DNA Biosensor Research Articles

Sensitive and rapid detection of influenza A virus for disease surveillance using dual-probe electrochemical biosensor

Influenza A virus (IAV) can cause influenza, a highly infectious zoonotic respiratory disease, and early detection is essential to prevent and control its rapid spread in the population. Given the limitations of traditional detection methods in clinical laboratories, we report a large surface TPB-DVA COFs (TPB: 1,3,5-Tris(4-aminophenyl) benzene, DVA: 1,4-Benzenedicarboxaldehyd, COFs: Covalent organic frameworks) nanomaterial modified electrochemical DNA biosensor, which has dual-probe specific recognition and signal amplification. The biosensor enables quantitative detection of influenza A viruses' complementary DNA (cDNA) from 10 fM to 1 × 103 nM (LOD = 5.42 fM) with good specificity and high selectivity. The reliability of the biosensor and portable device was verified by comparing the virus concentrations in animal tissues with those measured by digital droplet PCR (ddPCR) (P > 0.05). Moreover, the potential for influenza surveillance in this work was demonstrated by detecting the tissue samples from mice at different stages of infection. In summary, the good performance of this electrochemical DNA biosensor we proposed suggested it has the potential to be a rapid detection device for the influenza A virus, which could assist doctors or other professionals in obtaining rapid and accurate results for outbreak investigation and disease diagnosis.

Construction of a 3D rigidified DNA nanodevice for anti-interference and reinforced biosensing by turning nuclease into a catalyst

The practical application of DNA biosensors is impeded by numerous limitations in complicated physiological environments, particularly the susceptibility of common DNA components to nuclease degradation, which has been recognized as a major barrier in DNA nanotechnology. In contrast, the present study presents an anti-interference and reinforced biosensing strategy based on a 3D DNA-rigidified nanodevice (3D RND) by converting a nuclease into a catalyst. 3D RND is a well-known tetrahedral DNA scaffold containing four faces, four vertices, and six double-stranded edges. The scaffold was rebuilt to serve as a biosensor by embedding a recognition region and two palindromic tails on one edge. In the absence of a target, the rigidified nanodevice exhibited enhanced nuclease resistance, resulting in a low false-positive signal. 3D RNDs have been proven to be compatible with 10% serum for at least 8 h. Once exposed to the target miRNA, the system can be unlocked and converted into common DNAs from a high-defense state, followed by polymerase- and nuclease-co-driven conformational downgrading to achieve amplified and reinforced biosensing. The signal response can be improved by approximately 700% within 2 h at room temperature, and the limit of detection (LOD) is approximately 10-fold lower under biomimetic conditions. The final application to serum miRNA-mediated clinical diagnosis of colorectal cancer (CRC) patients revealed that 3D RND is a reliable approach to collecting clinical information for differentiating patients from healthy individuals. This study provides novel insights into the development of anti-interference and reinforced DNA biosensors.

Absorbance biosensors-based hybrid mathbf {MoS_{2}} nanosheets for Escherichia coli detection

Detecting Escherichia coli is essential in biomedical, environmental, and food safety applications. In this paper, we have developed a simple, rapid, sensitive, and selective E. coli DNA sensor based on the novel hybrid-type text {MoS}_text {2} and mathrm {(NH_4)_6Mo_7O_{24}} nanosheets. The sensor uses the absorbance measurement to distinguish among the DNA of E. coli, Vibrio proteolyticus, and Bacillus subtilis when implemented in conjunction with text {NH}_text {2}-probes. Our experiments showed that the absorbance increased when sensors detected E. coli DNA, whereas it decreased when sensors detected V. proteolyticus and B. subtilis DNA. To the best of authors’ knowledge, there are no reports using the novel hybrid-mathrm {MoS_2} and mathrm {(NH_4)_6Mo_7O_{24}} materials for differentiating three types of DNA using cost-effective and rapid absorbance measurements. In addition, the label-free E. coli DNA biosensor exhibited a linear response in the range of 0 fM to 11.65 fM with a limit of detection of 2 fM. The effect of text {NH}_text {2}-probes on our sensors’ working performance is also investigated. Our results will facilitate further research in pathogen detection applications, which have not been fully developed yet.

Smartphone conducted DNA portable quantitative detection platform based on photonic crystals chip and magnetic nanoparticles

It is of great significance to develop a highly sensitive and intuitive virus detection tool. A portable platform is constructed for quantitative detection of viral DNA based on the principle of fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and graphene oxide nanosheets (GOs) in this work. To implement a high sensitivity and low detection limit, GOs are modified by magnetic nanoparticles to prepare magnetic graphene oxide nanosheets (MGOs). Among them, the application of MGOs can not only eliminate the background interference, but also amplify the fluorescence intensity to a certain extent. Whereafter, a simple carrier chip based on photonic crystals (PCs) is introduced to realize a visual solid-phase detection, which also amplifies the luminescence intensity of the detection system. Finally, under the application of the 3D printed accessory and smartphone program of red-green-blue (RGB) evaluation, the portable detection can be completed simply and accurately. In a word, this work proposes a portable DNA biosensor with the triple functions of quantification, visualization and real-time detection can be used as a high-quality viral detection strategy and clinical diagnosis method.

The application of a DNA nanobiosensor based on MXene/MWCNTs/PPY nanocomposite as a sensitive detection method of CEACAM5 for diagnosis of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is responsible for the high mortality rate related to gynecologic cancers. Due to the late-stage diagnosis, a 5-year survival rate is expected in only about 30% of patients. In this study, an electrochemical DNA biosensor based on MXene/MWCNTs/PPY nanocomposite was designed and introduced as an alternative method for Real-time PCR based detection methods of tumor markers. The biosensor specifically detected CEACAM5 as an ovarian cancer-related tumor marker in 26 real clinical samples. The statistical analysis results represented that the biosensor is able to differentiate between the case and control groups with 93.3 % sensitivity and high specificity. Moreover, the biosensor revealed a wide linear range of 5 × 10-11 − 5 × 10-7 M and good stability for up to 20 days. In conclusion, the fabricated biosensor provides a fast, low-cost, simple, and novel method for EOC detection.

Label-free, ultra-low detection limit DNA biosensor using high quality optical microcavity functionalized by DNA tetrahedral nanostructure probes

Abstract This study proposes and demonstrates a novel label-free DNA biosensor using high quality optical microcavity functionalized by 3D DNA nanostructure probes. To achieve ultra-low limit DNA biosensing, optical sensing interface of the hollow-core, thin wall-thickness microcavity was functionalized by self-assembled DNA tetrahedral nanostructure (DTN) probes with size of 17 bp and length of ∼5.8 nm. High efficiency binding of the DTN probes with the optical sensor interface and the target ssDNA are achieved. Whispering gallery mode (WGM) spectra with high-Q factor of >107 are excited and traced for DNA detection inside the microfluidic channel of the microcavity, with a small sample volume of nL. Incorporation of nanoscale DTN probes onto surface of the optical microcavity makes it an effective way for increasing efficient probe density and eliminating entanglement between DNA probes, thus ∼1000 times lower detection limit is achieved as compared to using 1D ssDNA probes. Due to its desirable merits of label-free, ultra-low LoD, real time and compact size, the proposed DNA biosensor has broad application prospects in bioengineering and medical diagnosis.

Rolling-Circle-Amplification-Assisted DNA Biosensors for Sensitive and Specific Detection of Hypochlorous Acid and Myeloperoxidase

Hypochlorous acid (HClO) is a common reactive oxygen species (ROS), with a high chemical reactivity. Myeloperoxidase (MPO) is an enzyme that catalyzes in vivo redox reactions between H2O2 and Cl− to produce HClO. Abnormal levels of HClO and MPO may lead to oxidative stress, irreversible tissue damage and, thus, serious diseases; they are thus becoming important biomarkers and therapeutic targets. In this work, using HClO-induced site-specific cleavage of phosphorothioate-modified DNA to trigger rolling circle amplification (RCA), RCA-assisted biosensors have been developed for the highly sensitive and specific detection of HClO and MPO. Only two DNA oligonucleotides are used in the sensing systems. The powerful signal-amplification capability of RCA endows the sensing systems with a high sensitivity, and the specific fluorescent response of thioflavin T (ThT) to G-quadruplexes in RCA products makes a label-free signal output possible. The proposed biosensors were demonstrated to work well not only for the sensitive and specific quantitation of HClO and MPO with detection limits of 1.67 nM and 0.33 ng/mL, respectively, but also for the screening and inhibitory capacity evaluation of MPO inhibitors, thus holding great promise in disease diagnosis and drug analysis.

The Optimization of a Label-Free Electrochemical DNA Biosensor for Detection of Sus scrofa mtDNA as Food Adulterations

Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b S. scrofa gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box–Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5–1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.

A graphitic nano-onion/molybdenum disulfide nanosheet composite as a platform for HPV-associated cancer-detecting DNA biosensors

An electrochemical DNA sensor that can detect human papillomavirus (HPV)-16 and HPV-18 for the early diagnosis of cervical cancer was developed by using a graphitic nano-onion/molybdenum disulfide (MoS2) nanosheet composite. The electrode surface for probing DNA chemisorption was prepared via chemical conjugation between acyl bonds on the surfaces of functionalized nanoonions and the amine groups on functionalized MoS2 nanosheets. The cyclic voltammetry profile of an 1:1 nanoonion/MoS2 nanosheet composite electrode had an improved rectangular shape compared to that of an MoS2 nanosheet elecrode, thereby indicating the amorphous nature of the nano-onions with sp2 distancing curved carbon layers that provide enhanced electronic conductivity, compared to MoS2 nanosheet only. The nanoonion/MoS2 sensor for the DNA detection of HPV-16 and HPV-18, respectively, was measured at high sensitivity through differential pulse voltammetry (DPV) in the presence of methylene blue (MB) as a redox indicator. The DPV current peak was lowered after probe DNA chemisorption and target DNA hybridization because the hybridized DNA induced less effective MB electrostatic intercalation due to it being double-stranded, resulting in a lower oxidation peak. The nanoonion/MoS2 nanosheet composite electrodes attained higher current peaks than the MoS2 nanosheet electrode, thereby indicating a greater change in the differential peak probably because the nanoonions enhanced conductive electron transfer. Notably, both of the target DNAs produced from HPV-18 and HPV-16 Siha and Hela cancer cell lines were effectively detected with high specificity. The conductivity of MoS2 improved by complexation with nano-onions provides a suitable platform for electrochemical biosensors for the early diagnosis of many ailments in humans.Graphical

An Ultrasensitive Voltammetric Genosensor for the Detection of Bacteria Vibrio cholerae in Vegetable and Environmental Water Samples.

In view of the presence of pathogenic Vibrio cholerae (V. cholerae) bacteria in environmental waters, including drinking water, which may pose a potential health risk to humans, an ultrasensitive electrochemical DNA biosensor for rapid detection of V. cholerae DNA in the environmental sample was developed. Silica nanospheres were functionalized with 3-aminopropyltriethoxysilane (APTS) for effective immobilization of the capture probe, and gold nanoparticles were used for acceleration of electron transfer to the electrode surface. The aminated capture probe was immobilized onto the Si-Au nanocomposite-modified carbon screen printed electrode (Si-Au-SPE) via an imine covalent bond with glutaraldehyde (GA), which served as the bifunctional cross-linking agent. The targeted DNA sequence of V. cholerae was monitored via a sandwich DNA hybridization strategy with a pair of DNA probes, which included the capture probe and reporter probe that flanked the complementary DNA (cDNA), and evaluated by differential pulse voltammetry (DPV) in the presence of an anthraquninone redox label. Under optimum sandwich hybridization conditions, the voltammetric genosensor could detect the targeted V. cholerae gene from 1.0 × 10-17-1.0 × 10-7 M cDNA with a limit of detection (LOD) of 1.25 × 10-18 M (i.e., 1.1513 × 10-13 µg/µL) and long-term stability of the DNA biosensor up to 55 days. The electrochemical DNA biosensor was capable of giving a reproducible DPV signal with a relative standard deviation (RSD) of <5.0% (n = 5). Satisfactory recoveries of V. cholerae cDNA concentration from different bacterial strains, river water, and cabbage samples were obtained between 96.5% and 101.6% with the proposed DNA sandwich biosensing procedure. The V. cholerae DNA concentrations determined by the sandwich-type electrochemical genosensor in the environmental samples were correlated to the number of bacterial colonies obtained from standard microbiological procedures (bacterial colony count reference method).

Universal CRISPR/Cas12a-associated aptasensor suitable for rapid detection of small proteins with a plate reader.

With the discovery of the collateral cleavage activity, CRISPR/Cas12a has recently been identified as a key enabling approach in novel DNA biosensor development. Despite its remarkable success in nucleic acid detection, realizing a universal CRISPR/Cas biosensing system for non-nucleic acid targets remains challenging, particularly at extremely high sensitivity ranges for analyte concentrations lower than the pM level. DNA aptamers can be designed to bind to a range of specific target molecules, such as proteins, small molecules, and cells, with high affinity and specificity through configuration changes. Here, by harnessing its diverse analyte-binding ability and also redirecting the specific DNA-cutting activity of Cas12a to selected aptamers, a simple, sensitive, and universal biosensing platform has been established, termed CRISPR/Cas and aptamer-mediated extra-sensitive assay (CAMERA). With simple modifications to the aptamer and guiding RNA of Cas12a RNP, CAMERA demonstrated 100fM sensitivity for targeting small proteins, such as IFN-γ and insulin, with less than 1.5-h detection time. Compared with the gold-standard ELISA, CAMERA achieved higher sensitivity and a shorter detection time while retaining ELISA's simple setup. By replacing the antibody with an aptamer, CAMERA also achieved improved thermal stability, allowing to eliminate the requirement for cold storage. CAMERA shows potential to be used as a replacement for conventional ELISA for a variety of diagnostics but with no significant changes for the experimental setup.

Recent Aptamer-Based Biosensors for Cd2+ Detection.

Cd2+, a major environmental pollutant, is heavily toxic to human health. Many traditional techniques are high-cost and complicated; thus, developing a simple, sensitive, convenient, and cheap monitoring approach is necessary. The aptamer can be obtained from a novel method called SELEX, which is widely used as a DNA biosensor for its easy acquisition and high affinity of the target, especially for heavy metal ions detection, such as Cd2+. In recent years, highly stable Cd2+ aptamer oligonucleotides (CAOs) were observed, and electrochemical, fluorescent, and colorimetric biosensors based on aptamers have been designed to monitor Cd2+. In addition, the monitoring sensitivity of aptamer-based biosensors is improved with signal amplification mechanisms such as hybridization chain reactions and enzyme-free methods. This paper reviews approaches to building biosensors for inspecting Cd2+ by electrochemical, fluorescent, and colorimetric methods. Finally, many practical applications of sensors and their implications for humans and the environment are discussed.

Enzyme-Triggered DNA Sensor Technology for Spatially-Controlled, Cell-Selective Molecular Imaging.

ConspectusWith unparalleled programmability, DNA has evolved as a powerful scaffold for engineering intricate and dynamic systems that can perform diverse tasks. By allowing serial detection of molecular targets in complex cellular milieus, increasingly sophisticated DNA sensors have not only promoted significant advances in unveiling the fundamental mechanisms of various pathophysiological processes but also provided a useful toolkit for disease diagnostics based on molecular signatures. Despite much progress, an inherent limitation of DNA-based sensors is that they often lack spatial control and cell-type selectivity for the sensing activity because of their "always active" design mechanism. Since most molecular targets of interests are not exclusive to disease cells, they are also shared by normal cells, the application of such biosensors for disease-specific imaging is limited by inadequate signal-to-background ratios due to indistinguishable signal response in both disease and normal cells. Therefore, imparting biosensors with spatial controllability remains a key issue to achieve molecular imaging with high sensitivity and cell specificity.As a biocatalyst, enzyme has been found to be closely related with the pathological conditions of numerous diseases. For example, many nucleases, protease, and kinases have been identified overexpressed in disease cells and considered as important biomarkers of cancer, inflammation, and neurological diseases. Recently, we have envisioned that such pathophysiology-associated enzymes could be leveraged as endogenous triggers to achieve spatial control over the molecular imaging activity of the DNA-based sensors with improved cell-specificity. In this Account, we outline the research efforts from our group on the development of endogenous enzyme-triggered, DNA-based sensor technology that enables spatially controlled, cell-type selective molecular imaging. With programmable DNA design and further engineering of enzymatically cleavable sites, a series of DNAzyme- and aptamer-based sensors have been developed for enzyme-controlled imaging of various molecular targets (e.g., metal ions and small molecules) in a cancer cell-selective manner. In particular, by introduction of PNA as bridge molecules to engineer DNA-based sensors with functional peptides, the conceptual design of protease-activated DNA biosensors has been established for spatioselective molecular imaging in cancer cells and extracellular tumor microenvironments. Furthermore, enzyme-triggered signal amplification approaches, such as enzymatically activated molecular beacon and catalytic hairpin assembly, have been developed for spatially selective RNA imaging in specific disease cells (e.g., inflammatory cells and cancer cells), which enables enhanced disease-site specificity and thus improved signal-to-background ratio. The signal amplification strategy is further expanded to cell-selective amplified imaging of non-RNA species through the combination with functional DNA design. Finally, the challenges and potential future directions in this burgeoning field are discussed. We hope this Account offers insights into rational design of enzymatically controlled, DNA-based sensor platforms for opening new frontiers in spatially resolved, cell-selective molecular imaging. We believe that the continuing advances in DNA-based molecular sensing technology together with the discoveries of diverse disease-associated enzymes will promise to usher a new era of diagnosis.

Comparison of DNA-Gold Nanoparticle Conjugation Methods: Application in Lateral Flow Nucleic Acid Biosensors.

Lateral flow nucleic acid biosensors (LFNABs) have attracted extensive attention due to their rapid turnaround time, low cost, and results that are visible to the naked eye. One of the key steps to develop LFNABs is to prepare DNA-gold nanoparticle (DNA-AuNP) conjugates, which affect the sensitivity of LFNABs significantly. To date, various conjugation methods-including the salt-aging method, microwave-assisted dry heating method, freeze-thaw method, low-pH method, and butanol dehydration method-have been reported to prepare DNA-AuNP conjugates. In this study, we conducted a comparative analysis of the analytical performances of LFNABs prepared with the above five conjugation methods, and we found that the butanol dehydration method gave the lowest detection limit. After systematic optimization, the LFNAB prepared with the butanol dehydration method had a detection limit of 5 pM for single-strand DNA, which is 100 times lower than that of the salt-aging method. The as-prepared LFNAB was applied to detect miRNA-21 in human serum, with satisfactory results. The butanol dehydration method thus offers a rapid conjugation approach to prepare DNA-AuNP conjugates for LFNABs, and it can also be extended to other types of DNA biosensors and biomedical applications.

Development of a Single-Molecule Nanobalance Ratiometric Electrochemical DNA Biosensor Using a Triblock Poly-Adenine Probe.

The development of electrochemical DNA biosensors has been limited by their reliability and reproducibility due to many interfering factors such as electrode properties, DNA surface densities, and complex biological samples. In this work, we developed a nanobalance polyA hairpin probe (polyA-HP), which was effectively assembled onto the gold electrode surface through the affinity between the central polyA fragment and the Au surface. One flanking probe of the polyA-HP captured the target sequence together with a MB-labeled signal probe, and the other flanking probe captured a reference probe simultaneously. The MB signal related to the amount of target was normalized by the reference Fc signal; thus, the signal-to-noise (S/N) was as high as 2000, and the reproducibility was remarkably improved to 2.77%, even facing deliberately changed experiment conditions. By designing a hairpin structure at the terminal of the polyA-HP, the selectivity and specificity were dramatically improved for the analysis of mismatched sequences. The analysis performance of biological samples was dramatically improved after normalization, which is critical for its practicability. Our novel biosensor is a universal single-molecule platform for ratiometric biosensors with excellent performance in real samples, indicating great potential for next-generation high-precision electrochemical sensors.

Light-Up Split Broccoli Aptamer as a Versatile Tool for RNA Assembly Monitoring in Cell-Free TX-TL Systems, Hybrid RNA/DNA Origami Tagging and DNA Biosensing.

Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a 'bio-orthogonal' hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets.

A dual-blocker aided and dual-label-free electrochemical biosensor based on mbHCR/rGO nanocomplexes for ultrasensitive DNA detection

Heterogeneous electrochemical DNA biosensors have attracted huge attention due to their enhanced signal sensitivity, compared to homogeneous biosensors. However, the high cost of probe labeling and the reduced recognition efficiency associated with current heterogeneous electrochemical biosensors confine their potential applications. In the present work, a dual-blocker assisted and dual-label-free heterogeneous electrochemical strategy based on multi-branched hybridization chain reaction (mbHCR) and reduced graphene oxide (rGO) was fabricated for ultrasensitive detection of DNA. The target DNA could trigger the mbHCR of two DNA hairpin probes, resulting in the generation of multi-branched long chain of DNA duplexes with bidirectional arms. One direction of the multi-branched arms in the mbHCR products were then bound to the label-free capture probe on the gold electrode through multivalent hybridization with enhanced recognition efficiency. The other direction of multi-branched arms in mbHCR product could adsorb rGO via π-π stacking interactions. Two DNA blockers were ingeniously designed to block the binding of excessive H1-pAT on electrode and to prevent the adsorption of rGO by residual unbound capture probes. As a result, with the electrochemical reporter methylene blue selectively intercalated into the long chain of DNA duplex and absorbed on rGO, a remarkable electrochemical signal rise was observed. Thus, a dual-blocker aided and dual-label-free electrochemical strategy for ultrasensitive DNA detection is readily realized with the merit of cost-effective. The as-developed dual-label-free electrochemical biosensor has great potential to be employed in nucleic acid related medical diagnostics.

Development of a QCM-based biosensor for the detection of non-small cell lung cancer biomarkers in liquid biopsies

Lung cancer is the main malignant cancer reported worldwide, with one of the lowest survival rates. Deletions in the Epidermal Growth Factor Receptor (EGFR) gene are often associated with non-small cell lung cancer (NSCLC), a common subtype of lung cancer. The detection of such mutations provides key information for the diagnosis and treatment of the disease; therefore, the early screening of such biomarkers is of vital importance. The need for fast, reliable, and early detection means applied to NSCLC has led to the development of highly sensitive devices that can detect cancer-associated mutations. Such devices, known as biosensors, are a promising alternative to more conventional detection methods and can potentially alter the way cancer is diagnosed and treated. In this study, we report the development of a DNA-based biosensor, namely a quartz crystal microbalance (QCM), applied to the detection of NSCLC, from liquid biopsies samples. The detection, as is the case of most DNA biosensors, is based on the hybridization between the NSCLC-specific probe and the sample DNA (containing specific mutations associated with NSCLC). The surface functionalization was performed with a blocking agent (dithiothreitol) and thiolated-ssDNA strands. The biosensor was able to detect specific DNA sequences in both synthetic and real samples. Aspects such as reutilization and regeneration of the QCM electrode were also studied.

Calf thymus ds-DNA intercalation with pendimethalin herbicide at the surface of ZIF-8/Co/rGO/C3N4/ds-DNA/SPCE; A bio-sensing approach for pendimethalin quantification confirmed by molecular docking study

Pendimethalin (PND) is a herbicide that is regarded to be possibly carcinogenic to humans and toxic to the environment. Herein, we fabricated a highly sensitive DNA biosensor based on ZIF-8/Co/rGO/C3N4 nanohybrid modification of a screen-printed carbon electrode (SPCE) to monitor PND in real samples. The layer-by-layer fabrication pathway was conducted to construct ZIF-8/Co/rGO/C3N4/ds-DNA/SPCE biosensor. The physicochemical characterization techniques confirmed the successful synthesis of ZIF-8/Co/rGO/C3N4 hybrid nanocomposite, as well as the appropriate modification of the SPCE surface. The utilization of ZIF-8/Co/rGO/C3N4 nanohybrid as a modifier was analyzed using. The electrochemical impedance spectroscopy results showed that the modified SPCE exhibited significantly lowered charge transfer resistance due to the enhancement of its electrical conductivity and facilitation of the transfer of charged particles. The proposed biosensor successfully quantified PND in a wide concentration range of 0.01–35 μM, with a limit of detection (LOD) value of 8.0 nM. The PND monitoring capability of the fabricated biosensor in real samples including rice, wheat, tap, and river water samples was verified with a recovery range of 98.2–105.6%. Moreover, to predict the interaction sites of PND herbicide with DNA, the molecular docking study was performed between the PND molecule and two sequence DNA fragments and confirmed the experimental findings. This research sets the stage for developing highly sensitive DNA biosensors that will be used to monitor and quantify toxic herbicides in real samples by fusing the advantages of nanohybrid structures with crucial knowledge from a molecular docking investigation.

Feasibility of a DNA biosensor assay based on loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the visual detection of Ascaridia galli eggs in faecal samples

ABSTRACT Ascaridia galli is an important nematode that causes ascaridiasis in free-range and indoor system chicken farms. Infection with A. galli may damage the intestinal mucosa and inhibit nutrient absorption, leading to a reduced growth rate, weight loss and a decreased egg production. Consequently, A. galli infection is a significant health problem in chickens. In this study, we developed a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for the visual detection of A. galli eggs in faecal samples. The LAMP-LFD assay consists of six primers and one DNA probe that recognize the internal transcribed spacer 2 (ITS2) region; it can be performed within 70 min and the results can be interpreted with the naked eye. Using the LAMP-LFD assay developed in this study, A. galli DNA was specifically amplified without any cross-reactions with other related parasites (Heterakis gallinarum, Raillietina echinobothrida, R. tetragona, R. cesticillus, Cotugnia sp., Echinostoma miyagawai) and definitive hosts (Gallus gallus domesticus, Anas platyrhynchos domesticus). The minimum detectable DNA concentration was 5 pg/μl, and the detectable egg count was 50 eggs per reaction. The assay can be performed in a water bath, without the need for post-mortem morphological investigations and laboratory instruments. It is therefore a viable alternative for the detection of A. galli in chicken faeces and can replace classical methods in field screening for epidemiological investigations, veterinary health and poultry farming management. RESEARCH HIGHLIGHTS This is the first study using the LAMP-LFD assay for Ascaridia galli detection. The results can be observed by the naked eye. The developed assay can be used to detect Ascaridia galli eggs in faecal samples.