Dual enzyme electrochemical coding for detecting DNA hybridization

Enzyme-based hybridization assays for the simultaneous electrochemical measurements of two DNA targets are described. Two encoding enzymes, alkaline phosphatase and beta-galactosidase, are used to differentiate the signals of two DNA targets in connection to chronopotentiometric measurements of their electroactive phenol and alpha-naphthol products. These products yield well-defined and resolved peaks at +0.31 V (alpha-naphthol) and +0.63 V (phenol) at the graphite working electrode (vs. Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding target. The dual target detection capability is coupled to the amplification feature of enzyme tags (to yield fmol detection limits) and with an efficient magnetic removal of non-hybridized nucleic acids. Proper attention is given to the choice of the substrates (for attaining well resolved peaks), to the activity of the enzymes (for obtaining similar sensitivities), and to the selection of the enzymes (for minimizing cross interferences). The new bioassay is illustrated for the simultaneous detection of two DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. Prospects for electrochemical coding of multiple DNA targets are discussed.     

Electrochemical stripping detection of DNA hybridization based on cadmium sulfide nanoparticle tags

We report on the detection of DNA hybridization in connection to cadmium sulfide nanoparticle tracers and electrochemical stripping measurements of the cadmium. A nanoparticle-promoted cadmium precipitation is used to enlarge the nanoparticle tag and amplify the stripping DNA hybridization signal. In addition to measurements of the dissolved cadmium ion we demonstrate solid-state measurements following a ‘magnetic’ collection of the magnetic-bead/DNA-hybrid/CdS-tracer assembly onto a thick-film electrode transducer. The new protocol combines the amplification features of nanoparticle/polynucleotides assemblies and highly sensitive stripping potentiometric detection of cadmium, with an effective magnetic isolation of the duplex. The low detection limit (100 fmol) is coupled to good reproducibility (RSD=6%). Prospects for using binary inorganic colloids for multi-target detection are discussed.     

Lead Sulfide Nanoparticle as Oligonucleotides Labels for Electrochemical Stripping Detection of DNA Hybridization

We report a method for the detection of DNA hybridization in connection to lead sulfide (PbS) nanoparticle tags and electrochemical stripping measurement of the lead. A kind of lead sulfide nanoparticle with free carboxyl groups on its surface was synthesized in aqueous solution. The nanoparticle was used as a marker to label a sequence‐known oligonucleotide, which was then employed as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were monitored by the oxidation dissolution of the lead sulfide anchored on the hybrids and the indirect determination of the lead ions by anodic stripping voltammetry (ASV). The detection limit is 0.3 pmol L^?1 of target oligonucleotides. The PbS nanoparticle combining its easy conjugation to the DNA molecule with the highly sensitive stripping voltammetry detection of lead shows its promising application in the electrochemical DNA hybridization analysis assay.     

Simultaneous electrochemical measurement of metal and organic propellant constituents of gunshot residues

The simultaneous electrochemical measurement of heavy-metal and organic propellants relevant to gunshot residues (GSRs) is demonstrated. Cyclic voltammetry (CV) and cyclic square-wave stripping voltammetry (C-SWV) are shown to detect, in a single run, common propellants, such as nitroglycerin (NG) and dinitrotoluene (DNT), along with the heavy metal constituents of GSR, antimony (Sb), lead (Pb), zinc (Zn) and barium (Ba). The voltammetric detection of the stabilizer diphenylamine (DPA) along with inorganic constituents has also been examined. The resulting electrochemical signatures combine -in a single voltammogram- the response for the various metals and organic species, based on the reduction and oxidation peaks of the constituents. Cyclic square-wave voltammetry at the glassy carbon electrode (GCE), involving an intermittent accumulation at the reversal potentials of -0.95 V (for Sb, Pb, DNT and NG) and -1.3 V (for Sb, Pb, Zn and DPA) is particularly useful to offer distinct electrochemical signatures for these constituents of GSR mixtures, compared to analogous cyclic voltammetric measurements. Simultaneous voltammetric measurements of barium (at thin-film Hg GCE) and DNT (at bare GCE) are also demonstrated in connection to intermittent accumulation at the reversal potential of -2.4 V. Such generation of unique, single-run, information-rich inorganic/organic electrochemical fingerprints holds considerable promise for 'on-the-spot' field identification of individuals firing a weapon, as desired for diverse forensic investigations.     

Amplified label-free electrical detection of DNA hybridization

A new protocol is described for amplifying label-free electrochemical measurements of DNA hybridization based on the enhanced accumulation of purine nucleobases in the presence of copper ions . Such electrical DNA assays involve hybridization of the target to inosine-substituted oligonucleotide probes (captured on magnetic beads), acidic dipurinization of the hybrid DNA, and adsorptive chronopotentiometric stripping measurements of the free nucleobases in the presence of copper ions. Both amplified adenine and guanine peaks can be used for detecting the DNA hybridization. The dramatic signal amplification advantage of this type of detection has been combined with efficient magnetic removal of non-complementary DNA, use of microliter sample volumes and disposable transducers. Factors influencing the signal enhancement were assessed and optimized. A detection limit of 40 fmol (250 pg) was obtained with 10 min hybridization and 5 min adsorptive-accumulation times. The advantages of this procedure were demonstrated by its application in the detection of DNA segments related to the BRCA1 breast cancer gene. The copper enhancement holds great promise not only for the detection of DNA hybridization, but also for trace measurement of nucleic acids.     

Multianalyte Electrochemical Biosensor Based on Aptamer‐ and Nanoparticle‐Integrated Bio‐Barcode Amplification

In the present work, a signal‐on electrochemical sensing strategy for the simultaneous detection of adenosine and thrombin is developed based on switching structures of aptamers. An Au electrode as the sensing surface is modified with two kinds of thiolated capture probes complementary to the linker DNA that contains either an adenosine aptamer or thrombin aptamer. The capture probes hybridize with their corresponding linker DNA, which has prehybridized with the reporter DNA loaded onto the gold nanoparticles (AuNPs). The AuNP contained two kinds of bio‐barcode DNA: one is complementary to the linker DNA (reporter), whereas the other is not (signal) and is tagged with different metal sulfide nanoparticles. Thus a “sandwich‐type” sensing interface is fabricated for adenosine and thrombin. With the introduction of adenosine and thrombin, the aptamer parts bind with their targets and fold to form the complex structures. As a result, the bio‐barcoded AuNPs are released into solution. The metal sulfide nanoparticles are measured by anodic stripping voltammetry (ASV), and the concentrations of adenosine and thrombin are proportional to the signal of either metal ion. With the dual amplification of the bio‐barcoded AuNP and the preconcentration of metal ions through ASV technology, detection limits as low as 6.6×10^?12 M for adenosine and 1.0×10^?12 M for thrombin are achieved. The sensor exhibits excellent selectivity and detectability in biological samples.     

Interaction of DNA with echinomycin at the mercury electrode surface as detected by impedance and chronopotentiometric measurements

The capacitance measurement (dependence of the differential capacitance C of the electrode double layer on potential E, C-E curves), electrochemical impedance spectroscopy (frequency response of the impedance Z of the electrode double layer-EIS) and constant current chronopotentiometry (dependence of dt/dE on potential at constant current, chronopotentiometric stripping analysis-CPSA) have been used for electrochemical study of echinomycin and its interaction with single-stranded (ss) and double-stranded (ds) DNA at the hanging mercury drop electrode (HMDE). The capacitance measurement showed that echinomycin gives a pseudocapacitance redox peak strongly dependent on the a.c. voltage frequency at the potential of -0.53 V. This peak is observed with dsDNA-echinomycin complex as well, but not with ssDNA treated by echinomycin. Similar results were obtained using CPSA measurements. Thus capacitance measurements and CPSA can distinguish with the aid of the bis-intercalator echinomycin the single-stranded and double helical form of DNA adsorbed at the mercury electrode surface. Impedance measurement in connection with adsorptive transfer technique can find the differences between ssDNA and dsDNA, which promise to use this technique for detection of dsDNA in hybridisation reactions.     

Cadmium sulfide nanocluster-based electrochemical stripping detection of DNA hybridization

A novel, sensitive electrochemical DNA hybridization detection assay, using cadmium sulfide (CdS) nanoclusters as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the CdS nanocluster oligonucleotide DNA probe, followed by the dissolution of the CdS nanoclusters anchored on the hybrids and the indirect determination of the dissolved cadmium ions by sensitive anodic stripping voltammetry (ASV) at a mercury-coated glassy carbon electrode (GCE). The results showed that only a complementary sequence could form a double-stranded dsDNA-CdS with the DNA probe and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. The combination of the large number of cadmium ions released from each dsDNA hybrid with the remarkable sensitivity of the electrochemical stripping analysis for cadmium at mercury-film GCE allows detection at levels as low as 0.2 pmol L(-1) of the complementary sequence of DNA.     

Ex situ Voltammetry and Chronopotentiometry of Doxorubicin at a Pyrolytic Graphite Electrode: Redox and Catalytic Properties and Analytical Applications

Ex situ (adsorptive transfer stripping) electrochemical techniques in connection with basal‐plane PGE have been applied to the study of redox and catalytic properties of doxorubicin (DOX). Cyclic and square‐wave voltammetry and constant current chronopotentiometric stripping (CPS) analysis were used to follow reversible reduction of DOX quinone moiety around ?0.5 V and its coupling to catalytic oxygen reduction. CPS was for the first time used for sensitive ex situ determination of the DOX using the catalytic signal around ?0.5 V in the presence of oxygen, allowing detection of femtomole amounts of DOX. We show that specific interaction of DOX with double‐stranded DNA can easily be monitored using the catalytic CPS signal.     

Magnetically-induced solid-state electrochemical detection of DNA hybridization

A magnetic triggering of a solid-state electrical transduction of DNA hybridization is described. Positioning of an external magnet below the thick-film electrode attracts the DNA/particle network and enables the solid-state electrochemical stripping detection of the silver tracer. TEM imaging indicates that the hybridization event results in a three-dimensional aggregate structure in which duplex segments link the metal nanoparticles and magnetic spheres, and that most of this assembly is covered with the silver precipitate. This leads to a direct contact of the metal tag with the surface (in connection to the magnetic collection) and enables the solid-state electrochemical transduction (without prior dissolution and subsequent electrodeposition of the metal), using oxidative dissolution of the silver tracer. No such aggregates (and hence magnetic "collection") are observed in the presence of noncomplementary DNA, that is, without the linking hybrid. The new method couples high sensitivity of silver-amplified assays with effective discrimination against excess of closely related nucleotide sequences (including single-base imperfections). Such direct electrical detection of DNA/metal-particle assemblies can bring new capabilities to the detection of DNA hybridization, and could be applied to other bioaffinity assays.     

Electrochemical Processes of Sulfide in NaCl Electrolyte Solutions on Mercury Electrode

The electrochemical processes of sulfide in NaCl electrolyte solutions were studied using stripping cyclic voltammetry, differential capacity and chronoamperometric measurements. Some new effects were observed and are here discussed. At potentials around ?0.1 to ?0.3 V a pair of Faradaic adsorptive peaks, which can only be observed in the presence of chloride, is attributed to the formation of HgCl ion from the deposited layer of HgS. A second pair of peaks of purely capacitive character was observed at ?0.4 V. This is the result of the reorientation and partial desorption or transformation of HgS layers. The formation and dissolution of HgS layers on mercury has been well investigated previously. We observed some new effects after depositing approximately ten layers of HgS and attributed this to the formation of polysulfides at positive potentials.     

Adsorptive Stripping Voltammetric Detection of Single‐Stranded DNA at Electrochemically Modified Glassy Carbon Electrode

Electrochemically modified glassy carbon electrode (GCE) was used to study the electrochemical oxidation and detection of denatured single‐stranded (ss) DNA by means of adsorptive stripping voltammetry. The modification of GCE, by electrochemical oxidation at +1.75 V (vs.SCE) for 10 min and cyclic sweep between +0.3 V and ?1.3 V for 20 cycles in pH 5.0 phosphate buffer, results in 100‐fold improvement in sensitivity for ssDNA detection. We speculated that the modified GCE has a high affinity to single‐stranded DNA through hydrogen bond (specific static adsorption). Single‐stranded DNA can accumulate at the GCE surface at open circuit and produce a well‐defined oxidation peak corresponding to the guanine residues at about +0.80 V in pH 5.0 phosphate buffer, while the native DNA gives no signal under the same condition. The peak currents are proportional to the ssDNA concentration in the range of 0–18.0 μg mL^?1. The detection limit of denatured ssDNA is ca. 0.2 μg mL^?1 when the accumulation time is 8 min at open circuit. The accumulation mechanism of ssDNA on the modified GCE was discussed.     

Label-free bioelectronic detection of aptamer–protein interactions

We demonstrate for the first time the utility of nucleic acid aptamers for electrochemical detection of proteins. Highly specific and sensitive label-free detection of the target protein is achieved by combining aptamer-coated magnetic beads and chronopotentiometric stripping measurements of the captured protein (in connection to the intrinsic electroactivity of the protein). Lysozyme has thus been detected selectively in a mixture containing a large excess of six proteins and amino acids (both electroactive and non-electroactive), with a detection limit of 350 fmol (7 nM). While aptamer-based electronic sensors are in their infancy, such devices offer attractive opportunities for electrochemical detection of proteins and for developing proteomic chips.     

A Graphene‐based Electrochemical Sensor for the Individual,Selective and Simultaneous Determination of Total Chlorogenic Acids,Vanillin and Caffeine in Food and Beverage Samples

An electrochemical sensor based on the electrocatalytic activity of graphene (GR) was prepared, and used for the individual, selective and simultaneous determination of 5‐O‐Caffeoylquinic acid (5‐CQA) that is major compound of chlorogenic acids in coffee, vanillin (VAN) and caffeine (CAF). The electrochemical behaviors of these compounds on GR modified glassy carbon electrode (GR/GCE) were investigated by cyclic voltammetry and square‐wave adsorptive stripping voltammetry. By using stripping conditions after 30 s accumulation under open‐circuit voltage, the electrochemical oxidation peaks appeared at +0.53, 0.83 and 1.39 V in phosphate buffer pH 2.5, and good linear current responses were obtained with detection limits of 4.4×10⁻⁹, 5.0×10⁻⁷, and 3.0×10⁻⁷ M for 5‐CQA, VAN and CAF, respectively. The potential applicability of the proposed method was illustrated in commercial food and beverage samples.     

磷钼钒杂多酸-聚邻苯二胺修饰碳糊电极示差脉冲溶出伏安法测定对苯二酚和邻苯二酚

在由磷钼钒杂多酸和邻苯二胺组成的支持电解质中,以0.100V.s-1扫速在碳糊电极上,于0.800～-0.800V范围内循环扫描20周后,制得磷钼钒杂多酸-聚邻苯二胺修饰碳糊电极。循环伏安法研究发现:对苯二酚(HQ)和邻苯二酚(CC)在该修饰电极上均出现了一对氧化还原峰;示差脉冲溶出伏安法研究发现:HQ和CC分别在0.140V和0.288V处出现二次微分溶出峰,两峰电位差为0.148V。HQ和CC的线性范围均为1.50×10-7～7.50×10-4 mol.L-1,检出限(3S/N)均为6.80×10-8 mol.L-1。据此提出了示差脉冲溶出伏安法测定模拟水样中HQ和CC,测得回收率在94.0%～106.0%之间。     

Intrinsic Electrocatalysis of RNA as a Label‐free and Reagent‐less Tool for Detection of MicroRNAs

We show for the first time that RNA catalyzes hydrogen evolution reaction at mercury‐containing electrodes. We previously showed that DNA is electrocatalytically active, and so we compared heights and potentials of the RNA chronopotentimetric stripping (CPS) peaks with analogous signals of DNA of the same sequence and found out they were very similar. RNA peaks showed differences depending on the RNA base composition. Catalytic nature of CPS peak enabled detection of 25 pM microRNA in electrochemical cell, or 500 pM microRNA in a 5 μL solution drop (corresponding to 2.5 fmole of microRNA). This finding opens the door for simple, label‐free and reagent‐less analysis of low concentrations of RNA molecules.     

Genomagnetic electrochemical assays of DNA hybridization

An electrochemical genomagnetic hybridization assay has been developed to take advantage of a new and efficient magnetic separation/mixing process, the amplification feature of enzyme labels, and single-use thick-film carbon transducers operated in the pulse-voltammetric mode. It represents the first example of coupling a magnetic isolation with electrochemical detection of DNA hybridization. The new protocol employs an enzyme-linked sandwich solution hybridization, with a magnetic-particle labeled probe hybridizing to a biotinylated DNA target that captures a streptavidin-alkaline phosphatase (AP). The alpha-naphthol product of the enzymatic reaction is quantitated through its well-defined, low-potential (+0.1 V vs. Ag/AgCl) differential pulse-voltammetric peak at the disposable screen-printed electrode. The efficient magnetic isolation is particularly attractive for electrical detection of DNA hybridization which is commonly affected by the presence of non-hybridized nucleic acid adsorbates. The new biomagnetic processing combines such magnetic separation with a low-volume magnetic mixing, and allows simultaneous handling of 12 samples. The attractive bioanalytical behavior of the new enzyme-linked genomagnetic electrical assay is illustrated for the detection of DNA segments related to the breast-cancer BRCA1 gene.     

DNA Hybridization at Magnetic Nanoparticles with Electrochemical Stripping Detection

A simple and practical method for electrochemical DNA hybridization assay has been developed to take advantage of magnetic nanoparticles for ssDNA immobilization and zinc sulfide nanoparticle as oligonucleotide label. Magnetic nanoparticles were prepared by coprecipitation of Fe²⁺ and Fe³⁺ with NH₄OH, and then amino silane was coated onto the surface of magnetite nanoparticles. The magnetic nanoparticles have the advantages of easy preparation, easy surface modification and low cost. The target ssDNA with the phosphate group at the 5′ end was then covalently immobilized to the amino group of magnetite nanoparticles by forming a phosphoramidate bond in the presence of 1‐ethyl‐3‐(3‐dimeth‐ylaminopropyl)carbodiimide (EDAC). The zinc sulfide (ZnS) nanoparticle‐labeled oligonucleotides probe was used to identify the target ssDNA immobilized on the magnetic nanoparticles based on a specific hybridization reaction. The hybridization events were assessed by the dissolution of the zinc sulfide nanoparticles anchored on the hybrids and the indirect determination of the dissolved zinc ions by anodic stripping voltammetry (ASV) at a mercury film glassy carbon electrode (GCE). The proposed method couples the high sensitivity of anodic stripping analysis for zinc ions with effective magnetic separation for eliminating nonspecific adsorption effects and offers great promise for DNA hybridization analysis.     

Direct Electrochemical Monitoring of RNase Activity

Here we present a highly sensitive, rapid and simple electrochemical assay of RNase based on coupling magnetic separation of the enzymatically treated RNA with stripping potentiometric detection of the purine nucleobases. A detection limit of 1×10^?8 U RNase (ca. 4 pg/mL) is obtained in connection to a 60 min enzymatic digestion. The attractive performance of this direct indicator‐free electrochemical assay offers great promise for a wide range of molecular biology and water quality applications.     

Simultaneous determination of guanine and adenine in DNA using an electrochemically pretreated glassy carbon electrode

A simple and reliable method based on adsorptive stripping at an electrochemically pretreated glassy carbon electrode (GCE) was proposed for simultaneous or individual determination of guanine and adenine in DNA. The detection sensitivity of guanine and adenine was improved greatly by activating the GCE electrochemically. After accumulation on pretreated GCE at open circuit for 5 min or at the potential of +0.3 V for 120 s, guanine and adenine produced well-defined oxidation peaks at about +0.8 and +1.1 V, respectively in pH 5 phosphate buffer. The detection limit for individual measurement of guanine and adenine was 4.5 ng ml⁻¹ (3×10⁻⁸ mol l⁻¹) and 4 ng ml⁻¹ (3×10⁻⁸ mol l⁻¹), respectively. Acid-denatured DNA showed two oxidation peaks corresponding to guanine and adenine residues in the same buffer. The proposed method can be used to estimate the guanine and adenine contents in DNA with good selectivity in a linear range of 0.25-5 μg ml⁻¹.