Sensitive and selective DNA probe based on “turn-on” photoluminescence of C-dots@RGO

In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA have been used for the detection of DNA. The photoluminescence of C-dots@RGO is quenched by MB, which is restored in the presence of a target DNA. The combination of the C-dots@RGO, MB, and a DNA probe is selective for perfectly matched DNA over mismatched DNA, mainly because relative to single-stranded DNA, double-stranded DNA intercalates more strongly with MB, but interacts more weakly with RGO. In the presence of a target DNA, MB intercalates with the as-formed double-stranded DNA and is released from the surface of C-dots@RGO, leading to “turn-on” photoluminescence. The practicality of this assay has been validated by the determination of tumor suppressor gene BRCA1, with linearity over the concentration range from 25 to 250 nM and a limit of detection (LOD, at a signal-to-noise ratio of 3) of 14.6 nM. The C-dots@RGO probe provides higher specificity towards target DNA than towards common salts, carbohydrates, amino acids, and proteins found in real samples. Having the advantages of simplicity, cost-effectiveness, selectivity, and sensitivity, the DNA-P/C-dots@RGO–MB probe on microwells has been successfully employed for the detection of DNA, suggesting its potential for multiple analyses of DNA targets when various DNA probes are employed.     

Electrochemical Determination of Label Free BRCA Hybridization by Single Use Antioxidant Modified Electrode

A novel DNA probe based on caffeic acid modified disposable pencil graphite electrodes were developed for the first time for the electrochemical determination of breast cancer gene sequence (BRCA) hybridization. Amino‐linked BRCA probe highly immobilized onto the caffeic acid modified electrode by means of the interaction between the amino group of BRCA probe and the carboxyl group of caffeic acid compared to the bare electrode. 44 % signal enhancement in guanine oxidation signal was measured by caffeic acid modified electrode. Besides, these probes exhibited high selectivity towards its complementary DNA sequences (target). Hybridization between probe and target (BRCA1) was studied to evaluate the selectivity of the probes for complementary, non‐complementary and mismatch sequences. The selectivity was also tested in the presence of mixture containing the target and one base mismatch BRCA sequences in the same ratio (1 : 1). It can be said this probe can select its complementary from the mixture.     

A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodyna mics aspects.The optimization of the assista nt strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90%.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1(rs1799949,c.2082 CT).The limit of detection to the mutation abundance in the DNA three-way junction structure system(0.2%) was one order lower compared with that in the double-stranded DNA structure system(2%).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

Plasmonics nanoprobes: detection of single-nucleotide polymorphisms in the breast cancer BRCA1 gene

This paper describes the application of plasmonics-based nanoprobes that combine the modulation of the plasmonics effect to change the surface-enhanced Raman scattering (SERS) of a Raman label and the specificity of a DNA hairpin loop sequence to recognize and discriminate a variety of molecular target sequences. Hybridization with target DNA opens the hairpin and physically separates the Raman label from the metal nanoparticle thus reducing the plasmonics effect and quenching the SERS signal of the label. We have successfully demonstrated the specificity and selectivity of the nanoprobes in the detection of a single-nucleotide polymorphism (SNP) in the breast cancer BRCA1 gene in a homogenous solution at room temperature. In addition, the potential application of plasmonics nanoprobes for quantitative DNA diagnostic testing is discussed.     

Application of graphene oxide nanosheets as probe oligonucleotide immobilization platform for DNA sensing

In this work, a simple and novel electrochemical biosensor based on a glassy carbon electrode (GCE) modified with graphene oxide nanosheets (GO) was developed for detection of DNA sequences. The morphology of prepared nanoplatform was investigated by scanning electron microscopy, infrared (FTIR) and UV/Vis absorption spectra. The fabrication processes of electrochemical biosensor were characterized with cyclic voltammetry and electrochemical impedance spectroscopy (EIS) in an aqueous solution. The optimization of experimental conditions such as immobilization of the probe BRCA1 and its hybridization with the complementary DNA was performed. Due to unique properties of graphene oxide nanosheets such as large surface area and high conductivity, a wide liner range of 1.0 × 10^?17–1.0 × 10^?9 M and detection limit of 3.3 × 10^?18 M were obtained for detection of BRCA1 5382 mutation by EIS technique. Under the optimum conditions, the proposed biosensor (ssDNA/GO/GCE) revealed suitable selectivity for discriminating the complementary sequences from non-complementary sequences, so it can be applicable for detection of breast cancer.     

Graphene Doped Mn2O3 Nanofibers as a Facile Electroanalytical DNA Point Mutation Detection Platform for Early Diagnosis of Breast/Ovarian Cancer

This paper demonstrates a simple, label‐free detection methodology for detecting single point DNA mutations. Single point mutation detection is a key enabler for diagnosis and prevention of several genetic disorders that manifest into cancers. Specifically for this purpose, herein, an electrochemical biosensor utilizing electrospun graphene doped manganese III oxide nanofibers (GMnO) is developed. The charge transfer resistance offered by GMnO is extremely sensitive to the localized change in the conductivity. This sensitivity, attributed to the low band gap of Mn₂O₃ and high charge transfer kinetics of graphene, is explored in the proposed mutation detection platform. As a proof of concept, ultrasensitive detection of BRCA1 gene specific point mutation is demonstrated. The target specific single stranded probe DNA is immobilized onto GMnO modified glassy carbon working electrodes via chemisorption. Post target‐DNA hybridization, differential pulse voltammetry is employed to facilitate detection of targeted point mutation, wherein, difference in peak currents is used to distinguish the target DNA as normal or mutant. Efficiency of the proposed method is evaluated against a target concentration ranging from 10 pM−1 μM. With respect to the mutated target DNA, the LoD of the proposed device is found to be 0.8±0.069 pM. The proposed approach can be extended for detecting any mutation/hybridization of interest by simply adapting an appropriate functionalization protocol.     

Carrier-resolved technology for homogeneous and multiplexed DNA assays in a 'one-pot reaction'

For clinical diagnosis, a small number of targets (2-10 biomarkers) are often all that is required for disease assessment and accurate early disease diagnosis. In the current paper we have developed novel, carrier-resolved, single-label-based multiplexed assays for the simultaneous detection and quantification of a limited number of DNA targets associated with breast cancer. In contrast to current encoding strategies, every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physico-chemical signature. Moreover, a simple chemiluminescence setup is employed to read the carrier code instead of expensive and complicated flow-cytometer or imaging-systems commonly used for multiplexed assays. Herein we demonstrate a new protocol using three homogeneous carriers, i.e. thermo-sensitive poly(N-isopropylacrylamide) (PNIP), polystyrene beads, and magnetic beads respectively. This new methodology allowed for the simultaneous determination of three oligonucleotide sequences (60 bases in length) associated with the breast cancer gene (BRCA1) and showed high selectivity and attomolar-femtomolar sensitivity. The mixture of three different capture probe conjugates first hybridizes with three corresponding target sequences, sandwiches with three biotinylated DNAs, and then reacts with peroxidase-streptavidin polymer in a single vessel without any washing, leading to the development of a 'one-pot reaction system'. Only one washing step in our protocol is required prior to detection leading to our whole procedure being simple and efficient. The results show that the hybridization response to sample mixtures containing increasing levels of each target is proportional to the amount of corresponding DNA targets, indicating minimal cross-interferences. The work presented here validates the design and concept of a system for the detection of a limited number of DNA targets and provides the foundation for the development of highly sensitive techniques with increased multi-analyte capabilities.     

A Sandwich‐Type Electrochemical Biosensor for Detection of BCR/ABL Fusion Gene Using Locked Nucleic Acids on Gold Electrode

In this study, a sandwich‐type electrochemical enzyme‐based LNA‐modified DNA biosensor was developed to detect relative gene in chronic Myelogenous Leukemia first. This biosensor is based on a ‘sandwich’ detection strategy, which involves a pair of probes (a capture probe immobilized at the electrode surface and a reporter probe labeled biotin as an affinity tag for avidin‐HRP) modified LNA. Since biotin can be connected with avidin‐HRP, this biosensor offers an enzymatically amplified electrochemical current signal for the detection of target DNA. This new pattern exhibits high sensitivity and selectivity, and this biosensor has been used for an assay of PCR real sample with satisfactory result.     

Molecular biosensing system based on intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219-498) was utilized in the detection of p53 (residues 311-393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311-393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219-498). Dansyl-X, SE-conjugated BRCA1 (219-498) detected p53 (311-393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change.     

Bacillus sp. immobilized on Amberlite XAD-4 resin as a biosorbent for solid phase extraction of thorium prior to UV-vis spectrometry determination

A DNA biosensor is presented that is based on gold interdigitated nanoelectrodes of 100 nm width and 250 nm pitch. A single-strand oligonucleotide acts as the capture probe. Because of its nanometer dimensions, the device shows improved sensitivity when compared to similar systems. This encouraged us to perform a direct and unlabelled detection. After incubation with the DNA target, the impedance spectrum was recorded between 1 kHz and 10 MHz to obtain the net capacitance change. The use of a biotinylated DNA signalling probe permitted the integration of an amplification stage in a sandwich format that employs streptavidin-modified gold nanoparticles. The strategy was preliminarily tested by detecting the breast cancer related BRCA1 gene, where the noncomplementary, wild and mutated forms were easily differentiated at a concentration level of 3 μM (corresponding to a 30 pmol quantity).     

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 × 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.     

The Fanconi anemia pathway and ubiquitin

Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin. To date, 12 FA gene products have been identified, which cooperate in a common DNA damage-activated signaling pathway regulating DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA damage, mediates the monoubiquitylation of the FA protein FANCD2. Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these factors are required for cellular resistance to DNA crosslinking agents. The inactivation of the FA pathway has also been observed in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the treatment of cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Label-free selective DNA detection with high mismatch recognition by PNA beacons and ion exchange HPLC

Two 11mer peptide nucleic acid (PNA) beacons were synthesized and tested for the detection of full-matched or single mismatched DNA. Fluorescent measurements carried out in solution showed only partial discrimination of the mismatched sequence, while using anion-exchange HPLC, in combination with fluorimetric detection, allowed DNA analysis to be performed with high sensitivity and extremely high sequence selectivity. Up to >90 : 1 signal discrimination in the presence of one single mismatched base was observed. The analysis was tested on both short and long DNA oligomers. Detection of DNA obtained from PCR amplification was also performed allowing the selective detection of the target sequence in complex mixtures. Label free detection of the DNA with high sequence selectivity is therefore possible using the present approach.     

An Electrochemical DNA Biosensor for the Detection of the Apa I Polymorphism in the Vitamin D Receptor Gene Using Meldola's Blue as a Hybridization Indicator

Electrochemical detection of nucleic acid base mismatches related to Apa I single nucleotide polymorphism (SNP) in the vitamin D receptor gene was performed successfully using 7‐dimethyl‐amino‐1,2‐benzophenoxazinium salt (Meldola's blue, MDB) with 10.9 pmol/100 μL of detection limit. MDB reduction signals obtained from probe, mismatch(probe‐SNP containing target) and hybrid(probe‐target) modified pencil graphite electrode(PGE) increased respectively. The sensor was able to clearly distinguish perfect match from mismatch DNA in a 30 min. detection time. Several factors affecting on the hybridization and indicator response are studied to maximize sensitivity and selectivity. The advantages of the biosensor are discussed in comparison with previous electrochemical assays for DNA hybridization.     

Electrochemical coding technology for simultaneous detection of multiple DNA targets

Nucleic-acid hybridization assays based on the use of different inorganic-colloid (quantum dots) nanocrystal tracers for the simultaneous electrochemical measurements of multiple DNA targets are described. Three encoding nanoparticles (zinc sulfide, cadmium sulfide, and lead sulfide) are used to differentiate the signals of three DNA targets in connection to stripping-voltammetric measurements of the heavy metal dissolution products. These products yield well-defined and resolved stripping peaks at -1.12 V (Zn), -0.68 V (Cd), and -0.53 V (Pb) at the mercury-coated glassy-carbon electrode (vs Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding DNA target. The multi-target detection capability is coupled to the amplification feature of stripping voltammetry (to yield femtomole detection limits) and with an efficient magnetic removal of nonhybridized nucleic acids to offer high sensitivity and selectivity. The protocol is illustrated for the simultaneous detection of three DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. The new electrochemical coding is expected to bring new capabilities for DNA diagnostics, and for bioanalysis, in general.     

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.     

Carbon-nanotube-modified glassy carbon electrodes for amplified label-free electrochemical detection of DNA hybridization

The preparation and attractive performance of carbon-nanotube modified glassy-carbon (CNT/GC) electrodes for improved detection of purines, nucleic acids, and DNA hybridization are described. The surface-confined multiwall carbon-nanotube (MWCNT) facilitates the adsorptive accumulation of the guanine nucleobase and greatly enhances its oxidation signal. The advantages of CNT/GC electrodes are illustrated from comparison to the common unmodified glassy carbon, carbon paste and graphite pencil electrodes. The dramatic amplification of the guanine signal has been combined with a label-free electrical detection of DNA hybridization. Factors influencing the enhancement of the guanine signal are assessed and optimized. The performance characteristics of the amplified label-free electrochemical detection of DNA hybridization are reported in connection to measurements of nucleic-acid segments related to the breast-cancer BRCA1 gene.     

An Electrochemical Sensor for the Detection of Cu2+ Based on Gold Nanoflowers‐modifed Electrode and DNAzyme Functionalized Au@MIL‐101 (Fe)

In this study, we developed an electrochemical sensor for sensitive detection of Cu²⁺ based on gold nanoflowers (AuNFs)‐modified electrode and DNAzyme functionalized Au@MIL‐101(Fe) (MIL: Materials of Institute Lavoisier). The AuNFs‐modified indium tin oxide modified conductive glass electrode(AuNFs/ITO) prepared via electrodeposition showed improved electronic transport properties and provided more active sites to adsorb large amounts of oligonucleotide substrate (DNA1) via thiol‐gold bonds. The stable Au@MIL‐101(Fe) could guarantee the sensitivity because of its intrinsic peroxidase mimic property, while the Cu²⁺‐dependent DNA‐cleaving DNAzyme linked to Au@MIL‐101(Fe) achieved the selectivity toward Cu²⁺. After the DNAzyme substrate strand (DNA2) was cleaved into two parts due to the presence of Cu²⁺, the oligonucleotide fragment linked to MIL‐101(Fe) was able to hybridize with DNA1 adsorbed onto the surface of AuNFs/ITO. Due to the peroxidase‐like catalytic activity of MIL‐101(Fe) and the affinity recognition property of DNAzyme toward Cu²⁺, the electrochemical biosensor showed a sensitive detection range from 0.001 to 100 μM, a detection limit of 0.457 nM and a high selectivity, demonstrating its potential for Cu²⁺ detection in real environmental samples.     

Dual linear ion trap/orthogonal acceleration time-of-flight mass spectrometer with improved precursor ion selectivity

A new hybrid mass spectrometer based on dual linear ion traps (LITs) and an orthogonal acceleration time-of-flight mass spectrometer (oaTOF), that can achieve MS(n) analysis and high-mass-accuracy detection with high sensitivity, has been developed. Dual-LIT was necessary because, in a single LIT plus oaTOF combination, the LIT pressure favorable for high precursor selectivity in MS(n) analysis (less than 1 mTorr) was far different from an optimum damping pressure (50-100 mTorr) for efficient connection to the TOF mass spectrometer. A dual-LIT solved this problem of inconsistency of the optimum pressures by using the first LIT for MS(n) analysis and the second LIT for collisional damping. This dual-LIT/TOF instrument achieved high-sensitivity MS(n) analysis with high precursor-ion selectivity.