Using molecular beacons to probe molecular interactions between lactate dehydrogenase and single-stranded DNA

The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.9 x 10(-7) M(-1). We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.     

Single-molecular AFM probing of specific DNA sequencing using RecA-promoted homologous pairing and strand exchange

The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA.     

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.     

A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons

We have prepared a novel optical fiber evanescent wave DNA biosensor using a newly developed molecular beacon DNA probe. The molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization with target DNA/RNA molecules. Biotinylated MBs have been designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. The DNA sensor based on a MB does not need labeled analyte or intercalation reagents. It can be used to directly detect, in real-time, target DNA/RNA molecules without using competitive assays. The sensor is rapid, stable, highly selective, and reproducible. We have studied the hybridization kinetics of the immobilized MB by changing the ionic strength of the hybridization solution and target DNA concentration. Our result shows divalent cations play a more important role than monovalent cations in stabilizing the MB stem hybrids and in accelerating the hybridization reaction with target DNA/RNA molecules. The concentration detection limit of the MB evanescent wave biosensor is 1.1 nM. The MB DNA biosensor has been applied to the analysis of specific gamma-actin mRNA sequences amplified by polymerase chain reaction.     

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.     

Improved sensitivity for the electrochemical biosensor with an adjunct probe

Despite their promising applications in the biomedical research, the development of electrochemical biosensors with improved sensitivity and low detection limit has remained a great challenge. Here, we demonstrate a new approach to improve the sensitivity of the electrochemical biosensor by simply introducing an adjunct probe into its construction. This signal-on biosensor consists of a thiol-functionalized capture probe attached on the gold electrode surface, an electrochemical sign (methyl blue, MB)-modified reporter probe which is complementary to the capture probe, and an adjunct probe attached nearby the capture probe. The adjunct probe functions as a fixer to immobilize the element of reporter probe which is displaced by the target DNA and protein, increasing the chance of the dissociative reporter probe to collide with the electrode surface and facilitating the electron transfer. The biosensor with an adjunct probe exhibits improved sensitivity and a large dynamic range for DNA and the thrombin assay and can even distinguish 1-base mismatched target DNA. Importantly, the use of this biosensor is not limited to such and is viable for sensitive detection of numerous biomolecules, including RNA, proteins, and small molecules such as cocaine.     

Small-volume detection of Plasmodium falciparum CSP gene using a 50-microm-diameter cavity with self-contained electrochemistry

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1(0)P, which was of two different sequences we call 1(0)P(a) and 1(0)P(b)) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1(0)P(a), 47 bases were complementary to T1 and T2 at 543-590, while on 1(0)P(b), 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2(0)P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.     

Detection of Single DNA Molecule Hybridization on a Surface by Atomic Force Microscopy

Improving the detection of DNA hybridization is a critical issue for several challenging applications encountered in microarray and biosensor domains. Herein, it is demonstrated that hybridization between complementary single‐stranded DNA (ssDNA) molecules loosely adsorbed on a mica surface can be achieved thanks to fine‐tuning of the composition of the hybridization buffer. Single‐molecule DNA hybridization occurs in only a few minutes upon encounters of freely diffusing complementary strands on the mica surface. Interestingly, the specific hybridization between complementary ssDNA is not altered in the presence of large amounts of nonrelated DNA. The detection of single‐molecule DNA hybridization events is performed by measuring the contour length of DNA in atomic force microscopy images. Besides the advantage provided by facilitated diffusion, which promotes hybridization between probes and targets on mica, the present approach also allows the detection of single isolated DNA duplexes and thus requires a very low amount of both probe and target molecules.     

Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum hsp70 mRNA

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Cryptosporidium parvum in water has been developed. The target molecule was a 121-nucleotide sequence from the C. parvum heat shock protein 70 (hsp70 mRNA from U71181 gene). This analyte offers the possibility of distinguishing dead from live oocysts. The assay involves covalent attachment of a primary DNA probe via its 5'-amine-terminus to self-assembled monolayers of mercaptoundecanoic acid to a gold surface. The primary DNA probe was used to capture the target (sequence 1039-1082 of U71181 gene for the mRNA), by hybridization to a 20-base complementary sequence on the target (at sequence 1063-1082). A secondary DNA probe labeled with alkaline phosphatase (AP) was then hybridized to base sequence 1039-1062 on the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution, in which gold-coated silicon wafer modified with the complete assembly of the assay components was incubated, is linear with concentration of the target (5-50 microg/mL, where P1 and P2-AP concentrations are 50 microg/mL). A detection limit of 2 microg/mL (or 146 nM) of the DNA target was obtained. Cross-reactivity tests showed high selectivity for heat-shocked C. parvum. No signal was obtained for either the synthetic DNA for hsp70 of Campylobacter lari, Escherichia coli, Giardia lamblia, Salmonella typhimurium, and Listeria monocytogenes or for the products of heat-shocked whole organisms of E. coli, G. lamblia, Staphylococcus aureus, and Cryptosporidium muris.     

Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.     

Imaging gold nanoparticles for DNA sequence recognition in biomedical applications

The hybridization of single-stranded oligonucleotide-derivatized gold nanoparticles (Au nanoprobes) with double stranded complementary DNA was directly observed by atomic force microscopy (AFM). This specific interaction is the basis for an Au nanoprobe-based homogeneous assay for specific DNA sequence detection, based on salt-induced particle aggregation that is prevented when a complementary target is present. For long DNA targets (linearized plasmid DNA) complicated hybridized target DNA-Au-nanoprobes structures were formed, that were interpreted as the basis for stability of the Au nanoprobes against salt-induced aggregation. For shorter DNA targets (PCR amplified fragments) hybridization with the Au nanoprobes occurred, in the majority of cases, in the expected location of the DNA target fragment containing the specific sequence. The formation of the observed DNA hybridized structures provides evidence at the molecular level for specific hybridization to the target sequence as the method of binding of the Au nanoprobes.     

Combining a nanowire SERRS sensor and a target recycling reaction for ultrasensitive and multiplex identification of pathogenic fungi

Development of a rapid, sensitive, and multiplex pathogen DNA sensor enables early diagnosis and, subsequently, the proper treatment of infectious diseases, increasing the possibility to save the lives of infected patients. Here, the development of an ultrasensitive and multiplex pathogen DNA detection method that combines a patterned Au nanowire (NW)-on-film surface-enhanced resonance Raman scattering (SERRS) sensor with an exonuclease III-assisted target DNA recycling reaction is reported. Multiple probe DNAs are added to the target DNA solution, and among them, only the complementary probe DNA is selectively digested by exonuclease III, resulting in the decrease in its concentration. The digestion process is repeated by recycling of target DNAs. The decrease of the complementary probe DNA concentration is detected by SERRS. Combining the high sensitivity of the NW-on-film sensor and the target recycling reaction significantly improves DNA detection performance, resulting in the detection limit of 100 fM corresponding to 3 amole. By positioning Au NWs at specific addresses, multiple pathogen DNAs can be identified in a single step. Clinical sample tests with multiple genomic DNAs of pathogens show the potential of this sensor for practical diagnosis of infectious diseases.     

High sensitivity DNA detection based on regioselectively decorated electrocatalytic nanoparticles

Self-assembled monolayers (SAMs) of dodecanethiol have been formed on gold electrodes to produce nanoscale defects. These defects define nucleation sites for the electrodeposition of mushroom shaped platinum nanoparticles (PtNPs). The top surfaces of these PtNPs have been selectively functionalized with single stranded probe DNA. These regioselectively modified particles were desorbed by applying a current jump to yield nanoparticles capable of biorecognition on the top curved side and efficient electrocatalysis on the nonfunctionalized lower surface. A second electrode was functionalized with single stranded capture DNA that has a sequence that is complementary to the pathogen, Staphylococcus aureus but leaves a section of the target available to bind the probe strand immobilized on the PtNPs. Following hybridization of the target and capture strands, the surface was exposed to the probe DNA labeled electrocatalytic PtNPs. Target binding was detected by monitoring the current associated with the reduction of hydrogen peroxide in a solution of 0.01 M H(2)SO(4). Calibration plots of the log[DNA] versus faradaic current were linear from 10 pM to 1 μM and picomolar concentrations could be detected without the need for amplification of the target, for example, using PCR or NASBA. As well as a wide dynamic range, this detection strategy has an excellent ability to discriminate DNA mismatches and a high analytical sensitivity.     

DNA detection on plastic: surface activation protocol to convert polycarbonate substrates to biochip platforms

A mild and efficient surface activation protocol to convert polycarbonate (PC) substrates, e.g., plastic bases of compact disks, to biochip platforms for DNA probe immobilization and target detection is described. The preparation procedure (activation, patterning, and coupling) is simple and effective; the on-chip hybridization is sensitive and selective. Particularly, UV/ozone treatment of PC sheets produces a hydrophilic surface with a high density of reactive carboxylic acid groups [(4.8 +/- 0.2) x 10-10 mol/cm2] in less than 10 min at ambient conditions, and no significant aging or physical damage to the substrate is observed. Covalent immobilization of DNA probes via both passive (reagent-less photopatterning and coupling in bulk solution phase) and flow-through (creation of microarrays with microfluidic channel plates) procedures has been demonstrated. Subsequent hybridization shows uniform and strong fluorescent signals for complementary target DNA and allows clear discrimination between fully complementary targets and strands with a single base-pair mismatch. The surface chemistry described herein will facilitate the development of disposable plastic biochips (not limited to DNA microarrays) and the fabrication of biomedical devices that are readable with conventional optical drives.     

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.     

Application of graphene–pyrenebutyric acid nanocomposite as probe oligonucleotide immobilization platform in a DNA biosensor

A stable and uniform organic–inorganic nanocomposite that consists of graphene (GR) and pyrenebutyric acid (PBA) was obtained by ultrasonication, which was characterized by scanning electron microscopy (SEM) and UV–vis absorption spectra. The dispersion was dropped onto a gold electrode surface to obtain GR–PBA modified electrode (GR–PBA/Au). Electrochemical behaviors of the modified electrode were characterized by cyclic voltammetry and electrochemical impedance spectroscopy using [Fe(CN)₆]^3 ?/4 ? as the electroactive probe. A novel DNA biosensor was constructed based on the covalent coupling of amino modified oligonucleotides with the carboxylic group on PBA. By using methylene blue (MB) as a redox-active hybridization indicator, the biosensor was applied to electrochemically detect the complementary sequence, and the results suggested that the peak currents of MB showed a good linear relationship with the logarithm values of target DNA concentrations in the range from 1.0 × 10^? 15 to 5.0 × 10^? 12 M with a detection limit of 3.8 × 10^? 16 M. The selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences.     

Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system

A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples.     

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.     

Label-free and high-resolution protein/DNA nanoarray analysis using Kelvin probe force microscopy

Using the scanning probe technique known as Kelvin probe force microscopy it is possible to successfully devise a sensor for charged biomolecules. The Kelvin probe force microscope is a tool for measuring local variations in surface potential across a substrate of interest. Because many biological molecules have a native state that includes the presence of charge centres (such as the negatively charged backbone of DNA), the formation of highly specific complexes between biomolecules will often be accompanied by local changes in charge density. By spatially resolving this variation in surface potential it is possible to measure the presence of a specific bound target biomolecule on a surface without the aid of special chemistries or any form of labelling. The Kelvin probe force microscope presented here is based on an atomic force microscopy nanoprobe offering high resolution (<10 nm), sensitivity (<50 nM) and speed (>1,100 microm s(-1)), and the ability to resolve as few as three nucleotide mismatches.