Inducible alkylation of DNA by a quinone methide-peptide nucleic acid conjugate

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate, but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alternatively, alkylation of noncomplementary sequences is only possible when a template strand is present to colocalize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self-adducts formed between the PNA and its attached QM remained active and reversible over more than 8 days in aqueous solution prior to reaction with a chosen target added subsequently.     

Novel optical solid-state glucose sensor using immobilized glucose oxidase

Meso-tetra(4-carboxyphenyl)porphine (CTPP(4)) binds reversibly to immobilized glucose oxidase (GOD), resulting in an absorbance peak for the CTPP(4)-GOD complex at 427nm. The absorbance intensity of the 427nm peak is reduced upon exposure to glucose, which causes the dissociation of CTPP(4) from GOD. The change in absorbance at 427nm shows linear dependence on glucose concentration from 20 to 200mg/dL (1.1-11.1mM).     

Amperometric glucose sensor based on catalytic reduction of dissolved oxygen at soluble carbon nanofiber

This work shows excellent catalytic activity of soluble carbon nanofiber (CNF), which was obtained with a simple nitric acid treatment, toward the electroreduction of dissolved oxygen at a low operating potential. Thus the CNF was applied in the construction of amperometric biosensors for oxidase substrates using glucose oxidase as a model. The good dispersion of CNF led to convenient preparation and acceptable repeatability of the proposed sensors. UV-vis spectra, Fourier transform infrared spectra, X-ray photoelectron spectra and titration curves demonstrated that the good dispersion resulted from the large numbers of surface oxygen-rich groups produced in the treatment process. The membrane of CNF showed good stability and provided fast response to dissolved oxygen with a linear range from 0.1 to 78 microM and detection limit of 0.07 microM. The proposed glucose biosensor could monitor glucose ranging from 10 to 350 microM with detection limit of 2.5 microM and sensitivity of 36.3 nA cm(-2) microM(-1). The coefficients of variation for intra-assay were 4.7 and 3.2% at glucose concentrations of 20 and 210 microM, respectively. The use of a low operating potential (-0.3 V) and Nafion membrane produced good selectivity toward the glucose detection. CNF-based biosensors would provide wide range of bioelectrochemical applications in different fields.     

Amperometric dimethyl sulfoxide sensor using dimethyl sulfoxide reductase from Rhodobacter sphaeroides

An amperometric dimethyl sulfoxide (DMSO) sensor was constructed based on DMSO reductase (DMSO-R). DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was immobilized by BSA-glutaraldehyde cross-linking at the surface of a glassy carbon electrode. Mediators were added to the sample solution in a free form. Several mediators (methyl viologen (MV), benzyl viologen (BV), neutral red (NR), safranin T (ST), FMN, phenazine methosulfate (PMS)), which can donate electrons to DMSO-R, were examined with the DMSO-R immobilized electrode. Among them MV was selected as a model mediator because of its wide linear response range and fast response time. The response current was effected by the measurement temperature but hardly effected by the pH of the sample solution. The response current was increased with the measurement temperature up to 50 degrees C. A response current was observed at 1 microM DMSO and the response time was 20 s under the optimum conditions. The response was observed for approximately 2 weeks. By the reduction of Schiff base in the cross-linking layer the response range became narrower but most of the response current was retained at 300 microM of DMSO for more than 5 weeks.     

Amperometric glucose sensor based on coimmobilization of glucose oxidase and Poly(p-phenylenediamine) at a platinum microdisk electrode

A miniaturized glucose biosensor in which glucose oxidase (GOD) and poly(p-phenylenediamine) (poly-PPD) were coimmobilized at the surface of a platinum microdisk electrode was developed and used successfully for amperometric determination of glucose. The performance of sensors prepared at different monomer concentrations and polymerization potentials with different media was investigated in detail. It was found that similarly to poly(o-phenylenediamine) (poly-OPD), (poly-PPD) noticeably eliminated the electrochemical interference of ascorbic acid, uric acid, and l-cysteine. The amperometric response of glucose with the biosensor under optimal conditions exhibited a linear relationship in the range of 5.0 x 10(-5) to 3.0 x 10(-3) M with correlation coefficient 0.9995. According to the Michaelis-Menten equation, the apparent Michaelis constant for glucose and the maximum steady-state current density of the poly-PPD/GOD-modified microelectrode were 3.94 mM and 607.5 microA cm(-2), respectively. The current density of the sensor responding to glucose in the linear range can reach 160 microA cm(-2) mM(-1), which is far greater than that obtained using poly-OPD and poly(phenol) film. In addition, the stability of the sensor was examined over a 2-month period.     

Amperometric sensor based on ferrocene-modified multiwalled carbon nanotube nanocomposites as electron mediator for the determination of glucose

A kind of nanocomposite with good dispersion in water was prepared through covalent adsorption of ferrocenecarboxaldehyde on multiwalled carbon nanotubes (MWNTs) for electrical communication between glucose oxidase (GOD) and electrode. The ferrocene-modified multiwalled carbon nanotube nanocomposites (MWNTs-Fc) could be conveniently cast on electrode surfaces. With the aid of chitosan, GOD was then immobilized on the nanostructure film to form a reagentless amperometric sensor for glucose determination. FTIR spectra and cyclic voltammetry were used to characterize the nanocomposites. The presence of both ferrocene as mediator of electron transfer and MWNTs as conductor enhanced greatly the enzymatic response to the oxidation of glucose. The novel biosensor exhibited a fast response toward glucose with a detection limit of 3.0 × 10⁻⁶ mol/L and the linear range extended up to 3.8 × 10⁻³ mol/L.     

Preparation of lyophilized pyrroloquinoline quinone glucose dehydrogenase using trehalose as an additive

Of 8 compounds tested as additives, trehalose at 100 mM was the most effective for the preparation of lyophilized pyrroloquinoline quinone glucose dehydrogenase (PQQGDH). Lyophilized PQQGDH retained about 80% of initial activity after 1 h at 80°C and can be stored at 28°C for more than a month without any significant decrease in enzymatic activity. It is thus suitable for use as a biosensor component.     

Development of glucose sensor using two-photon adsorbed photopolymerization

A novel glucose sensor was constructed, and its analytical potential examined. A chip-type three-electrode system for use in a flow-type electrochemical glucose sensor was fabricated using a UV lithography technique on a glass slide. An Ag/AgCl reference electrode was made by electroplating silver onto a Pt electrode and dipping in a saturated KCl solution for 30 min. In addition, a glucose-sensing electrode was fabricated using a two-photon adsorbed photopolymerization technique with a photo-reactive resin containing a glucose oxidase enzyme, ferrocene mediator, non-ionic surfactant, and carbon nanotubes. The cyclic voltammetry of the potassium ferrocyanide in the Pt sensor system showed a stable electrode condition. The response of the modified Pt sensor confirms the feasibility of using a two-photon adsorbed photopolymerization technique for the easy fabrication of functional biosensors.     

Amperometric glucose biosensor based on lipid film

A novel glucose biosensor based on cast lipid film was developed. This model of biological membrane was used to supply a biological environment on the surface of the electrode, moreover it could greatly reduce the interference and effectively exclude hydrophilic electroactive material from reaching the detecting surface. TTF was selected as a mediator because of its high electron-transfer efficiency, and it was incorporated in the lipid film firmly. Glucose oxidase was immobilized in hydrogel covered on the lipid film. The effects of pH, operating potential were explored for the optimum analytical performance by using amperometric method. The response time of the biosensor was less than 20 s, and the linear range is up to 10 mmol l(-1) (corr. coeff. 0.9932) with the detection limit of 2 x 10(-5) mol l(-1). The biosensor also exihibited good stability and reproducibility.     

Flexible glucose sensor using CVD-grown graphene-based field effect transistor

In this paper, a new concept to achieve improved probe-target recognition has been devised by introducing a novel class of DNA-functionalized three-dimensional (3D), stand-free, and nanostructured electrodes. The gold nanofibrous electrodes were created using MHZ ultrafast laser material processing in air at ambient conditions. The developed nanofibrous DNA biosensor was characterized by cyclic voltammetry with the use of ferrocyanide as an electrochemical redox indicator. Currently, electrochemical signal enhancement which is of great significance for improving the sensitivity in DNA detection remains a great challenge. Through, enhanced surface area-to-volume ratio and more efficient arrangement of probe molecules on nanofibrous electrodes, our newly developed electrode system overcomes some of the sensitivity challenges of the existing systems. This nanofiber-based system realizes femtomolar (fM) sensitivity toward complementary target DNA, and demonstrates a very wide dynamic range (from 1 fM to 1 nM).     

DNA hybridization electrochemical sensor using conducting polymer

We report the use of poly(thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (PTAE) for application to electrochemical hybridization sensor. A synthetic route for the thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (TAE) is described, which is used as a monomer of conducting polymer sensor. A direct chemical substitution of probe oligonucleotide to good leaving group site in the PTAE is carried out on the conducting polymer film. A biological recognition can be monitored by comparison with the electrochemical signal (cyclic voltammogram) of single and double strand state oligonucleotide. The sensitivity of the electrochemical sensor is 0.62 microA/nmole and the detection limit is 1 nmole. The oxidation current of double strand state oligonucleotide is a half of that of single strand, that is corresponding to the decrease of electrochemical activity of conducting polymer with increase of stiffness of side group of the polymer. The oxidation current decreasing ratios of perfect matched and single nucleotide mismatched samples are 52 and 25-30%, respectively. The more decreasing ratio is attributable to the more steric hindrance of single nucleotide mismatched sample.     

Amperometric acetylcholine sensor catalyzed by nickel anode electrode

An amperometric method was using a nickel catalytic electrode in aqueous base solution for detecting acetylcholine (ACh). A sensing mechanism was developed in which ACh was hydrolyzed in base aqueous solution to produce the acetic anion and choline. The alcohol group of choline was oxidized to the corresponding carboxylic acid by Ni(OH)2/NiOOH catalytic system. The amperometric response resulted from the current generated by ACh oxidation in response to step changes in ACh concentration. The potential window of limiting current of ACh anodic oxidation at the Ni interface was determined in NaOH electrolyte. The effect of NaOH electrolyte concentration on sensitivity was also discussed. At the optimum operating condition, the method exhibits a good linear relationship between the response current and the ACh concentration. The response time of the ACh sensing system was 10 s. Scanning electrochemical microscopy (SECM) with platinum micro-tips was used to investigate the diffusion layer thickness of Ni electrode.     

Amperometric glucose biosensor utilizing FAD-dependent glucose dehydrogenase immobilized on nanocomposite electrode

Amperometric glucose biosensors utilizing commercially available FAD-dependent glucose dehydrogenases from two strains of Aspergillus species are described. Enzymes were immobilized on nanocomposite electrode consisting of multi-walled carbon nanotubes by entrapment between chitosan layers. Unlike the common glucose oxidase based biosensor, the presented biosensors appeared to be O(2)-independent. The optimal amount of enzymes, working potential and pH value of working media of the glucose biosensors were determined. The biosensor utilizing enzyme isolated from Aspergillus sp. showed linearity over the range from 50 to 960 μM and from 70 to 620 μM for enzyme from Aspergillus oryzae. The detection limits were 4.45 μM and 4.15 μM, respectively. The time of response was found to be 60 s. The biosensors showed excellent operational stability - no loss of sensitivity after 100 consecutive measurements and after the storage for 4 weeks at 4 °C in phosphate buffer solution. When biosensors were held in a dessicator at room temperature without use, they kept the same response ability at least after 6 months. Finally, the results obtained from measurements of beverages and wine samples were compared with those obtained with the enzymatic-spectrophotometric and standard HPLC methods, respectively. Good correlation between results in case of analysis of real samples and good analytical performance of presented glucose biosensor allows to use presented concept for mass production and commercial use.     

Amperometric measurement of copper ions with a deputy substrate using a novel Saccharomyces cerevisiae sensor

The first microbial biosensor to detect Cu2+ by an amperometric method has been developed. For this purpose, recombinant Saccharomyces cerevisiae strains are suitable as the microbial component. These strains contain plasmids with the Cu2+-inducible promoter of the CUP1-gene from Saccharomyces cerevisiae fused to the lacZ-gene from E. coli. On this sensor the CUP1 promoter is first induced by the Cu2+-containing probe and subsequently lactose is used as a deputy substrate to make the measurement. If Cu2+ is present in the sample, these recombinant strains are able to utilize lactose as a carbon source, which leads to alterations in the oxygen consumption of the cells. The sensor measured Cu2+ in a concentration range between 0.5 and 2 mM CuSO4. In addition, an indirect amperometric measurement principle was developed which allows the detection of samples containing Cu2+ and fast biodegradable substances.     

Amperometric glucose biosensor based on single-walled carbon nanohorns

The biosensing application of single-walled carbon nanohorns (SWCNHs) was demonstrated through fabrication of an amperometric glucose biosensor. The biosensor was constructed by encapsulating glucose oxidase in the Nafion-SWCNHs composite film. The cyclic voltammograms for glucose oxidase immobilized on the composite film displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of -0.453 V. The biosensor had good electrocatalytic activity toward oxidation of glucose. To decrease detection potential, ferrocene monocarboxylic acid was used as a redox mediator. The mediated glucose biosensor shows a linear range from 0 to 6.0 mM. The biosensor shows high sensitivity (1.06 microA/mM) and stability, and can avoid the commonly coexisted interference. Because of impressive properties of SWCNHs, such as high purity and high surface area, SWCNHs and their composites are expected to be promising material for biomolecular immobilization and biosensing applications.     

A chromatographic tool for preparing combinatorial quinone–thiol conjugate libraries

Quinones are well established as key players in the production of reactive oxygen species within cellular environments. Many factors govern their cytotoxicity but most studies have been restricted to a few, core, derivatives. A new strategy for the in situ production of quinone derivatives has been developed such that libraries of diverse functionality can be rapidly created without recourse to extensive synthetic procedures. The approach relies upon nucleophilic addition by reduced thiol derivatives to the quinone core within a pre-culture assay mixture and provides a generic strategy that exploits the large reservoir of commercial thiols currently available. A readily accessible chromatographic method has been developed that allows the derivatisation process to be easily monitored and the purity of the resulting one pot preparation to be assessed. The viability of the combinatorial approach has been fully validated through comparison with a range of quinone-S-conjugates prepared using conventional bench synthesis. The latter have been fully characterised.     

Amperometric determination of sodium nitrite by a microbial sensor

Summary A microbial sensor was prepared to determine sodium nitrite. This microbial sensor consisted of immobilized Nitrobacter sp. and an oxygen electrode. When a sample solution containing sodium nitrite was tested, nitrite was changed to NO₂ gas in the buffer (pH 2.0) and the current of the electrode decreased with time until a steady state was reached. The steady state current was attained within 10 min and the maximum decrease in current was obtained at 30°C and pH 2.0. A linear relationship was observed between the current decrease and the sodium nitrite concentration below 0.59 mM, the minimum sodium nitrite concentration that could be determined was 0.01 mM. The current decrease was reproducible (5% relative error). The current output of the sensor was almost constant for more than 21 days and 400 assays.     

Calibration in dogs of a subcutaneous miniaturized glucose sensor using a glucose meter for blood glucose determination.

The feasibility of calibrating a glucose sensor by using a wearable glucose meter for blood glucose determination and moderate variations of blood glucose concentration was assessed. Six miniaturized glucose sensors were implanted in the subcutaneous tissue of conscious dogs, and the parameters used for the in vivo calibration of the sensor (sensitivity coefficient and extrapolated current in the absence of glucose) were determined from values of blood glucose and sensor response obtained during glucose infusion. (1) Venous plasma glucose level and venous total blood glucose level were measured simultaneously on the same sample, using a Beckman analyser and a Glucometer II, respectively. The regression between plasma glucose (x) and whole blood glucose (y) was y = 1.12x-0.08 mM (n = 114 values, r = 0.96, p = 0.0001). The error grid analysis indicated that the use of a Glucometer II for blood glucose determination was appropriate in dogs. (2) The in vivo sensitivity coefficients were 0.57 +/- 0.11 nA mM-1 when determined from plasma glucose, and 0.51 +/- 0.07 nA mM-1 when determined from whole blood glucose (t = 1.53, p = 0.18, n.s.). The background currents were 0.88 +/- 0.57 nA when determined from plasma glucose, and 0.63 +/- 0.77 nA when determined from whole blood glucose (t = 0.82, p = 0.45, n.s.). (3) The regression equation of the estimation of the subcutaneous glucose level obtained from the two methods was y = 1.04x + 0.56 mM (n = 171 values, r = 0.98, p = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Amperometric, screen-printed, glucose biosensor for analysis of human plasma samples using a biocomposite water-based carbon ink incorporating glucose oxidase

This paper describes the optimisation of a screen-printing water-based carbon ink containing cobalt phthalocyanine (CoPC) and glucose oxidase (GOD) for the fabrication of a glucose biosensor. To optimise the performance of the biosensor, the loadings of the electrocatalyst (CoPC) and enzyme (GOD) were varied. It was found that the maximum linear range was achieved with a CoPC loading of 20% (m/m, relative to the mass of carbon) and a GOD loading of 628 U per gram of carbon. In our studies we chose to employ chronoamperometry, as this technique is commonly used for commercial devices. The optimum operating applied potential was found to be +0.5 V, following an incubation period of 60 s. The optimum supporting electrolyte was found to be 0.05 M phosphate buffer at pH 8.0, which resulted in a linear range of 0.2-5 mM, the former represents the detection limit. The sensitivity was 1.12 microA mM(-1). The effect of temperature was also investigated, and it was found that 40 degrees C gave optimal performance. The resulting amperometric biosensors were evaluated by measuring the glucose concentrations for 10 different human plasma samples containing endogenous glucose and also added glucose. The same samples were analysed by a standard spectrophotometric method, and the results obtained by the two different methods were compared. A good correlation coefficient (R(2) = 0.95) and slope (0.98) were calculated from the experimental data, indicating that the new devices hold promise for biomedical studies.