Design of a multiwalled carbon nanotube–Nafion–cysteamine modified tyrosinase biosensor and its adaptation of dopamine determination

In this work, a multiwalled carbon nanotube (MWCNT)–Nafion–cysteamine (CA) modified tyrosinase biosensor brings a new and original perspective to biosensor technology intended for the development of dopamine determination. Dopamine measurements were done at 0.2 V with the amperometric method by the developed biosensor system. In addition, in this study dopamine determination was carried out by using the differential pulse voltammetry method between potentials of 0.4 and −0.15 V. In the optimization studies of the biosensor, some parameters such as optimal pH, optimal temperature, optimal enzyme amount, and effect of MWCNT concentration were investigated. Afterward, in the characterization studies, some parameters such as linearity and reproducibility were determined. In the reproducibility experiment, an average value of 1.026 μM, a standard deviation of ±0.03975, and a coefficient of variation of 3.8% were determined for a 1-μM dopamine concentration (n = 15). Determination of dopamine was carried out in drug samples by the developed biosensor.     

Electrochemical oxidation behavior of guanine and adenine on graphene–Nafion composite film modified glassy carbon electrode and the simultaneous determination

The electrochemical behavior of guanine and adenine on the graphene and Nafion composite film modified glassy carbon electrode was investigated by differential pulse voltammetry (DPV). The results indicated that the modified electrode exhibited an excellent electrocatalytic activity towards the oxidation of guanine and adenine, testified by the increased oxidation peak current and decreased oxidation potential. The experimental conditions were optimized. The separation of the two oxidation peaks was 0.364 V in 0.1 M pH 4.4 acetate buffer solution (ABS). Based on this, a novel electrochemical method was proposed to simultaneously determine guanine and adenine with the detection limit of 0.58 (guanine) and 0.75 (adenine) μM (S/N = 3). The proposed method was applied to determine guanine and adenine in milk powder, urine and herring sperm DNA samples with satisfactory results. The value of (G + C)/(A + T) in herring sperm DNA was calculated to be 0.8065. The fabricated electrode showed excellent reproducibility, stability and anti-interference.     

Electrochemical selectivity enhancement by using monosuccinyl β-cyclodextrin as a dopant for multi-wall carbon nanotube-modified glassy carbon electrode in simultaneous determination of quercetin and rutin

Monosuccinyl β-cyclodextrin (succinyl-β-CD) was synthesized and the selectivity to quercetin and rutin of the succinyl-β-CD-modified, multi-wall carbon nanotube (MWNT)-coated, glassy carbon electrode [(succinyl-β-CD + MWNT)/GCE] was investigated. ¹H NMR and MALDI-MS data confirmed molecular structure of the synthesized succinyl-β-CD. As a dopant in carboxylated MWNT-modified electrode, succinyl-β-CD clearly separated the peak potential (E_p) of quercetin from that of rutin. The measured peak potential separation (ΔE_p) was 110 mV. More favorable complexation between succinyl-β-CD and quercetin may enhance relative selectivity to quercetin of the (succinyl-β-CD + MWNT)/GCE in quercetin-rutin mixture as compared to the β-CD-modified GCE.     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.     

ZnO–CuxO/polypyrrole nanocomposite modified electrode for simultaneous determination of ascorbic acid,dopamine, and uric acid

Novel zinc oxide (ZnO) nanosheets and copper oxide (Cu_xO, CuO, and Cu₂O) decorated polypyrrole (PPy) nanofibers (ZnO–Cu_xO–PPy) have been successfully fabricated for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The morphology and structure of ZnO–Cu_xO–PPy nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy. Compared with the bare glassy carbon electrode (GCE), PPy/GCE, Cu_xO–PPy/GCE, and ZnO–PPy/GCE, ZnO–Cu_xO–PPy/GCE exhibits much higher electrocatalytic activities toward the oxidation of AA, DA, and UA with increasing peak currents and decreasing oxidation overpotentials. Cyclic voltammetry (CV) results show that AA, DA, and UA could be detected selectively and sensitively at ZnO–Cu_xO–PPy/GCE with peak-to-peak separation of 150 and 154 mV for AA–DA and DA–UA, respectively. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.2 to 1.0 mM, 0.1 to 130.0 μM, and 0.5 to 70.0 μM, respectively. The lowest detection limits (signal/noise = 3) were 25.0, 0.04, and 0.2 μM for AA, DA, and UA, respectively. With good selectivity and sensitivity, the current method was applied to the determination of DA in injectable medicine and UA in urine samples.     

Biomimetic synthesis and characterization of cobalt nanoparticles using apoferritin, and investigation of direct electron transfer of Co(NPs)–ferritin at modified glassy carbon electrode to design a novel nanobiosensor

Oxyhydroxy cobalt CoO(OH) nanoparticles (Co-NPs) were prepared in horse spleen apoferritin (HsAFr) cavity. Transmission electron microscopy revealed the particle size was 5.5-6 nm. Mineralization effect on HsAFr was investigated by fluorescence and far-UV circular dichroism (far-UV CD) spectroscopies. The far-UV CD experiments indicated an increase in the α-helical content after mineralization. Intrinsic fluorescence data showed that mineralization acts as a quencher of HsAFr. For the first time, direct electron transfer between Co(NPs)-HsAFr and a glassy carbon electrode in the thin film of dihexadecylphosphate (DHP) was investigated by cyclic voltammetry (CV) to design a biosensor. The anionic surfactant DHP was used to achieve direct electron-transfer between Co(NPs)-HsAFr molecules and the GC electrode surface. CV result showed clearly a pair of well-defined and quasi-reversible redox peaks arise from Co(NPs)-HsAFr embedded in DHP film. This novel biosensor can be used in medical and industrial fields to detect different analytes.     

Salivary chenodeoxycholic acid and its glycine-conjugate: Their determination method using LC–MS/MS and variation of their concentrations with increased saliva flow rate

Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC–MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-μl sample and the limits of quantification for CDCA and GCDCA were 25 and 50 pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing.     

Synthesis of enantiomerically enriched drug precursors and an insect pheromone via reduction of ketones using commercially available carbonyl reductase screening kit “Chiralscreen® OH”

Commercially available “Chiralscreen® OH” starter kit containing five types of carbonyl reductases (E001, E007, E031, E039, and E078) was used for the reduction of several aromatic and aliphatic ketones to obtain enantiomerically enriched drug precursors and an insect pheromone. Almost stereochemically pure secondary alcohols, used in the synthesis of drugs such as (R)-rasagiline mesylate, (S)-rivastigmine, (R)-chlorphenesin carbamate, and (R)-mexiletine, and the insect pheromone (4S,5R)-sitophilure, were conveniently obtained. The enzymes worked well with ketones containing at least one non-bulky substituent at the carbonyl group. The diverse stereochemical preference of the above five carbonyl reductases was clarified.     

Novel detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> β-<Emphasis Type="SmallCaps">d</Emphasis>-glucuronidase activity using a microbially-modified glassy carbon electrode and its potential for faecal pollution monitoring

The electrochemical detection of Escherichia coli β-d-glucuronidase activity as a means of monitoring water pollution by faecal material was investigated using separate Moraxella- and Pseudomonas putida-modified glassy carbon electrodes. The former was more sensitive and selective. The Moraxella-modified biosensor was 100 times more rapid and sensitive than the spectrophotometric detection of β-d-glucuronidase activity. The experimental limit of detection of the biosensor was two c.f.u. per 100 ml polluted water sample within 20 min. The biosensor gave a linear response to commercial β-d-glucuronidase concentration between 0.2 ng and 2 μg ml⁻¹. The biosensor detected activity of β-d-glucuronidase from viable but non-culturable (VBNC) cells and can therefore serve as a presence or absence device for rapid water quality monitoring.