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.     

A metal-chelating piezoelectric sensor chip for direct detection and oriented immobilization of polyHis-tagged proteins

A metal-chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis-tagged proteins has been demonstrated. The chip was prepared by covalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriacetic acid (NTA) ligand onto an activated alkanethiol-modified PZ crystal. The resulting chip effectively captured Ni2+ ions onto its NTA surface, as disclosed by the resonant frequency shift of the crystal and an X-ray photoelectron spectroscopy analysis. The real-time frequency analysis revealed that the bare NTA chip was nonfouling, regenerable, and highly reusable during continuous repetitive injections of ion solutions and binding proteins. In addition, the chip displayed good long-term reusability and storage stability. The individual binding studies of a polyHis-tagged glutathione-S-transferase and its native untagged form on various metal-charged chips revealed that Co2+, Cu2+, and Ni2+ ions each had different immobilization ability on the NTA surface, as well as their binding ability and selectivity with the tagged protein. As a result, the tagged protein immobilized on the Ni2+-charged chip can actively be bound with its antibody and substrate. Further, the quantitative analyses of the tagged protein in crude cell lysate with a single Ni2+-charged chip and of its substrate with a protein-coated chip were also successfully demonstrated. Therefore, this study initiates the possibilities of oriented, reversible, and universal immobilization of any polyHis-tagged protein and its functional study using a real-time PZ biosensor.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Surface immobilization of DNA aptamers for biosensing and protein interaction analysis

To utilize aptamers as molecular recognition agents in biosensors and biodiagnostics, it is important to develop strategies for reliable immobilization of aptamers so that they retain their biophysical characteristics and binding abilities. Here we report on quartz crystal microbalance (QCM) measurements and atomic force microscope (AFM)-based force spectroscopy studies to evaluate aptasensors fabricated by different modification strategies. Gold surfaces were modified with mixed self assembled monolayers (SAMs) of aptamer and oligoethylene glycol (OEG) thiols (HS-C(11)-(EG)(n)OH, n=3 or 6) to impart resistance to nonspecific protein adsorption. By affinity analysis, we show that short OEG thiols have less impact on aptamer accessibility than longer chain thiols. Backfilling with OEG as a step subsequent to aptamer immobilization provides greater surface coverage than using aptamer and OEG thiol to form a mixed SAM in one-step. Immunoglobulin E and vascular endothelial growth factor (VEGF) were studied as target proteins in these experiments. Binding forces obtained by these strategies are similar, demonstrating that the biophysical properties of the aptamer on the sensors are independent from the immobilization strategy. The results present mixed SAMs with aptamers and co-adsorbents as a versatile strategy for aptamer sensor platforms including ultrasensitive biosensor design.     

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO₂ surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 μs, respectively, about 10⁹ times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1 V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.     

Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated.     

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance

A direct human ferritin immunosensor was developed using anti-human ferritin monoclonal antibodies (MAbs) immobilized on the gold surface of a self-assembled surface plasmon resonance (SPR) apparatus. A kind of self-assembled monolayer (SAM) prepared by cystamine-glutaraldehyde method was applied to immobilize the MAbs. The reusability of the sensor chip adopting the SAM was found to be better than the other immobilization methods including adsorption, protein A, concanavalin A method. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1M HCl solution. A linear relationship existed between the angle shifts (millidegrees) and the log values of ferritin concentrations in the range from 0.2 to 200 ng/ml in buffer and human serum. When used for 15 days, the angle shifts were all >95% of those on the response at the first day. A 10 M NaOH solution was used for clearing nonspecific binding in human serum. Correlation coefficient was 0.991 between this SPR method and chemiluminescent immunoassay for determination of ferritin in clinical human serum samples. The SPR sensor offers advantages of simplicity of immobilization, high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.     

Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein

A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.     

Evaluation of two- and three-dimensional streptavidin binding platforms for surface plasmon resonance spectroscopy studies of DNA hybridization and protein-DNA binding

Surface plasmon resonance (SPR) spectroscopy has been used to study DNA assembly, DNA hybridization, and protein-DNA interactions on two streptavidin (SA) sensor chips. On one chip, SA molecules are immobilized on a biotin-exposed surface, forming an ordered two-dimensional (2D) SA monolayer. The other chip, BIAcore's SA chip, contains SA molecules immobilized within a three-dimensional (3D) carboxylated dextran matrix. Compared to the 2D chip, the 3D SA matrix allows for a slower immobilization rate of biotinylated DNA due to diffusion limitation in the dextran matrix, but with twice the amount of the immobilized DNA due to the greater number of reactive sites, which in turn enables a higher sensitivity for DNA hybridization detection. Interestingly, having a greater DNA probe dispersion in the 3D matrix does not induce a higher DNA hybridization efficiency. In a study of protein binding to immobilized DNA (estrogen receptor to estrogen response elements), aiming at assessing the DNA sequence dependent protein binding behavior, the 2D and 3D chips produce different binding characteristics. On the 2D chip, the protein binding exhibits a better selectivity to the specific sequences, regardless of binding stringency (e.g. salt concentration), whereas on the 3D chip, the liquid handling system needs to be optimized in order to minimize transport limitations and to detect small affinity differences. Through this study we demonstrate that the physicochemical structure of SPR chips affects the apparent binding behaviors of biomolecules. When interpreting SPR binding curves and selecting a sensor chip, these effects should be taken into account.     

Surface-plasmon-resonance-based biosensor with immobilized bisubstrate analog inhibitor for the determination of affinities of ATP- and protein-competitive ligands of cAMP-dependent protein kinase

Interactions between adenosine-oligoarginine conjugates (ARC), bisubstrate analog inhibitors of protein kinases, and catalytic subunits of cAMP-dependent protein kinase (cAPK Calpha) were characterized with surface-plasmon-resonance-based biosensors. ARC-704 bound to the immobilized kinase with subnanomolar affinity. The immobilization of ARC-704 to the chip surface via streptavidin-biotin complex yielded a high-affinity surface (K(D)=16nM). The bisubstrate character of ARC-704 was demonstrated with various ligands targeted to ATP-binding pocket (ATP and inhibitors H89 and H1152P) and protein-substrate-binding domain of Calpha (RIIalpha and GST-PKIalpha) in competition assays. The experiments performed on surfaces with different immobilization levels of ARC-704 produced similar results. The closeness of the obtained affinities of the tested compounds to the inhibitory potencies and affinities of the compounds measured with other methods demonstrates the applicability of the chip with the immobilized biligand inhibitor for the characterization of both ATP- and substrate protein-competitive ligands of basophilic protein kinases.     

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.     

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.     

Probing fundamental film parameters of immobilized enzymes--towards enhanced biosensor performance. Part I--QCM-D mass and rheological measurements

Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers.In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization.     

Preparation and evaluation of a peptide nucleic acid gene chip system associated with surface plasmon resonance

The aim of this study was to construct a gene chip system based on a surface plasmon resonance technique, where peptide nucleic acid (PNA) oligomers are used as probes. Since the self-assembled monolayer (SAM) technology offers good control at the molecular level, we prepared 2D surface chemistry via SAM for probe attachments. PNA, which was designed according to the bioinformatics, was immobilized on the SAM-modified chip, and subsequently, relevant parameters of the experiment were ensured and optimized. Our results suggest that the ion strength and pH value of the buffer solution do not play significant roles in PNA or its complementary strand hybridization. The PNA probe binds to its complementary nucleic acid strand with a higher sensitivity and specificity compared to those of a traditional DNA probe. The PNA probe combined with surface plasmon resonance (SPR) technology has the benefits of being a label-free and in-real time monitor, as well as having improved hybridization and stability efficiency, which highlight the PNA gene chip detection system as a promising biosensor for clinical applications.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.     

2'-MeO-RNA containing oligodeoxyribonucleotide primers can prevent template-independent base extension on microarrays

DNA microarrays require tens of thousands of deoxyoligonucleotides to be registered in an addressable fashion through immobilization, so that they have the high-throughput capability of analyzing a large number of samples simultaneously in a minimal volume of each reagent. However, using immobilized DNA molecules on microarrays can impose certain technical problems for some assays. For example, high background noise has been observed in using immobilized oligonucleotide microarrays (DNA chip) for primer extension reactions. This noise may be associated with the reactions of secondary structures formed by the adjacent primers physically constrained on the surface. Single-base extension (SBE) of arrayed primers on a chip has been extensively used in mini-sequencing to examine single nucleotide polymorphisms (SNP). Some primers appeared to be extendable in the absence of any template and thus competed against the base extension directed by. the assay target such as genomic DNA. In this article, a method is reported that is capable of reducing template-independent extension by the substitution of a 2'-methoxyribonucleotide in the otherwise oligodeoxyribonucleotide primer. The surrogate compound placed at the 5'-end of the putative secondary structure sequence of a given primer was able to inhibit template-independent extension and to improve data quality of surface-attached primer extension assays.     

Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

Kinetics and thermodynamics of DNA hybridization on gold nanoparticles

Hybridization of single-stranded DNA immobilized on the surface of gold nanoparticles (GNPs) into double stranded DNA and its subsequent dissociation into ssDNA were investigated. Melting curves and rates of dissociation and hybridization were measured using fluorescence detection based on hybridization-induced fluorescence change. Two distribution functions, namely the state distribution and the rate distribution, were proposed in order to take interfacial heterogeneity into account and to quantitatively analyze the data. Reaction and activation enthalpies and entropies of DNA hybridization and dissociation on GNPs were derived and compared with the same quantities in solution. Our results show that the interaction between GNPs and DNA reduces the energetic barrier and accelerates the dissociation of adhered DNA. At low surface densities of ssDNA adhered to GNP surface, the primary reaction pathway is that ssDNA in solution first adsorbs onto the GNP, and then diffuses along the surface until hybridizing with an immobilized DNA. We also found that the secondary structure of a DNA hairpin inhibits the interaction between GNPs and DNA and enhances the stability of the DNA hairpin adhered to GNPs.     

2′-MeO-RNA Containing Oligodeoxyribonucleotide Primers Can Prevent Template-independent Base Extension on Microarrays

DNA microarrays require tens of thousands of deoxyoligonucleotides to be registered in an addressable fashion through immobilization, so that they have the high-throughput capability of analyzing a large number of samples simultaneously in a minimal volume of each reagent. However, using immobilized DNA molecules on microarrays can impose certain technical problems for some assays. For example, high background noise has been observed in using immobilized oligonucleotide microarrays (DNA chip) for primer extension reactions. This noise may be associated with the reactions of secondary structures formed by the adjacent primers physically constrained on the surface. Single-base extension (SBE) of arrayed primers on a chip has been extensively used in mini-sequencing to examine single nucleotide polymorphisms (SNP). Some primers appeared to be extendable in the absence of any template and thus competed against the base extension directed by the assay target such as genomic DNA. In this article, a method is reported that is capable of reducing template-independent extension by the substitution of a 2′-methoxyribonucleotide in the otherwise oligodeoxyribonucleotide primer. The surrogate compound placed at the 5′-end of the putative secondary structure sequence of a given primer was able to inhibit template-independent extension and to improve data quality of surface-attached primer extension assays.