DNA Probes For Detection Research Articles

Hybridization induced fluorescence turn-on of AIEgen–oligonucleotide conjugates for specific DNA detection

We report a two-armed AIE probes for specific DNA detection, and the signal output can be further enhanced when two probes hybridize to each.

Fluorine-modified bisbenzimide derivative as a molecular probe for bimodal and simultaneous detection of DNAs by (19)F NMR and fluorescence.

3,5-Bis(trifluoromethyl)benzene modified bisbenzimide H 33258 was synthesized as a (19)F magnetic resonance-based DNA detection probe. The chemical shift and fluorescence of the probe were significantly changed by the addition of hairpin DNAs having an AATT sequence. The probe enables (19)F NMR/fluorescence bimodal detection of the model DNA double strands simultaneously.

One-step conjugation of aminoferrocene to phosphate groups as electroactive probes for electrochemical detection of sequence-specific DNA

A straightforward electrochemical DNA biosensing approach based on exploiting organometallic compound, aminoferrocene (AFC), as electroactive probes was firstly demonstrated, where the probes could be directly labeled to the free phosphate groups of the hybridized PNA/DNA heteroduplexes merely through one-step conjugation in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. Briefly, mercapto-terminated peptide nucleic acid (PNA) was firstly immobilized onto gold electrode and used as the capture probes for the specific recognition of target single-stranded DNA (ssDNA). After hybridization, AFC probes were directly labeled to the free 5′-terminal phosphate groups, which were activated by EDC and imidazole, of the hybridized PNA/DNA heteroduplexes, and then they were exploited as the electroactive probes to monitor the hybridization. As the captured ssDNA was labeled with AFC in the stoichiometric ratio of 1:1, thus the electrochemical analysis of the proportionally labeled AFC based on differential pulse voltammetry (DPV) enabled a quantitative determination of sequence-specific DNA. Under optimal conditions, the approach presented a good linear relationship between the current intensities and logarithm of ssDNA concentrations in the range from 0.1nM to 100nM with a detection limit of 93pM, and it rendered satisfactory analytical performance in serum samples. Furthermore, it exhibited excellent specificity toward single-nucleotide polymorphism (SNP) and precluded complicated protocols. More importantly, the simplicity of this approach together with its compatibility with standard micro-fabrication techniques makes it great potential in practical applications, especially in microarray areas where simple procedures are preferred.

Label-free molecular beacons for biomolecular detection.

Biomolecular detection and imaging methods provide quantitative measurements essential for biological research. In this context, molecular beacon based sensors have emerged as powerful, no-wash imaging agents, providing target-specific fluorescent activation for nucleic acids, proteins, and small molecules. Conventional molecular beacons require double-labeled DNA sequences, which are costly and time-consuming to prepare. To address this issue, we developed DNA based label-free molecular beacons consisting of two regions: a signal-generating region based on human telomeric G-quadruplex sequence that activates Thioflavin T fluorescence and a target recognition sequence designed to interact in a molecular beacon format. We demonstrated the utility of these probes for the selective detection of DNA, RNA, and protein. Multiple probes were applied against a single target to achieve improved brightness in fluorescence detection of nucleic acid targets. This label-free strategy provides a straightforward, cost-effective alternative to fluorescently labeled oligonucleotides in biomolecular detection and imaging.

Ultrasensitive electroanalysis of low-level free microRNAs in blood by maximum signal amplification of catalytic silver deposition using alkaline phosphatase-incorporated gold nanoclusters.

An ultrasensitive sandwich-type analysis method has been initially developed for probing low-level free microRNAs (miRNAs) in blood by a maximal signal amplification protocol of catalytic silver deposition. Gold nanoclusters (AuNCs) were first synthesized and in-site incorporated into alkaline phosphatase (ALP) to form the ALP-AuNCs. Unexpectedly, the so incorporated AuNCs could dramatically enhance the catalysis activities of ALP-AuNCs versus native ALP. A sandwiched hybridization protocol was then proposed using ALP-AuNCs as the catalytic labels of the DNA detection probes for targeting miRNAs that were magnetically caught from blood samples by DNA capture probes, followed by the catalytic ligation of two DNA probes complementary to the targets. Herein, the ALP-AuNC labels could act as the bicatalysts separately in the ALP-catalyzed substrate dephosphorylation reaction and the AuNCs-accelerated silver deposition reaction. The signal amplification of ALP-AuNCs-catalyzed silver deposition was thereby maximized to be measured by the electrochemical outputs. The developed electroanalysis strategy could allow for the ultrasensitive detection of free miRNAs in blood with the detection limit as low as 21.5 aM, including the accurate identification of single-base mutant levels in miRNAs. Such a sandwich-type analysis method may circumvent the bottlenecks of the current detection techniques in probing short-chain miRNAs. It would be tailored as an ultrasensitive detection candidate for low-level free miRNAs in blood toward the diagnosis of cancer and the warning or monitoring of cancer metastasis in the clinical laboratory.

Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence

In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5nM, Mao et al. Anal. Chem. 2009, 81, 1660–1668). Another important feature is that the preparation of MWCNT–DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT–antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3′-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria.

Changing trends in diagnostics of trypanosomosis in animals

Animal trypanosomosis presents special problems with regard to diagnosis since the clinical signs are not pathognomonic and standard parasitological techniques are not sufficiently sensitive. Formol-gel and mercuric chloride tests using serum of infected animals were adopted as a routine diagnostic tool for trypanosomosis, however, these chemical tests suffer from inherent problem of non specificity. To overcome this problem, alternative methods of diagnosis were developed, which detected antibody responses to antigens of infecting trypanosomes. Indirect immunofluorescent antibody test, enzyme immunoassay (ELISA) and the card agglutination test for trypanosomosis (CATT) were found to be useful tests for the diagnosis of Trypanosoma evansi infections in view of their sensitivity and specificity. However, antibody detection tests failed to distinguish between current and past infections because of persistent antibody titres. Recently, development of assays for the detection of circulating trypanosomal antigens in infected animals has circumvented this problem since antigen-positivity indicates existing infection. Latex agglutination test, being simple to perform, rapid, convenient, cost-effective could be quite suitable for field-level diagnosis and screening of trypanosomosis. Presently molecular diagnostic techniques like polymerase chain reaction (PCR) and DNA probes for detection of parasitic DNA have been used more efficiently as these indicate a sure sign of an active infection. These techniques eliminate the possibilities of cross-reaction and offer high specificity and sensitivity for detection of trypanosomosis in animals.

A Supercharged Fluorescent Protein as a Versatile Probe for Homogeneous DNA Detection and Methylation Analysis

AbstractSupercharged proteins are a new class of functional proteins with exceptional stability and potent ability to deliver bio‐macromolecules into cells. As a proof‐of‐principle, a novel application of supercharged proteins as a versatile biosensing platform for nucleic acid detection and epigenetics analysis is presented. Taking supercharged green fluorescent protein (ScGFP) as the signal reporter, a simple turn‐on homogenous method for DNA detection has been developed based on the polyionic nanoscale complex of ScGFP/DNA and toehold strand displacement. This assay shows high sensitivity and potent ability to detect single‐base mismatch. Furthermore, combined with bisulfite conversion, this ScGFP‐based assay was further applied to analyze site‐specific DNA methylation status of genomic DNA extracted from real human colon carcinoma tissue sample with ultrahigh sensitivity (4 amol methylated DNA).

A supercharged fluorescent protein as a versatile probe for homogeneous DNA detection and methylation analysis.

Supercharged proteins are a new class of functional proteins with exceptional stability and potent ability to deliver bio-macromolecules into cells. As a proof-of-principle, a novel application of supercharged proteins as a versatile biosensing platform for nucleic acid detection and epigenetics analysis is presented. Taking supercharged green fluorescent protein (ScGFP) as the signal reporter, a simple turn-on homogenous method for DNA detection has been developed based on the polyionic nanoscale complex of ScGFP/DNA and toehold strand displacement. This assay shows high sensitivity and potent ability to detect single-base mismatch. Furthermore, combined with bisulfite conversion, this ScGFP-based assay was further applied to analyze site-specific DNA methylation status of genomic DNA extracted from real human colon carcinoma tissue sample with ultrahigh sensitivity (4 amol methylated DNA).

Novel styryldehydropyridocolinium derivative as turn-on fluorescent probe for DNA detection

Novel styryldehydropyridocolinium derivative L was prepared and found to have low fluorescence quantum yield in water; however, upon addition of DNA, the fluorescence intensity increased by a factor of 60-fold. Meanwhile, it also showed rapid cellular uptake properties in cell imaging due to its cationic structure favored for cell membrane permeability.

Phosphorescent quantum dots/doxorubicin nanohybrids based on photoinduced electron transfer for detection of DNA

MPA-capped Mn-doped ZnS QDs/DXR nanohybrids (MPA: 3-mercaptopropionic acid; QDs: quantum dots; DXR: cetyltrimethyl ammonium bromide) were constructed via photoinduced electron transfer (PIET) and then used as a room-temperature phosphorescence (RTP) probe for detection of DNA. DXR as a quencher will quench the RTP of Mn-doped ZnS QDs via PIET, thereby forming Mn-doped ZnS QDs/DXR nanohybrids and storing RTP. With the addition of DNA, it will be inserted into DXR and thus DXR will be competitively desorbed from the surface of Mn-doped ZnS QDs, thereby releasing the RTP of Mn-doped ZnS QDs. Based on this, a new method for DNA detection was built. The sensor for DNA has a detection limit of 0.039mgL−1 and a linear range from 0.1 to 14mgL−1. The present QDs-based RTP method does not need deoxidants or other inducers as required by conventional RTP detection methods, and avoids interference from autofluorescence and the scattering light of the matrix that are encountered in spectrofluorometry. Therefore, this method can be used to detect the DNA content in body fluid.

Universal Electrochemical Probe for Genotyping

DNA analysis is a fundamental topic of research leading to many studies related to the detection of disease-causing microbes, gene variations causing genetic disorders, DNA mapping, and evolutionary links between ancient and modern remains. This study uses stem-loop folded DNA (SLF-DNA) probe immobilized on the surface of an electrode as a part of tri-component sensor for detection of specific DNA. The advantages over traditional methods such as Southern and Northern Blots, or PCR amplification include the possibility to detect low quantities of unlabeled target analyte, high specificity of nucleic acid recognition and possibility to use the same immobilized DNA probe for detection of many different sequences.In our approach hairpin shaped oligonucleotide (SLF-DNA) is immobilized onto a gold electrode and the signal of specific tag is measured. Importantly, immobilized SLF-DNA does not directly hybridize with the target analyte, but via the two adaptor strands. This design allows application of the same electrode in the same immobilized adaptor strand to be used for the detection of almost any target, relatively inexpensive adaptor should be changes to recognize a new DNA/RNA analyte. These electrochemical biosensors are simple to use, low cost, low power, portable with fast response times, and high specificity and sensitivity.

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.

The frequency characteristics analysis of series photodetector frequency circuit system based on circuit parameters of quartz crystal and its application for bacterial DNA identification

The circuit parameters of quartz crystal were employed for frequency sensitivity analysis of series photodetector frequency circuit system. The influence of circuit parameters of quartz crystal on the oscillation frequency and response sensitivity were theoretically derived and experimentally verified. On the basis of optimal circuit parameters, the DNA probe detection limit 2pmol/L can be measured by 49.4MHz sensor system. In comparison with the conventional fluorescence technique, the frequency method showed that the detection limits of DNA probe AH642 with Cy5 fluorescence dye and DNA probe VA180 with Cy5 fluorescence dye were lower than the conventional fluorescence technique by 2–3 orders; meanwhile, through the feature of probe uniqueness, Aeromonas hydrophila DNA and fluorescence probe AH642-Cy5 can be successfully judged for hybridization reaction. Moreover, Vibro alginolyticus DNA and fluorescence probe VA180-Cy5 can be successfully judged for hybridization reaction.

Time-Resolved Probes Based on Guanine/Thymine-Rich DNA-Sensitized Luminescence of Terbium(III)

In this study, we have developed a novel strategy to highly sensitize the luminescence of terbium(III) (Tb(3+)) using a designed guanine/thymine-rich DNA (5'-[G3T]5-3') as an antenna ligand, in which [G3T]5 improved the luminescence of Tb(3+) by 3 orders of magnitude due to energy transfer from nucleic acids to Tb(3+) (i.e., antenna effect). Furthermore, label-free probes for the luminescent detection of biothiols, Ag(+), and sequence-specific DNA in an inexpensive, simple, and mix-and-read format are presented based on the [G3T]5-sensitized luminescence of Tb(3+) (GTSLT). The long luminescence lifetime of the probes readily enables time-resolved luminescence (TRL) experiments. Hg(2+) can efficiently quench the luminescence of Tb(3+) sensitized by [G3T]5 (Tb(3+)/[G3T]5); however, biothiols are readily applicable to selectively grab Hg(2+) for restoration of the luminescence of Tb(3+)/[G3T]5 initially quenched by Hg(2+), which can be used for "turn on" detection of biothiols. With the use of cytosine (C)-rich oligonucleotide c[G3T]5 complementary to [G3T]5, the formed [G3T]5/c[G3T]5 duplex cannot sensitize the luminescence of Tb(3+). However, in the presence of Ag(+), Ag(+) can combine the C base of c[G3T]5 to form C-Ag(+)-C complexes, leading to the split of the [G3T]5/c[G3T]5 duplex and then release of [G3T]5. The released [G3T]5 acts as an antenna ligand for sensitizing the luminescence of Tb(3+). Therefore, the Tb(3+)/[G3T]5/c[G3T]5 probe can be applied to detect Ag(+) in a "turn on" format. Moreover, recognition of target DNA via hybridization to a molecular beacon (MB)-like probe (MB-[G3T]5) can unfold the MB-[G3T]5 to release the [G3T]5 for sensitizing the luminescence of Tb(3+), producing a detectable signal directly proportional to the amount of target DNA of interest. This allows the development of a fascinating label-free MB probe for DNA sensing based on the luminescence of Tb(3+). Results and methods reported here suggest that a guanine/thymine-rich DNA-sensitized luminescence probe of Tb(3+) represents a new opportunity for versatile background-free biosensing applications.

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0×10−9M to 2.0×10−8M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10×10−10M. Additionally, the specificity and hybridization response of this simple sensor were investigated.

Photoluminescence studies of a Terbium(III) complex as a fluorescent probe for DNA detection

The photoluminescence properties of a Tb(III) complex of the form [Tb(phen)2Cl3·OH2] (phen=1,10-phenanthroline) in different solvents are presented. It shows the characteristic luminescence of the corresponding Ln3+ ion in the visible region. The emission intensity of this complex in coordinating solvent is higher than non-coordinating one. The suggested mechanism for the energy transfer between the ligand and Tb3+ ion is the intramolecular energy transfer mechanism. The interactions of the Tb(III) complex with fish salmon DNA are studied by fluorescence spectroscopy, circular dichroism study and viscosity measurements. The results of fluorescence titration reveal that DNA strongly quenches the intrinsic fluorescence of the complex through a static quenching procedure. The binding constant (Kb) of the above metal complex at 25°C is determined by the fluorescence titration method and it is found to be (8.06±0.01)×103M−1. The thermodynamic parameters (ΔH0>0, ΔS0>0 and ΔG0<0) indicate that the hydrophobic interactions play a major role in DNA–Tb complex association. The results support the claim that the title complex bonds to FS-DNA by a groove mode.

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

AbstractIn this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10−13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.

Preparation of Ag/NaA zeolite modified carbon paste electrode as a DNA biosensor

In this research, we reported a method for synthesis of NaA zeolite. NaA zeolite has been successfully prepared within 30min by microwave heating without using any conventional hydrothermal treatment and due to the exchange process the silver-loaded NaA microsized zeolites was produced. The synthesized zeolite was characterized using X-ray diffraction, scanning electronic microscopy and FT-IR techniques. The modified carbon paste electrode was prepared by incorporation of Ag(I) zeolite in the carbon paste matrix. Differential pulse voltammetry (DPV) was employed for development of electrochemical DNA hybridization biosensors based on Ag/NaA zeolite modified carbon paste electrode (Ag/ZMCPE) and methylene blue (MB) as electroactive label was used. The sensors rely on immobilization of a 15-mer single stranded oligonucleotide probe for detection of target DNA, as a model. The hybridization event was evaluated by DPV. Moreover, Ag/ZMCPE showed better advantages than NaA zeolite modified carbon paste electrode (ZMCPE). Under optimized experimental conditions, limit of detection was calculated 4.00×10−12M.