Selective DNA Detection Research Articles

Quantum dots modified with the Al(III)-pefloxacin complex as a novel bioprobe for the sensitive turn-on and dual-fluorescence detection of dsDNA

We describe a novel fluorescent bioprobe for the sensitive and selective detection of double-stranded DNA (dsDNA). It consists of quantum dots (Q-dots) whose fluorescence is quenched (through photoinduced electron transfer) on covering them with the Al(III)-pefloxacin complex. It is found, however, that dsDNA has an affinity for this complex that is so strong that it can break up the binding to the quantum dots, this leading to a complete recovery of the fluorescence of both the Q-dots and the Al(III)-pefloxacin complex. Dual fluorescence (peaking at 420 and 547 nm) is observed under a single excitation wavelength of 350 nm. Neither ribonucleic acid, bovine serum albumin, nor biologically relevant metal ions are capable of giving this effect. The detection limit (3σ/k) for herring sperm DNA is 15.3 μg L−1 if determined via the fluorescence of the Q-dots, and 48 μg L−1 via the Al(III)-pefloxacin complex. The method was applied to the determination of hsDNA in synthetic samples and gave excellent results.

Use of a capacitive affinity biosensor for sensitive and selective detection and quantification of DNA—A model study

A capacitive DNA-sensor model system was used to monitor the capture of complementary single-stranded DNAs. The sensor chip consisted of a gold electrode, which was carefully insulated with a polytyramine layer and covalently tagged with 25-mer oligo-C. As low as 10−11 moles per liter of target oligo-G could be detected by injecting 250μL of sample. Elevated temperature was used to reduce non-specific hybridization. Less than 10% of non-target 25-mer oligo-T interacted nonspecifically with the oligo-C probes when hybridization process was performed at 50°C. Studying the relationship of length of the analyte to the signal strength, the output from the capacitive DNA-sensor increased to almost the double; from 50 to 88-nFcm−2, when a 25-mer oligo-G was used instead of a 15-mer. By sandwich hybridization at room temperature, it was possible to further increase the signal, from 78-nFcm−2 for the target 50-mer oligo-G alone, to 114-nFcm−2.

Synthesis and evaluation of new BODIPY-benzofuroquinoline conjugates for sensitive and selective DNA detection

The sensitive and selective detection of nucleic acids is important for basic research and many applied fields. Herein, a series of new BODIPY-benzofuroquinoline conjugates were designed, synthesized and evaluated as DNA intercalating dyes. All compounds were characterized by using 1H, 13C NMR, IR, UV–Vis and fluorescence spectroscopy, and DNA binding properties of these conjugates to calf thymus DNA were studied by using fluorescence titration, UV titration, isothermal titration calorimetry and CD analysis. Significant enhancement of the fluorescent quantum yield was observed for all the conjugates in the presence of calf thymus DNA, and one compound showed excellent sensitivity and selectivity offering its potential application as a DNA specific fluorescent probe. Our results showed that these conjugates could intercalate into calf thymus DNA with high binding affinities. The properties of these dyes as fluorescent probes for living cells imaging were also investigated.

2,6-Bis(arylsulfonyl)anilines as Fluorescent Scaffolds through Intramolecular Hydrogen Bonds: Solid-State Fluorescence Materials and Turn-On-Type Probes Based on Aggregation-Induced Emission.

A series of 2,6-bis[aryl(alkyl)sulfonyl]anilines were synthesized by nucleophilic aromatic substitution of 2,6-dichloronitrobenzene with various aryl or alkyl thiolates (benzyl-, phenyl-, 2-naphthyl-, and 2-aminophenyl thiolate), followed by hydrogenation and subsequent oxidation. All prepared 2,6-bis[aryl-(alkyl)sulfonyl]anilines showed high fluorescence emissions in the solid state; X-ray structures revealed well-defined intramolecular hydrogen bonds, which served to immobilize the rotatable amino group and generate a fluorescence enhancement in addition to improved photostability. Moreover, absorption and fluorescence spectra showed redshifts in the order of benzyl<phenyl<2-naphthyl in solution and the solid state, and DFT calculations confirmed charge transfer from the central aniline unit to the side aryl groups through the sulfonyl bridges, which together indicated the push-pull effect with an extended π-conjugated system comprising the 2,6-bis(arylsulfonyl)aniline unit. Furthermore, 2,6-bis(2-aminophenylsulfonyl)aniline, with rotatable amino groups at the flanking aryl units, was affected by fluorescence quenching in solution. This nonemissive state showed an enhanced environmental sensitivity to solvent polarity and viscosity, along with a "turn-on" fluorescence response in frozen solvent and in the aggregated form. 2,6-Bis(2-aminophenylsulfonyl)aniline behaved as a turn-on fluorescence probe for selective detection of DNA based on its aggregation-induced emission.

PCR-Free Ultrasensitive Capacitive Biosensor for Selective Detection and Quantification of Enterobacteriacea DNA

This paper presents a flow-based ultrasensitive capacitive biosensor for the detection of bacterial DNA. The used sensor chip consists of a gold electrode, insulated with a polytyramine layer and covalently tagged with a DNA capture probe. The hybridization of target DNA to the capture probe resulted in sensor response. The sensor response was linear vs. log concentration in the range 1.0 × 10-12 to 1.0 × 10-7 moles per litre with a detection limit of 6.5 × 10-13 M. An alternative approach to bacterial DNA sample preparation for a flow-based analysis is also reported. The approach involved application of a thermostable ssDNA binding protein to prevent re-annealing of a heatdenatured target DNA prior to analysis. During analysis, formamide was integrated in the running buffer to denature ET SSB. E. coli DNA corresponding to 10 cells per millilitre of sample was detected in 15 min by this DNA-sensor. The sensor chip could be re-used up to 20 times with RSD of < 6%. The DNA-sensor chip was able to discriminate between Enterobacteriaceae (E. coli) and Lactobacillaceae (L. reuteri) DNAs. The reported DNA-sensor lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.

A sensitive electrochemical DNA biosensor based on silver nanoparticles-polydopamine@graphene composite

A novel electrochemical biosensor for sensitive and selective DNA detection was constructed based on polydopamine@graphene composite functionalized with silver nanoparticles (AgNPs-Pdop@Gr). The Pdop@Gr was prepared by simple chemical route and AgNPs further deposited over the Pdop@Gr by mildly stirring to form AgNPs-Pdop@Gr composite films, which was considered as potential sensing platform. DNA labeled at 5′ end using 6-mercapto-1-hexhane (HS-ssDNA) was immobilized on AgNPs-Pdop@Gr surface to form ssDNA-S-AgNPs-Pdop@Gr and hybridization sensing was done in phosphate buffer. Differential pulse voltammetry was applied to monitor the DNA hybridization event using methylene blue as an electrochemical indicator. Under optimum conditions, the peak currents of methylene blue were linear with the logarithm of the concentrations of complementary DNA from 1.0×10−13 to 1.0×10−8M with a detection limit of 3.2×10−15M (3σ/S). The biosensor also displayed high selectivity to differentiate one-base mismatched DNA. All these excellent performances of the developed biosensor indicated that this sensing platform could be easily extended to the detection of other nucleic acids.

A sensitive biosensing strategy for DNA detection based on graphene oxide and T7 exonuclease assisted target recycling amplification

A fluorescence biosensing strategy based on graphene oxide (GO) was reported for simple, rapid, sensitive, and selective DNA detection by T7 exonuclease assisted target recycling amplification. Due to the super fluorescence quenching efficiency of GO, the fluorescein amiditelabeled signal probe was firstly adsorbed onto the surface of GO and the fluorescence was quenched. Owing to its excellent selectivity for double-stranded DNA, T7 exonuclease was chosen as a signal-amplifying biocatalyst to improve the detection sensitivity. In the presence of target DNA, the signal probe could bind with target DNA and form a DNA duplex structure to trigger the digestion of the signal probe by T7 exonuclease, leading to the recycling of target DNA and the increasing of fluorescence intensity. Upon the recycling use of target DNA, this method achieved a high sensitivity towards target DNA with a detection limit of 0.3 pmol/L, which was lower than previously reported for GO-based DNA biosensors. Moreover, it does not require complex modifications of the molecular beacon and time-consuming thermal cycling procedures. Thus, the simple strategy provides a universal biosensing platform for DNA detection and it could find wide applications in DNA damage analysis and diagnostics.

Rationally Designed Nucleobase and Nucleotide Coordinated Nanoparticles for Selective DNA Adsorption and Detection

Nanomaterials for DNA adsorption are useful for sequence-specific DNA detection. Current materials for DNA adsorption employ electrostatic attraction, hydrophobic interaction, or π-π stacking, none of which can achieve sequence specificity. Specificity might be improved by involving hydrogen bonding and metal coordination. In this work, a diverse range of nucleobase/nucleotide (adenine, adenosine, adenosine 5'-triphosphate (ATP), adenosine 5'-monophosphate (AMP), and guanosine 5'-triphosphate (GTP)) coordinated materials containing various metal ions (Au(III), Ag(I), Ce(III), Gd(III), and Tb(III)) are prepared. In most cases, nanoparticles are formed. These materials have different surface charges, and positively charged particles only show nonspecific DNA adsorption. Negatively charged materials give different adsorption kinetics for different DNA sequences, where complementary DNA homopolymers are adsorbed faster than other sequences. Therefore, the bases in the coordinated materials can still form base pairs with the DNA. The adsorption strength is mainly controlled by the metal ions, where Au shows the strongest adsorption while lanthanides are weaker. These materials can be used as sensors for DNA detection and can also deliver DNA into cells with no detectable toxicity. By tuning the nanoparticle formulation, enhanced detection can be achieved. This study is an important step toward rational design of materials to achieve specific interactions between biomolecules and synthetic nanoparticle surfaces.

A label-free electrochemical biosensor for highly sensitive and selective detection of DNA via a dual-amplified strategy

In this work, by combining the enzymatic recycling reaction with the DNA functionalized gold nanoparticles (AuNPs)-based signal amplification, we have developed an electrochemical biosensor for label-free detection of DNA with high sensitivity and selectivity. In the new designed biosensor, a hairpin-structured probe HP was designed to hybridize with target DNA first, and an exonuclease ExoIII was chosen for the homogeneous enzymatic cleaving amplification. The hybridization of target DNA with the probe HP induced the partial cleavage of the probe HP by ExoIII to release the enzymatic products. The enzymatic products could then hybridize with the hairpin-structured capture probe CP modified on the electrode surface. Finally, DNA functionalized AuNPs was further employed to amplify the detection signal. Due to the capture of abundant methylene blue (MB) molecules by both the multiple DNAs modified on AuNPs surface and the hybridization product of capture DNA and enzymatic products, the designed biosensor achieved a high sensitivity for target DNA, and a detection limit of 0.6pM was obtained. Due to the employment of two hairpin-structured probes, HP and CP, the proposed biosensor also exhibited high selectivity to target DNA. Moreover, since ExoIII does not require specific recognition sequences, the proposed biosensor might provide a universal design strategy to construct DNA biosensor which can be applied in various biological and medical samples.

Ultrasensitive and label-free electrochemical DNA biosensor based on water-soluble electroactive dye azophloxine-functionalized graphene nanosheets

This paper reported an ultrasensitive and label-free electrochemical DNA biosensing platform based on a newly synthesized water-soluble electroactive dye azophloxine-functionalized graphene nanosheets (AP-GNs). Azophloxine can be attached on the graphene surface through synergistic noncovalent charge-transfer and π–π stacking interactions with graphene. The attachment of azophloxine on the graphene surface not only acts as a stabilizer for graphene but also as the in situ probe for the electrochemical DNA detection. Under optimum conditions, this biosensor exhibited high sensitivity and low detection limit for detecting DNA. The linear range is from 1.0×10−15 to 1.0×10−11molL−1 and the detection limit is 4.0×10−16molL−1. Furthermore, this biosensor showed extraordinary capability for single nucleotide polymorphisms (SNPs) assay. The results demonstrate that this AP-GNs based biosensor has potential application in sensitive and selective DNA detection.

An electrochemical DNA biosensor based on gold nanorods decorated graphene oxide sheets for sensing platform

A simple electrochemical sensor for sensitive and selective DNA detection was constructed based on gold nanorods (Au NRs) decorated graphene oxide (GO) sheets. The high-quality Au NRs–GO nanocomposite was synthesized via the electrostatic self-assembly technique, which is considered a potential sensing platform. Differential pulse voltammetry was used to monitor the DNA hybridization event using methylene blue as an electrochemical indicator. Under optimal conditions, the peak currents of methylene blue were linear with the logarithm of the concentrations of complementary DNA from 1.0×10−9 to 1.0×10−14M with a detection limit of 3.5×10−15M (signal/noise=3). Moreover, the prepared electrochemical sensor can effectively distinguish complementary DNA sequences in the presence of a large amount of single-base mismatched DNA (1000:1), indicating that the biosensor has high selectivity.

Graphene nanonet for biological sensing applications

We report a simple but efficient method to fabricate versatile graphene nanonet (GNN)-devices. In this method, networks of V2O5 nanowires (NWs) were prepared in specific regions of single-layer graphene, and the graphene layer was selectively etched via a reactive ion etching method using the V2O5 NWs as a shadow mask. The process allowed us to prepare large scale patterns of GNN structures which were comprised of continuous networks of graphene nanoribbons (GNRs) with chemical functional groups on their edges. The GNN can be easily functionalized with biomolecules for fluorescent biochip applications. Furthermore, electrical channels based on GNN exhibited a rather high mobility and low noise compared with other network structures based on nanostructures such as carbon nanotubes, which was attributed to the continuous connection of nanoribbons in GNN structures. As a proof of concept, we built DNA sensors based on GNN channels and demonstrated the selective detection of DNA. Since our method allows us to prepare high-performance networks of GNRs over a large surface area, it should open up various practical biosensing applications.

Selective, Highly Sensitive, and Rapid Detection of Genomic DNA by Using Gated Materials: Mycoplasma Detection

Financial support from the Spanish Government (MAT2009-14564-C04-01 and SAF2010 15512), the Generalitat Valenciana (PROM-ETEO/2009/016 and 2010/005) is gratefully acknowledged. E. C. thanks the Ministerio de Educacion for a fellowship. L. M. thanks Generalitat Valenciana for her Post-Doc VALI + D contract.

Molecular sensing with magnetic nanoparticles using magnetic spectroscopy of nanoparticle Brownian motion

Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedical applications. Here we use functionalized mNPs to develop a highly sensitive, versatile sensing strategy required in practical biological assays and potentially in vivo analysis. We demonstrate a new sensing scheme based on magnetic spectroscopy of nanoparticle Brownian motion (MSB) to quantitatively detect molecular targets. MSB uses the harmonics of oscillating mNPs as a metric for the freedom of rotational motion, thus reflecting the bound state of the mNP. The harmonics can be detected in vivo from nanogram quantities of iron within 5s. Using a streptavidin–biotin binding system, we show that the detection limit of the current MSB technique is lower than 150pM (0.075pmole), which is much more sensitive than previously reported techniques based on mNP detection. Using mNPs conjugated with two anti-thrombin DNA aptamers, we show that thrombin can be detected with high sensitivity (4nM or 2pmole). A DNA–DNA interaction was also investigated. The results demonstrated that sequence selective DNA detection can be achieved with 100pM (0.05pmole) sensitivity. The results of using MSB to sense these interactions, show that the MSB based sensing technique can achieve rapid measurement (within 10s), and is suitable for detecting and quantifying a wide range of biomarkers or analytes. It has the potential to be applied in variety of biomedical applications or diagnostic analyses.

The role of reactivity in DNA templated native chemical PNA ligation during PCR

DNA templated fluorogenic reactions have been used as a diagnostic tool for the sequence specific detection of nucleic acids; and it has been shown that the native chemical ligation between thioester- and 1,2-aminothiol-modified PNA probes is amongst the most selective DNA detection methods reported. We explored whether a DNA templated reaction can be interfaced with the polymerase chain reaction (PCR). This endeavor posed a significant challenge. The reactive groups involved must be sufficiently stable to tolerate the high temperature applied in the PCR process. Nevertheless, the ligation reaction must proceed very rapidly and sequence specifically within the short time available in the annealing and primer extension steps before denaturation is used after approx. 1min to commence the next PCR cycle. This required a careful optimization of the ternary complex architecture as well as adjustments of probe length and probe reactivities. Our results point to the prime importance of the ligation architecture. We show that once suitable annealing sites have been identified less reactive probe sets may be preferable if sequence specificity is of major concern. The reactivity tuning enabled the development of an in-PCR ligation, which was used for the single nucleotide specific typing of the V600E (T1799A) point mutation in the human BRaf gene. Showcasing the efficiency and sequence specificity of native chemical PNA ligation, attomolar template proofed sufficient to trigger signal while a 1000-fold higher load of single mismatched template failed to induce appreciable signal.

Selective DNA detection at Zeptomole level based on coulometric measurement of gold nanoparticle-mediated electron transfer across a self-assembled monolayer

A selective DNA sensing with zeptomole detection level is developed based on coulometric measurement of gold nanoparticle (AuNPs)-mediated electron transfer (ET) across a self-assembled monolayer on the gold electrode. After immobilization of a thiolated hairpin-structured DNA probe, an alkanethiol monolayer was self-assembled on the resultant electrode to block [Fe(CN)6]3−/4− in a solution from accessing the electrode. In the presence of DNA target, hybridization between the DNA probe and the DNA target breaks the stem duplex of DNA probe. Consequently, stem moiety at the 3′-end of the DNA probes was removed from the electrode surface and made available for hybridization with the reporter DNA-AuNPs conjugates (reporter DNA-AuNPs). The thiolated reporter DNA matches the stem moiety at the 3′-end of the DNA probe. AuNPs were then enlarged by immersing the electrode in a growth solution containing HAuCl4 and H2O2 after the reporter DNA-AuNPs bound onto the electrode surface. The enlarged AuNPs on the electrode restored the ET between the electrode and the [Fe(CN)6]3−/4−, as a result, amplified signals were achieved for DNA target detection using the coulometric measurement of Fe(CN)6 3− electro-reduction by prolonging the electrolysis time. The quantities of ET on the DNA sensor increased with the increase in DNA target concentration through a linear range of 3.0 fM to 1.0 pM when electrolysis time was set to 300 s, and the detection limit was 1.0 fM. Correspondingly, thousands of DNA (zeptomole) copies were detected in 10-μL samples. Furthermore, the DNA sensor showed excellent differentiation ability for single-base mismatch.

Label‐Free Electrical Detection of DNA Hybridization on Graphene using Hall Effect Measurements: Revisiting the Sensing Mechanism

AbstractThere is broad interest in using graphene or graphene oxide sheets as a transducer for label‐free and selective electrical detection of biomolecules such as DNA. However, it is still not well explored how the DNA molecules interact with and influence the properties of graphene during the detection. Here, Hall effect measurements based on the Van der Pauw method are used to perform single‐base sequence selective detection of DNA on graphene sheets, which are prepared by chemical vapor deposition. The sheet resistance increases and the mobility decreases with the addition of either complementary or one‐base mismatched DNA to the graphene device. The hole carrier concentration of the graphene devices increases significantly with the addition of complementary DNA but it is less affected by the one‐base mismatched DNA. It is concluded that the increase in hole carrier density, indicating p‐doping to graphene, is better correlated with the DNA hybridization compared to the commonly used parameters such as conductivity change. The different electrical observations of p‐doping from Hall effect measurements and n‐doping from electrolyte‐gated transistors can be explained by the characteristic morphology of partially hybridized DNA on graphene and the mismatch between DNA chain length and Debye length in electrolytes.

Imidazolium-based 1,1′-bi-2-naphthol fluorescent probe for ratiometric and selective detection of DNA in water

A water-soluble imidazolium-based BINOL receptor R-1 was exploited, which could selectively sense DNA over other biologically relevant anions including nucleotides under physiological conditions. Upon addition of DNA, R-1 displayed notably blue-shifted and ratiometric fluorescence enhancement.

PNA-assembled graphene oxide for sensitive and selective detection of DNA

DNA detection based on peptide nucleic acid (PNA)-DNA hybridization is emerging as an important method in the area of DNA microarrays and biosensors because PNA shows remarkable hybridization properties. In this work, we provide a novel, simple, sensitive, and selective strategy based on a PNA-graphene oxide (GO) assembled biosensor for fluorescence turn-on detection of DNA, in which the new nanomaterial GO was used as a scaffold for PNA and a quencher for the fluorophore. The PNA-GO assembled biosensor is capable of distinguishing sequence specificity including complementary, one-base mismatched and non-complementary targets. Moreover, the results show that the biosensor is able to detect target DNA down to hundreds of picomolar. This sensing platform has been demonstrated to be highly sensitive and specific, and we expect that it will find great applications in the field of biomedicine and disease diagnostics.

A novel quantum dots-based OFF–ON fluorescent biosensor for highly selective and sensitive detection of double-strand DNA

Abstract We have developed a novel biosensor for the rapid determination of double-strand (ds) DNA based on the photoinduced electron transfer (PET) from glutathione (GSH) capped CdTe quantum dots (QDs) to praseodymium(III)–rutin (Pr 3+ –rutin) complex. The biosensor was based on a fluorescence “OFF–ON” mode. To provide the platform for dsDNA detection, the Pr 3+ –rutin complex was first employed as an effective fluorescence quencher to QDs through PET. Because of its strong binding affinity with Pr 3+ –rutin complex, dsDNA can break up the low fluoresced ionic ensemble, set free the luminescent QDs. Thus, the recognition of dsDNA by Pr 3+ –rutin complex can be realized through the restoration of QDs fluorescence. The relative recovered fluorescence intensity is directly proportional to the concentration of herring sperm (hs) DNA between 0.0874 μg mL −1 and 20 μg mL −1 , and the detection limit (3 δ / K ) is 0.0262 μg mL −1 . The entire experimental process does not require any chemical modification and coupling of CdTe QDs and dsDNA, greatly simplifying the experimental process. Some possible reaction mechanisms are discussed.