一种基于脱氧核酶的铅离子电致化学发光传感器

本文设计了一种基于脱氧核酶(DNAzyme)检测Pb2+的电致化学发光(Electrocheluminescent,ECL)传感器。将对Pb2+特异性识别的DNAzyme通过金-巯键固定于金电极表面,并与标记有二氧化硅包埋的钌联吡啶(Ru-SNPs)的底物DNA链发生杂交,形成双链DNA(ds-DNA)传感器。Pb2+不存在时,由于Ru-SNPs靠近电极表面,产生强的ECL信号。当Pb2+存在时,DNAzyme催化底物链断裂,Ru-SNPs远离电极表面,导致ECL信号下降。实验结果表明ECL强度与Pb2+浓度在0.2-1.0 nmol/L范围内呈良好的线性关系,检测限可达0.04 nmol/L,其他二价金属离子对其基本无干扰。     

DNAzyme-based colorimetric sensing of lead (Pb(2+)) using unmodified gold nanoparticle probes

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, have been intensively studied due to their potential applications in therapeutics and sensors. Taking advantage of the high specificity of 17E DNAzyme for Pb(2+), highly sensitive and selective fluorescent, electrochemical and colorimetric sensors have been developed for Pb(2+). In this work, we report a simple, sensitive and label-free 17E DNAzyme-based sensor for Pb(2+) detection using unmodified gold nanoparticles (GNPs) based on the fact that unfolded single-stranded DNA could be adsorbed on the citrate protected GNPs while double-stranded DNA could not. By our method the substrate cleavage by the 17E DNAzyme in the presence of Pb(2+) could be monitored by color change of GNPs, thereby Pb(2+) detection was realized. The detection of Pb(2+) could be realized within 20?min, with a detection limit of 500?nM. The selectivity of our sensor has been investigated by challenging the sensing system with other divalent metal ions. Since common steps such as modification and separation could be successfully avoided, the sensor developed here could provide a simple, cost-effective yet rapid and sensitive measurement tool for Pb(2+) detection and may prove useful in the development of sensors for clinical toxicology and environmental monitoring in the future.     

A two-channel microfluidic sensor that uses anodic electrogenerated chemiluminescence as a photonic reporter of cathodic redox reactions

This paper describes a new approach for sensing electrochemically active substrates in microfluidic systems. This two-electrode sensor relies on electrochemical detection at one electrode and electrogenerated chemiluminescent (ECL) reporting at the other. Each microfabricated indium tin oxide electrode is located in a separate microfluidic channel, but the channels are connected downstream of the electrodes to maintain a complete electrical circuit. Because of laminar flow, there is no bulk mixing of the fluids in the detecting and reporting channels. This approach allows the ECL reaction to be physically and chemically decoupled from the sensing channel of the device, which greatly expands the number of analytes that can be detected. However, because the cathode and anode are connected, electron-transfer processes occurring at the sensing electrode are electrically coupled to the ECL reaction. Charge balance permits the ECL light output to be quantitatively correlated to electrochemical reductions at the cathode. The system is used to detect Fe(CN)6(3-), Ru(NH3)6(3+), and benzyl viologen and report their presence via Ru(bpy)3(2+) (bpy = bipyridine) luminescence. Each different redox target initiates ECL at a unique potential bias related to its standard redox potential. The influence of the concentrations of Ru(bpy)3(2+) and the target analytes is discussed.     

Amplified surface plasmon resonance and electrochemical detection of Pb2+ ions using the Pb2+-dependent DNAzyme and hemin/G-quadruplex as a label

The hemin/G-quadruplex nanostructure and the Pb(2+)-dependent DNAzyme are implemented to develop sensitive surface plasmon resonance (SPR) and electrochemical sensing platforms for Pb(2+) ions. A complex consisting of the Pb(2+)-dependent DNAzyme sequence and a ribonuclease-containing nucleic acid sequence (corresponding to the substrate of the DNAzyme) linked to a G-rich domain, which is "caged" in the complex structure, is assembled on Au-coated glass surfaces or Au electrodes. In the presence of Pb(2+) ions, the Pb(2+)-dependent DNAzyme cleaves the substrate, leading to the separation of the complex and to the self-assembly of the hemin/G-quadruplex on the Au support. In one sensing platform, the Pb(2+) ions are analyzed by following the dielectric changes at the surface as a result of the formation of the hemin/G-quadruplex label using SPR. This sensing platform is further amplified by the immobilization of the sensing complex on Au NPs (13 nm) and using the electronic coupling between the NPs and the surface plasmon wave as an amplification mechanism. This method enables the sensing of Pb(2+) ions with a detection limit that corresponds to 5 fM. The second sensing platform implements the resulting hemin/G-quadruplex as an electrocatalytic label that catalyzes the electrochemical reduction of H(2)O(2). This method enables the detection of Pb(2+) with a detection limit of 1 pM. Both sensing platforms reveal selectivity toward the detection of Pb(2+) ions.     

Impedimetric immobilized DNA-based sensor for simultaneous detection of Pb2+, Ag+, and Hg2+

An unlabeled immobilized DNA-based sensor was reported for simultaneous detection of Pb(2+), Ag(+), and Hg(2+) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)(6)](4-/3-) as redox probe, which consisted of three interaction sections: Pb(2+) interaction with G-rich DNA strands to form G-quadruplex, Ag(+) interaction with C-C mismatch to form C-Ag(+)-C complex, and Hg(2+) interaction with T-T mismatch to form T-Hg(2+)-T complex. Circular dichroism (CD) and UV-vis spectra indicated that the interactions between DNA and Pb(2+), Ag(+), or Hg(2+) occurred. Upon DNA interaction with Pb(2+), Ag(+), and Hg(2+), respectively, a decreased charge transfer resistance (R(CT)) was obtained. Taking advantage of the R(CT) difference (ΔR(CT)), Pb(2+), Ag(+), and Hg(2+) were selectively detected with the detection limit of 10 pM, 10 nM, and 0.1 nM, respectively. To simultaneously (or parallel) detect the three metal ions coexisting in a sample, EDTA was applied to mask Pb(2+) and Hg(2+) for detecting Ag(+); cysteine was applied to mask Ag(+) and Hg(2+) for detecting Pb(2+), and the mixture of G-rich and C-rich DNA strands were applied to mask Pb(2+) and Ag(+) for detecting Hg(2+). Finally, the simple and cost-effective sensor could be successfully applied for simultaneously detecting Pb(2+), Ag(+), and Hg(2+) in calf serum and lake water.     

Electrogenerated chemiluminescence sensors using Ru(bpy)3(2+) doped in silica nanoparticles

A novel electrogenerated chemiluminescence (ECL) sensor based on Ru(bpy)3(2+)-doped silica (RuDS) nanoparticles conjugated with a biopolymer chitosan membrane was developed. These uniform RuDS nanoparticles (approximately 40 nm) were prepared by a water-in-oil microemulsion method and were characterized by electrochemical and transmission electron microscopy technology. The Ru(bpy)3(2+)-doped interior maintained its high ECL efficiency, while the exterior nanosilica prevented the luminophor from leaching out into the aqueous solution due to the electrostatic interaction. This is the first attempt to branch out the application of RuDS nanoparticles into the field of ECL, and since a large amount of Ru(bpy)3(2+) was immobilized three-dimensionally on the electrode, the Ru(bpy)3(2+) ECL signal could be enhanced greatly, which finally resulted in the increased sensitivity. This sensor shows a detection limit of 2.8 nM for tripropylamine, which is 3 orders of magnitude lower than that observed at a Nafion-based ECL sensor. Furthermore, the present ECL sensor displays outstanding long-term stability.     

Computer-Readable DNAzyme Assay on Disc for ppb-Level Lead Detection

A method for the convenient detection of lead at the parts-per-billion (ppb)-level has been developed; it uses a conventional compact disc (CD) as the platform for preparing DNAzyme assays and an unmodified optical drive of ordinary desktop/laptop computers as the readout device. In particular, by immobilization of Pb(2+)-specific DNAzyme sensing constructs on the "transparent side" of a conventional CD-R via mild surface reactions, the Pb(2+) concentration can be determined by a free diagnostic program that checks the error distribution on the CD (i.e., it extracts the number of errors in a prerecorded audio file). The reading errors increase monotonically over a wide range of Pb(2+) concentrations (from 10 nM to 1 mM), and the selectivity is confirmed by testing several other divalent cations (Zn(2+), Ba(2+), Mg(2+), Ca(2+), Cu(2+), and Hg(2+)).     

"Outer-sphere to inner-sphere" redox cycling for ultrasensitive immunosensors

This paper reports chemical-chemical (CC) and electrochemical-chemical-chemical (ECC) redox cycling, for use in ultrasensitive biosensor applications. A triple chemical amplification approach using an enzymatic reaction, CC redox cycling, and ECC redox cycling is applied toward electrochemical immunosensors of cardiac troponin I. An enzymatic reaction, in which alkaline phosphatase converts 4-aminophenyl phosphate to 4-aminophenol (AP), triggers CC redox cycling in the presence of an oxidant and a reductant, and electrochemical signals are measured with ECC redox cycling after an incubation period of time in an air-saturated solution. To obtain high, selective, and reproducible redox cycling without using redox enzymes, two redox reactions [the reaction between AP and the oxidant and the reaction between the oxidized form of AP (4-quinone imine, QI) and the reductant] should be fast, but an unwanted reaction between the oxidant and reductant should be very slow. Because species that undergo outer-sphere reactions (OSR-philic species) react slowly with species that undergo inner-sphere reactions (ISR-philic species), highly OSR-philic Ru(NH(3))(6)(3+) and highly ISR-philic tris(2-carboxyethyl)phosphine (TCEP) are chosen as the oxidant and reductant, respectively. The OSR- and ISR-philic QI/AP couple allows fast redox reactions with both the OSR-philic Ru(NH(3))(6)(3+) and the ISR-philic TCEP. Highly OSR-philic indium-tin oxide (ITO) electrodes minimize unwanted electrochemical reactions with highly ISR-philic species. Although the formal potential of the Ru(NH(3))(6)(3+)/Ru(NH(3))(6)(2+) couple is lower than that of the QI/AP couple, the endergonic reaction between Ru(NH(3))(6)(3+) and AP is driven by the highly exergonic reaction between TCEP and QI (via a coupled reaction mechanism). Overall, the "outer-sphere to inner-sphere" redox cycling in the order of highly OSR-philic ITO, highly OSR-philic Ru(NH(3))(6)(3+)/Ru(NH(3))(6)(2+) couple, OSR- and ISR-philic QI/AP couple, and highly ISR-philic TCEP allows high, selective, and reproducible signal amplification. The electrochemical data obtained by chronocoulometry permit a lower detection limits than those obtained by cyclic voltammetry. The detection limit of an immunosensor for troponin I in serum, calculated from the anodic charges in chronocoulometry, is ca. 10 fg/mL.     

Graphene-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb2+ with a high selectivity

On the basis of the remarkable difference in affinity of graphene (GO) with ssDNA containing a different number of bases in length, we for the first time report a GO-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb(2+). A FAM-labeled DNAzyme-substrate hybrid acted as both a molecular recognition module and signal reporter and GO as a superquencher. By taking advantage of the super fluorescence quenching efficiency of GO, our proposed biosensor exhibits a high sensitivity toward the target with a detection limit of 300 pM for Pb(2+), which is lower than previously reported for catalytic beacons. Moreover, with the choice of a classic Pb(2+)-dependent GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the newly designed sensing system also shows an obviously improved selectivity than previously reported methods. Moreover, the sensing system was used for the determination of Pb(2+) in river water samples with satisfying results.     

Chemiluminescence detection using regenerable tris(2,2'-bipyridyl)ruthenium(II) immobilized in Nafion.

The development of a detection method based on the electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+], immobilized in a Nafion film coated on an electrode is discussed. Control of the electrode potential controls creation of the reactive reagent Ru(bpy)3(3+) which reacts with certain analytes to yield chemiluminescence emission of intensity proportional to the analyte concentration. The reaction results in Ru(bpy)3(3+) being converted to Ru(bpy)3(2+), which then is recycled to Ru(bpy)3(3+) again at the electrode. This sensor has been used in flow injection to determine oxalate, alkylamines, and NADH. Detection limits are 1 microM, 10 nM, and 1 microM, respectively, with working ranges extending over 4 decades in concentration. Sensitivity is constant over the wide pH range from 3 to 10. With oxalate, and to a small extent with amines, emission intensities increase with increasing ionic strength; this was shown to be a phenomenon related to the Nafion film and not to the chemiluminescence reaction. Emission intensities increase with temperature. The sensor remains stable for several days with suitable storage conditions. Significant amounts of Ru(bpy)3(3+) are shown to be capable of storage within the film.     

Microfluidic electrochemical aptameric assay integrated on-chip: a potentially convenient sensing platform for the amplified and multiplex analysis of small molecules

Aptamers are artificial oligonucleotides that have been widely employed to design biosensors (i.e., aptasensors). In this work, we report a microfluidic electrochemical aptamer-based sensor (MECAS) by constructing Au-Ag dual-metal array three-electrode on-chip for multiplex detection of small molecules. In combination with the microfluidic channels covering on the glass chip, different targets are transported to the Au electrodes integrated on different positions of the chip. These electrodes are premodified by different kinds of aptamers, respectively, to fabricate different sensing interfaces which can selectively capture the corresponding target. It is an address-dependent sensing platform; thus, with the use of only one electrochemical probe, multitargets can be recognized and detected according to the readout on a corresponding aptamer-modified electrode. In the sensing strategy, the electrochemical probe, [Ru(NH(3))(6)](3+) (RuHex), which can quantitatively bind to surface-confined DNA via electrostatic interaction, was used to produce chronocoulometric signal; Au nanoparticles (AuNPs) were used to improve the sensitivity of the sensor by amplifying the detection signals. Moreover, the sensing interface fabrication, sample incubation, and electrochemical detection were all performed in microfluidic channels. By using this detection chip, we achieved the multianalysis of two model small molecules, ATP, and cocaine, in mixed samples within 40 min. The detection limit of ATP was 3 × 10(-10) M, whereas the detection limit of cocaine was 7 × 10(-8) M. This Au-Ag dual metal electrochemical chip detector integrated MECAS was simple, sensitive, and selective. Also it is similar to a dosimeter which accumulates signal upon exposure. It held promising potential for designing electrochemical devices with high throughput, high automation, and high integration in multianalysis.     

Electrochemical performance of diamond thin-film electrodes from different commercial sources

The electrochemical properties of two commercial (Condias, Sumitomo) boron-doped diamond thin-film electrodes were compared with those of two types of boron-doped diamond thin film deposited in our laboratory (microcrystalline, nanocrystalline). Scanning electron microscopy and Raman spectroscopy were used to characterize the electrode morphology and microstructure, respectively. Cyclic voltammetry was used to study the electrochemical response, with five different redox systems serving as probes (Fe(CN)(6)(3)(-)(/4)(-), Ru(NH(3))(6)(3+/)(2+), IrCl(6)(2)(-)(/3)(-), 4-methylcatechol, Fe(3+/2+)). The response for the different systems was quite reproducibile from electrode type to type and from film to film for electrodes of the same type. For all five redox systems, the forward reaction peak current varied linearly with the scan rate(1/2) (nu), indicative of electrode reaction kinetics controlled by mass transport (semi-infinite linear diffusion) of the reactant. Apparent heterogeneous electron-transfer rate constants, k degrees (app), for all five redox systems were determined from deltaE(p)-nu experimental data, according to the method described by Nicholson (Nicholson, R. S. Anal. Chem. 1965, 37, 1351.). The rate constants were also verified through digital simulation (DigiSim 3.03) of the voltammetric i-E curves at different scan rates. Good fits between the experimental and simulated voltammograms were found for scan rates up to 50 V/s. k degrees (app) values of 0.05-0.5 cm/s were observed for Fe(CN)(6)(3)(-)(/4)(-), Ru(NH(3))(6)(3+/2+), and IrCl(6)(2)(-)(/3)(-) without any extensive electrode pretreatment (e.g., polishing). Lower k degrees (app) values of 10(-)(4)-10(-)(6) cm/s were found for 4-methylcatechol and Fe(3+/2+). The voltammetric responses for Fe(CN)(6)(3)(-)(/4)(-) and Ru(NH(3))(6)(3+/2+) were also examined at all four electrode types at two different solution pH (1.90, 7.35). Since the hydrogen-terminated diamond surfaces contain few, if any, ionizable carbon-oxygen functionalities (e.g., carboxylic acid, pK(a) approximately 4.5), the deltaE(p), i(p)(ox), and i(p)(red) values for the two systems were, for the most part, unaffected by the solution pH. This is in contrast to the typical behavior of oxygenated, sp(2) carbon electrodes, such as glassy carbon.     

Characterization of batch-microfabricated scanning electrochemical-atomic force microscopy probes

A procedure for the batch microfabrication of scanning electrochemical-atomic force microscopy (SECM-AFM) probes is described. The process yields sharp AFM tips, incorporating a triangular-shaped electrode (base width 1 microm, height 0.65 microm) at the apex. Microfabrication was typically carried out on (1)/(4) 3-in. wafers, yielding 60 probes in each run. The measured spring constant of the probes was in the range 1-1.5 N m(-1). To date, processing has been carried out twice successfully, with an estimated success rate for the fabrication process in excess of 80%, based on field emission-scanning electron microscopy imaging of all probes and current-voltage measurements on a random selection of approximately 30 probes. Steady-state voltammetric measurements for the reduction of Ru(NH(3))(6)(3+) in aqueous solution indicate that the electrode response is well-defined, reproducible, and quantitative, based on a comparison of the experimental diffusion-limited current with finite element simulations of the corresponding mass transport (diffusion) problem. Topographical imaging of a sputtered Au film with the SECM-AFM probes demonstrates lateral resolution comparable to that of conventional Si(3)N(4) AFM probes. Combined electrochemical-topographical imaging studies have been carried out on two model substrates: a 10-microm-diameter disk ultramicroelectrode (UME) and an array of 1-microm-diameter UMEs, spaced 12.5 microm apart (center to center). In both cases, an SECM-AFM probe was first employed to image the topography of the substrates. The tip was then moved back a defined distance from the surface and use to detect Ru(NH(3))(6)(2+) produced at the substrate, biased at a potential to reduce Ru(NH(3))(6)(3+), present in bulk solution, at a diffusion-controlled rate (substrate generation-tip collection mode). These studies establish the success of the batch process for the mass microfabrication of SECM-AFM tips.     

Immobilization of a catalytic DNA molecular beacon on Au for Pb(II) detection

A Pb(II)-specific DNAzyme fluorescent sensor has been modified with a thiol moiety in order to immobilize it on a Au surface. Self-assembly of the DNAzyme is accomplished by first adsorbing the single-thiolated enzyme strand (HS-17E-Dy) followed by adsorption of mercaptohexanol, which serves to displace any Au-N interactions and ensure that DNA is bound only through the S-headgroup. The preformed self-assembled monolayer is then hybridized with the complementary fluorophore-containing substrate strand (17DS-Fl). Upon reaction with Pb(II), the substrate strand is cleaved, releasing a fluorescent fragment for detection. Fluorescence intensity may be correlated with original Pb(II) concentration, and a linear calibration was obtained over nearly four decades: 10 microM > or = [Pb(II)] > or = 1 nM. The immobilized DNAzyme is a robust system; it may be regenerated after cleavage, allowing multiple sensing cycles. In addition, drying of fully assembled DNAzyme before reaction with Pb(II) does not significantly affect analytical performance. These results demonstrate that, in comparison with solution-based schemes, immobilization of the DNAzyme sensor onto a Au surface lowers the detection limit (from 10 to 1 nM), maintains activity and specificity, and allows sensor regeneration and long-term storage. Realization of Pb(II) detection through an immobilized DNAzyme is the first important step toward creation of a stand-alone, portable Pb(II) detection device such as those immobilizing DNAzyme recognition motifs in the nanofluidic pores of a microfluidic-nanofluidic hybrid multilayer device.     

Sensitive immunoassay of a biomarker tumor necrosis factor-alpha based on poly(guanine)-functionalized silica nanoparticle label

A novel electrochemical immunosensor for the detection of tumor necrosis factor-alpha (TNF-alpha) based on poly(guanine)-functionalized silica nanoparticles (NPs) label is presented. The detection of mouse TNF-alpha via immunological reaction is based on a dual signal amplification: (1) a large amount of guanine residues introduced on the electrode surface through sandwich immunoreaction and poly(guanine)-functionalized silica NP label; (2) Ru(bpy)3(2+)-induced catalytic oxidation of guanine, which results in great enhancement of anodic current. The synthesized silica NP conjugates were characterized with atomic force microscopy, X-ray photoelectron spectroscopy, and electrochemistry. These experiments confirmed that poly(guanine) and avidin were immobilized on the surface of silica NPs. The performance of the electrochemical immunosensor was evaluated and some experiment parameters (e.g., concentration of Ru(bpy)3(2+), incubation time of TNF-alpha, etc.) were optimized. The detection limit for TNF-alpha is found to be 5.0 x 10(-11) g mL(-1) (2.0 pM), which corresponds to 60 amol of TNF-alpha in 30 microL of sample. This immunosensor based on the poly(guanine)-functionalized silica NP label offers great promise for rapid, simple, cost-effective analysis of biological samples.     

Large-amplitude Fourier transformed high-harmonic alternating current cyclic voltammetry: kinetic discrimination of interfering Faradaic processes at glassy carbon and at boron-doped diamond electrodes

Significant advantages of Fourier transformed large-amplitude ac higher (second to eighth) harmonics relative to responses obtained with conventional small-amplitude ac or dc cyclic voltammetric methods have been demonstrated with respect to (i) the suppression of capacitive background currents, (ii) the separation of the reversible reduction of [Ru(NH(3))(6)](3+) from the overlapping irreversible oxygen reduction process under conditions where aerobic oxygen remains present in the electrochemical cell, and (iii) the kinetic resolution of the reversible [Ru(NH(3))(6)](3+/2+) process in mixtures of [Fe(CN)(6)](3-) and [Ru(NH(3))(6)](3+) at appropriately treated boron-doped diamond electrodes, even when highly unfavorable [Fe(CN)(6)](3-) to [Ru(NH(3))(6)](3+) concentration ratios are employed. Theoretical support for the basis of kinetic discrimination in large-amplitude higher harmonic ac cyclic voltammetry is provided.     

Aptamer-based biosensors for label-free voltammetric detection of lysozyme

This paper reports a simple electrochemical approach for the detection of the ubiquitous protein lysozyme using aptamer-modified electrodes. Anti-lysozyme DNA aptamers were immobilized on gold surfaces by means of self-assembly, for which the surface density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH3)6]3+) bound to the surface via electrostatic interaction with the DNA phosphate backbone. Upon incubation of the electrode with a solution containing lysozyme, the CV response of surface-bound [Ru(NH3)6]3+ changed substantially, and the relative decrease in the integrated charge of the reduction peak can be tabulated as a quantitative measure of the protein concentration. It is significant that the on-chip protein/aptamer binding constant and the optimized surface density to achieve the best detection limit can be evaluated. This biosensor is label-free and offers an alternative, sensitive, and versatile method for protein detection, which is beneficial to the ever-growing interests of fabricating portable bioanalytical devices with simple electrical readout protocols.     

纳米Fe2O3-还原氧化石墨烯复合材料的制备及对双酚A的检测

双酚A（BPA）被广泛应用于食品包装材料中,它会引起人体内分泌失调,并导致免疫和生殖系统异常,因此对生活用水中BPA的检测十分重要。本文采用一步水热法合成纳米Fe₂O₃-还原氧化石墨烯（Fe₂O₃-rGO）复合材料并进行表征,基于Fe₂O₃-rGO复合材料构建电化学传感器Fe₂O₃-rGO/玻碳电极,用于检测水样中的BPA。通过FTIR、XRD和SEM分析,表明纳米Fe₂O₃粒子成功附着到rGO上;采用微分脉冲伏安法（DPV）进行BPA的电化学检测,结果显示BPA在0.1~100 μmol/L范围内呈现良好的线性关系,检出限为0.033 μmol/L（信噪比为3）。同时,Fe₂O₃-rGO/玻碳电极电化学传感器对电活性物质和常见金属离子具有良好的抗干扰能力,且实样检测结果理想。      

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 16. Sensing by fluorescence

A fluorescence spectroelectrochemical sensor capable of detecting very low concentrations of metal complexes is described. The sensor is based on a novel spectroelectrochemical sensor that incorporates multiple internal reflection spectroscopy at an optically transparent electrode (OTE) coated with a selective film to enhance detection limits by preconcentrating the analyte at the OTE surface. Nafion was used as the selective cation exchange film for detecting Ru(bpy)(3)(2+), the model analyte, which fluoresces at 605 nm when excited with a 441.6-nm HeCd laser. The unoptimized linear dynamic range of the sensor for Ru(bpy)(3)(2+) is between 1 x 10(-)(11) and 1 x 10(-)(7) M with a calculated 2 x 10(-)(13) M detection limit. The sensor employs extremely thin films ( approximately 12 nm) without significantly sacrificing its sensitivity. The sensor response is demonstrated with varying film thicknesses. A state-of-the-art flow cell design allows variable cell volumes as low as approximately 4 microL. Fluorescence of the sample can be controlled by electromodulation between 0.7 and 1.3 V. Sensor operation is not reversible for the chosen model film (Nafion) and sample (Ru(bpy)(3)(2+)) but it can be regenerated with ethanol for multiple uses.     

Use of polymeric indicator for electrochemical DNA sensors: poly(4-vinylpyridine) derivative bearing [Os(5,6-dimethyl-1,10-phenanthroline)2Cl]2+

A poly(4-vinylpyridine) (PVP) derivative bearing redox-active osmium complexes, PVP-[Os(5,6-dmphen)(2)Cl](2+) (5,6-dmphen = 5,6-dimethyl-1,10-phenanthroline), was employed as a hybridization indicator for electrochemical DNA sensors. PVP-[Os(5,6-dmphen)(2)Cl](2+) exhibited approximately 1000 times higher sensitivity than the corresponding monomeric analogue, [Os(5,6-dmphen)(3)](2+), in DNA determination due to polymeric effects. The detection limit of the present sensor was approximately 0.5 amol. Another merit of the polymeric indicator is that the redox potential was found to be +360 mV (vs Ag/AgCl), which is significantly lower than that reported for the monomeric analogue (+672 mV). The polymeric indicator was applicable to the discrimination of single- and double-base-mismatched DNAs from fully matched target DNA. The polymeric indicator can be removed from the electrode surface by rinsing the electrode in a high-temperature buffer for 6 min, and thus, the polymeric indicator-based DNA sensor can be used repeatedly.