A microfluidic-based electrochemical biochip for label-free diffusion-restricted DNA hybridization analysis

DNA hybridization detection in microfluidic devices can reduce sample volumes, processing times, and can be integrated with other measurements. However, as device footprints decrease and their complexity increase, the signal-to-noise ratio in these systems also decreases and the sensitivity is thereby compromised. Device miniaturization produces distinct properties and phenomena with greater influence at the micro-scale than at the macro-scale. Here, a diffusion-restriction model was applied to a miniaturized biochip nanovolume reactor to accurately characterize DNA hybridization events that contribute to shifts in both charge transfer resistance and diffusional resistance. These effects are shown to play a significant role in electrochemical impedance spectroscopy (EIS) analyses at these length scales. Our highly functional microfluidic biosensor enables the detection of ssDNA targets selectively, with a calculated detection limit of 3.8 nM, and cross-reactivity of 13% following 20 min incubation with the target. This new biosensing approach can be further modeled and tested elucidating diffusion behavior in miniaturized devices and improving the performance of biosensors.     

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.     

Electrical frequency dependent characterization of DNA hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of electrochemical impedance spectroscopy (EIS) and quartz crystal microgravimetry (QCM). Synthetic 5'-amino terminated single stranded oligonucleotides (ssDNA) were attached to the exposed glass surface between the digits of microlithographically fabricated interdigitated microsensor electrodes using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA immobilization was achieved to the surface of the gold driving electrodes of AT-cut quartz QCM crystals using 3-mercaptopropyl-trimethoxysilane. Significant changes in electrochemical impedance values (both real and imaginary components) (11% increase in impedance modulus at 120 Hz) and resonant frequency values (0.004% decrease) were detected as a consequence of hybridization of the bound ssDNA upon exposure to its complement under hybridization conditions. Non-complementary (random) sequence sowed a modest decrease in impedance and a non-detectable change in resonant frequency. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA, has been demonstrated. The potential for development of label-free, low density DNA microarrays is demonstrated and is being pursued.     

Frequency dependent and surface characterization of DNA immobilization and hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued.     

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH–ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

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).     

Ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based DNA amplification

The present study describes an ultrasensitive protein biochip that employs nanogap electrodes and self-assembled nanoparticles to electrically detect protein. A bio-barcode DNA technique amplifies the concentration of target antigen at least 100-fold. This technique requires the establishment of conjugate magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) through binding between monoclonal antibodies (2B2), the target antigen, and polyclonal antibodies (GP). Both GP and capture ssDNA (single-strand DNA) bonds to bio-barcode ssDNA are immobilized on the surface of AuNPs. A denature process releases the bio-barcode ssDNAs into the solution, and a hybridization process establishes multilayer AuNPs over the gap surface between electrodes. Electric current through double-layer self-assembled AuNPs is much greater than that through self-assembled monolayer AuNPs. This significant increase in electric current provides evidence that the solution contains the target antigen. Results show that the protein biochip attains a sensitivity of up to 1 pg/μL.     

Metallic oxide CdIn2O4 films for the label free electrochemical detection of DNA hybridization

DNA functionalised semiconductor metallic oxide electrodes have been developed for the direct electrochemical detection of DNA hybridization, without labelling or the introduction of a redox couple. Conductive CdIn(2)O(4) thin films with controlled properties were deposited on glass substrates using an aerosol pyrolysis technique. The films exhibit a polycrystalline microstructure with a surface roughness of 1.5 nm (r.m.s.) and an electrical resistivity ranging between 1 and 3 x 10(-3) Omega cm. These electrodes were functionalised using hydroxylation and silanisation steps, to allow the binding of DNA probe sequences (20 bases). The electrical detection of DNA hybridization with complementary sequences has been performed using electrochemical impedance spectrometry (EIS) measuring the variation of impedance before and after hybridization. Two set-ups were used, a standard set-up including three electrodes and a set-up including two symmetrical electrodes. In both configurations, a significant increase of the impedance modulus, more particularly of the real part of the impedance (160-225% according to the electrochemical cell used) has been obtained over a frequency range of 10-10(5)Hz. DNA hybridization has also been systematically confirmed using the fluorescence spectrometry. This study emphasizes the high sensitivity of the CdIn(2)O(4) as a working electrode for the detection of biological events occurring at the electrode surface.     

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

Single-Stranded DNA Catalyzes Hybridization of PCR-Products to Microarray Capture Probes

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.     

Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Detection and modelling of DNA hybridization by EIS measurements. Mention of a polythiophene matrix suitable for electrochemically controlled gene delivery

A conducting polymer sensor for direct label-free DNA detection based on a polythiophene bearing an electroactive linker group is investigated. DNA hybridization is studied by electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM) techniques. Modelling of DNA hybridization by EIS measurements exhibits the contribution of nucleic acid to a superficial p-doping process. A 675-mer single-stranded DNA is produced using asymmetric PCR from a DNA sequence of a transposable element mariner and hybridized to the previously immobilized probe. Electrochemical stimulus leads to the release "on demand" of DNA fragments and the amount delivery permits to do PCR amplification.     

Innovative integrated system for real-time measurement of hybridization and melting on standard format microarrays

Despite the great popularity and potential of microarrays, their use for research and clinical applications is still hampered by lengthy and costly design and optimization processes, mainly because the technology relies on the end point measurement of hybridization. Thus, the ability to monitor many hybridization events on a standard microarray slide in real time would greatly expand the use and benefit of this technology, as it would give access to better prediction of probe performance and improved optimization of hybridization parameters. Although real-time hybridization and thermal denaturation measurements have been reported, a complete walk-away system compatible with the standard format of microarrays is still unavailable. To address this issue, we have designed a biochip tool that combines a hybridization station with active mixing capability and temperature control together with a fluorescence reader in a single compact benchtop instrument. This integrated live hybridization machine (LHM) allows measuring in real time the hybridization of target DNA to thousands of probes simultaneously and provides excellent levels of detection and superior sequence discrimination. Here we show on an environmental single nucleotide polymorphism (SNP) model system that the LHM enables a variety of experiments unachievable with conventional biochip tools.     

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.     

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

Biophysical methods for biochip analysis. Use of wide field digital fluorescent microscopy

This paper discusses an issue on the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitive throughput recording analyses by biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides the higher rate of the analysis compared to laser scanning devices. With this analyzer a scope where biological microchips can be applied becomes wider, the development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for the analysis performance can be reduced.