) were between 80.28 and 103.13%, and the repeatability (RSD ≤ 5.10%) and reproducibility (RSD ≤ 7.68%) were accepted. This developed method was applied to determine phenolic compounds in environmental water samples. There is a positive result only for 2-naphthol with concentration of 0.38 ng mL(-1) in seawater sample.

An electrochemiluminescence biosensor for sensitive and selective detection of Hg2+ based on π–π interaction between nucleotides and ferrocene–graphene nanosheets

A solid-state electrochemiluminescence (ECL) biosensor based on a DNA-modified electrode platform that depends on the variation of π-π interaction before and after the binding of target analytes is put forward. The single-stranded DNA (ssDNA) probe was successfully assembled on the surface of a glassy carbon electrode (GCE), which was pre-modified with Ru(bpy)3 2+ complex and gold nanoparticles (GNPs). The ssDNA probe could strongly adsorb graphene due to the strong π-π interaction between nucleotides and graphene (GN), while in the presence of Hg2+, the conformational transformation of DNA from a single-stranded to a double-stranded structure resulted in inhibited adsorption of GN. With thymine (T)-rich ssDNA as a Hg2+ probe, we prepared the ECL biosensor by using ferrocene-graphene (Fc-GN) as a quenching unit to quench the ECL emission of Ru(bpy)3 2+, and the Hg2+ can be detected by quenching efficiency transformation when the Fc-GN gets away from Ru(bpy)3 2+. The biosensor exhibited a sensitive response to various ranges of concentration of Hg2+ with a detection limit of 18 pM. The ECL biosensor held great promise in the highly sensitive and selective detection of Hg2+ in natural water.

Trace analysis of three antihistamines in human urine by on-line single drop liquid-liquid-liquid microextraction coupled to sweeping micellar electrokinetic chromatography and its application to pharmacokinetic study.

A rapid and efficient dual preconcentration method of on-line single drop liquid-liquid-liquid microextraction (SD-LLLME) coupled to sweeping micellar electrokinetic chromatography (MEKC) was developed for trace analysis of three antihistamines (mizolastine, chlorpheniramine and pheniramine) in human urine. Three analytes were firstly extracted from donor phase (4 mL urine sample) adjusted to alkaline condition (0.5 M NaOH). The unionized analytes were subsequently extracted into a drop of n-octanol layered over the urine sample, and then into a microdrop of acceptor phase (100 mM H(3)PO(4)) suspended from a capillary inlet. The enriched acceptor phase was on-line injected into capillary with a height difference and then analyzed directly by sweeping MEKC. Good linear relationships were obtained for all analytes in a range of 6.25 × 10(-6) to 2.5 × 10(-4)g/L with correlation coefficients (r) higher than 0.987. The proposed method achieved limits of detections (LOD) varied from 1.2 × 10(-7) to 9.5 × 10(-7)g/L based on a signal-to-noise of 3 (S/N=3) with 751- to 1372-fold increases in detection sensitivity for analytes, and it was successfully applied to the pharmacokinetic study of three antihistamines in human urine after an oral administration. The results demonstrated that this method was a promising combination for the rapid trace analysis of antihistamines in human urine with the advantages of operation simplicity, high enrichment factor and little solvent consumption.

Combining a loop-stem aptamer sequence with methylene blue: a simple assay for thrombin detection by resonance light scattering technique

An ingenious sensing strategy for detecting thrombin in human serum has been developed on the basis of a hairpin DNA sequence and resonance light scattering (RLS) technique. A thrombin aptamer sequence was embedded inside the hairpin DNA strand (H-eTBA), which was designed to be the loop-stem structure. Moreover, methylene blue (MB) was utilized as the RLS signal indicator according to its different affinity to single or double stranded DNA. Upon the addition of thrombin, the thrombin aptamer inside H-eTBA interacted specifically with thrombin. Thus the conformation of H-eTBA would change. After the introduction of the DNA strand (CTBA), which was complementary to H-eTBA, the amount of double stranded DNA would decrease as a consequence. Later when MB solution was added, the RLS signal would present various response values based on different amounts of thrombin. The determination of thrombin in human serum could be obtained with a detection limit of 0.32 nM and this specific sensor could be applied to detect thrombin practically. Furthermore, this aptasensor showed quite good selectivity and simplicity toward thrombin. Finally, the proposed sensing method showed its superiority with selectivity and practicability, which could be used as a simple platform for thrombin detection.

Label-free fluorescence turn on detection of tiopronin with tunable dynamic range based on the ensemble of Alizarin Red S/copper ion

In this study, a new type of rapid, label-free fluorescence turn-on assay for detection of tiopronin using Alizarin Red S (ARS)/copper ion ensemble is developed. ARS is high fluorescence in BR buffer solution. But, the fluorescence of ARS can be significantly quenched by copper ions due to ground-state complexation. However, in the presence of tiopronin, copper ions were released from the ARS and thus restored the fluorescence of ARS. The assay has several important features. First, the system is simple in design, fast in operation and is more convenient and promising than other methods. Second, the proposed assay eliminated the separation process and sophisticated instrumentations. Third, the detection process can be seen with the naked eye and can be easily adapted to automated high-throughput screening. At last, the assay has high sensitivity and selectivity for tiopronin and the detection limit is 0.8 ng/mL which is lower than or at least comparable to the previous methods. Moreover, the dynamic range of the sensor can be tuned simply by adjusting the concentration of copper ions. Importantly, the protocol offers high selectivity for the determination of tiopronin in pharmaceutical tablets, injection and biological samples with satisfactory results. Thus, the assay shows great potential applications in the fields of pharmaceuticals and clinical analysis.