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1.
Talanta ; 143: 19-26, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26078123

ABSTRACT

The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed.


Subject(s)
Alkaline Phosphatase/metabolism , Biosensing Techniques/methods , DNA/analysis , Intercalating Agents/chemistry , RNA, Ribosomal, 16S/analysis , Biotinylation , DNA/chemical synthesis , DNA/chemistry , DNA Probes/chemistry , Electrochemistry , Escherichia coli/genetics , Escherichia coli/isolation & purification , Limit of Detection , Oligonucleotide Array Sequence Analysis , Proflavine/chemistry , RNA, Ribosomal, 16S/chemistry , Streptavidin/metabolism
2.
Faraday Discuss ; 149: 11-22; discussion 63-77, 2011.
Article in English | MEDLINE | ID: mdl-21413171

ABSTRACT

A 32-electrode microelectrode array modified with a self-assembled monolayer of a thiolated DNA capture strand and 11-mercapto-l1-undecanol was used for the detection of multi-resistant Staphylococcus aureus (MRSA) upon hybridization of the complementary target DNA. In the proposed assay strategy the obtained double-stranded DNA (dsDNA) is at first non-covalently labeled by intercalation of a proflavine derivative which is functionalized via a flexible spacer with biotin moieties. Subsequent to this epost-labelling a avidin/alkaline phosphatase conjugate is bound to the biotin moieties thus introducing a reporter group at sites bearing dsDNA. Hybridization and hence the presence of MRSA DNA is detected via oxidation ofp-aminophenol enzymatically generated from p-aminophenylphosphate. The assay strategy was carefully evaluated using ferrocene-modified target strands. An increase in sensitivity of the proposed label-free DNA assays based on a careful design of the sensing interface and the implemented enzymatic amplification was achieved.


Subject(s)
Biotin/chemistry , DNA, Bacterial/genetics , Intercalating Agents/chemistry , Methicillin-Resistant Staphylococcus aureus/genetics , Nucleic Acid Hybridization/methods , Staphylococcal Infections/diagnosis , DNA, Bacterial/chemistry , Humans , In Situ Hybridization , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Microelectrodes , Potentiometry/methods
3.
Biosens Bioelectron ; 26(4): 1414-9, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-20729052

ABSTRACT

DNA sensor arrays integrated on CMOS chips allow fully electronic readout of biological information. Compared to state-of-the-art optical methods optical setups are not needed. Key features of fully electronic systems are robust and easy operation. These features enable applications in new fields and markets like diagnosis in doctors' offices, food control, etc. In this article we present a fully integrated system-on-chip design of a digital CMOS DNA-chip, which represents a cost optimized, robust and user friendly solution. Design issues of the chip components are discussed. Measurement results of electrical, electrochemical and DNA tests are presented and demonstrate the functionality. The realized DNA sensor chip is based on chronocoulometric measurement. The scheme of the chip is implemented in a 0.35 µm standard CMOS technology and is extended by an additional backend process dedicated to the gold electrodes. The whole chip with a total of 384 sensor positions captures an area of 15.8 mm(2) and dissipates less than 102 mW. Due to the chip's fully automatic working mode, a complete electrochemical DNA detection can be done in multiple of milliseconds for the whole sensor array. Several electrochemical analysis, such as cyclic voltammetry and chronocoulometric can be done, making the chip multifunctional and flexible but still easy to handle.


Subject(s)
Biosensing Techniques/instrumentation , DNA/analysis , DNA/genetics , Oligonucleotide Array Sequence Analysis/instrumentation , Biosensing Techniques/methods , Electrochemical Techniques , Equipment Design , Oligonucleotide Array Sequence Analysis/methods , Semiconductors
4.
Anal Bioanal Chem ; 398(6): 2617-23, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20632166

ABSTRACT

An electrochemical method for the detection of Epstein-Barr virus (EBV) infections is described. The method relies on an immunoassay with electrochemical read-outs based on recombinant antigens. The antigens are immobilised on an Au electrode surface and used to complementarily bind antibodies from serum samples found during different stages of infection with EBV. Thiol chemistry under formation of self-assembled monolayers functions as a means to immobilise the antigens at the Au electrodes. A reporter system consisting of a secondary antibody labelled with alkaline phosphatase is used for electrochemical detection. The feasibility of the assay design is demonstrated and the assay performance is tested against the current gold standard in EBV detection. Close correlation is obtained for the results found for the developed electrochemical immunoassay and a standard line assay. Moreover, the electrochemical immunoassay is combined with a nanoporous electrode system allowing signal amplification by means of redox recycling. An amplification factor of 24 could be achieved.


Subject(s)
Antibodies, Viral/blood , Biosensing Techniques/methods , Electrochemical Techniques/methods , Herpesvirus 4, Human/immunology , Aniline Compounds/chemistry , Antigens, Viral/chemistry , Biosensing Techniques/instrumentation , Electrochemical Techniques/instrumentation , Gold/chemistry , Humans , Immunoassay/methods , Nanopores , Organophosphorus Compounds/chemistry , Oxidation-Reduction , Recombinant Proteins/chemistry , Substrate Specificity , Surface Properties
5.
Chembiochem ; 10(7): 1193-9, 2009 May 04.
Article in English | MEDLINE | ID: mdl-19353601

ABSTRACT

Sensible DNA: An electrochemical DNA assay based on specific Salmonella spp. capture probes and enzyme labeling with alkaline phosphatase was optimized by using a 48-electrode microarray and scanning electrochemical microscopy (SECM). SECM was further used to evaluate potential amplification strategies due to redox cycling. Due to insufficient detection limits and selectivity, electrochemical DNA sensors are not yet used as everyday tools in diagnostics. Here, we present an electrochemical DNA assay that is based on specific Salmonella spp. capture probes. Our optimization strategies and the specific features of related electrochemical DNA sensor arrays, which are comprised of a chip with 48 gold electrodes, are also described. A ssDNA monolayer is formed by chemisorption of the thiol-modified capture strand on the different gold electrodes of the array after spotting with a needle spotter. The assay parameters were optimized for the use of minimum amounts of sample and reagents and short assay times. Scanning electrochemical microscopy (SECM) has been used to visualize the local activity of an enzyme label used for amplified hybridization detection at high lateral resolution. The potential of SECM to further amplify the sensor signal by means of redox cycling is demonstrated by using single-stranded DNA capture probe modified gold microelectrodes as SECM tips. The detection limit of the proposed DNA sensor is shown to be in the femtomolar range without redox cycling amplification.


Subject(s)
Biosensing Techniques/methods , DNA/analysis , Microscopy, Electron, Scanning/methods , Alkaline Phosphatase/metabolism , DNA Probes/chemistry , Electrochemical Techniques , Electrodes , Gold/chemistry , Microarray Analysis , Oxidation-Reduction , Salmonella/chemistry , Salmonella/genetics
6.
Langmuir ; 21(17): 7848-53, 2005 Aug 16.
Article in English | MEDLINE | ID: mdl-16089391

ABSTRACT

The detection of DNA hybridization is of central importance to the diagnosis and treatment of genetic diseases. Due to cost limitations, small and easy-to-handle testing devices are required. Electrochemical detection is a promising alternative to evaluation of chip data with optical readout. Independent of the actual readout principle, the hybridization process still takes a lot of time, hampering daily use of these techniques, especially in hospitals or doctor's surgery. Here we describe how direct local electrical heating of a DNA-probe-modified gold electrode affects the surface hybridization process dramatically. We obtained a 140-fold increase of alternating current voltammetric signals for 20-base ferrocene-labeled target strands when elevating the electrode temperature during hybridization from 3 to 48 degrees C while leaving the bulk electrolyte at 3 degrees C. At optimum conditions, a target concentration of 500 pmol/L could be detected. Electrothermal regeneration of the immobilized DNA-probe strands allowed repetitive use of the same probe-modified electrode. The surface coverage of DNA probes, monitored by chronocoulometry of hexaammineruthenium(III), was almost constant upon heating to 70 degrees C. However, the hybridization ability of the probe self-assembled monolayer declined irreversibly when using a 70 degrees C hybridization temperature. Coupling of heated electrodes and highly sensitive electrochemical DNA hybridization detection methods should enhance detection limits of the latter significantly.


Subject(s)
DNA/analysis , Nucleic Acid Hybridization/methods , Temperature , Electrochemistry , Electrodes , Gold/chemistry , Sensitivity and Specificity , Surface Properties , Time Factors
7.
Biosens Bioelectron ; 20(5): 925-32, 2004 Nov 15.
Article in English | MEDLINE | ID: mdl-15530789

ABSTRACT

The supposed repelling mode of scanning electrochemical microscopy (SECM) allows truly label-free electrochemical recognition of the presence and hybridisation of nucleic acids that are immobilised on conducting DNA chips. Basically, the SECM-based detection of single- and double-stranded DNA profits from the electrostatic repulsion between deprotonated phosphate groups at the backbone of the oligonucleotides and a free-diffusing negatively charged redox mediator (e.g. [Fe(CN)(6)](3-/4-)). In electrolytes of proper pH and ionic strength, this coulomb interaction is heavily influencing the diffusion properties of the mediator in the vicinity of the surface-anchored DNA strands. This charge interaction modulates the diffusional mass transport for the charged redox species in the DNA modified regions, and thus locally decreases the positive feedback currents measured with a SECM tip placed within the electrochemical nearfield of the chip surface. This approach was used to study arrays of synthetic 20-base oligonucleotide probes that were immobilised on monolayer-modified gold surfaces. Evidence is provided that the density of probes, the ionic strength of solution and the tip-to-sample distance have a strong impact on the capability of the repelling mode of SECM to visualise probe spots and hybridisation while the concentration of the chosen mediator did not significantly affect detection.


Subject(s)
DNA, Single-Stranded/chemistry , DNA, Single-Stranded/ultrastructure , Electrochemistry/methods , In Situ Hybridization/methods , Microscopy, Electron, Scanning/methods , Oligonucleotide Array Sequence Analysis/methods , Coated Materials, Biocompatible/chemistry , DNA, Single-Stranded/analysis , Static Electricity
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