A new BOD estimation method employing a double-mediator system by ferricyanide and menadione using the eukaryote Saccharomyces cerevisiae

A new biochemical oxygen demand (BOD) sensing method employing a double-mediator (DM) system coupled with ferricyanide and a lipophilic mediator, menadione and the eukaryote Saccharomyces cerevisiae has been developed. In this study, a stirred micro-batch-type microbial sensor with a 560 μL volume and a two-electrode system was used. The chronamperometric response of this sensor had a linear response between 1 μM and 10 mM hexacyanoferrate(II) (r² = 0.9995, 14 points, n = 3, average of relative standard deviation and R.S.D._av = 1.3%). Next, the optimum conditions for BOD estimation by the DM system (BOD_DM) were investigated and the findings revealed that the concentration of ethanol, used to dissolve menadione, influenced the sensor response and a relationship between the sensor output and glucose glutamic acid concentration was obtained over a range of 6.6-220 mg O₂ L⁻¹ (five points, n = 3, R.S.D._av 6.6%) when using a reaction mixture incubated for 15 min. Subsequently, the characterization of this sensor was studied. The sensor responses to 14 pure organic substances were compared with the conventional BOD₅ method and other biosensor methods. Similar results with the BOD biosensor system using Trichosporon cutaneum were obtained. In addition, the influence of chloride ion, artificial seawater and heavy metal ions on the sensor response was investigated. A slight influence of 20.0 g L⁻¹ chloride ion and artificial seawater (18.4 g L⁻¹ Cl⁻) was observed. Thus, the possibility of BOD determination for seawater was suggested in this study. In addition, no influence of the heavy metal ions (1.0 mg L⁻¹ Fe³⁺, Cu²⁺, Mn²⁺, Cr³⁺ and Zn²⁺) was observed. Real sample measurements using both river water and seawater were performed and compared with those obtained from the BOD₅ method. Finally, stable responses were obtained for 14 days when the yeast suspension was stored at 4 °C (response reduction, 93%; R.S.D. for 6 testing days, 9.1%).     

Evaluation of Redox Activity of Human Myocardium‐derived Mesenchymal Stem Cells by Scanning Electrochemical Microscopy

In this study the redox activity of human myocardium‐derived mesenchymal stem cells (hmMSC) were investigated by redox‐competition (RC‐SECM) and generation‐collection (GC‐SECM) modes of scanning electrochemical microscopy (SECM), using 2‐methylnaphthalene‐1,4‐dione (menadione, MD) as a redox mediator. The redox activity of human healthy and dilated hmMSCs was evaluated by measuring reduction of MD. Measurements were performed by approaching and retracting the UME from the surface of growing hmMSC cells. The current study shows that the RC‐SECM mode can be applied to investigate integrity of cell membranes, whereas the most promising results were observed by using the GC‐SECM mode and applying the Hill's equation for the calculation/fitting of dependencies of electrical current vs menadione concentration. The calculated apparent Michaelis constant (K_M) for the production of menadiol (MDH₂) in the pathological hmMSC cells was 14.4 folds higher compared to that of the healthy hmMSC revealing the lover redox activity of pathological cells. Moreover, the calculated Hill's coefficient n shows a negative cooperative binding between MD and healthy hmMSC and positive cooperative binding between MD and pathological hmMSC. It means that healthy hmMSC is of lower affinity to MD, which is also related to the better membrane integrity of healthy cells. Data of this study demonstrate that SECM can be applied to investigate intracellular redox and membrane changes ongoing in human dilated myocardium‐derived hmMSC in order to improve their functioning and further regenerative potential.     

Accurately measuring respiratory activity of single living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current i_p on this first scan curve is composed of i_p1, which corresponds to the cell respiratory activity and i_p2, which corresponds to the cell topography. In order to isolate the i_p2 component, the cell is killed by exposing it to 1.0 × 10⁻³ mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, i_p2 is obtained from the second SECM scan curve. Finally, i_p1 corresponding to the respiratory activity of the living cell can be accurately calculated using i_p1 = i_p − i_p2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN₃ and KCN.     

Respiration activity of Escherichia coli entrapped in a cone-shaped microwell and cylindrical micropore monitored by scanning electrochemical microscopy (SECM)

The metabolic activity of E. coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide. The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate. A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well. The ferrocyanide production rate of a single E. coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system.     

Cytokine assay on a cellular chip by combining collagen gel embedded culture with scanning electrochemical microscopy

A novel cytokine assay has been designed using a cellular chip by combining a collagen gel embedded cell culture technique with scanning electrochemical microscopy-enzyme linked immunosorbent assay (SECM-ELISA). An array of cell-collagen gel mixture (2 μL) was spotted on an antibody-coated chip and incubated for 0.5-24 h. The very small trace amounts of cytokines produced by the activated leukocytes on the chip were effectively entrapped within the collagen gel matrix, and these were collected with the immobilized antibodies on the chip. The chip was further treated with horseradish peroxidase (HRP)-labeled antibodies via the sandwich method after removing the cell-collagen gel spots from the chip. Scanning electrochemical microscopy (SECM) was used to quantitatively evaluate the cytokines from the activated leukocytes produced on the chip, and the SECM images were obtained to visualize the position and concentration of IL-1β secreted from THP-1 and HL-60 cell lines at concentration levels of 10-350 pg mL⁻¹. Based on the chemiluminescence method, the sensitivity of the cytokine assay system in combination with SECM-ELISA is comparable to that of the marketed cytokine assay kit; further, the sample volume required for a single assay is drastically reduced.     

Surface patterning using scanning electrochemical microscopy to locally trigger a “click” chemistry reaction

We report on the surface micropatterning of conductive surfaces via the electrochemical triggering of a click reaction, the copper(I) catalyzed azide–alkyne cycloaddition reaction (CuAAC) by SECM via a two-step approach: (i) functionalization on the entire surface with azido-aryl groups by using the diazonium approach followed by (ii) the covalent linkage of alkyne-bearing ferrocene by CuAAC within a local area by SECM. More precisely, the click reaction was triggered by Cu(I) catalyst generation for 30 min at the SECM tip positioned ≈ 10 μm above the azido-aryl modified surface. The dimension of the spot obtained under these conditions was ≈ 75 μm. The electrochemical imaging by SECM of the ultra thin area locally clicked with ferrocene moieties was made thanks to the electrocatalytic properties of the ferrocene modified surface towards ferrocyanide electrooxidation. This local clicking procedure opens the gate to further controlled functionalization of restricted small substrates.     

Sol-gel derived urease-based optical biosensor for the rapid determination of heavy metals

A urease optical biosensor for the determination of heavy metals based on sol-gel immobilization technique was developed. A fluorescent dye, FITC-dextran, was encapsulated and parameters including optical properties of the probe, relative enzyme activity, initial pH value and the buffer concentration for substrate preparation were investigated. In sol-gel immobilization, 1 mM Tris-HCl at pH 7.1 provided a sufficient buffer capacity for metal ion analysis as well as the enzyme activity maintenance. Also, two analytical procedures, incubated and un-incubated systems, were compared to understand the sensitivity and applicability to heavy metal analysis. The developed optical biosensor showed high reproducibility and the relative standard deviation (R.S.D.) of 5.1% (n=10) was obtained. Also, eight measurements can be completed automatically within 36 min. The biosensor has high sensitivity to Cu(II) and Cd(II) and an analytical range of 10-230 μM with a detection limit of 10 μM was achieved. Moreover, biological and environmental samples were examined to evaluate the applicability of the developed biosensor. A 19-82% of inhibition was observed when 20-45 μM metal ions were amended into tested samples, revealing that the developed system has the potential for the determination of heavy metals in real samples.     

Enzyme-based determination of cholesterol using the quartz crystal acoustic wave sensor

We have used the AT-cut quartz crystal sensor to measure in real-time the total cholesterol concentration in buffer and serum, using the trienzyme system of cholesterol esterase (ChE), cholesterol oxidase (ChOx) and horseradish peroxidase (HRP). The hydrogen peroxide produced from the ChE-ChOx reaction oxidises diaminobenzidine (DAB), in the presence of HRP. The response of the sensor to cholesterol is optimal in the presence of 0.1% (v/v) Triton X-100 at 0.2 U/ml ChOx, and 1 U/ml ChE. A response is obtained in less than 25 min. Using the optimal concentrations of the reagents, the linear range for free cholesterol and low density lipoprotein (LDL) cholesterol determination was between 50 and 300 μM, and 25 and 400 μM, respectively. It was found that the concentration of high density lipoprotein (HDL) cholesterol could not be determined because it solubilised the oxidised DAB, leading to poor adsorption at the crystal surface. We obtained a response to the use of cholesterol in serum at 300 μM, demonstrating that this biosensor could be used for cholesterol determination in clinical samples.     

Grafted Azure A modified electrodes as disposable β-nicotinamide adenine dinucleotide sensors

We report the in situ generation of aryl diazonium cations of Azure A, a redox-active phenothiazine dye, by reaction between the corresponding aromatic aminophenyl group and sodium nitrite in 0.1 M HCl. The subsequent electrochemical reduction of these dye diazonium salts gives rise to conductive electrografted films onto screen-printed carbon (SPC) electrodes. The resulting Azure A films have a very stable and reversible electrochemical response and exhibit potent and persistent electrocatalytic behavior toward NADH oxidation. We have optimized the electrografting conditions in order to obtain SPC modified electrodes with high and stable electrocatalytic response. The kinetic of the reaction between the NADH and the redox active centers in the Azure A film has been characterized using cyclic voltammetry and single step chronoamperometry. The catalytic currents were proportional to the concentration of NADH giving rise to linear calibration plots up to a concentration of 0.5 mM with a detection limit of 0.57 ± 0.03 μM and a sensitivity of 9.48 A mol cm⁻² μM⁻¹. The precision of chronoamperometric determinations was found to be 2.3% for five replicate determinations of 3.95 μM NADH. The great stability of such modified electrodes makes them ideal for their application in the development of biosensing platforms based on dehydrogenases.     

Simultaneous determination of cell aging and ATP release from erythrocytes and its implications in type 2 diabetes

Glucose-6-phosphate dehydrogenase (G6PD) is a determinant in the antioxidant status of the red blood cell (RBC) and is also used as an indicator of cell age. However, it is unknown if the relationship among antioxidant status, cell age, and RBC-derived adenosine triphosphate (ATP) occurs immediately or over a period of time. Therefore, the development of a simultaneous determination of G6PD activity (via the determination of nicotinamide adenine dinucleotide phosphate (NADPH)) in RBCs and the determination of deformation-induced RBC-derived ATP is described. The NADPH and ATP were determined while undergoing a chemically induced aging process via inhibition of G6PD with dehydroepiandroesterone (DHEA). Upon incubation with DHEA for 30 min, NADPH levels measured in a flow stream decreased to 7.96 ± 1.10 μM from an original value of 13.20 ± 1.80 μM in a 0.02% solution of RBCs. In order to demonstrate a direct relationship between G6PD activity and deformation-induced ATP release from RBCs, a simultaneous microflow determination of G6PD activity and ATP release was performed. Upon inhibition with DHEA, NADPH levels decreased to 8.62 ± 0.29 μM from its original value of 12.73 ± 0.50 μM while ATP release decreased from 0.21 ± 0.07 μM to 0.06 ± 0.02 μM. These values were validated by an examination of NADPH levels in, and ATP release from, RBC fractions containing younger and older cells (separated by cell density centrifugation). This determination provides evidence that antioxidant status in the RBC and its ability to release ATP, a known stimulus of nitric oxide production, are closely related.     

Scanning Electrochemical Microscopy for Electrochemical Detection of Single‐base Mismatches by Tagging Ferrocenecarboxylic Acid as a Redox Probe to DNA

Scanning electrochemical microscopy (SECM) was employed for sensitive detection of single base mismatches (SBMs) in a sandwiched dsDNA. Ferrocenecarboxylic acid (Fc), covalently conjugated to the dsDNA, was oxidized to Fc⁺ via the DNA‐mediated charge transfer from the underlying gold substrate, and reduced back to Fc by SECM tip generated ferrocyanide. The electrocatalytic oxidation of SECM tip‐generated ferrocyanide was sensitive to presence, as well as the type of SBMs. Apparent standard rate constants (k⁰_app) values for different SBMs, both near the electrode surface and far from it, were evaluated by SECM. The method can detect SBMs independent of their position in dsDNA.     

Fabrication of microbial chip using collagen gel microstructure

A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli(E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.     

An inhibition type amperometric biosensor based on tyrosinase enzyme for fluoride determination

In this study, a new biosensor based on the inhibition of tyrosinase for the determination of fluoride is described. To construct the biosensor tyrosinase was immobilized by using gelatine and cross-linking agent glutaraldehyde on a Clark type dissolved oxygen (DO) probe covered with a teflon membrane which is sensitive for oxygen. The phosphate buffer (50 mM, pH 7.0) at 30 °C were established as providing the optimum working conditions. The method is based on the measurement of the decreasing of dissolved oxygen level of the interval surface that related to fluoride concentration added into reaction medium in the presence of catechol. Inhibitor effect of fluoride results in decrease in dissolved oxygen concentration. The biosensor response depends linearly on fluoride concentration between 1.0 and 20 μM with a response time of 3 min.In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity and storage stability were carried out. From the experiments, the average value (x), Standard deviation (S.D) and coefficient of variation (C.V %) were found as 10.5 μM, ± 0.57 μM, 5.43%, respectively for 10 μM fluoride standard.     

Development of a long-life capillary enzyme bioreactor for the determination of blood glucose

A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability than previous systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53 mm i.d. fused-silica capillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxide (H₂O₂). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactor into a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumption compared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 °C) stability for 20 weeks, the glucose biosensing system was prepared using an optimal GOx concentration (200 mg/mL). This exhibited an FIA peak response of 7 min and a detection limit of 10 μM (S/N = 3) with excellent reproducibility (coefficient of variation, CV < 0.75%). It also had a linear working range from 10¹ to 10⁴ μM. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samples were analyzed using this system, and these correlated favorably (correlation coefficient, r² = 0.935) with results for the same samples obtained using a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.     

A novel quantification method for analysis of twenty natural amino acids in human serum based on N-phosphorylation labeling using reversed-phase liquid chromatography–tandem mass spectrometry

A novel method based on the strategy of N-phosphorylation labeling is described for quantification of twenty natural amino acids in human serum by reversed-phase liquid chromatography–electrospray tandem mass spectrometry (RP-LC/ESI-MS). The derivatization reaction was easily performed in one-pot reaction under mild conditions within 30 min. The reaction mixture was then evaporated to dryness, redissolved, desalted by C18 SPE. The twenty N-phosphoryl amino acids were separated on an RP-C18 column within 20 min by isocratic elution (0.1% formic acid–acetonitrile, v/v 7:3). At the same time, multiple reaction monitoring (MRM) MS enabled quantitation of twenty natural amino with the LOD of 0.0005–0.15 μM and LOQ of 0.0020–0.5 μM in human serum. The linear range was from 0.025 to 25 μM (except Cys and Trp) with R > 0.99. The recovery range was determined to be 85.5–117.4% with the relative standard deviation (RSD) in the range of 1.3–13.9%. All twenty amino acids were successfully detected in human serum samples with the concentration from 5.7 to 577.9 μM, which indicates potential of the developed method for determination of amino acids in complex biological samples, hence for screening of amino acid metabolite related diseases.     

PdNi- and Pd-coated electrodes prepared by electrodeposition from ionic liquid for nonenzymatic electrochemical determination of ethanol and glucose in alkaline media

Nonenzymatic electrochemical determination of ethanol and glucose was respectively achieved using PdNi- and Pd-coated electrodes prepared by electrodeposition from the novel metal-free ionic liquid (IL); N-butyl-N-methylpyrrolidinium dicyanamide (BMP-DCA). BMP-DCA provided an excellent environment and wide cathodic limit for electrodeposition of metals and alloys because many metal chlorides could dissolve in this IL where the reduction potentials of Pd(II) and Ni(II) indeed overlapped, leading to the convenience of potentiostatic codeposition. In aqueous solutions, the reduction potentials of Pd(II) and Ni(II) are considerably separated. The bimetallic PdNi coatings with atomic ratios of ∼80/20 showed the highest current for ethanol oxidation reaction (EOR). Ethanol was detected by either cyclic voltammetry (CV) or hydrodynamic amperometry (HA). Using CV, the dependence of EOR peak current on concentration was linear from 4.92 to 962 μM with a detection limit of 2.26 μM (σ = 3), and a linearity was observed from 4.92 to 988 μM using HA (detection limit 0.83 μM (σ = 3)). The Pd-coated electrodes prepared by electrodeposition from BMP-DCA showed electrocatalytic activity to glucose oxidation and CV, HA, and square-wave voltammetry (SWV) were employed to determine glucose. SWV showed the best sensitivity and linearity was observed from 2.86 μM to 107 μM, and from 2.99 mM to 10.88 mM with detection limits of 0.78 μM and 25.9 μM (σ = 3), respectively. For glucose detection, the interference produced from ascorbic acid, uric acid, and acetaminophen was significantly suppressed, compared with a regular Pt disk electrode.     

Simultaneous separation of nine surfactants of various types by HPLC with evaporative light scattering detection

A simultaneous separation of cationic, amphoteric and nonioinc nine surfactants (DMDS, DMDP, DMDM, DMDL, BZC, CDE, A/O, SUNC, IMD) has been performed by a reverse phase-HPLC method utilizing a single J'sphere ODS (250 mm × 4.6 mm, 4 μm) column and a methanol-water containing 0.2% TFA eluent system within 60 min. The observed precision in determination of concentration was less than 5% R.S.D., which revealed that ELSD was an effective tool to detect the various studied surfactants of low volatility without chromophore. In addition, the detection limits were in the concentration range of 3.5-10 μg/mL, and the calibration curves, i.e. the log-log plots, were linear in the working range of 5-4600 μg/mL with the slopes of 1.321-1.668. The application of the analytical procedure to three household products without pretreatment supported that the presented chromatographic method was simple to be practical for a routine analysis of commercial products.     

A PVC-membrane bulk optode for gallium(III) ion determination

An optical probe responsive to gallium(III) ion has been developed. The gallium sensing system was prepared by incorporating 4-(p-nitrophenyl azo)-pyrocatechol (NAP) as ionophore in a plasticized PVC membrane containing tributylphosphate (TBP) as plasticizer. The sensing membrane in contact with gallium ion at pH 3.5, changes color from yellow-brown to pink-brown. Under optimum conditions, the proposed membrane displayed a linear range of 5-83 μM with a limit of detection of 4 μM. The response time of the membrane was within 10-15 min depending on the concentration of Ga³⁺ ions. The selectivity of the probe towards gallium determination was found to be very good. Experimental results showed that the probe could be used as an effective tool in analyzing the gallium content of water samples.     

Graphene-loaded nanofiber-modified electrodes for the ultrasensitive determination of dopamine

A novel and highly sensitive electrochemical system based on electrospun graphene/polyaniline/polystyrene (G/PANI/PS) nanofiber-modified screen-printed carbon electrodes has been developed for dopamine (DA) determination. A dramatic increase (9 times) in the current signal for the redox reaction of a standard, ferri/ferrocyanide [Fe(CN)₆]^3−/4− couple was found when compared to an unmodified electrode. This modified electrode also exhibited favorable electron transfer kinetics and excellent electrocatalytic activity toward the oxidation of DA. When used together with square wave voltammetry (SWV), DA can be selectively determined in the presence of the common interferents (i.e. ascorbic acid and uric acid). Under optimal conditions, a very low limit of detection (0.05 nM) and limit of quantification (0.30 nM) were achieved for DA. In addition, a wide dynamic range of 0.1 nM to 100 μM was found for this electrode system. Finally, the system can be successfully applied to determine DA in complex biological environment (e.g. human serum, urine) with excellent reproducibility.     

Intrinsic peroxidase-like catalytic activity of nitrogen-doped graphene quantum dots and their application in the colorimetric detection of H2O2 and glucose

In this paper, the highly intrinsic peroxidase-like catalytic activity of nitrogen-doped graphene quantum dots (N-GQDs) is revealed. This activity was greatly dependent on pH, temperature and H₂O₂ concentration. The experimental results showed that the stable N-GQDs could be used for the detection of H₂O₂ and glucose over a wide range of pH and temperature, offering a simple, highly selective and sensitive approach for their colorimetric sensing. The linearity between the analyte concentration and absorption ranged from 20 to 1170 μM for H₂O₂ and 25 to 375 μM for glucose with a detection limit of 5.3 μM for H₂O₂ and 16 μM for glucose. This assay was also successfully applied to the detection of glucose concentrations in diluted serum and fruit juice samples.