Photoelectrochemical oxidation of DNA by ruthenium tris(bipyridine) on a tin oxide nanoparticle electrode

Selective photoelectrochemical oxidation of DNA was achieved by ruthenium tris(bipyridine) immobilized on a tin oxide nanoparticle electrode. The metal complex was covalently attached to a protein, avidin, which adsorbed strongly on the tin oxide electrode by electrostatic interaction. Upon irradiation with 473-nm light, anodic photocurrent was generated in a blank electrolyte and was enhanced significantly after addition of poly(guanadylic acid) (poly-G) into the electrolyte. The current increased progressively with the nucleotide concentration, suggesting the enhancement effect was related to poly-G. The action spectrum indicates that the photocurrent was initiated by light absorption of the ruthenium compound immobilized on the electrode. Among the various polynucleotides examined, poly-G produced the largest photocurrent increase, followed by poly-A, single-stranded DNA, chemically damaged DNA, and double-stranded DNA, whereas poly-C and poly-U showed little effect. The combined experimental data support the hypothesis that the photoexcited Ru2+ species injects an electron into the semiconductor and produces Ru3+, which is then reduced back to Ru2+ by guanine and adenine bases in DNA, resulting in the recycling of the metal complex and enhanced photocurrent. The photoelectrochemical reaction can be employed as a new method for the detection of DNA damage.     

Biochemical detection for direct bead surface analysis

A continuous-flow biochemical detection system is presented which recognizes biologically active compounds immobilized to solid phases. This approach can be used to screen, for example, solid-phase combinatorial libraries for lead compounds. Biochemical detection is performed by mixing a plug of a solid-phase suspension with labeled affinity protein. During a short reaction time, the labeled affinity protein will only bind to ligands, i.e., compounds with biological activity. Hereafter, the free and bound labels are separated by means of a hollow fiber module. Quantitation of the free label is performed with a conventional flow-through fluorescence detector. Total assay time amounts to less than 3 min. Biochemical detection for direct bead surface analysis was developed for two model systems. The first model system used fluorescence-labeled avidin as affinity protein and its ligands biotin and iminobiotin immobilized to agarose as analytes. The second model system used fluorescence-labeled antisheep (Fab)(2) fragments as affinity protein and different IgGs immobilized to agarose as analytes. The feasibility of this approach for recognition of solid-phase immobilized ligands was documented by screening 50 samples with a 100% hit rate.     

Quantification of 8-oxodGuo lesions in double-stranded DNA using a photoelectrochemical DNA sensor

Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO(2) nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ～1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine-DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe(2+)/200 μM H(2)O(2). Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.     

A new method of immobilization of proteins on activated ester terminated alkanethiol monolayers towards the label free impedancemetric detection

This paper investigates a new immobilization procedure for biological molecules that is based on the formation of reactive ω-functionalized-self-assembled thiol monolayers onto a gold electrode. The homogeneous self-assembled monolayer was characterized by X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The SAM modified gold electrode showed a clear peak corresponding to S_2p that characterized the Au-thiolate bond, while cyclic voltammetry and electrochemical impedance spectroscopy measurements, in 10 mM phosphate buffer pH 7, in the presence of Fe(CN)₆^{− 3/− 4} as redox probe, showed that these monolayers were densely packed and prevented electron transfer towards the gold surface. These homogeneous SAMs were used to immobilize biotin hydrazide by covalent attachment, after the nucleophilic attack of the amino group of biotin hydrazide on the ω-activated ester function of thiols. The biotin–avidin interaction was then examined as a model for an affinity biosensor with electrochemical impedance spectroscopy. A Randles equivalent circuit was used for the interpretation of impedance data and the change in the interfacial properties at the modified-electrode/electrolyte interface were monitored through charge-transfer resistance variation. The proposed affinity biosensor showed a detection range that was linear between 200 and 800 ng/ml for avidin. In order to improve the sensitivity the technique of mixed self-assembled monolayers was adopted. Mixed SAMs were elaborated by co-adsorption of two differently substituted thiols, one was substituted by a reactive group that was used to react with the amino group of biotin hydrazide, whereas the other was substituted by an hydroxyl group that was chosen to mimic protein resistance. In this study, we started with a 1:3 activated ester:hydroxyl-terminated alkanethiol ratio. The results obtained with the mixed SAMs appeared to be better than those obtained with the homogeneous SAMs, and the corresponding affinity biosensor presented two detection ranges that were linear between 20 and 100 ng/ml and between 100 and 1200 ng/ml, respectively, with two different slopes.     

ZnS nanoparticles electrodeposited onto ITO electrode as a platform for fabrication of enzyme-based biosensors of glucose

The electrochemical and photoelectrochemical biosensors based on glucose oxidase (GOD) and ZnS nanoparticles modified indium tin oxide (ITO) electrode were investigated. The ZnS nanoparticles were electrodeposited directly on the surface of ITO electrode. The enzyme was immobilized on ZnS/ITO electrode surface by sol–gel method to fabricate glucose biosensor. GOD could electrocatalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. The reduction peak current decreased linearly with the addition of glucose, which could be used for glucose detection. Moreover, ZnS nanoparticles deposited on ITO electrode surface showed good photocurrent response under illumination. A photoelectrochemical biosensor for the detection of glucose was also developed by monitoring the decreases in the cathodic peak photocurrent. The results indicated that ZnS nanoparticles deposited on ITO substrate were a good candidate material for the immobilization of enzyme in glucose biosensor construction.     

Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label

Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-biotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 x 10(-8) M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.     

Selective photoelectrochemical detection of DNA with high-affinity metallointercalator and tin oxide nanoparticle electrode

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.     

Pyruvate carboxylase as a model for oligosubstituted enzyme-ligand conjugates in homogeneous enzyme immunoassays

Theoretical models suggest that the detection capabilities of homogeneous enzyme immunoassays can be improved by the use of oligosubstituted enzyme-ligand conjugates rather than the traditionally used multisubstituted ones. The natural form of pyruvate carboxylase contains four covalently bound biotins (one per subunit) and it can be considered as an oligosubstituted enzyme-biotin conjugate. The enzyme is nearly completely inhibited in the presence of the natural binder for biotin, avidin. When the enzyme is incubated with avidin and free biotin, a competition occurs between the free biotin and the prosthetic group of the enzyme for the avidin. Steep dose-response curves are obtained by relating the observed inhibition to the free biotin concentration. By variation of the amount of avidin or enzyme in the assay, the detection limits of the system can be altered allowing for sensitive determinations over a wide range of biotin concentrations. Such data from real sample analysis of several vitamin supplements are reported.     

High-performance liquid chromatographic postcolumn reaction detection based on a competitive binding system

Postcolumn reactions are typically employed to improve detection in high-performance liquid chromatography (HPLC) separation techniques. This study proposes the use of competitive binding principles in designing novel postcolumn reaction schemes. The feasibility of this approach was tested by using the HPLC determination of biotin and biocytin as a model system. The effluent from the HPLC column was merged with a reagent stream containing avidin, whose binding sites were occupied by the dye HABA (2-[4'-hydroxyphenylazo]benzoic acid). HABA was displaced by the analytes from the avidin-HABA complex and the free dye was determined with a UV-vis detector at 345 nm. The procedure was optimized with respect to reactor design, reagent concentrations, and the flow rate of reagent solution. Analytical characteristics of the developed procedure were determined and compared with the direct detection of biotin and biocytin at 220 nm. The postcolumn reaction scheme improved the selectivity and sensitivity of the detection of biotin and biocytin while maintaining similar detection limits.     

透明纳米TiO2薄膜的制备及其紫外光电特性研究

采用溶胶-凝胶法于ITO导电玻璃和石英玻璃基底上制备了均匀透明的TiO2多孔纳米薄膜.XRD和AFM测试结果表明,构成薄膜的TiO2粒子为锐钛矿相,粒径约为50nm.紫外-可见(UV-vis)吸收光谱显示,制得的TiO2薄膜对紫外光表现出很强的吸收特性.借助标准三电极体系进行的光电化学测试结果表明,在紫外光照射下,制得的薄膜电极可产生稳定的阳极光生电流,且电流大小显著依赖于照射光的波长(λ),λ=320nm的光产生的光电流最大.另外,薄膜电极中的光电流大小与电极电位(U)有关.高于0.6V的电位可有效抑制光生电子向电解液的注入,并增大光生电子向导电基底的扩散速度,从而提高阳极光电流的大小.实验结果表明,纳米TiO2薄膜在紫外光传感器的制备方面具有较好的应用潜力.     

Development of binding assays in microfabricated picoliter vials: an assay for biotin

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.     

Poly(3-hexylthiophene)/TiO2 nanoparticle-functionalized electrodes for visible light and low potential photoelectrochemical sensing of organophosphorus pesticide chlopyrifos

A dramatic visible light photoelectrochemical sensing platform for the detection of pesticide molecules at zero potential (versus saturated calomel electrode) was first constructed using poly(3-hexylthiophene)-functionalized TiO(2) nanoparticles. Poly(3-hexylthiophene) (P3HT) was synthesized via chemical oxidative polymerization with anhydrous FeCl(3) as the oxidant, 3-hexylthiophene as the monomer, and chloroform as the solvent, and the functional TiO(2) nanoparticles were facilely prepared by blending TiO(2) nanoparticles and P3HT in chloroform solution. The resulting photoelectrocatalysts were characterized by scanning electron microscopy, Raman spectroscopy, and X-ray diffractometry. Under visible light irradiation, P3HT generated the transition from the valence band to the conduction band, delivering the excited electrons into the conduction band of TiO(2) and then to the glassy carbon electrode. Simultaneously, a positive charged hole (h(+)) of TiO(2) may form and migrate to the valence band of P3HT, which can react with H(2)O to generate (?)OH, and then it converted chlopyrifos into chlopyrifos(?) that promoted the amplifying photocurrent response. On the basis of the proposed photoelectrochemical mechanism, a methodology for sensitive photoelectrochemical sensing for chlopyrifos at zero potential was thus developed. Under optimal conditions, the proposed photoelectrochemical method could detect chlopyrifos ranging from 0.2 to 16 μmol L(-1) with a detection limit of 0.01 μmol L(-1) at a signal-to-noise ratio of 3. The photoelectrochemical sensor had an excellent specificity against the other pesticides and could be successfully applied to the detection of reduced chlopyrifos in green vegetables, showing a promising application in photoelectrochemical sensing.     

4-(Dimethylamino)butyric acid labeling for electrochemiluminescence detection of biological substances by increasing sensitivity with gold nanoparticle amplification

4-(Dimethylamino)butyric acid (DMBA) labeling combined with gold nanoparticle amplification for electrochemiluminescence (ECL) determination of a biological substance (bovine serum albumin (BSA) and immunoglobulin G (IgG) as models) was presented. After DMBA, an analogue of tripropylamine, was tagged on the (anti)analytes, an ECL signal related to the content of the analytes was generated when the analyte tagged with DMBA was in contact with tris(2,2'-bipyridine)ruthenium (Ru(bpy)(3)2+) solution and a potential was applied. To improve the adsorption capacity, a gold nanoparticle layer was first combined into the surface of the 2-mm-diameter gold electrode. For the determination of BSA, avidin was covalently conjugated to a self-assembled monolayer of 3-mercaptopropanoic acid on the gold nanoparticle layer. Biotinylated BSA-DMBA was then immobilized on the gold nanoparticle layer of the gold electrode via the avidin-biotin reaction. IgG was tested via a typical sandwich-type immobilization method. ECL signals were generated from the electrodes immobilized with BSA or IgG by immersing them in a 1 mmol L-1 Ru(bpy)(3)2+ solution and scanning from 0.5 to 1.3 V versus Ag/AgCl. With gold nanoparticle amplification, the ECL peak intensity was proportional to the concentration over the range 1-80 and 5-100 microg/mL for BSA and IgG consuming 50 microL of sample, respectively. A 10- and 6-fold sensitivity enhancement was obtained for BSA and IgG over their direct immobilization on an electrode using DMBA labeling. The relative standard deviations of five replicate determinations of 10 microg/mL BSA and 20 microg/mL IgG were 8.4 and 10.2%, respectively. High biocompatibility and low cost were the main advantages of the present DMBA labeling technique over the traditional Ru(bpy)(3)2+ labeling.     

Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize

The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.     

A biotinylated conducting polypyrrole for the spatially controlled construction of an amperometric biosensor.

A new biotin derivative functionalized by an electropolymerizable pyrrole group has been synthesized. The electrooxidation of this biotin pyrrole has allowed the formation of biotinylated conducting polypyrrole films in organic electrolyte. Gravimetric measurements based on a quartz crystal microbalance, modified by the biotinylated polymer, revealed an avidin-biotin-specific binding at the interface of polymer-solution. The estimated mass increase corresponded to the anchoring of 1.5 avidin monolayers on the polypyrrole surface. In addition, the subsequent grafting of biotinylated glucose oxidase was corroborated by electrochemical permeation studies. Enzyme multilayers composed of glucose oxidase or polyphenol oxidase were elaborated on the electrode surface modified by the biotinylated polypyrrole film. The amperometric response of the resulting biosensors to glucose or catechol has been studied at +0.6 or -0.2 V vs SCE, respectively.     

Highly sensitive photoelectrochemical immunoassay with enhanced amplification using horseradish peroxidase induced biocatalytic precipitation on a CdS quantum dots multilayer electrode

Herein we demonstrate the protocol of a biocatalytic precipitation (BCP)-based sandwich photoelectrochemical (PEC) horseradish peroxidase (HRP)-linked immunoassay on the basis of their synergy effect for the ultrasensitive detection of mouse IgG (antigen, Ag) as a model protein. The hybrid film consisting of oppositely charged polyelectrolytes and CdS quantum dots (QDs) is developed by the classic layer by layer (LbL) method and then employed as the photoactive antibody (Ab) immobilization matrix for the subsequent sandwich-type Ab-Ag affinity interactions. Improved sensitivity is achieved through using the bioconjugates of HRP-secondary antibodies (Ab(2)). In addition to the much enhanced steric hindrance compared with the original one, the presence of HRP would further stimulate the BCP onto the electrode surface for signal amplification, concomitant to a competitive nonproductive absorption that lowers the photocurrent intensity. As a result of the multisignal amplification in this HRP catalyzed BCP-based PEC immunoassay, it possesses excellent analytical performance. The antigen could be detected from 0.5 pg/mL to 5.0 ng/mL with a detection limit of 0.5 pg/mL.     

多孔硅表面组装胆固醇分子印迹膜的光电化学

光电化学刻蚀出多孔硅（PS）用含有SnCl2、SbCl3和Ce（NO3）3的乙醇溶液浸泡后,经过400℃10min热处理,表面形成Ce—Sb共掺的SnO2光透导电薄层（TOCL）。在TOCL的表面组装含有胆固醇分子的十二烷基硫醇薄膜,萃取其中的胆固醇分子后形成筛孔分子印迹电极（SHE）。该电极与不同浓度的胆固醇乙醇溶液作用后,光电化学方法袁征了分子印迹电极对胆固醇浓度响应特性。实验结果表明分子印迹电极与吸附胆固醇的浓度具有较好的关联。特别指出的是,该筛孔分子印迹电极有望在不需要掺比电极以及零偏电压条件下实现对胆固醇的检测。     

Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry

It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation.     

Liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplification of antigen--antibody or oligonucleotide--DNA sensing processes by the precipitation of an insoluble product on electrodes

Liposomes labeled with biotin and the enzyme horseradish peroxidase (HRP) are used as a probe to amplify the sensing of antigen-antibody interactions or oligonucleotide-DNA binding. The HRP-biocatalyzed oxidation of 4-chloro-1-naphthol (1) in the presence of H2O2, and the precipitation of the insoluble product 2 on electrode supports, are used as an amplification route for the sensing processes. The anti-dinitrophenyl antibody (DNP-Ab) is sensed by a dinitrophenyl-L-cysteine antigen monolayer associated with an Au electrode. A biotinylated anti-IgG-antibody (Fc-specific) is linked to the antigen-DNP-Ab complex, and the biotin-labeled HRP-liposomes associate with the assembly through an avidin bridge. The biocatalyzed precipitation of 2 on the electrode increases the electron-transfer resistances at the electrode-solution interface or the electrode resistance itself. The binding events of the different proteins on the electrode and the biocatalyzed precipitation of 2 on the conductive support are followed by Faradaic impedance spectroscopy or constant-current chronopotentiometry. DNP-Ab concentrations as low as 1 x 10(-11) g x mL(-1) can be detected by this method. The labeled liposomes were also used for the amplified detection of DNA 3. The oligonucleotide 4, complementary to a part of the target DNA 3 that is a model nucleic acid sequence for the Tay-Sachs genetic disorder, is assembled on an Au electrode. Hybridization of the analyte 3 followed by the association of the biotin-tagged oligonucleotide 5 yields a three-component double-stranded assembly. Sensing of the analyte 3 is amplified by the association of avidin, the labeled liposomes, and the subsequent biocatalyzed precipitation of 2 on the electrodes. The DNA 3 is detected with a sensitivity that corresponds to 6.5 x 10(-13) M. Faradaic impedance spectroscopy and chronopotentiometry were employed to follow the stepwise assembly of the systems and the electronic transduction of the detection of the analyte DNA 3.     

Micrometer dimension derivatization of biosensor surfaces using confocal dynamic patterning.

Using laser scanning confocal optics in conjunction with avidin/biotin technology, micrometer-sized patterns of biomolecules were fabricated on glassy-carbon and fused-silica surfaces. Photoactive biotin was immobilized using the 325-nm line of a Helium-Cadmium laser, which was focused through a 25x or 100x quartz microscope objective. A three-dimensional piezoelectric micromanipulator was used to position the sample surface in the focal plane of the microscope objective and to create patterns on the focused surface. Biotin patterns with line widths of 5-20 microns were produced by varying the scan speed of the micromanipulator while exposing the surface to the laser. The integrity of the immobilized biotin was confirmed by subsequent derivatization with fluorescently labeled avidin. Fluorescence microscopy with a cooled charge coupled device (CCD) imaging system was used to visualize the distribution of biotin and fluorescent avidin within the patterns created by the laser.