Combination of aptamer with gold nanoparticles for electrochemical signal amplification: application to sensitive detection of platelet-derived growth factor

In this paper, we report a novel electrochemical detection approach for platelet-derived growth factor (PDGF) via "sandwich" structure and gold nanoparticles (Au-NPs) mediated amplification technique. The "sandwich" structure is fabricated based on the fact that PDGF has two aptamer-binding sites, which makes it possible for one PDGF molecule to connect with two aptamers simultaneously. It is found that this electrochemical system with "sandwich" structure and Au-NPs can significantly amplify the signal of electrochemical probe [Ru(NH(3))(5)Cl](2+) for PDGF detection, and thus increase the detection sensitivity significantly. As a result, this PDGF detection approach obtains an extraordinarily low detection limit of 1 x 10(-14)M for purified samples, 1 x 10(-12)M for contaminated-ridden samples or undiluted blood serum. This detection approach can also exhibit good stability and excellent specificity.     

Electrochemical DNA sensing using osmium complexes as hybridization indicators

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.     

Comprehensive study of interactions between DNA and new electroactive Schiff base ligands. Application to the detection of singly mismatched Helicobacter pylori sequences

N,N'-Bis(3,4-dihydroxybenzylidene)-1,2-diaminobenzene (3,4-DHS) and N,N'-bis(2,5-dihydroxybenzylidene)-1,2-diaminobenzene (2,5-DHS) have been used as electrochemical probes in DNA sensing. These ligands, containing ortho and para quinone functional groups, respectively, as well as planar aromatic domains, are capable of binding to double stranded DNA (ds-DNA) more efficiently than to single stranded DNA (ss-DNA). Emphasis has been placed on the elucidation of the nature of the interaction by combining spectroscopic and electrochemical techniques. From spectrophotometric titration experiments, the binding constants of 3,4-DHS and 2,5-DHS with ds-DNA were found to be (9.0+/-0.3) x 10(3) and (3.3+/-0.2) x 10(3)M(-1), respectively. These values are consistent with a binding mode dominated by interactions with the minor groove of ds-DNA. The electroactivity of the quinone moiety in 3,4-DHS bound to DNA could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. After hybridization with the complementary target sequence, 3,4-DHS was accumulated within the double stranded DNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range where the quinone moiety is redox active. Using this approach, complementary target sequences of H. pylori can be quantified over the range of 8.9-22.2 microM with a detection limit of 8.3+/-0.4 microM and a linear correlation coefficient of 0.989. In addition this approach is capable of detecting hybridization of complementary sequences containing a single mismatch.     

Voltammetric detection of trimethylamine using immobilized trimethylamine dehydrogenase on an electrodeposited goldnanoparticle electrode

A voltammetric enzyme electrode was developed based on nicotinamide-independent trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), which catalyses the oxidation of trimethylamine (TMA) to dimethylamine and formaldehyde. A quaternized osmium hydrogel polymer, poly(vinylimidazole-[Os(4,4′-dimethyl-2,2′-bipyridine)₂Cl]^+/2+) with ethylamine (PVI-Os-EA), was prepared as a potential redox mediator in an electrochemical biosensor. TMA was detected using TMADH that was co-immobilized with an osmium hydrogel polymer on electrodeposited gold nanoparticles (Au-NPs) on screen-printed carbon electrodes (SPCEs). The Au-NPs deposited onto SPCEs provided about a three times higher electrochemical response compared to that of a planar gold electrode. As TMA was catalyzed by wired TMADH, the electrical signal was monitored at 0.3 V versus Ag/AgCl by cyclic voltammetry and chronoamperometry. The anode currents increased linearly in proportion to the TMA concentration over the 0 ∼ 2.5 mM range with a detection limit of 1 μM (R = 0.9972).     

Adamantane/beta-cyclodextrin affinity biosensors based on single-walled carbon nanotubes

One challenging goal for the development of biosensors is the conception of three-dimensional biostructures on electrode surfaces. With the aim to develop 3D architectures based on single-walled carbon nanotubes (SWCNTs) frameworks a novel adamantane-pyrrole monomer was synthesized. After electrochemical polymerization at 0.95V in acetonitrile, the resulting polypyrrole film provided affinity interactions with beta-cyclodextrin. SWCNT coatings were thus functionalized with poly(adamantane-pyrrole) and applied to the anchoring of glucose oxidase (GOX), modified with beta-cyclodextrin. By using this affinity system adamantine-cyclodextrin, beta-cyclodextrin-modified gold nanoparticles were attached onto the functionalized SWCNT deposit as intermediate layer. This allows the immobilization of adamantane-tagged GOX. The responses of these biosensors to glucose were measured by potentiostating the modified electrodes at 0.7V versus saturated calomel electrode (SCE) in order to oxidize the enzymatically generated hydrogen peroxide in the presence of glucose and oxygen. The highest sensitivity and maximum current density were recorded for the configuration based on beta-cyclodextrin-modified gold particles as intermediate layer between adamantine-functionalized SWCNTs and GOX (31.02 mAM(-1)cm(-2) and 350 microAcm(-2), respectively). The similar configuration without SWCNTs exhibits a sensitivity and J(max) of 0.98 mAM(-1)cm(-2) and 75 microAcm(-2), respectively. The resulting supramolecular assemblies were characterized by scanning electron microscopy (SEM). Advantages and disadvantages of the different preparation methods and the performance of each affinity sensor setup are discussed in detail.     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis

Attomole (10(-18)mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16 amole detection of 1563 nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the "alarm signal" range (from 1 to 100 cells per 100mL).     

New antibody immobilization method via functional liposome layer for specific protein assays

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.     

Electrochemical synthesis of gold nanostructure modified electrode and its development in electrochemical DNA biosensor

In this article, gold nanostructure modified electrodes were achieved by a simple one-step electrodeposition method. The morphologies of modified electrodes could be easily controlled by changing the pH of HAuCl₄ solution. The novel nanoflower-like particles with the nanoplates as the building blocks could be interestingly obtained at pH 5.0. The gold nanoflower modified electrodes were then used for the fabrication of electrochemical DNA biosensor. The DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. The DNA immobilization and hybridization on gold nanoflower modified electrode was studied with the use of [Ru(NH₃)₆]³⁺ as a hybridization indicator. The electrochemical DNA biosensor shows a good selectivity and sensitivity toward the detection of target DNA. A detection limit of 1 pM toward target DNA could be obtained.     

Electrochemical properties of cytochroms P450 using nanostructured electrodes: direct electron transfer and electro catalysis

The present study demonstrates direct electron transfer between cytochromes P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51b1) on the one hand and screen-printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB) on the other. Electro detection of heme proteins was possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, and lanosterol) and with P450 inhibitor (ketoconazole) were analyzed using cyclic voltammetry (CV), square wave voltammetry (SWV) differential pulse voltammetry (DPV), and amperometry.     

Voltammetric detection of single base-pair mismatches and quantification of label-free target ssDNA using a competitive binding assay

The application of electrochemical techniques for DNA detection is motivated by their potential to detect hybridisation events in a more rapid, simplistic and cost-effective manner compared to conventional optical assays. Here, we present an electrochemical DNA sensor for the specific and quantitative detection of single-stranded DNA (ssDNA). Probe oligonucleotides were immobilised onto thin gold film electrodes by a 5'-thiol-linker. Hybridisation was detected by means of the electroactive redox-marker methylene blue (MB) covalently attached to the 5'-end of the target ssDNA and voltammetric techniques. MB-labeled target ssDNA was recognised down to 30 pmol. By application of a competitive binding assay, non-labeled ssDNA was detected down to 3 pmol. In addition, the DNA-modified electrodes were capable of sensing single base-pair mismatches at different positions within the sequence of the hybridised double-stranded DNA (dsDNA).     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Electrochemical impedance spectroscopy for study of amyloid beta-peptide interactions with (-) nicotine ditartrate and (-) cotinine

Immobilization of amyloid beta (Abeta) (1-40) peptide on Au-colloid modified gold electrodes has been studied. Colloidal Au was self-assembled onto gold electrodes through the thiol groups of 1,6-hexanedithiol monolayer. Next, buffered aqueous solution of Abeta (1-40) peptide existing in the beta-sheet structure in the acidic media was dropped on the electrode surface. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The changes of the resistance of the layer with deposited Abeta (1-40) peptide, occurred under stimulation by different concentration of (-) nicotine ditartrate and (-) cotinine were measured with EIS and were used for the calculation of association constants. The gentle measuring conditions applied in electrochemical impedance spectroscopy, together with suitable environment for biomolecules immobilization created by Au-colloid, might be recommended as the analytical tool for assessing the effectiveness of potential drugs used in Alzheimer's disease (AD) therapy.     

Design and simple routes of synthesis of oligonucleotide conjugates for studies of DNA triple helix formation

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2',3'-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

DESIGN AND SIMPLE ROUTES OF SYNTHESIS OF OLIGONUCLEOTIDE CONJUGATES FOR STUDIES OF DNA TRIPLE HELIX FORMATION

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2′,3′-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1).     

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10pg/ml and 1ng/ml with limit of detection 5pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.     

L-半胱氨酸修饰碳纳米管的合成、表征及电化学性质

通过对碳纳米管氧化,合成了L-半胱氨酸修饰碳纳米管。运用红外、差热-热重分析、透射电镜对该复合物进行了表征。借助循环伏安法研究了其电化学性质。结果表明,碳纳米管的掺入极大地提高了L-半胱氨酸在金电极表面的电子传输速率和电流响应,同时也有利于L-半胱氨酸的电氧化,对L-半胱氨酸的氧化具有催化作用。     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.