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.     

Unlabeled hairpin-DNA probe for the detection of single-nucleotide mismatches by electrochemical impedance spectroscopy

An unlabeled hairpin-DNA probe was used for the detection of eight single-nucleotide mismatches by electrochemical impedance spectroscopy (EIS). Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened and forms a duplex DNA. Accordingly, the film thickness is increased, which causes differences in the electrical properties of the film before and after hybridization. Randles equivalent circuits were employed to evaluate the EIS result. The differences in the charge-transfer resistance DeltaR(CT) between hairpin DNA (before hybridization) and duplex DNA (after hybridization) shows the consequence of a large structural rearrangement from hairpin to duplex. If a single-nucleotide mismatch is present in the center of the duplex, the difference in charge-transfer resistance DeltaR(CT) between B-DNA in the absence and presence of Zn(2+) allows the unequivocal detection of all eight single-nucleotide mismatches. The detection limit was measured, and DeltaR(CT) allows the discrimination of a single-nucleotide mismatch with the concentration of the target strand as low as 10 pM.     

Structure-function relationships of three kinds of immobilized DNA probes in the discrimination abilities of single nucleotide variation

We have compared here the nucleic acid hybridization abilities of three kinds of immobilized probes with different structures. Under the conventional structural design, we found that the share-stem hairpin structure probe (SHP) was more easily hybridized with the target than the linear probe and conventional hairpin shaped probe (HP). The HP probe had the lowest hybridization ability. However, it was shown a contrary result in the single-nucleotide mismatch discrimination ratio. Subsequently, we increased the Tm of the two hairpin-shaped probes and found both of the two probes were improved on the hybridization specificities even though the hybridization abilities were decreased. The result of the hybridization temperature optimization experiment showed that at 45 degrees C, the Dr Ratio (mismatch discrimination ratio) of HP-28T and SHP-32T were both as high as 8.5 while the ratios of linear probe were -0.3-0.7. The results suggested that the immobilized shared stem hairpin shaped probes also had the ability to discriminate single-nucleotide variation under proper design.     

Electrochemical detection of DNA hybridization using biometallization

We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.     

Direct detection of DNA conformation in hybridization processes

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.     

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.     

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.     

Monitoring molecular beacon/DNA interactions using atomic force microscopy

The molecular beacon (MB) is a new fluorescence probe containing a single-stranded oligonucleotide with a probe sequence embedded in complementary sequences that form a hairpin stem. Due to the inherent fluorescent signal transduction mechanism, an MB functions as a sensitive probe with a high signal-to-background ratio for real-time monitoring and provides a variety of exciting opportunities in DNA, RNA, and protein studies. To better understand the properties of MBs, the specific interactions between MB and target DNA (complementary and one-base mismatch) have been directly investigated by atomic force microscopy. The interaction force between a linear DNA probe and the target DNA was also detected and compared to that between MB and target DNA. The results demonstrate the high specificity of the MB/target DNA compared to the linear DNA/target DNA interaction.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.     

"Off-On" Electrochemical Hairpin-DNA-Based Genosensor for Cancer Diagnostics

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.     

Sensitive and homogeneous protein detection based on target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification

Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.     

DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.     

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.     

Dynamic DNA hybridization on a chip using paramagnetic beads

Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

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.     

Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.     

Magnetic particle-based sandwich sensor with DNA-modified carbon nanotubes as recognition elements for detection of DNA hybridization

In this contribution, we design a visual sensor for DNA hybridization with DNA probe-modified magnetic particles (MPs) and multiwalled carbon nanotubes (MWNTs) without involving a visual recognition element such as fluorescent/chemiluminescent reagents. It was found that DNA probe-modified MWNTs, which could be dispersed in aqueous medium and have strong light scattering signals under the excitation of a light beam in the UV-vis region, could connect with DNA probe-modified MPs together in the presence of perfectly complementary target DNA and form a sandwich structure. In a magnetic field, the formed MP-MWNT species can easily be removed from the solution, resulting in a decrease of light scattering signals. Thus, a magnetic particle-based sandwich sensor could be developed to detect DNA hybridization by measuring the light scattering signals with DNA-modified MWNTs as recognition elements. Experiments showed that the DNA-modified MPs sensor could be reused at least 17 times and was stable for more than 6 months.     

Detection of human immunodeficiency virus type 1 DNA sequence using plasmonics nanoprobes

This paper describes the use of plasmonics-based nanoprobes that act as molecular sentinels for DNA diagnostics. The plasmonics nanoprobe comprises a metal nanoparticle and a stem-loop DNA molecule tagged with a Raman label. The nanoprobe utilizes the specificity and selectivity of the DNA hairpin probe sequence to detect a specific target DNA sequence of interest. In the absence of target DNA, the stem-loop configuration maintains the Raman label in proximity to the metal nanoparticle, inducing an intense surface-enhanced Raman scattering (SERS) effect that produces a strong Raman signal upon laser excitation. Upon hybridization of a complementary target DNA sequence to the nanoprobe, the stem-loop configuration is disrupted, causing the Raman label to physically separate from the metal nanoparticle, thus quenching the SERS signal. The usefulness and potential application of the plasmonics nanoprobe for diagnosis is demonstrated using the gag gene sequence of the human immunodeficiency virus type 1 (HIV-1). We successfully demonstrated the specificity and selectivity of the plasmonics nanoprobes to detect PCR amplicons of the HIV gene. The potential for combining the spectral selectivity and high sensitivity of the SERS process with inherent molecular specificity of DNA hairpins to diagnose molecular target sequences in homogeneous solutions is discussed.     

Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation

DNA methylation catalyzed by methylase plays an important role in many biological events. However, traditional methods of methylase activity analysis by gel electrophoresis were laborious and discontinuous. In this paper, we report a new strategy to study methylase activity using fluorescent probes coupled with enzyme-linkage reactions. A hairpin DNA probe is prepared with a fluorophore and a quencher linked at the 5'- and 3'-terminus of the probe. A disturbance of the stem sequence by DNA methylation would cause the separation of the fluorophore and the quencher, resulting in the restoration of the fluorescence. We used DNA adenine methylation (Dam) methyltransferase (MTase) and Dpn I endonuclease, both having a 5'-G-A-T-C-3' recognition sequence. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and Dpn I cut the sequence of 5'-G-Am-T-C-3'. The fluorescence of the hairpin probe was restored when it was cleaved by Dpn I endonuclease during the course of methylation. Unlike traditional methods, this assay was done in real time and could be used to monitor the dynamic process of methylation. Our method is easy, simple, and nonradioactive, yet as efficient as gel electrophoresis in detecting the activity of methylase. It also had the potential to screen suitable inhibitor drugs for Dam methylase.