Patterned assembly of genetically modified viral nanotemplates via nucleic acid hybridization

The patterning of nanoparticles represents a significant obstacle in the assembly of nanoscale materials and devices. In this report, cysteine residues were genetically engineered onto the virion surface of tobacco mosaic virus (TMV), providing attachment sites for fluorescent markers. To pattern these viruses, labeled virions were partially disassembled to expose 5' end RNA sequences and hybridized to virus-specific probe DNA linked to electrodeposited chitosan. Electron microscopy and RNAase treatments confirmed the patterned assembly of the virus templates onto the chitosan surface. These findings demonstrate that TMV nanotemplates can be dimensionally assembled via nucleic acid hybridization.     

Electrochemical assay of nonlabeled DNA chip and SNOM imaging by using streptavidin-biotin interaction

The assay of DNA biosensor-based nucleic acid recognition using microfabrication technology provides for high sensitivity, good surface coverage and reproducibility. We have achieved efficient immobilization and hybridization of nonlabeled DNA using cyclic voltammetry (CV), square wave voltammetry (SWV) and scanning near-field optical microscopy (SNOM) techniques. The increased electrochemical response observed following the immobilization of biotinlyated ssDNA probe suggests that nucleic acid is a somewhat better medium for electronic transfer. We demonstrated the high coverage of immobilized FITC-labeled biotinylated DNA probe on a streptavidin-modified surface using SNOM imaging. SNOM imaging of FITC-labeled complementary DNA also exhibited fluorescent light spots of hybridization distributed throughout. No fluorescent light was observed with the hybridization of non-complementary DNA.     

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.     

Electrokinetically controlled DNA hybridization microfluidic chip enabling rapid target analysis

Biosensors and more specifically biochips exploit the interactions between a target analyte and an immobilized biological recognition element to produce a measurable signal. Systems based on surface nucleic acid hybridization, such as microarrays, are particularly attractive due to the high degree of selectivity in the binding interactions. One of the drawbacks of this reaction is the relatively long time required for complete hybridization to occur, which is often the result of diffusion-limited reaction kinetics. In this work, an electrokinetically controlled DNA hybridization microfluidic chip will be introduced. The electrokinetic delivery technique provides the ability to dispense controlled samples of nanoliter volumes directly to the hybridization array (thereby increasing the reaction rate) and rapidly remove nonspecific adsorption, enabling the hybridization, washing, and scanning procedures to be conducted simultaneously. The result is that all processes from sample dispensing to hybridization detection can be completed in as little as 5 min. The chip also demonstrates an efficient hybridization scheme in which the probe saturation level is reached very rapidly as the targets are transported over the immobilized probe site enabling quantitative analysis of the sample concentration. Detection levels as low as 50 pM have been recorded using an epifluorescence microscope.     

A universal nucleic acid sequence biosensor with nanomolar detection limits

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.     

Label-free determination of picogram quantities of DNA by stripping voltammetry with solid copper amalgam or mercury electrodes in the presence of copper

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of the DNA sensors. We show that subnanomolar concentrations (related to monomer content) of unlabeled DNA can be determined using copper solid amalgam electrodes or hanging mercury drop electrodes in the presence of copper. DNA is first treated with acid (e.g., 0.5 M perchloric acid), and the acid-released purine bases are directly determined by the cathodic stripping voltammetry. Volumes of 5-3 microL of acid-treated DNA can easily be analyzed, thus making possible the determination of picogram and subpicogram amounts of DNA corresponding to attomole and subattomole quantities of 1000-base pair DNA. Application of this determination in DNA hybridization detection is demonstrated using surface H for the hybridization (superparamagnetic beads with covalently attached DNA probe) and the mercury electrodes only for the determination of DNA selectively captured at surface H.     

Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-aminobenzenesulfonic acid-modified glassy carbon electrode

In this study, an electrochemical DNA biosensor was developed for detection of the breakpoint cluster region gene and the cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia by using 18-mer locked, nucleic acid-modified, single-stranded DNA as the capture probe. The capture probe was covalently attached on the sulfonic-terminated aminobenzenesulfonic acid monolayer-modified glassy carbon electrode through the free amines of DNA bases based on the acyl chloride cross-linking reaction. The covalently immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA (dsDNA) on the LNA/4-ABSA/GCE surface. Differential pulse voltammetry was used to monitor the hybridization reaction on the capture probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated that, in pH 7.0 Tris-HCl buffer solution, the peak current was linear with the concentration of complementary strand in the range of 1.0 x 10 (-12)1.1 x 10 (-11) M with a detection limit of 9.4 x 10 (-13) M. This new method demonstrates its excellent specificity for single-base mismatch and complementary dsDNA after hybridization, and this probe has been used for assay of PCR real sample with satisfactory results.     

Bulk acoustic wave affinity biosensor for genetically modified organisms detection

Bulk acoustic waves have been applied as affinity sensors. In particular, a nucleic acid sensor for hybridization studies has been developed and applied for detecting DNA target sequences in solution. A DNA probe is immobilized on the sensor surface while the target sequence is free in solution; the interaction between the two complementary strands (hybridization) is followed in real-time, without the use of any label. The system has been applied to analytical problems, i.e., genetically modified organisms (GMOs) detection. The probe was complementary to characteristic DNA sequences present in GMOs. The probe sequences were internal to the sequence of 35S promoter and Nos terminator that are inserted sequences in the genome of the GMO regulating the transgene expression. Two different probe immobilization procedures were characterized to improve the performances of a piezoelectric crystal DNA sensor for GMOs detection: 1) thiol-dextran-streptavidin-biotin procedure and 2) thiol-derivatized probe and blocking thiol procedure. The system has been optimized using synthetic oligonucleotides. The probe immobilization step was monitored by a surface plasmon resonance system.     

On-chip transduction of nucleic acid hybridization using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1.     

Electrochemical imaging of localized sandwich DNA hybridization using scanning electrochemical microscopy

Imaging of localized hybridization of nucleic acids immobilized on gold-DNA chip was performed by means of the feedback mode of scanning electrochemical microscopy (SECM). Thiol-tethered oligodeoxynucleotide (HS-ODN) probes, spotted on a gold surface, were hybridized with unmodified target sequence via sandwich hybridization with a biotinylated signaling probe. Spots where sequence-specific hybridization had occurred were developed by adding a streptavidin-alkaline phosphatase conjugate and biocatalyzed precipitation of an insoluble and insulating product. As a consequence, the surface conductivity of the spotted region of the chip where hybridization had taken place changed. These changes in conductivity were sensitively detected by the SECM tip. The proposed method allows imaging of a DNA array in a straightforward way. Analysis of real samples was also performed coupling this method with polymerase chain reaction. The imaging of 60 nM PCR amplicon (255 bp) was demonstrated.     

Reusable electrochemical sensing platform for highly sensitive detection of small molecules based on structure-switching signaling aptamers

Aptamers are nucleic acids that have high affinity and selectivity for their target molecules. A target may induce the structure switching from a DNA/DNA duplex to a DNA/target complex. In the present study, a reusable electrochemical sensing platform based on structure-switching signaling aptamers for highly sensitive detection of small molecules is developed using adenosine as a model analyte. A gold electrode is first modified with polytyramine and gold nanoparticles. Then, thiolated capture probe is assembled onto the modified electrode surface via sulfur-gold affinity. Ferrocene (Fc)-labeled aptamer probe, which is designed to hybridize with capture DNA sequence and specifically recognize adenosine, is immobilized on the electrode surface by hybridization reaction. The introduction of adenosine triggers structure switching of the aptamer. As a result, Fc-labeled aptamer probe is forced to dissociate from the sensing interface, resulting in a decrease in redox current. The decrement of peak current is proportional to the amount of adenosine. The present sensing system could provide both a wide linear dynamic range and a low detection limit. In addition, high selectivity, good reproducibility, stability, and reusability are achieved. The recovery test demonstrates the feasibility of the designed sensing system for an adenosine assay.     

Enzyme-based colorimetric detection of nucleic acids using peptide nucleic acid-immobilized microwell plates

The development of label-free or nonlabeling assays for nucleic acids is important in basic biological research and biomedical diagnosis. In this study, we have developed an enzyme-based colorimetric assay for nucleic acids, which combines the robustness of nonlabeling of DNA and RNA samples and the adequate sensitivity of enzymatic reactions. The core of this assay is the use of neutral peptide nucleic acid (PNA) as capture probe and the electrostatic adsorption of horseradish peroxidase (HRP) on hybridized, negatively charged nucleic acids to report the hybridization events, through HRP-catalyzed color reactions of 3,3',5,5'-tetramethylbenzidine and H(2)O(2). The proposed assay has been validated with fully complementary and single base-mismatched DNAs of different chain lengths. The proposed assay has also been validated with total RNA samples extracted from two human cancer cell lines (A 549 lung cancer cell and HeLa cell) for microRNA detection in real samples. Through extensive optimizations of HRP adsorption and nucleic acid hybridization conditions, detection limits of 0.1-0.2 nM for DNA (depending on chain length) and approximately 2 microg of total RNA have been achieved. Surface plasmon resonance spectroscopy has been used to elucidate the HRP adsorption and PNA-nucleic acid hybridizations through real-time measurements and to provide guidance for the development of the colorimetric assay.     

Electronic detection of target nucleic acids by a 2,6-disulfonic acid anthraquinone intercalator

A DNA hybridization biosensor based on long-range electron transfer that is capable of detecting DNA single-base mismatch is presented. A mixed self-assembled monolayer of single-stranded DNA (ss-DNA), thiolated at the 3' end, and 6-mercapto-1-hexanol was formed on a gold surface. This probe ss-DNA-modified gold surface was incubated in 2,6-disulfonic acid anthraquinone (AQDS) intercalator solution, rinsed, and placed in an AQDS-free buffer solution, whereupon voltammetric experiments were performed. No voltammetric peaks were observed for probe ss-DNA-modified gold electrodes. Upon DNA hybridization and incubation in AQDS, clear voltammetric peaks, consistent with the oxidation and reduction of AQDS, were observed. The absence of AQDS electrochemistry for ss-DNA-modified surfaces clearly shows the electrochemistry is due to long-range electron transfer through the DNA duplex. No peak currents were observed when the probe ss-DNA-modified surface was exposed to noncomplementary target DNA, but there was a diminution in current signal upon hybridization with C-A mismatched and a G-A mismatched targets.     

Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of Lamivudine resistance

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized.     

Bottom-up assembly of large-area nanowire resonator arrays

Directed-assembly of nanowire-based devices will enable the development of integrated circuits with new functions that extend well beyond mainstream digital logic. For example, nanoelectromechanical resonators are very attractive for chip-based sensor arrays because of their potential for ultrasensitive mass detection. In this letter, we introduce a new bottom-up assembly method to fabricate large-area nanoelectromechanical arrays each having over 2,000 single-nanowire resonators. The nanowires are synthesized and chemically functionalized before they are integrated onto a silicon chip at predetermined locations. Peptide nucleic acid probe molecules attached to the nanowires before assembly maintain their binding selectivity and recognize complementary oligonucleotide targets once the resonator array is assembled. The two types of cantilevered resonators we integrated here using silicon and rhodium nanowires had Q-factors of approximately 4,500 and approximately 1,150, respectively, in vacuum. Taken together, these results show that bottom-up nanowire assembly can offer a practical alternative to top-down fabrication for sensitive chip-based detection.     

Carbon nanocapsules as an effective sensing platform for fluorescence-enhanced nucleic acid detection

In this communication, we demonstrate the proof of concept that carbon nanocapsules (CNCs) can be used as an effective fluorescent sensing platform for nucleic acid detection with selectivity down to single-base mismatch. The detection is accomplished by two steps: (1) CNC adsorbs and quenches the fluorescence of the dye-labeled single-stranded DNA (ssDNA) probe; (2) in the presence of the target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the CNC surface, leading to recovery of the dye fluorescence.     

Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.     

Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides

To establish a quantitative, corroborative understanding of observed correlations between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples, we have characterized amine-terminated, single-stranded DNA probes attached to amine-reactive commercial microarray slides and complementary DNA target hybridization using fluorescence imaging, X-ray photoelectron spectroscopy (XPS) and 32P-radiometric assays. Importantly, we have reproduced DNA probe microarray immobilization densities in macroscopic spotted dimensions using high ionic strength, high-concentration DNA probe solutions to permit direct XPS surface analysis of DNA surface chemistry with good reliability and reproducibility. Target capture hybridization efficiency with complementary DNA exhibited an optimum value at intermediate DNA probe immobilization densities. The macroscopic array model provides a new platform for the study of DNA surface chemistry using highly sensitive, quantitative surface analytical techniques (e.g., XPS, ToF-SIMS). Sensitive 32P-DNA radiometric density measurements were calibrated with more routine XPS DNA signals, facilitating future routine DNA density determinations without the use of a hazardous radioactive assay. The objective is to provide new insight into different surface chemistry influences on immobilized DNA probe environments that affect target capture efficiency from solution to improve microarray assay performance.     

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.     

Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA

In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.