Characteristic structure and environment in FAD cofactor of (6-4) photolyase along function revealed by resonance Raman spectroscopy

A pyrimidine-pyrimidone (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD) are major DNA lesions induced by ultraviolet irradiation, and (6-4) photolyase, an enzyme with flavin adenine dinucleotide (FAD) as a cofactor, repairs the former specifically by light illumination. We investigated resonance Raman spectra of (6-4) photolyase from Arabidopsis thaliana having neutral semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm(-1) in H(2)O, whose frequency is the lowest among various flavoenzymes, apparently splits into two comparable bands at 1594 and 1608 cm(-1) in D(2)O, and similarly, that at 1522 cm(-1) in H(2)O does into three bands at 1456, 1508, and 1536 cm(-1) in D(2)O. This D(2)O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were observed at significantly low frequencies (1621, 1576 cm(-1)) and with band splittings (1508/1493, 1346/1320 cm(-1)). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.     

Manganese Oxide Nanoparticles/Reduced Graphene Oxide as Novel Electrochemical Platform for Immobilization of FAD and its Application as Highly Sensitive Persulfate Sensor

In this study, manganese oxide nanoparticles/reduced graphene oxide(MnOxNPs/rGO) was used as support for strong immobilization of flavin adenine dinucleotide(FAD). A thin film of rGO cast on the electrode surface, followed by performing electrodeposition of MnOxNPs at applied constant potential of +1.4 V vs. Ag/AgCl for 200 s. Finally, FAD was electrodeposited onto the rGO/MnOxNPs film by potential cycling between 1.0 to ?1.0 V in solution containing 1 mg ml^?1 FAD. Electrochemical properties and catalytic activity of GCE/rGO‐MnOxNPs/FAD toward persulfate (S₂O₈^2?) reduction was investigated. Under optimized condition, the concentration calibration range, detection limit, and sensitivity were 0.1 μM–2 mM, 90 nM and 125.8 nA/μM, respectively, using hydrodynamic amperometry technique.     

Characterisation of poly(neutral red) modified carbon film electrodes; application as a redox mediator for biosensors

The polymer redox mediator, poly(neutral red) (PNR), has been synthesised and characterised electrochemically to investigate the best electropolymerisation and mediation conditions for application in enzyme biosensors and to clarify the mechanism of action. Neutral red was electropolymerised by potential cycling on carbon film electrode substrates by allowing the monomer to be oxidised during the full 20 cycles of polymerisation or reducing the positive limit of the potential window after the first 2 cycles to impede monomer oxidation with a view to obtaining longer polymer chains and a lesser degree of branching. Comparison was made with glassy carbon substrates. The PNR films on carbon film electrodes were characterised using cyclic voltammetry and electrochemical impedance spectroscopy, as well as in glucose biosensors prepared with PNR. Glucose oxidase enzyme was immobilised by encapsulation in silica sol-gel and compared with that obtained by cross-linking with glutaraldehyde. The biosensors were evaluated by chronoamperometry in 0.1 M phosphate buffer saline solution, pH 7.0, and showed evidence of electron transfer between the enzyme cofactor flavin adenine dinucleotide and PNR dissolved in the enzyme layer competing with PNR-mediated electrochemical degradation of H₂O₂ formed during the enzymatic process. This paper is dedicated to Professor Dr. Algirdas Vaskelis on the occasion of his 70th birthday.     

Preparation and Characterization of GCE Coatings Combining DDDMAB and PDADMAC with FAD and HCM for Electrocatalytic Detection of Oxygen and Sulfur Oxoanions

Electrochemically active hybrid coatings based on cationic films, didodecyldimethylammonium bromide (DDDMAB), and poly(diallyldimethylammonium chloride) (PDADMAC) are prepared on glassy carbon electrode surface by cycling the film‐covered electrode repetitively in a pH 7 solution containing flavin adenine dinucleotide (FAD), and anionic hexacyanometalate (HCM) complexes, Fe(CN)₆^3? and Ru(CN)₆^4?. Cyclic voltammetric features of hybrid coatings resemble that of electron transfer process of surface‐confined redox species. Electrochemical quartz crystal microbalance (EQCM) was used to monitor the deposition of FAD on DDDMAB film. Cyclic voltammetric peak potentials of modified electrode were found to be shifted to more negative region with increasing pH of contacting solution with a slope value of 63.3mV per pH unit. The electrocatalytic behavior of FAD‐modified DDDMAB‐coated GCE and hybrid film electrodes was tested towards reduction of oxygen, S₂O₈^2?, SO₅^2? and oxidation of SO₃^2?. The application of FAD‐modified DDDMAB‐coated GCE for S₂O₈^2? estimation was demonstrated in amperometric mode. The sensitivity and detection limit (S/N=3) were 267.6 μA mM^?1 and 2×10^?6 M, respectively.     

新型聚中性红膜修饰碳糊电极的制备、表征及应用

运用一种新型的化学引发-电聚合方式将中性红膜固定到碳糊电极表面,制备出聚中性红薄膜修饰碳糊电极(PNR/CPE)。利用循环伏安法(CV)和交流阻抗谱(EIS)对修饰电极的电化学性能进行研究,借助于扫描电子显微镜(SEM)对修饰电极表面进行表征,并采用红外吸收光谱法(IR)和紫外可见吸收光谱法(UV-Vis)对PNR薄膜结构进行测试。结果表明,中性红成功地固定在碳糊电极表面,修饰电极的表面呈现特定的立体化结构,表面的电活性位点增多,电催化性能增大。在优化条件下,将该电极应用于鲱鱼精DNA(hs DNA)的检测,PNR电极上出现了1对较强的氧化还原峰,峰电流与其浓度在1.0×10~(-6)~8.0×10~(-5)mol/L范围内呈良好的线性关系,检出限为1.0×10~(-7)mol/L。     

Effects of electrolyte and pH on the behavior of cross-linked films of ferrocene-modified poly(ethylenimine)

Ferrocene redox polymers based on the coupling of ferrocenecarboxaldehyde to both linear and branched poly(ethylenimine) (PEI) have been prepared to investigate the effects of pH, electrolyte, and cross-linking on electron charge transport and film swelling. The redox behavior of both ferrocene-modified linear PEI and ferrocene-modified branched PEI was investigated by cyclic voltammetry, while electron diffusion coefficients reported for PEI-based redox polymers were determined by electrochemical impedance spectroscopy. In phosphate solutions at pH>7, cross-linked films of both redox polymers exhibited multiple redox wave behavior and were unstable. In contrast, in non-phosphate solutions, cross-linked films exhibited stable electrochemical behavior and fast electron transfer in solutions with pH<11. Gel swelling experiments suggested that the multiple wave behavior and instability exhibited in either phosphate solutions or at high pH in non-phosphate solutions were related to a combination of film collapse and electrolyte binding within the hydrogel. The electron diffusion coefficients for these polymers are on the order of 10-8 (mol cm(-2) s(-1/2)), which are approximately 40 times greater than other ferrocene-modified polymers. Incorporation of the enzyme, glucose oxidase, into these films demonstrated that these redox polymers were able to electrically communicate with the enzyme's flavin adenine dinucleotide (FAD) redox centers. Glucose sensors based on these films exhibited enzyme saturation current densities that ranged from 240 to 480 microA/cm2 in response to glucose, which were dependent upon the supporting electrolyte and pH. The sensitivity of these sensors at 5 mM glucose ranged from 10 to 48 microA.cm(-2).mM(-1).     

多璧碳管/聚中性红修饰电极的制备及对鸟嘌呤和腺嘌呤的应用研究

结合纳米材料的电催化特性和中性红聚合物薄膜的分子识别能力, 以玻碳电极为基体制备了多壁碳管/聚中性红(MWNT/PNR)修饰电极, 并用表面扫描电镜和循环伏安法进行了表征. 实验表明, 该修饰电极对腺嘌呤(A)和鸟嘌呤(G)都表现出了良好的电催化性能. 在最佳条件下, 用示差脉冲伏安法对A和G进行了测定, 其氧化峰电流于A和G的浓度分别在0.01～4 μmol/L和0.01～8 μmol/L范围内呈良好的线性关系, 检测限均为5×10-9 mol/L (S/N=3). 该修饰电极可以用来同时测定DNA中的A和G.     

Adsorption behavior of dinucleotides on bare and ru-modified glassy carbon electrode surfaces

The interactive behavior of flavin adenine dinucleotide (FAD) with a bare glassy carbon electrode (GCE) and a Ru-modified GCE was investigated. The reduction of FAD at a GCE/ruthenium-modified GCE surface is a quasi-reversible, surface-controlled process, and our data implied that the attachment of FAD onto the surface is caused by nonspecific adsorption instead of covalent linkage, in which the adenine ring of FAD adopts a flat orientation on the GCE surface in neutral and dilute solutions in order to maximize the pi-pi stacking with the carbon surface and reorients to a perpendicular orientation as the surface gets more crowded. FAD desorption during the exchange with nicotinamide adenine dinucleotide (NAD+) is one order of magnitude slower than desorption in the absence of NAD+, which indicates a strong interaction between FAD and NAD+. General knowledge of the interactive behavior of NAD+ on a FAD-adsorbed GCE provides useful information for the design of a modified electrode surface for the generation of NADH from NAD+.     

聚中性红/纳米二氧化硅复合修饰电极直接测定抗坏血酸

用滴涂法和电化学聚合法制备了聚中性红/纳米二氧化硅修饰电极(PNR/nano-SiO2/GCE),并用循环伏安法和交流阻抗法研究了修饰电极表面的电化学行为。实验表明,该修饰电极对抗坏血酸(AA)表现出良好的电催化氧化性能,探讨了复合修饰电极协同增效作用的机理。用线性扫描伏安法研究了AA浓度与峰电流之间线性关系,在pH2.0的磷酸盐缓冲溶液中,AA氧化峰电流在1.8×10-6～5.0×10-3mol/L浓度范围内呈良好的线性关系,检出限为5.4×10-7mol/L(S/N=3)。该修饰电极制备简单,可用于药品及果蔬食品中抗坏血酸的直接测定。     

Development of Amperometric Glucose Biosensor Based on Reconstitution of Glucose Oxidase on Polymeric 3‐Aminophenyl Boronic Acid Monolayer

An amperometric biosensor for determining glucose based on deflavination of the enzyme glucose oxidase and subsequent reconstitution of the apo‐protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The GOx‐reconstituted electrode exhibited excellent electrocatalytic activities towards the reduction and oxidation of hydrogen peroxide as well. The prepared biosensor showed an excellent performance for glucose at +0.5 V with a high sensitivity (5.94 μA/mM) and relatively good response time (～12 s) in a wide concentration range of 1–17 mM (correlation coefficient of 0.9998). The applicability to blood analysis was also evaluated.     

Amperometric Biosensors for Glucose Based on Layer‐by‐Layer Assembled Functionalized Carbon Nanotube and Poly (Neutral Red) Multilayer Film

Abstract

Multiwalled carbon nanotubes (MWNTs) were treated with a mixture of concentrated sulfuric and nitric acid to introduce carboxylic acid groups to the nanotubes. Conducting polymer film was prepared by electrochemical polymerization of neutral red (NR). By using a layer‐by‐layer method, homogeneous and stable MWNTs and poly (neutral red) (PNR) multilayer films were alternately assembled on glassy carbon (GC) electrodes. With the introduction of PNR, the MWNTs/PNR multilayer film system showed synergy between the MWNTs and PNR, with a significant improvement of redox activity due to the excellent electron‐transfer ability of carbon nanotubes (CNTs) and PNR. The electropolymerization is advantageous, providing both prolonged long‐term stability and improved catalytic activity of the resulting modified electrodes. The MWNTs/PNR multilayer film modified glassy carbon electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. As compared to MWNTs and PNR‐modified GC electrodes, the magnitude of the amperometric response of the MWNTs/PNR composite‐modified GC electrode is more than three‐fold greater than that of the MWNTs modified GC electrode, and nine‐fold greater than that of the PNR‐modified GC electrode. With the immobilization of glucose oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to glucose has been constructed. In pH 6.98 phosphate buffer, nearly interference‐free determination of glucose has been realized at ?0.2 V vs. SCE with a linear range from 50 µM to 10 mM and response time <10s. The detection limit was 10 µM glucose (S/N=3).     

Detection of flavins by liquid chromatography using an electrochemical detector with two electrodes in series

A liquid chromatographic method for the selective detection of riboflavin (vitamin B₂) and flavin adenine dinucleotide (FAD) utilizing a thin-layer amperometric detector with two electrodes in series is described. The upstream electrode was held at ?0.4 V (vs. SCE) with the downstream electrode at +0.1 V (vs. SCE). The linear ranges are 40 ng?10 μg for riboflavin and 200 ng?8 μg for FAD and the limits of detection are 8 and 40 ng, respectively. The interference of thirteen different vitamins on the spectrophotometric or electrochemical detection of FAD and vitamin B₂ was studied and no interference was found using the two-electrode detector. The effects of light on riboflavin and FAD is discussed. The method is convenient, rapid and economic, and has high selectivity.     

New insights into the ultrafast photophysics of oxidized and reduced FAD in solution

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (～5.4 ps), the open (extended) conformation of the monomer (～2.8 ns), and the dimer (～27 ps). In the case of the stacked conformation of the monomer, we show that intramolecular electron transfer from the adenine to the isoalloxazine ring occurs with a time constant of 5.4 ps and is followed by charge recombination on a faster time scale, namely, 390 fs. We additionally demonstrate that deprotonated reduced flavin (FADH(-)) undergoes biphotonic ionization under high excitation fluence and dissociates into a hydrated electron and the neutral semiquinone radical FADH(?).     

Electrocatalysis and Amperometric Detection of the Reduced Form of Nicotinamide Adenine Dinucleotide at Toluidine Blue/Zinc Oxide Coated Electrodes

Thin toluidine blue (TBO) and zinc oxide (ZnO) hybrid films have been grown on glassy carbon electrode (GCE) and indium tin oxide coated (SnO₂) glass electrodes by using cyclic voltammetry (CV). Scanning electron microscopy (SEM) images revealed spherical and beads‐like shape of highly oriented TBO/ZnO hybrid films. Energy dispersive spectrometry (EDS) results declared that the films composed mainly of Zn and O. Moreover, TBO/ZnO hybrid films modified electrode is electrochemically active, dye molecules were not easily leached out from the ZnO matrix and the hybrid films can be considered for potential applications as sensor for amperometric determination of reduced nicotinamide adenine dinucleotide (NADH) at 0.0 V. A linear correlation between electrocatalytic current and NADH concentration was found to be in the range between 25 μM and 100 μM in phosphate buffer. In addition, we observed that dopamine, ascorbic acid and uric acid are not interference in amperometric detection of NADH in this proposed method. In addition, TBO/ZnO hybrid film modified electrode was highly stable and its response to the NADH also remained relentless.     

Oxygen uptake after electron transfer from amines, amino acids and ascorbic acid to triplet flavins in air-saturated aqueous solution

The photolysis of lumichrome, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) was studied in air-saturated aqueous solution at room temperature in the presence of appropriate electron donors: ascorbic acid, aromatic amino acids or amines, e.g. ethylenediaminetetraacetate (EDTA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide radical into hydrogen peroxide. The quantum yield of oxygen uptake increases with the donor concentration, e.g. up to 0.3 for riboflavin, FMN or FAD in the presence of EDTA or ascorbic acid (0.3-10mM). The formation of H(2)O(2) is initiated by quenching of the acceptor triplet state by the electron donor and subsequent reaction of the semiquinone radical with oxygen. Specific properties of flavins are discussed including the radicals involved and the pH and concentration dependences. The quantum yield of photodegradation is low under air, but substantial under argon, where the major product absorbing in the visible spectral range is the corresponding hydroquinone.     

Electrochemical biosensors based on redox carbon nanotubes prepared by noncovalent functionalization with 1,10-phenanthroline-5,6-dione

To improve the electrocatalytic activities of carbon nanotubes (CNT) towards the oxidation of nicotinamide adenine dinucleotide (NADH), we derive them with a redox mediator, 1,10-phenanthroline-5,6-dione (PD), by the noncovalent functionalization method. The redox carbon nanotubes (PD/CNT/GC) show excellent electrocatalytic activities towards the oxidation of NADH (catalytic reaction rate constant, k(h) = 7.26 × 10(3) M(-1) s(-1)), so the determination of NADH can be achieved with a high sensitivity of 8.77 μA mM(-1) under the potential of 0.0 V with minimal interference. We also develop an amperometric ethanol biosensor by integration of alcohol dehydrogenase (ADH) within the redox carbon nanotubes (PD/CNT/GC). The ethanol biosensor exhibits a wide linear range up to 7 mM with a lower detection limit of 0.30 mM as well as a high sensitivity of 10.85 nA mM(-1).     

Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana

The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FAD(ox), ca. 38.5%), in the semiquinone form (FADH., ca. 5%), and in the fully reduced neutral form (FAD(red)H2) or fully reduced anionic form (FAD(red)H-, ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. F?rster-type energy transfer from MTHF to FAD(ox) is observed. Photo-excitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FAD(ox) to FAD(red)H2 (or FAD(red)H-, FAD(ox) photo-cycle of moderate efficiency), reversible reduction of FADH. to FAD(red)H2 (or FAD(red)H-, FADH. photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H2 (or FAD(red)H-) to permanent FAD(red)H2 (or FAD(red)H-) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF', MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF').     

Involvement of electron transfer in the photoreaction of zebrafish Cryptochrome-DASH

Photoreaction of a blue-light photoreceptor Cryptochrome-DASH (Cry-DASH), a new member of the Cryptochrome family, from zebrafish was studied by UV-visible absorption spectroscopy in aqueous solutions at 293 K. Zebrafish Cry-DASH binds two chromophores, a flavin adenine dinucleotide (FAD) and a N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF) noncovalently. The bound FAD exists in the oxidized form (FAD(ox)) in the dark. Blue light converts FAD(ox) to the neutral radical form (FADH*). Formed FADH* is transformed to the fully reduced form FADH(2) (or FADH(-)) by successive light irradiation, or reverts to FAD(ox). FADH(2) (or FADH(-)) reverts to FADH* or possibly to FAD(ox) directly. The effect of dithiothreitol suggests a possible electron transfer between FAD in zebrafish Cry-DASH and reductants in the external medium. This is the first report on the photoreaction pathway and kinetics of a vertebrate Cry-DASH family protein.     

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β-d-glucose. The detection limit was 0.25 μM β-d-glucose for 50-μl samples. The electrode was kept at ?50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.     

Electrocatalytic oxidation of NADH based on polyluminol and functionalized multi-walled carbon nanotubes

This work presents that the electrocatalytic oxidation of NADH can be enhanced by the hybrid composites of polyluminol and functionalized multi-walled carbon nanotubes (MWCNTs). The hybrid composites can be easily prepared by the electropolymerization of luminol and the adsorption of functionalized MWCNTs. The modified electrode exhibits two redox couples which can show two electrocatalytic peaks at about 0.1 and 0.3 V (vs. Ag/AgCl) to NADH oxidation. The kinetic constant, k(kin), for the electrocatalytic oxidation of NADH, evaluated by chronoamperometry and voltammetry using a rotating disk electrode (RDE), provided values close to 10(5) M(-1) s(-1). At an applied potential of 0.1 V, the sensor provides a linear response range for NADH from 5 × 10(-6) up to 1.5 × 10(-4) M with a sensitivity of 183.9 μA mM(-1) cm(-2), and detection and quantification limits of 0.6 and 5 μM (S/N = 3), respectively.