Target DNA Concentration Research Articles

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing.

A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR deTection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect-ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip at isothermal conditions (37 °C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.

An electrochemical DNA biosensor fabricated from graphene decorated with graphitic nanospheres

Graphene decorated with graphitic nanospheres functionalized with pyrene butyric acid (PBA) is used for the first time to fabricate a DNA biosensor. The electrode was formed by attaching a DNA probe onto PBA, which had been stacked onto a graphene material decorated with graphene nanospheres (GNSs). The nanomaterial was drop-coated onto a carbon screen-printed electrode (SPE) to create the GNS-PBA modified electrode (GNS-PBA/SPE). A simple method was used to produce GNS by annealing graphene oxide (GO) solution at high temperature. Field emission scanning electron micrographs confirmed the presence of a spherical shape of GNS with a diameter range of 40–80 nm. A stable and uniform PBA-modified GNS (GNS-PBA) was obtained with a facile ultrasonication step. Thus allowing aminated DNA probes of genetically modified (GM) soybean to be attached to the nanomaterials to form the DNA biosensor. The GNS-PBA/SPE exhibited excellent electrical conductivity via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests using potassium ferricyanide (K3[Fe(CN)6]) as the electroactive probe. By employing an anthraquinone monosulfonic acid (AQMS) redox intercalator as the DNA hybridization indicator, the biosensor response was evaluated using the DPV electrochemical method. A good linear relationship between AQMS oxidation peak current and target DNA concentrations from 1.0 × 10−16 to 1.0 × 10−8 M with a limit of detection (LOD) of less than 1.0 × 10−16 M was obtained. Selectivity experiments revealed that the voltammetric GM DNA biosensor could discriminate complementary sequences of GM soybean from non-complementary sequences and hence good recoveries were obtained for real GM soybean sample analysis. The main advantage of using GNS is an improvement of the DNA biosensor analytical performance.

Tuning the dynamic range of an Au-nanoparticle-based DNA hybridization assay by optimizing particle size, concentration, and surface probe density

Our aim is to broaden the dynamic range of target DNA detection by an Au nanoparticle (AuNP) sandwich assay based on complementary DNA hybridization. We conducted electrophoresis band analysis for different sizes and concentrations of AuNPs bearing different numbers of surface-conjugated probe DNA molecules. We found that the AuNP concentration is critical to determining the maximum quantification limit (upper limit of the dynamic range). The number of immobilized probe DNAs per AuNP was also optimized to prevent errors in determining the target DNA concentration and to raise the upper limit. We examined AuNPs with diameters ranging from 15 to 40 nm at different concentrations and demonstrated tunability of the dynamic ranges spanning two to three orders of magnitude. The use of both 15 and 40 nm AuNPs can cover a dynamic range of over four orders of magnitude.

An Electroanalytical Paper-Based Wound Dressing Using ZIF-67/C3 N4 Nanocomposite Towards the Monitoring of Staphylococcus Aureus in Diabetic Foot Ulcer

Slow or non-healing wounds in chronic diabetic foot diseases are often associated with co-morbid circumstances, inevitably leading to lower limb amputations. The wound bed in chronic foot ulcers are prone to bacterial infections, with Staphylococcus aureus ( S. aureus ) acting as the major culprit in resisting the normal wound healing process. This study reports the development of a flexible screen-printed wound dressing based on a composite of Zeolitic Imidazolate Framework (ZIF 67) and carbon nitride (C3 N4) conjugated with S. aureus probe DNA for the detection of its corresponding target DNA. The nano-wound dressing was assessed using Electrochemical Impedance Spectroscopy (EIS) for the quantification of S. aureus target DNA within 1 fM to $10~\mu \text{M}$ . A direct proportionality between the charge transfer resistance ( $\text{R}_{\text {ct}}$ ) and S. aureus target DNA concentration. Circuit simulations detailing the quantifications of interfacial parameters, involved in the recognition event, were performed by curve fitting of Nyquist spectra in order to explain the variation of $\text{R}_{\text {ct}}$ with target DNA concentrations. The Limit of Detection (LoD), sensitivity and response time of the developed wound dressing-based sensor were found to be 0.46 fM, 0.25 $\text{k}\Omega $ /fM/mm2 and ~ 10 s respectively at 21.04 kHz, while being selective only towards S. aureus target DNA. The sensor was further validated in human serum samples and was shown to possess a shelf life of 35 days, thereby exhibiting tremendous potential to be implemented in connected health applications.

Spectrophotometric determination of single-stranded DNA with self-assembly hairpin DNA and silver nanoparticles

Nucleic acids containing genetic information are used as biomarkers for the diagnosis of genetic diseases. Herein, silver nanoparticles were utilized as probes to detect single stranded nucleic acids by self-assembly of DNA nanomaterials by spectrophotometry. The target DNA specifically triggered two hairpin DNA molecules to hybridize, generating linear double stranded DNA. This approach effectively prevents silver nanoparticles from aggregating in aqueous solutions of NaCl. Otherwise, silver nanoparticles aggregate without target DNA and hairpin DNA due to the low stability of double stranded DNA. The sensor response was shown to be proportional to the target DNA concentration from 5 to 200 nM with a detection limit equal to 5 nM. This simple platform for the specific determination of nucleic acids without enzymes, labels, and modification and demonstrates great prospects for bioanalysis.

Colorimetric Nucleic Acid Detection Based on Gold Nanoparticles with Branched DNA

Nucleic acid detection is becoming increasingly important in the diagnostics of genetic diseases for biological analysis. We herein propose gold nanoparticles as probe for colorimetric detection of nucleic acids with branched DNA nanostructures, which enables a novel and simple colorimetric biosensor. In our system, the target DNA specifically triggered two short-chain ssDNA probes to generate branched DNA nanostructures (Y-shape DNA), which prevent AuNPs from aggregation in aqueous NaCl solution. On the contrary, when the target DNA did not exist, gold nanoparticles were unstable and aggregated easily because there is no anti-aggregation function from Y-shape DNA. Sensor response was found to be proportional to the target DNA concentration from 5 to 100[Formula: see text]nM, with detection limits determined as 5[Formula: see text]nM. The developed platform is for colorimetric nucleic acid detection without enzymes, label and modification holds great promise for practical applications.

The rapid and visual detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus using multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor

BackgroundStaphylococcus aureus (S. aureus), including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), is an eminent human pathogen that can colonize the human host and cause severe life-threatening infections. The development of a reliable, simple and rapid assay for detecting S. aureus and identifying MRSA is important for diagnosis and follow-up treatment.MethodsA novel molecular diagnosis technique, named multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor (m-LAMP-LFB), was applied to detect all S. aureus species and identify MRSA. Two sets of primers were designed based on the femA gene (S. aureus-specific gene) and the mecA gene (encoding penicillin-binding protein 2a), and the multiple-LAMP products were analyzed using LFB. The m-LAMP-LFB amplification conditions, including the target DNA concentration, reaction temperature and time, were optimized. The sensitivity and specificity of the m-LAMP-LFB method were tested in the current study, and the multiple-LAMP-LFB technology was applied to detect the MSSA and MRSA strains from clinical samples.ResultsThe S. aureus- and MRSA-specific primers based on the femA and mecA genes allowed the multiple-LAMP technology to detect S. aureus and MRSA, respectively. The multiple-LAMP conditions were optimized at 63 °C for 40 min. The full process, including genomic DNA template preparation, LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the multiple-LAMP assay for femA and mecA detection was 100 fg of genomic DNA template per reaction. The specificity of m-LAMP-LFB detection was 100 %, and no cross-reactions to non-S. aureus strains were observed.ConclusionThe multiple-LAMP-LFB technique developed in the current study is a reliable, simple, rapid, specific and sensitive method to identify MSSA and MRSA infections for appropriate antibiotic therapy.

Interplay of Effective Surface Area, Mass Transport, and Electrochemical Features in Nanoporous Nucleic Acid Sensors.

Electrochemical biosensors transduce biochemical events (e.g., DNA hybridization) to electrical signals and can be readily interfaced with electronic instrumentation for portability. Nanostructuring the working electrode enhances sensor performance via augmented effective surface area that increases the capture probability of an analyte. However, increasing the effective surface area via thicker nanostructured electrodes hinders the analyte's permeation into the nanostructured volume and limits its access to deeper electrode surfaces. Here, we use nanoporous gold (np-Au) with various thicknesses and pore morphologies coupled with a methylene blue (MB) reporter-tagged DNA probe for DNA target detection as a model system to study the influence of electrode features on electrochemical sensing performance. Independent of the DNA target concentration, the hybridization current (surrogate for detection sensitivity) increases with the surface enhancement factor (EF), until an EF of ∼5, after which the sensor performance deteriorates. Electrochemical and fluorometric quantification of a desorbed DNA probe suggest that DNA permeation is severely limited for higher EFs. In addition, undesirable capacitive currents disguise the faradaic currents from the MB reporter at larger EFs that require higher square wave voltammetry (SWV) frequencies. Finally, a real-time hybridization study reveals that expanding the effective surface area beyond EFs of ∼5 decreases sensor performance.

Nucleic Acid Quantitation with Log-Linear Response Hybridization Probe Sets.

Concentrations of different nucleic acid species in biological samples span many orders of magnitude. A real-time polymerase chain reaction maps the concentration of a target nucleic acid sequence log-linearly into cycle threshold to enable quantitation with a wide dynamic range but suffers from enzymatic biases. Here, we present a general design for constructing hybridization probe sets with highly log-linear response curves to enable accurate enzyme-free quantitation across large ranges (more than 6 logs) of target DNA concentrations. The sensitivity of each component probe is accurately adjusted via formulation stoichiometry to reduce the standard error of target quantitation down to 7%. As a proof of concept, we show multiplexed quantitation of three microRNA species in total RNA of the human brain and liver.

A homogeneous electrochemical DNA sensor on the basis of a self-assembled thiol layer on a gold support and by using tetraferrocene for signal amplification.

An unmodified electrochemical biosensor has been constructed, which can directly detect DNA in homogeneous solution. The synthesized new compound tetraferrocene was used for signal amplification. The dual-hairpin probe DNA was tagged with a tetraferrocene at the 3' terminal and a thiol at the 5' terminal. Without being hybridized with target DNA, the loop of probe prevented the thiol from contacting the exposed gold electrode surface with an applied potential. After hybridization with the target DNA, the loop-stem structure of the probe was opened, which led to the formation of thehairpin DNA structure. Afterwards, the thiol easily contacted the electrode and accomplished potential-assisted Au-S self-assembly. Its current signal depends on the concentration of target DNA in the 1.8 × 10-13 to 1.8 × 10-9M concentration range, and the detection limit is 0.14pM. The technique is a meaningful study because of its high selectivity and sensitivity. Graphical abstract Schematic diagram of the electrochemical DNA sensor operation. Target DNA and probe DNA hybridization, resulting in the disappearance of the steric hindrance of the probe stem ring. A higher signal was generated when tetraferrocene reached the electrode. The electrochemical signals were determined by differential voltammetric pulses (DPV).

Thermodynamically designed target-specific DNA probe as an electrochemical hybridization biosensor

Applications of molecular techniques to elucidate identity or function using biomarkers still remain highly empirical and biosensors are no exception. In the present study, target-specific oligonucleotide probes for E. coli K12 were designed thermodynamically and applied in an electrochemical DNA biosensor setup. Biosensor was prepared by immobilization of a stem–loop structured probe, modified with a thiol functional group at its 5′ end and a biotin molecule at its 3′ end, on a gold electrode through self-assembly. Mercaptopropionic acid (MPA) was used to optimize the surface probe density of the electrode. Hybridization between the immobilized probe and the target DNA was detected via the electrochemical response of streptavidin-horseradish peroxidase in the presence of the substrate. The amperometric response showed a linear relationship with the target DNA concentration, ranging from 10 and 400 nM, with a correlation coefficient of 0.989. High selectivity and good repeatability of the biosensor showed that the thermodynamic approach to oligonucleotide probe design can be used in development of electrochemical DNA biosensors.

Investigating Catechol Surface Modification of TiO2 for the Development of High Performance Photoelectrochemical Biosensors

DNA biosensors are chemical sensors which are used to detect concentrations of specific DNA sequences or register DNA related biological events. There is significant interest in research into the design and optimization of DNA biosensors as they allow for rapid detection of cancers and infectious diseases 1,2. Photoelectrochemical (PEC) techniques can be applied to achieve high performance DNA biosensors with low cost and ultra-sensitivity. In this work, we explored the surface modification of TiO2 nanoparticles (NP) with various catechol molecules and used them to fabricate photoelectrodes for signal-off PEC DNA biosensors. Catechol modification provided dual functionality to the TiO2 NP photoelectrodes: it enhanced their PEC response and provided a molecular linker for attaching probes that capture target DNA.The surface modification of TiO2 NPs was done by adding aqueous solutions of catechol molecules to a suspension of TiO2. Adsorption of catechol on TiO2 took place very rapidly, indicated by a quick change in the colour of the suspension. The catechol-modified TiO2 suspensions were dropdeposited on indium tin oxide coated polyethylene terephthalate (ITO/PET) substrates and oven baked to produce photoelectrodes for the PEC DNA biosensor. Seven catechol molecules were used for surface modification: Caffeic acid (CA), 3,4-Dihydroxybenzaldehyde (DHBA), 3,4-Dihydroxybenzoic acid (DHB), 3,4-Dihydroxy-L-phenylalanine (DOPA), 3,4-Dihydroxyphenylacetic acid (DHPL), 2,3,4-Trihydroxybenzaldehyde (THBA), 2,3,4-Trihydroxybenzoic acid (THB). The photoelectrodes were optically characterized via ultraviolet/visible (UV/vis) spectroscopy and incident photon-to-electron conversion efficiency (IPCE). Additionally, the PEC current of the photoelectrodes were measured using chronoamperometry under white light excitation.Compared against unmodified TiO2, the UV/Vis spectroscopy and IPCE displayed increased light absorption in the visible range and red-shifting for the catechol-modified TiO2 photoelectrodes. This correlated to a great improvement in photocurrent generation. CA-modified TiO2 resulted in the largest increase in photocurrent, over 20 times from the unmodified TiO2 and therefore it was used as the basis for our PEC DNA biosensor. Single stranded DNA (ssDNA) probes were deposited onto the photoelectrodes, followed by target ssDNA deposition. The difference in PEC current after probe deposition and target deposition indicated the concentration of target DNA. A calibration curve was plotted with PEC current density as a function of target DNA concentration, which decreased logarithmically and ranged from 100 nM – 100 fM. A limit of detection (LOD) of 2.38 pM was estimated at a signalto-noise ratio of 3Sx/y (where Sx/y is the root-mean-square error (RMSE)).It is evident from our investigation that functionalizing TiO2 with catechol ligands greatly improves its optical properties which lead to a greater PEC current generation. In particular, high PEC current resulting from the combination of CA and TiO2 can form the basis for high-performance signal-off PEC DNA biosensors with a high dynamic range and low LOD. 1. Beltrán, A. P. & García, M., DNA biosensors and biomarkers to cancer detection. International Journal of Biosensors and Bioelectronics, 4(1), 20-21 (2018)2. Chua, A., Yean, C. Y., Ravichandran, M., Lim, B. & Lalitha, P., A Rapid DNA Biosensor for the Molecular Diagnosis of Infectious Disease. Biosensors and Bioelectronics, 26(9), 3825-3831 (2011)

Electrochemical detection of Mycobacterium tuberculosis IS6110 gene fragments based on the gold nanocrystals with uniform morphology and highly exposed high-index facets and target DNA-induced recycling amplification

This paper reports the synthesis of gold nanocrystals via the reduction of chloroauric acid using ascorbic acid in the mixed hexadecyltrimethylammonium chloride aqueous solution with potassium bromide and potassium iodide. The resulting gold nanocrystals have trioctahedral nanostructures with uniform morphology and highly exposed high-index facets. The nanocrystals unique structure promotes enhanced chemical activity and catalytic activity. Preparation of the electrochemical sensing platform for IS6110 gene involves the modification of gold nanocrystals on hairpin DNA 2 (H2) and thionine as the redox probe for target DNA-induced signal amplification. Target DNA (IS6110 gene) hybridizes with hairpin DNA 1 (H1) pre-modified on the gold electrode, which opens the hairpin structure of H1. This can hybridize with H2 to release target DNA, which is used in the next recycle. The utilization of target DNA-induced recycling allows one target DNA to transport many redox probes to the electrode surface promoting significant signal amplification. The detection signal is further enhanced by catalyzed redox reaction of thionine with gold nanocrystals. The differential pulse voltammetric signal increases with increasing concentration of target DNA in the range of 0.1-1.0 × 105 fM with detection limit of 0.031 fM. The method provides ultrahigh sensitivity, specificity and stability and was successfully applied in the detection of tuberculosis in human blood.

Reliable eDNA detection and quantification of the European weather loach (Misgurnus fossilis).

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies μl-1 , respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017-2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Qe ) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained.

Tunable Electrochemical Grafting of Diazonium for Highly Sensitive Impedimetric DNA Sensor

The development of a simple, rapid, and reliable method for detecting the virus at very low levels is therefore an important health objective. To that end, a gold surface was electrochemically modified by reducing 4-nitrobenzenediazonium tetrafluoroborate. The rate of radical formation and its polymerization were significantly affected by the reaction temperature, the concentration of the diazonium precursor solution, the number of cyclic voltammetry cycles, and the scan rate. The nitro functional group was subsequently electrochemically converted into the corresponding amine. In order to detect target DNA related to dengue virus, glutaraldehyde was used to link probe DNA to the amino functional group, after which the immobilized probe DNA was hybridized with various concentrations of target DNA. Ferri/ferrocyanide signals during each step in the overall transformation, including diazonium modification, reduction to the amine, probe DNA immobilization, and target DNA hybridization, were monitored by cyclic voltammetry and electrochemical impedance spectroscopy. We finally investigated the quantitative relationship between the change in charge-transfer resistance in response to target DNA hybridization and its concentration. This sensor responded linearly in the 100 pM to 1 nM concentration range, and its detection limit was determined to be 40.6 pM.

Establishing a Field-Effect Transistor Sensor for the Detection of Mutations in the Tumour Protein 53 Gene (TP53)-An Electrochemical Optimisation Approach.

We present a low-cost, sensitive and specific DNA field-effect transistor sensor for the rapid detection of a common mutation to the tumour protein 53 gene (TP53). The sensor consists of a commercially available, low-cost, field-effect transistor attached in series to a gold electrode sensing pad for DNA hybridisation. The sensor has been predominantly optimised electrochemically, particularly with respect to open-circuit potentiometry as a route towards understanding potential (voltage) changes upon DNA hybridisation using a transistor. The developed sensor responds sensitively to TP53 mutant DNA as low as 100 nM concentration. The sensor responds linearly as a function of DNA target concentration and is able to differentiate between complementary and noncomplementary DNA target sequences.

An impedimetric biosensor based on electrophoretically assembled ZnO nanorods and carboxylated graphene nanoflakes on anindium tin oxide electrode fordetection of the DNA of Escherichia coli O157:H7.

Aminopropyltrimethoxysilane (APTMS)-functionalized zinc oxide (ZnO) nanorods and carboxylated graphene nanoflakes (c-GNF) were used in acomposite that was electrophoretically deposited on anindium tin oxide (ITO) coated glass substrate. Themodified ITO electrodes were characterized using various microscopic and spectroscopic techniques which confirm the deposition of theAPTMS-ZnO/c-GNF composite. The electrodes have been used for the covalent immobilization of an Escherichia coli O157:H7 (E. coli)-specific DNA prob. Impedimetric studies revealed that the genesensor displays linearresponse in a wide range of target DNA concentration (10-16M to 10-6M) with a detection limit of 0.1 fM. The studies on thecross-reactivity to other water-borne pathogens show that the bioelectrodeis highly specific. Graphical abstractSchematic illustration for fabrication of nucleic acid biosensor for E. coli DNA detection using anITO electrodemodified with siloxane-functionalized zinc oxide (ZnO) nanorods and carboxylated graphene nanoflakes (c-GNFs).

A Novel Detection of Enterococcus faecalis Using Multiple Cross Displacement Amplification Linked with Gold Nanoparticle Lateral Flow Biosensor.

BackgroundEnterococcus faecalis, an opportunistic bacterial pathogen, is one of the most frequently isolated bacterial species and cause of serious nosocomial infections in recent decades. A reliable and rapid assay for E. faecalis detection is significant for the diagnosis and follow-up treatment.MethodsA novel assay method, named multiple cross displacement amplification linked with nanoparticle-based lateral flow biosensor (MCDA-LFB), was applied for detecting E. faecalis strains. A set of special 10 primers was designed according to E. faecalis-specific gene Ef0027. The MCDA amplification conditions, including the target DNA concentration, reaction temperature and time, were optimized. The sensitivity and specificity of MCDA method were tested in the current study, and then, the MCDA-LFB technology was applied to detect the E. faecalis strain from clinical samples.ResultsThe E. faecalis specific primers were valid for the establishment of MCDA-LFB technology forthe detection of E. faecalis based on the Ef0027 gene. The MCDA amplification condition was optimized at 62°C for 35 min. The MCDA products were directly sensed and displayed with a biosensor. The full process, comprising genomic DNA template preparation (approximately 30 mins), amplification of MCDA (35 mins), and the product identification (approximately 2 mins), could be achieved in 70 mins. The MCDA technique could detect as little as 10 fg per reaction system of pure E. faecalis genomic DNA. The specificity of E. faecalis-MCDA-LFB method is 100%, with no cross-reactions to non-E. faecalis strains.ConclusionThe MCDA-LFB technique established in the present study is a reliable, simple, rapid, sensitive and specific method to assay E. faecalis and can be applied for the detection of clinical samples.

Sensitive and selective fluorometric determination of DNA by using layered hexagonal nanosheets of a covalent organic framework prepared from p-phenylenediamine and benzene-1,3,5-tricarboxaldehyde.

A modified method is described for the preparation of amino-functionalized covalent organic framework nanosheets (COF-NSs). These consist of hexagonal layered sheets and were prepared from commercially available starting materials (p-phenylenediamine and benzene-1,3,5-tricarboxaldehyde). The interlayer stacking interactions between the ultra-thin COF-NSs became weak because the π stacking is destroyed by sonication. This result in the exfoliation of COF-NSs. As an application, the COF-NSs used for sensitive and selective fluorometric determination of DNA. To reach this goal, H1 and H2 hairpin-like DNA probes were chosen; H1 used Texas Red-labeled dye as a fluorescent probe. The addition of the COF-NSs, the hairpin probes was adsorbed onto the porous surface of the COFNSs. The π stacking and hydrogen-bond interactions between COFNSs and nucleic acid quench the fluorescence of the Texas red-labeled probe. The target DNA enables the recovery of the quenched fluorescence of the Texas red-labelled probe by triggering an inter-chain hybridization within hairpin probes. This results in a weaker interaction of double-stranded DNA (dsDNA) with the COFNSs. Consequently, the dsDNA detaches from the COFNSs, thereby recovering the dye's fluorescence (excitation/emission maxima at 590/612nm) with increasing target DNA concentration. The findings were applied to design a method for the determination of DNAthat has a 2 pMdetection limit. This is significantly lower than the limit of detection reported previously for 2D nanomaterial-based fluorometric DNA assays. Graphical abstractSchematic representation of 2D-covalent organic framework nanosheets (COF-NSs) probe act as a quencher allowing the highly sensitive and selective fluorescence turn-on detection for biomolecules. Here the H1 H2 are hairpin DNAs. H1 is associated with thefluorescent tag (red circle), while the "fluorescence off" state it denoted as ablack circle.

Block Copolymer‐Derived Recessed Nanodisk‐Array Electrodes as Platforms for Folding‐Based Electrochemical DNA Sensors

AbstractThis paper reports the characteristics of folding‐based electrochemical DNA sensors fabricated on recessed nanodisk‐array electrodes (RNEs). The RNEs were based on gold substrates coated with polystyrene‐block‐poly(methylmethacrylate)‐derived thin films (30 nm thick) comprising vertically‐oriented cylindrical nanopores (14, 20 or 30 nm in diameter). RNE‐based sensors were fabricated by modifying the underlying gold surface with a stem‐loop DNA probe with a terminal methylene blue (MB) tag. The electrochemical responses of such RNE‐based sensors to complementary single‐stranded 17‐mer DNA were compared with those of film‐free counterparts using cyclic voltammetry. RNE‐based sensors exhibited signal suppression at a significantly lower target DNA concentration than film‐free sensors, possibly due to the manipulated dynamic properties of the MB tag as suggested by its apparent electron transfer rate constant and/or enhanced hybridization within the nanopores. These results indicate that RNEs provide unique platforms for developing folding‐based electrochemical sensors with improved sensitivity.