Synthesis, DNA binding of bis-naphthyl ferrocene derivatives and the application as new electroactive indicators for DNA biosensor.

A series of bis-naphthyl ferrocene derivatives were synthesized and characterized. Based on the results obtained from UV-visible absorption titration and ethidium bromide (EB) displacement experiments, it was observed that the synthesized compounds exhibited a strong binding ability to dsDNA. In comparison to the viscosity curve of EB, the tested compounds demonstrated a bisintercalation binding mode when interacting with CT-DNA. Differential pulse voltammetry (DPV) was employed to assess the binding specificity of these indicators towards ssDNA and dsDNA. All tested indicators displayed more pronounced signal differences before and after hybridization between probe nucleic acids and target nucleic acids compared to Methylene Blue (MB). Among the evaluated compounds, compound 3j containing an ether chain showed superior performance as an indicator, making it suitable for constructing DNA-based biosensors. Under optimized conditions including probe ssDNA concentration and indicator concentration, this biosensor exhibited good sensitivity, reproducibility, stability, and selectivity. The limit of detection was calculated as 4.53 × 10-11 mol/L. Furthermore, when utilizing 3j as the indicator in serum samples, the biosensor achieved satisfactory recovery rates for detecting the BRCA1 gene.

Electrochemical DNA-nano biosensor for the detection of Goserelin as anticancer drug using modified pencil graphite electrode.

Goserelin is an effective anticancer drug, but naturally causes several side effects. Hence the determination of this drug in biological samples, plays a key role in evaluating its effects and side effects. The current studies have concentrated on monitoring Goserelin using an easy and quick DNA biosensor for the first time. In this study, copper(II) oxide nanoparticles were created upon the surface of multiwalled carbon nanotubes (CuO/MWCNTs) as a conducting mediator. The modified pencil graphite electrode (ds-DNA/PA/CuO/MWCNTs/PGE) has been modified with the help of polyaniline (PA), ds-DNA, and CuO/MWCNTs nanocomposite. Additionally, the issue with the bio-electroanalytical guanine oxidation signal in relation to ds-DNA at the surface of PA/CuO/MWCNTs/PGE has been examined to determination Goserelin for the first time. It also, established a strong conductive condition to determination Goserelin in nanomolar concentration. Thus, Goserelin's determining, however, has a 0.21 nM detection limit and a 1.0 nM-110.0 µM linear dynamic range according to differential pulse voltammograms (DPV) of ds-DNA/PA/CuO/MWCNTs/PGE. Furthermore, the molecular docking investigation highlighted that Goserelin is able to bind ds-DNA preferentially and supported the findings of the experiments. The determining of Goserelin in real samples has been effectively accomplished in the last phase using ds-DNA/PA/CuO/MWCNTs/PGE.

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling.

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63- reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63- for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63- and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ.

To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.

An attomolar-level electrochemical DNA biosensor based on target-triggered and entropy-driven catalytic amplification integrated with AuNPs@ZIF-8 nanocomposites for oral cancer overexpressed 1 detection.

It is of great interest and necessity to develop a nonenzymatic, simple but highly sensitive biosensor for early diagnosis of oral cancer. Present here is an electrochemical DNA biosensor which integrates a target-triggered, entropy-driven, nonenzymatic and isothermal amplification strategy with gold nanoparticles/zeolitic imidazolate frameworks-8 (AuNPs@ZIF-8) nanocomposites for ultra-sensitive detection of oral cancer-related biomarker (ORAOV 1) in saliva. It is worth noting that the nuclease is not involved in the whole reaction process, which is simple and flexible in design only using a series of linear single-stranded DNA, avoiding undesired secondary structure interference. Meanwhile, due to the synergistic effect of AuNPs and ZIF-8, AuNPs@ZIF-8 nanocomposites display high stability, excellent electrical conductivity and exceptional electrocatalytic activity, further enhancing the electrochemical signal and avoiding labeling electrochemical signal probes. Experimental results demonstrate that this electrochemical DNA biosensor has a wide linear range (1 fM ∼1 nM), a low limit of detection (163 aM), excellent specificity, superior reproducibility and stability to ORAOV 1. More importantly, the actual application of the newly developed electrochemical biosensor is exemplified in human saliva with satisfactory recoveries. Therefore, the newly developed electrochemical biosensor has a broad application prospect in the nondestructive and early screening of oral cancer.

Rational design and synthesis of triazene-amonafide derivatives as novel potential antitumor agents causing oxidative damage towards DNA through intercalation mode.

In this work, we rationally designed and synthesized two novel triazene-amonafide derivatives 2-(2-(diisopropylamino)ethyl)-5-(3,3-dimethyltriaz-1-en-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (D-11) and 5-(3,3-diethyltriaz-1-en-1-yl)-2-(2-(diisopropylamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (D-12) as potential antitumor agents. The DNA damage induced by the intercalation mode of D-11 (D-12) towards DNA was electrochemically detected through the construction of efficient biosensors. The consecutive processes of reversible redox of naphthylimide ring and irreversible oxidation of triazene moiety were elucidated on the surface of glassy carbon electrode (GCE) by CV, SWV, and DPV methods. Electrochemical biosensors were obtained through the immobilization of ctDNA, G-quadruplexes, poly(dG), and poly(dA), respectively, on the clean surface of GCE. After the incubation of biosensors with D-11 or D-12, the peaks of dGuo and dAdo decreased prominently, and the peak of 8-oxoGua appeared at +0.50 V, suggesting that the interaction between D-11 (D-12) and DNA could result in the oxidative damage of guanine. Unexpected, the as-prepared DNA biosensor possessed satisfactory anti-interference property and good practicability in real samples. UV-vis and fluorescence spectra, and gel electrophoresis assays were employed to further confirm the intercalation mode of D-11 (D-12) towards DNA base pairs. Moreover, D-11 was proved to exhibit stronger anti-proliferation activity than mitionafide and amonafide against both A549 and HeLa cell lines.

Detection of high-risk HPV 16 genotypes in cervical cancers using isothermal DNA amplification with electrochemical genosensor.

Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/µL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.

Free PCR virus detection via few-layer bismuthene and tetrahedral DNA nanostructured assemblies.

In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with H2O2, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 µM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification process.

Quantum Material-Based Self-Propelled Microrobots for the Optical "On-the-Fly" Monitoring of DNA.

Quantum dot-based materials have been found to be excellent platforms for biosensing and bioimaging applications. Herein, self-propelled microrobots made of graphene quantum dots (GQD-MRs) have been synthesized and explored as unconventional dynamic biocarriers toward the optical "on-the-fly" monitoring of DNA. As a first demonstration of applicability, GQD-MRs have been first biofunctionalized with a DNA biomarker (i.e., fluorescein amidite-labeled, FAM-L) via hydrophobic π-stacking interactions and subsequently exposed toward different concentrations of a DNA target. The biomarker-target hybridization process leads to a biomarker release from the GQD-MR surface, resulting in a linear alteration in the fluorescence intensity of the dynamic biocarrier at the nM range (1-100 nM, R2 = 0.99), also demonstrating excellent selectivity and sensitivity, with a detection limit as low as 0.05 nM. Consequently, the developed dynamic biocarriers, which combine the appealing features of GQDs (e.g., water solubility, fluorescent activity, and supramolecular π-stacking interactions) with the autonomous mobility of MRs, present themselves as potential autonomous micromachines to be exploited as highly efficient and sensitive "on-the-fly" biosensing systems. This method is general and can be simply customized by tailoring the biomarker anchored to the GQD-MR's surface.

DNA Sensor for the Detection of Brucella spp. Based on Magnetic Nanoparticle Markers.

Due to the limitations of conventional Brucella detection methods, including safety concerns, long incubation times, and limited specificity, the development of a rapid, selective, and accurate technique for the early detection of Brucella in livestock animals is crucial to prevent the spread of the associated disease. In the present study, we introduce a magnetic nanoparticle marker-based biosensor using frequency mixing magnetic detection for point-of-care testing and quantification of Brucella DNA. Superparamagnetic nanoparticles were used as magnetically measured markers to selectively detect the target DNA hybridized with its complementary capture probes immobilized on a porous polyethylene filter. Experimental conditions like density and length of the probes, hybridization time and temperature, and magnetic binding specificity, sensitivity, and detection limit were investigated and optimized. Our sensor demonstrated a relatively fast detection time of approximately 10 min, with a detection limit of 55 copies (0.09 fM) when tested using DNA amplified from Brucella genetic material. In addition, the detection specificity was examined using gDNA from Brucella and other zoonotic bacteria that may coexist in the same niche, confirming the method's selectivity for Brucella DNA. Our proposed biosensor has the potential to be used for the early detection of Brucella bacteria in the field and can contribute to disease control measures.

Multiplex Portable Biosensor for Bacteria Detection.

An advanced, cost-effective, and portable DNA biosensor capable of detecting multiple bacteria simultaneously has been developed. The biosensor comprises a fast and inexpensive potentiostat that controls the applied potential to a screen-printed electrochemical array platform functionalized with MoS2 flakes and bacterial DNA probes. The current response obtained by à la carte thionine functionalized carbon nanodots (Ty-CDs) is monitored as an electrochemical indicator of the hybridization event. The design of the potentiostat prioritizes achieving an optimal signal-to-noise ratio and incorporates a user-friendly interface compatible with various devices, including computers, mobile phones, and tablets. The device is compact, lightweight, and manufactured at a low cost. The key components of the potentiostat include a data acquisition board capable of analyzing multiple samples simultaneously and a controller board. The results of this study confirm the ability of the multiplex portable biosensor to successfully detect specific bacterial DNA sequences, demonstrating its reliability and superior performance compared with a traditional, more complex, and laboratory-oriented potentiostat.

Unraveling the Possibilities: Recent Progress in DNA Biosensing.

Due to the advantages of its numerous modification sites, predictable structure, high thermal stability, and excellent biocompatibility, DNA is the ideal choice as a key component of biosensors. DNA biosensors offer significant advantages over existing bioanalytical techniques, addressing limitations in sensitivity, selectivity, and limit of detection. Consequently, they have attracted significant attention from researchers worldwide. Here, we exemplify four foundational categories of functional nucleic acids: aptamers, DNAzymes, i-motifs, and G-quadruplexes, from the perspective of the structure-driven functionality in constructing DNA biosensors. Furthermore, we provide a concise overview of the design and detection mechanisms employed in these DNA biosensors. Noteworthy advantages of DNA as a sensor component, including its programmable structure, reaction predictility, exceptional specificity, excellent sensitivity, and thermal stability, are highlighted. These characteristics contribute to the efficacy and reliability of DNA biosensors. Despite their great potential, challenges remain for the successful application of DNA biosensors, spanning storage and detection conditions, as well as associated costs. To overcome these limitations, we propose potential strategies that can be implemented to solve these issues. By offering these insights, we aim to inspire subsequent researchers in related fields.

Multi-Walled Carbon Nanotube Array Modified Electrode with 3D Sensing Interface as Electrochemical DNA Biosensor for Multidrug-Resistant Gene Detection.

Drug resistance in cancer is associated with overexpression of the multidrug resistance (MDR1) gene, leading to the failure of cancer chemotherapy treatment. Therefore, the establishment of an effective method for the detection of the MDR1 gene is extremely crucial in cancer clinical therapy. Here, we report a novel DNA biosensor based on an aligned multi-walled carbon nanotube (MWCNT) array modified electrode with 3D nanostructure for the determination of the MDR1 gene. The microstructure of the modified electrode was observed by an atomic force microscope (AFM), which demonstrated that the electrode interface was arranged in orderly needle-shaped protrusion arrays. The electrochemical properties of the biosensor were characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Chronocoulometry (CC) was used for the quantitative detection of the MDR1 gene. Taking advantage of the good conductivity and large electrode area of the MWCNT arrays, this electrochemical DNA sensor achieved a dynamic range from 1.0 × 10-12 M to 1.0 × 10-8 M with a minimal detection limit of 6.4 × 10-13 M. In addition, this proposed DNA biosensor exhibited high sensitivity, selectivity, and stability, which may be useful for the trace analysis of the MDR1 gene in complex samples.

Rapid and Quantitative Detection of Lung Cancer Biomarker ENOX2 Using a Novel Aptamer in an Electrochemical DNA-Based (E-DNA) Biosensor.

To overcome early cancer detection challenges, diagnostic tools enabling more sensitive, rapid, and noninvasive detection are necessary. An attractive cancer target for diagnostic blood tests is human Ecto-NOX disulfide-thiol exchanger 2 (ENOX2), expressed in most human cancer types and regularly shed into blood sera. Here, we developed an electrochemical DNA-based (E-DNA) biosensor that rapidly detects physiologically relevant levels of ENOX2. To identify ENOX2-binding aptamers that could potentially be used in a biosensor, recombinantly expressed ENOX2 was used as a binding target in an oligonucleotide library pull-down that generated a highly enriched ENOX2-binding aptamer. This candidate aptamer sensitively bound ENOX2 via gel mobility shift assays. To enable this aptamer to function in an ENOX2 E-DNA biosensor, the aptamer sequence was modified to adopt two conformations, one capable of ENOX2 binding, and one with disrupted ENOX2 binding. Upon ENOX2 introduction, a conformational shift to the ENOX2 binding state resulted in changed dynamics of a redox reporter molecule, which generated a rapid, significant, and target-specific electrical current readout change. ENOX2 biosensor sensitivity was at or below the diagnostic range. The ENOX2 E-DNA biosensor design presented here may enable the development of more sensitive, rapid, diagnostic tools for early cancer detection.

Diagnostic genosensor for detection of rotavirus based on HFGNs/MXene/PPY signal amplification.

A novel genosensor was developed for rotavirus specific cDNA sequence detection. The genosensor was comprised of hierarchical flower-like gold nanostructures, MXene, and polypyrrole (HFGNs/MXene/PPY) nanocomposite as a signal amplification tag, specific antisense ssDNA oligonucleotide as a recognition bioelement, and methylene blue (MB) as a redox marker. The morphological and electrochemical features of the biosensor were first tested and optimized and the high performance of the platform was confirmed in terms of sensitivity and reproducibility. Then, 20 rotavirus RNA isolated from clinical and cell-cultured samples (10 positive and 10 negative confirmed by RT-PCR and electrophoresis methods) were evaluated by the genosensor. The analysis results revealed that the genosensor is able to differentiate successfully between the positive and negative control groups. The developed genosensor for rotavirus RNA detection presented an excellent limit of detection of ∼ 0.8 aM and a determination  range of  10-18 and 10-7 M. In addition, the ssDNA/HFGNs/MXene/PPY/GCE showed high selectivity and long-term stability of ~ 24 days. Therefore, this novel genosensor would be of great benefit for the clinical diagnosis of rotavirus.

Gemcitabine drug intercalation with ds-DNA at surface of ds-DNA/Pt-ZnO/SWCNTs/GCE biosensor: A DNA-biosensor for gemcitabine monitoring confirmed by molecular docking study.

Herein, a facile and highly sensitive electroanalytical tool for monitoring and quantifying the antineoplastic drug gemcitabine in real sample was provided. In this regard, a novel DNA-biosensor based on Pt-doped ZnO decorated single walled carbon nanotubes (Pt-ZnO/SWCNTs) hybrid nanomaterial modification of glassy carbon electrode (GCE) was fabricated. Ds-DNA (Calf Thymus), as a biological recognition element, was decorated onto nanomaterial-modified GCE via layer-by-layer fabrication strategy to attain ultimate biosensor ds-DNA/Pt-ZnO/SWCNTs/GCE. The characterizations confirmed the successful fabrication of hybrid nanomaterial, as well as the modification of electrode surface by fabricated nanomaterial. The electrochemical impedance spectroscopy (EIS) analysis revealed that the nanomaterial modification of GCE surface enhanced the electrical conductivity thanks to the synergistic effects of Pt-ZnO and SWCNTs structures, thereby boosted the electrocatalytic activity of the resultant biosensor. The electrochemical characterization results showed that the suggested biosensor is capable of detecting gemcitabine in a wide concentration range of 0.01-30.0 µM, with a detection limit of 5.0 nM. The intercalation binding mode of Gemcitabine inside guanine and cytosine rich region of DNA receptor was approved by molecular docking study. The results of the experimental data were well congruent with the molecular docking analysis, which showed that the binding mode of gemcitabine drug with ds-DNA was intercalation.

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).

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.

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.

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae.

Herein, we report results of the studies relating to the development of an impedimetric, magnetic bead-assisted supersandwich DNA hybridization assay for ultrasensitive detection of Neisseria gonorrhoeae, the causative agent of a sexually transmitted infection (STI), gonorrhea. First, a conductive ink was formulated by homogenously dispersing carboxylated multiwalled carbon nanotubes (cMWCNTs) in a stable emulsion of terpineol and an aqueous suspension of carboxymethyl cellulose (CMC). The ink, labeled C5, was coated onto paper substrates to fabricate C5@paper conductive electrodes. Thereafter, a magnetic bead (MB)-assisted supersandwich DNA hybridization assay was optimized against the porA pseudogene of N. gonorrhoeae. For this purpose, a pair of specific 5' aminated capture probes (SCP) and supersandwich detector probes (SDP) was designed, which allowed the enrichment of target gonorrheal DNA sequence from a milieu of substances. The SD probe was designed such that instead of 1:1 binding, it allowed the binding of more than one T strand, leading to a 'ladder-like' DNA supersandwich structure. The MB-assisted supersandwich assay was integrated into the C5@paper electrodes for electrochemical analysis. The C5@paper electrodes were found to be highly conductive by a four-probe conductivity method (maximum conductivity of 10.1 S·cm-1). Further, the biosensing assay displayed a wide linear range of 100 aM-100 nM (109 orders of magnitude) with an excellent sensitivity of 22.6 kΩ·(log[concentration])-1. The clinical applicability of the biosensing assay was assessed by detecting genomic DNA extracted from N. gonorrhoeae in the presence of DNA from different non-gonorrheal bacterial species. In conclusion, this study demonstrates a highly sensitive, cost-effective, and label-free paper-based device for STI diagnostics. The ink formulation prepared for the study was found to be highly thixotropic, which indicates that the paper electrodes can be screen-printed in a reproducible and scalable manner.