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Experimental methods capable of selectively probing water at the DNA minor groove, major groove, and phosphate backbone are crucial for understanding how hydration influences DNA structure and function. Chiral-selective sum frequency generation spectroscopy (chiral SFG) is unique among vibrational spectroscopies because it can selectively probe water molecules that form chiral hydration structures around biomolecules. However, interpreting chiral SFG spectra is challenging since both water and the biomolecule can produce chiral SFG signals. Here, we combine experiment and computation to establish a theoretical framework for the rigorous interpretation of chiral SFG spectra of DNA. We demonstrate that chiral SFG detects the N–H stretch of DNA base pairs and the O–H stretch of water, exclusively probing water molecules in the DNA first hydration shell. Our analysis reveals that DNA transfers chirality to water molecules only within the first hydration shell, so they can be probed by chiral SFG spectroscopy. Beyond the first hydration shell, the electric field-induced water structure is symmetric and, therefore, precludes chiral SFG response. Furthermore, we find that chiral SFG can differentiate chiral subpopulations of first hydration shell water molecules at the minor groove, major groove, and phosphate backbone. Our findings challenge the scientific perspective dominant for more than 40 years that the minor groove "spine of hydration" is the only chiral water structure surrounding the DNA double helix. By identifying the molecular origins of the DNA chiral SFG spectrum, we lay a robust experimental and theoretical foundation for applying chiral SFG to explore the chemical and biological physics of DNA hydration. [ABSTRACT FROM AUTHOR]

Coarse-grained models have emerged as valuable tools to simulate long DNA molecules while maintaining computational efficiency. These models aim at preserving interactions among coarse-grained variables in a manner that mirrors the underlying atomistic description. We explore here a method for testing coarse-grained vs all-atom models using stiffness matrices in Fourier space (q-stiffnesses), which are particularly suited to probe DNA elasticity at different length scales. We focus on a class of coarse-grained rigid base DNA models known as cgDNA and its most recent version, cgDNA+. Our analysis shows that while cgDNA+ closely follows the q-stiffnesses of the all-atom model, the original cgDNA shows some deviations for twist and bending variables, which are rather strong in the q → 0 (long length scale) limit. The consequence is that while both cgDNA and cgDNA+ give a suitable description of local elastic behavior, the former misses some effects that manifest themselves at longer length scales. In particular, cgDNA performs poorly on twist stiffness, with a value much lower than expected for long DNA molecules. Conversely, the all-atom and cgDNA+ twist are strongly length scale dependent: DNA is torsionally soft at a few base pair distances but becomes more rigid at distances of a few dozen base pairs. Our analysis shows that the bending persistence length in all-atom and cgDNA+ is somewhat overestimated. [ABSTRACT FROM AUTHOR]

This paper describes the design and optimization of a 10 ml cartridge for patient sample processing using a 3.5 GHz (empty resonant frequency) TM010 cylindrical microwave cavity. The cartridge has been designed to augment a novel approach for the rapid diagnosis of M. tuberculosis (the causative agent of Ttuberculosis), which uses the direct application of microwaves to a bacteria-containing sample to release pathogen-specific DNA. The target bacterial DNA is then captured and recovered using magnetic nanoparticles coated with pathogen-specific DNA probes. Excitation parameters were optimized using three surrogates for M. tuberculosis, namely, M. smegmatis, M. abscessus, and M. bovis suspended in water and simulated sputum. The paper also explores the mechanism of microwave-mediated DNA release from bacteria using scanning electron microscopy. Examination of bacteria exposed to microwaves at power levels known to mediate the release of DNA reveals no obvious signs of permanent cell disruption, suggesting that a more subtle interaction is taking place. Finally, the presence of microwave-liberated M. bovis DNA was able to be detected at a level of sensitivity comparable to that achieved using microscopy. [ABSTRACT FROM AUTHOR]

A ratiometric electrochemiluminescence (ECL) aptamer-based sensing platform was fabricated for prostate-specific antigen (PSA) determination. Activated CdS nanocrystals/multi-walled carbon nanotubes (CdS/MCNTs) and luminol-Pt/PAMAM nanocomposites (L-Pt/PAMAM NCs) were synthesized and used as cathodic and anodic ECL emitters, respectively. Amino group–modified aptamers were assembled on carboxylated magnetic beads, followed by hybridization with probe DNA functionalized L-Pt/PAMAM NCs. In the presence of PSA, the aptamer would bind specifically to the target PSA, thereby releasing L-Pt/PAMAM NCs. After magnetic separation, the separated L-Pt/PAMAM NCs would hybridize with capture DNA on CdS/MCNTs coated on glassy carbon electrode. This binding would lead to a decrease in cathodic ECL signal of CdS/MCNTs, due to the efficient energy transfer from CdS/MCNTs to L-Pt/PAMAM NCs. Meanwhile, L-Pt/PAMAM brought the anodic ECL signal from luminol. With the increase of PSA concentration, the ECL emission from luminol increased and the ECL emission from CdS/MCNTs decreased. The ratio of ECL intensity of luminol at 0.55 V and CdS/MCNTs at − 1.25 V could be used to quantify the concentration of PSA. This method enables sensitive and reliable detection of PSA over a wide range from 0.05 to 200 ng mL−1, and the detection limit is 0.02 ng mL−1. [ABSTRACT FROM AUTHOR]

The "Lab on a Chip" journal, published by The Royal Society of Chemistry, features various papers on micro- and nanoscale devices and applications. The current issue includes topics such as surface modification of paper-based microfluidic devices, reversible bonding in thermoplastic elastomer microfluidic platforms, and multiplexed ultrasensitive detection of cancer biomarkers. Other papers cover areas like neuroplasticity investigations, miRNA detection in skin interstitial fluid, and artificial intelligence performance in testing microfluidics for point-of-care applications. The journal also explores topics such as magnetophoresis in millifluidic systems, dynamic sampling from ex vivo adipose tissue, and inertial co-focusing of heterogeneous particles in hybrid microfluidic channels. [Extracted from the article]

MicroRNA (miRNA) is a type of short, non-coding nucleic acid molecule that plays essential roles in diagnosing and prognosing various types of cancer. MiRNA is abundantly present in skin interstitial fluid (ISF), providing real-time and localized physiological information. Hydrogel microneedle (HMN) patches enable miRNA collection in a fast, pain-free, minimally invasive, and user-friendly manner. In this study, we introduced a fluorescence-based HMN assay, namely the HMN-miR sensor, composed of methacrylated hyaluronic acid (MeHA) and a graphene oxide–probe DNA (GO.pDNA) conjugate for miR21 and miR210 detection. The HMN-miR sensor demonstrates excellent skin penetration efficiency, rapid ISF collection capability, and sufficient miRNA detection and sequence identification specificity. The HMN-miR sensor facilitates a new assay that, with further optimization, could be applied in future clinical settings. Its simple fabrication process and excellent biocompatibility give it significant potential for various clinical uses, such as personalized cancer treatment and monitoring the healing progress of burn wounds. [ABSTRACT FROM AUTHOR]

Chemical genomics is a powerful and increasingly accessible technique to probe gene function, gene–gene interactions, and antibiotic synergies and antagonisms. Indeed, multiple large-scale pooled datasets in diverse organisms have been published. Here, we identify an artifact arising from uncorrected differences in the number of cell doublings between experiments within such datasets. We demonstrate that this artifact is widespread, show how it causes spurious gene–gene and drug–drug correlations, and present a simple but effective post hoc method for removing its effects. Using several published datasets, we demonstrate that this correction removes spurious correlations between genes and conditions, improving data interpretability and revealing new biological insights. Finally, we determine experimental factors that predispose a dataset for this artifact and suggest a set of experimental and computational guidelines for performing pooled chemical genomics experiments that will maximize the potential of this powerful technique. Synopsis: A widespread artifact in bacterial chemical genomics caused by uncorrected differences in growth causes spurious correlations between mutant phenotypes. The authors present a facile post-hoc correction and demonstrate its utility for extracting new biology. Uncorrected differences in the number of cell doublings between chemical genomics experiments affect the measured condition-specific fitness of sick mutants. This artifact is widespread in published datasets, resulting in spurious gene-gene and drug-drug correlations. A simple post-hoc correction, presented with this manuscript, removes the effect of this artifact, enhancing the biological interpretability of the data. A widespread artifact in bacterial chemical genomics caused by uncorrected differences in growth causes spurious correlations between mutant phenotypes. The authors present a facile post-hoc correction and demonstrate its utility for extracting new biology. [ABSTRACT FROM AUTHOR]

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The latest pandemic resulting from the novel SARS-CoV-2 coronavirus has significantly affected public health, the worldwide economy, and social life. Metal organic frameworks (MOFs) are currently being implemented in biosensors for rapid and accurate detection of viruses thanks to their exceptional properties. This research aims to develop a Zeolitic Imidazolate Framework-8 (ZIF-8) based fluorescent biosensor for facile and rapid COVID-19 RNA sequence detection. ZIF-8 was characterized using several tests, such as FT-IR, TGA, and PXRD, to examine the MOF's crystalline structure and thermal stability. The results demonstrated high crystallinity and thermal stability up to a temperature of 550 °C. The experimental study showed that ZIF-8 is an excellent fluorescence quencher, with 78.39% quenching efficiency. Analyzing the adsorption mechanism of probe DNA into ZIF-8 revealed that they can form electrostatic and π–π stacking interactions, forming a P-DNA@ZIF-8 complex and that PET is more dominant than FRET in the quenching mechanism. This ZIF-8 biosensing platform showed high sensitivity towards COVID-19 RNA with an ultra-low limit of detection of 6.24 pM, a rapid detection time of 8 min, and high selectivity to COVID-19 RNA. Indeed, ZIF-8 experienced much lower fluorescence recovery when tested on two mismatched RNAs. The experimental results show the potential use of ZIF-8 as a novel biosensor for a rapid and sensitive COVID-19 diagnosis. [ABSTRACT FROM AUTHOR]

Background/Objectives: Vulvovaginal candidosis (VVC) is a common condition among women, with current diagnostic methods relying on clinical evaluation and laboratory testing. These traditional methods are often limited by the need for specialized training, variable performance, and lengthy diagnostic processes, leading to delayed treatment and inappropriate antifungal use. This review evaluates the efficacy of molecular diagnostic tools for VVC and provides guidance on their application in clinical practice. Methods: A literature search was conducted using PubMed to identify studies evaluating rapid diagnostic tests specifically for vulvovaginal Candida isolates. Inclusion criteria focused on studies utilizing molecular diagnostics for the detection of Candida species in VVC. Articles discussing non-vaginal Candida infections, non-English studies, and animal or in vitro research were excluded. Results: Twenty-three studies met the inclusion criteria, predominantly evaluating nucleid acid amplification tests/polymerase chain reaction (NAAT/PCR) assays and DNA probes. PCR/NAAT assays demonstrated high sensitivity and specificity (>86%) for VVC diagnosis, outperforming conventional diagnostic methods. Comparatively, DNA probes, while simpler, exhibited lower sensitivity. The included studies were mostly observational, with only one randomized controlled trial. Emerging diagnostic technologies, including artificial intelligence and integrated testing models, show promise for improving diagnostic precision and clinical outcomes. Conclusions: Molecular diagnostics offer a significant improvement in VVC management, though traditional methods remain valuable in resource-limited settings. [ABSTRACT FROM AUTHOR]

Simple Summary: Cervical cancer is the fourth most common cancer in women worldwide, with HPV being a prevalent cause. Recent automated signal detection methods in ISH assays have shown promise for detecting HPV DNA in cervical tissue. This study compared an ISH probe (Inform HPV II and III) to PCR assays in cervical tissue samples with varying degrees of cervical intraepithelial neoplasia (CIN) and normal cervix tissue. Findings indicated a significant association between ISH III levels and HPV outcomes; patients with positive outcomes had lower ISH III levels (MD = −7961.82, p = 0.005). However, automatic signal detection for ISH is limited in cervical tissue, making genotyping-based HPV testing more effective, as it allows for larger sample collection. ISH results should be interpreted alongside clinical history and morphology. Background: Cervical cancer is fourth the most common cancer in women worldwide. Due to the prevalence of human papillomavirus (HPV) in the population (80–90%), scientists are likely to discover even more associations of this pathogen with other diseases in the future. In recent years, In Situ Hybridization (ISH) assays that use automated signal-detecting methods in formalin-fixed, paraffin-embedded (FFPE) cervical tissue, such as the enzyme-categorized signal-detecting system, have shown a higher sensitivity. Objectives and Methods: To evaluate automatic signal detection of ISH assay for detecting HPV DNA, we compared the ability of an ISH probe, Inform HPV II and III (Ventana Medical Systems, Tucson, AZ), to that of PCR assays to detect HPV DNA in cervical tissue specimens with cervical intraepithelial neoplasia (CIN; CIN 1, 28 cases; CIN 2, 22 cases; and CIN 3, 20 cases) and normal cervix (2 cases). Results: Our findings showed a significant relation was confirmed between ISH III level and HPV outcome (positive/negative). Patients with positive HPV outcomes had significantly lower ISH III levels, MD = −7961.82 CI95 [−17,230.00; −199.21], p = 0.005. Conclusions: Automatic signal detection of ISH assay is not particularly applicable to cervical tissue material. A more useful method of confirming the presence of HPV in the cervix is the HPV test with genotyping, as it allows for collecting a larger amount of material from the cervical disc and canal. The interpretation of a positive or negative ISH test must be guided in the context of clinical history and morphology. [ABSTRACT FROM AUTHOR]

Surface-based biosensors have proven to be of particular interest in the monitoring of human pathogens by means of their distinct nucleic acid sequences. Genosensors rely on targeted gene/DNA probe hybridization at the surface of a physical transducer and have been exploited for their high specificity and physicochemical stability. Unfortunately, these sensing materials still face limitations impeding their use in current diagnostic techniques. Most of their shortcomings arise from their suboptimal surface properties, including low hybridization density, inadequate probe orientation, and biofouling. Herein, we describe and compare two functionalization methodologies to immobilize DNA probes on a glass substrate via a thermoresponsive polymer in order to produce genosensors with improved properties. The first methodology relies on the use of a silanization step, followed by PET-RAFT of NIPAM monomers on the coated surface, while the second relies on vinyl sulfone modifications of the substrate, to which the pre-synthetized PNIPAM was grafted to. The functionalized substrates were fully characterized by means of X-ray photoelectron spectroscopy for their surface atomic content, fluorescence assay for their DNA hybridization density, and water contact angle measurements for their thermoresponsive behavior. The antifouling properties were evaluated by fluorescence microscopy. Both immobilization methodologies hold the potential to be applied to the engineering of DNA biosensors with a variety of polymers and other metal oxide surfaces. [ABSTRACT FROM AUTHOR]

Three different Mn‐based peroxidase‐mimetic nanoparticles (NPs) were synthesized as nanozymes: (i) manganese (II)‐ferrite (MnFe2O4) fabricated by co‐precipitating MnCl2.4H2O and FeCl3.6H2O; (ii) MnO2 synthesized from precipitation of MnSO4 in basic medium; (iii) manganese oxide obtained by reducing KMnO4 with tris(2‐hydroxyethyl) amine, all characterized using STEM, EDXS, FTIR and XPS techniques. These NPs acted as peroxidases, revealed by using a DNA probe, on which the damage generated by peroxidase mimetic NPs was monitored spectrophotometrically by CUPRAC and 3,3′,5,5′‐tetramethylbenzidine (TMB) methods. Results were confirmed by RP‐HPLC and voltammetry, the latter involving an AuNPs‐modified glassy carbon electrode (GCE), multi‐walled carbon nanotubes (MWCNT) and Nafion combination. Colorimetric assay with CUPRAC rather than TMB better agreed with chromatographic/voltammetric findings in detecting both peroxidase activity and DNA hazard. Mn(III)‐containing manganese oxide from permanganate reduction had stronger peroxidase activity. The proposed method demonstrated protective effects of antioxidants (morin, catechin and quercetin) against oxidative DNA damage for the first time. [ABSTRACT FROM AUTHOR]

Understanding complex regulatory networks in plant systems requires elucidating the roles of various gene regulators under a spatial landscape. MicroRNA are key regulators that impart high information value through their tissue specificity and stability when using expression patterns for evaluating network outcomes. However, current techniques that utilize spatial multiplexing and quantitation of microRNA are limited to primarily mammalian systems. Here, we present a method to spatially resolve and quantify multiple endogenous microRNA in situ using ethanol fixed, paraffin embedded model plant species. This method utilizes target-specific microRNA capture along with universal ligating and labelling, all within functionalized hydrogel posts containing DNA probes in nanoliter well arrays. We demonstrate the platform's multiplexing capabilities through analyzing three endogenous microRNA in Arabidopsis thaliana rosettes which provide useful answers to fundamental plant growth and development from the unique expression patterns. The spatial tissue technique is also validated using non-spatial small RNA assays to demonstrate the versatility of the well array platform. Our new platform expands the toolkit of spatial omics technologies for plants. [ABSTRACT FROM AUTHOR]

Here, a high-sensitivity silver-based surface plasmon resonance biosensor by a coating of zinc oxide on silicon for detecting hybridization of DNA is reported numerically. By changing the surface plasmon resonance angle and the minimum reflectance, this proposed biosensor can examine the difference between matched and mismatched DNA strands, as well as the nucleotide bonding between double-stranded (dsDNA) helix structures, and recognize the hybridization of target DNAs by using probe DNA immobilized on zinc oxide in the proper orientation. Also, the methodology of this work can detect single nucleotide polymorphisms (SNP). Using the transfer matrix method, we investigated the performance parameters of the biosensor. We observed that our proposed sensor has a maximum sensitivity of 378.36 deg/RIU and a good quality factor of 71.183 RIU−1. As we can see, compared with other reported papers, our proposed biosensor shows much better performance. This paper uses the biocompatibility, the electrical properties, the chemical stability, and high affinity of both Si and ZnO in surface plasmon resonance biosensors for the hybridization of DNA. [ABSTRACT FROM AUTHOR]

Simple Summary: In the mammalian immune system, αβ T lymphocytes are a fundamental set of T cells capable of recognizing an extraordinary range of antigens, presented in the context of the major histocompatibility complex molecules. The function of αβ T cells is linked to the T-cell receptor, a heterodimeric cell surface protein composed of an α and β chain uniquely expressed on T cells. Unlike conventional proteins encoded by a single gene, α and β chains are encoded by a large set of noncontiguous TRA and TRB genes that are randomly assembled during a stochastic process known as V(D)J recombination, generating a large and diverse repertoire. The extent of diversification depends on the number of genes and their genomic organization at the TRA and TRB loci, which varies from species to species. In this paper, we describe the genomic organization and gene content of the horse TRB locus, based on the recently released horse genome. Compared to other mammalian species, the horse TRB locus was found to be the largest locus studied to date. A clonotype analysis of a transcriptomic dataset was also performed to assess the characteristics of the V(D)J somatic rearrangement. Overall, our data provide useful information for further functional studies in healthy and sick horses of different breeds. In this paper, we report a comprehensive and consistent annotation of the locus encoding the β-chain of the equine T-cell receptor (TRB), as inferred from recent genome assembly using bioinformatics tools. The horse TRB locus spans approximately 1 Mb, making it the largest locus among the mammalian species studied to date, with a significantly higher number of genes related to extensive duplicative events. In the region, 136 TRBV (belonging to 29 subgroups), 2 TRBD, 13 TRBJ, and 2 TRBC genes, were identified. The general genomic organization resembles that of other mammals, with a V cluster of 135 TRBV genes located upstream of two in-tandem aligned TRBD-J-C clusters and an inverted TRBV gene at the 3′ end of the last TRBC gene. However, the horse b-chain repertoire would be affected by a high number of non-functional TRBV genes. Thus, we queried a transcriptomic dataset derived from splenic tissue of a healthy adult horse, using each TRBJ gene as a probe to analyze clonotypes encompassing the V(D)J junction. This analysis provided insights into the usage of the TRBV, TRBD, and TRBJ genes and the variability of the non-germline-encoded CDR3. Our results clearly demonstrated that the horse β-chain constitutes a complex level of variability, broadly like that described in other mammalian species. [ABSTRACT FROM AUTHOR]

Early lung cancer screening by computed tomography is hampered by pulmonary nodules caused by massive COVID‐19 infections, necessitating an ultrasensitive approach for the early diagnosis of lung cancers at the single‐base level from circular tumor DNAs (ctDNAs). This study introduces an approach that merges DNA origami and DNA scissors technologies in a framework of surface plasmon resonance (SPR) biosensors. By combining the precision of DNA origami probes with the inherent single‐base resolution of DNA scissors, this method systematically addresses the limitations of conventional SPR techniques, resulting in enhanced detection accuracy. The synergistic interplay between DNA scissors and DNA origami enables the SPR biosensors to achieve unprecedented levels of sensitivity, precision, and practical utility. This efficacy allows the precise identification of mutations, demonstrated here by detection of the T790M mutation in the EGFR gene and the G12C mutation in the KRAS gene of non‐small cell lung cancer patients. With this technique, single‐base resolution as well as near zeptomolar‐level sensitivity is achieved. As a result, this discovery holds significant potential to advance the field of precision diagnostics. [ABSTRACT FROM AUTHOR]

Early diagnosis and treatment of gastric cancer (GC) play a vital role in improving efficacy, reducing mortality and prolonging patients' lives. Given the importance of early detection of gastric cancer, an electrochemical biosensor was developed for the ultrasensitive detection of miR-19b-3p by integrating MoS2-based nanozymes, hybridization chain reaction (HCR) with enzyme catalyzed reaction. The as-prepared MoS2-based nanocomposites were used as substrate materials to construct nanoprobes, which can simultaneously load probe DNA and HCR initiator for signal amplification. Moreover, the MoS2-based nanocomposites are also employed as nanozymes to amplify electrochemical response. The presence of miR-19b-3p induced the assembly of MoS2-based nanoprobes on the electrode surface, which can activate in-situ HCR reaction to load a large number of horseradish peroxidase (HRP) for signal amplification. Coupling with the co-catalytic ability of HRP and MoS2-based nanozymes, the designed electrochemical biosensor can detect as low as 0.7 aM miR-19b-3p. More importantly, this biosensor can efficiently analyze miR-19b-3p in clinical samples from healthy people and gastric cancer patients due to its excellent sensitivity and selectivity, suggesting that this biosensor has a potential application in early diagnosis of disease. [ABSTRACT FROM AUTHOR]

A novel platform has been developed for the sensitive label-free detection of mutation in the TP53 gene, one of the most prominent genes in cancer research. The platform is based on the electrochemical properties of functionalized multi-walled carbon nanotubes (f-MWCNTs), gold nanoparticles (AuNPs), and polypyrrole (PPy). The microscopic structure and morphology of the prepared films on an ITO electrode surface were characterized using X-ray diffractometry (XRD), field emission scanning electron microscopy (FE-SEM), and Fourier transform infrared (FTIR). The hybridization events specific to the TP53 gene were monitored using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The modified electrode, which incorporates vertically aligned f-MWCNTs arrays and AuNPs, exhibited enhanced probe DNA attachment density, improving the sensitivity of the DNA sensor. The combination of AuNPs/PPy with an in-situ produced f-MWCNTs array provides a viable strategy for developing a highly sensitive biosensor for fast mutation detection in human cancer types. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are potentially useful biomarkers for the evaluation of cancers and chemical toxicity. However, simply and easily operated detection techniques are increasingly needed to enable their use in the biomedical and environmental fields. In this study, we investigated sequence-specific miRNA detections by surface plasmon resonance (SPR) and electrochemical (EC) imaging measurements on SPR-EC chips possessing a Au electrode array modified with probe DNAs and by employing a miRNA-detection-selective signal amplification method. MiRNAs were sequence-specifically detected by SPR and EC imaging measurements on a single chip. SPR signals were enhanced by a combination process employing the sequencespecific hybridization of miRNAs with the probe DNA, the extension reaction of polyadenine [poly(A)] tails on the 3’ end of the miRNAs by poly(A) polymerase, the binding of T30-Au nanoparticles (AuNPs) to the poly(A) tails, and the reduction reaction of Ag+ catalyzed on the AuNPs, depositing a gray precipitate on the surface. The oxidation reaction of the accumulated Ag precipitation additionally provides EC signal amplification. This process considerably and sequence-specifically amplifies the SPR and EC signals. This setup constitutes a simple and feasible signal amplification method for the on-site detection of miRNA biomarkers. [ABSTRACT FROM AUTHOR]

AbstractCRISPR-based diagnostics (CRISPR/Dx) have revolutionized the field of molecular diagnostics. It enables home self-test, field-deployable, and point-of-care testing (POCT). Despite the great potential of CRISPR/Dx in diagnoses of biologically complex diseases, preamplification of the template often is required for the sensitive detection of low-abundance nucleic acids. Various amplification-free CRISPR/Dx systems were recently developed to enhance signal detection at sufficient sensitivity. Broadly, these amplification-free CRISPR/Dx systems are classified into five groups depending on the signal enhancement strategies employed: CRISPR/Cas12a and/or CRISPR/Cas13a are integrated with: (1) other catalytic enzymes (Cas14a, Csm6, Argonaute, duplex-specific nuclease, nanozyme, or T7 exonuclease), (2) rational-designed oligonucleotides (multivalent aptamer, tetrahedral DNA framework, RNA G-quadruplexes, DNA roller machine, switchable-caged guide RNA, hybrid locked RNA/DNA probe, hybridized cascade probe, or "U" rich stem-loop RNA), (3) nanomaterials (nanophotonic structure, gold nanoparticle, micromotor, or microbeads), (4) electrochemical and piezoelectric plate biosensors (SERS nanoprobes, graphene field-effect transistor, redox probe, or primer exchange reaction), or (5) cutting-edge detection technology platforms (digital bioanalysis, droplet microfluidic, smartphone camera, or single nanoparticle counting). Herein, we critically discuss the advances, pitfalls and future perspectives for these amplification-free CRISPR/Dx systems in nucleic acids detection. The continued refinement of these CRISPR/Dx systems will pave the road for rapid, cost-effective, ultrasensitive, and ultraspecific on-site detection without resorting to target amplification, with the ultimate goal of establishing CRISPR/Dx as the paragon of diagnostics. [ABSTRACT FROM AUTHOR]

This study reports the development of an innovative electrochemical bioplatform for the indirect determination of acrylamide (AM) based on its inhibitory effect on the hybridization of nucleic acids through the formation of AM‐guanine single‐stranded (ssDNA) adducts. The method combines the advantages of the formation and selective immunorecognition of DNA/RNA heteroduplex, with the use of MBs and amperometric transduction using disposable SPCEs. Increasing concentrations of acrylamide prevented the efficient hybridization of a synthetic biotinylated DNA probe immobilized on streptavidin‐coated magnetic beads with its complementary RNA target and the as‐formed heteroduplex recognition with a specific antibody and a secondary HRP‐labeled antibody used for electrochemical readout. The resulting magnetic bioconjugates were magnetically trapped on the surface of the working electrode of a SPCE and the amperometric transduction was carried out in the presence of the H2O2/HQ system, recording a cathodic response inversely proportional to the AM concentration. The developed bioplatform is more competitive with other reported methods in terms of simplicity and assay time, allowing the selective detection of 1 nM AM in 60 min. In addition, the bioplatform was applied to the analysis of potato crisp water extracts. [ABSTRACT FROM AUTHOR]

Reactive N‐hydroxy‐9‐azabicyclo[3.3.1]nonane (ABNOH) linked 2′‐deoxyuridine 5′‐O‐mono‐ and triphosphates were synthesized through a CuAAC reaction of ABNOH‐PEG4‐N3 with 5‐ethynyl‐dUMP or ‐dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto‐ABNO radical reacted with tryptophan (Trp) and Trp‐containing peptides to form a stable tricyclic fused hexahydropyrrolo‐indole conjugates. Similarly modified ABNOH‐linked nucleotides reacted with Trp‐containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNOH‐modified DNA probe was used for bioconjugations and crosslinking with Trp‐containing peptides or proteins. [ABSTRACT FROM AUTHOR]

We report non‐adiabatic dynamics, performed in the surface hopping formalism, of 2‐thienyl‐3‐hydroxychromone, an environment‐dependent luminescent organic DNA probe. In particular we have shown that the first shell solvent water molecules undergo a rather complex reorganization upon light excitation. This involves also the triggering of a water‐mediated proton transfer process which leads to the formation of the tautomeric structure. The presence of this solvent‐mediated transfer mechanism globally diminishes the intersystem crossing efficiency, and hence the population of the triplet state manifold, as compared to the non‐solvated systems. Our results also point out the non‐innocent role of solvent networks in tuning complex photophysical processes, while opening competitive relaxation channels. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a significant global health concern. However, there are limited effective treatments available for it. The use of natural products in the management and treatment of HCC is gaining more attention. Baicalein is a flavonoid compound that has been reported to have antitumor activities in HCC. However, the direct binding targets of baicalein are still unknown. Therefore, we used the DNA‐programmed affinity labeling method to identify the target of baicalein and validated its function in HCC cells. We set blank and competitive DNA probes as negative controls. The results showed that baicalein had 136 binding targets, of which 13 targets were differently expressed in HCC tissues. The enriched cellular process of these targets was apoptosis, which involved MAPK9. We tested the binding affinity of baicalein with MAPK9 as 89.7 nM (Kd) by surface plasmon resonance and analyzed the binding sites by virtual docking. Notably, the binding of baicalein with MAPK9 increased the protein levels of MAPK9 itself and the related downstream apoptosis signaling, triggering the apoptosis of HCC cells. However, the inhibitor of MAPK9, SP600125, blocked the baicalein‐induced apoptosis, and the amounts of MAPK9 and downstream molecules were also decreased, indicating that baicalein acted through MAPK9 to induce apoptosis of HCC cells. In conclusion, we used the DNA‐programmed affinity labeling method to identify the direct‐binding target MAPK9 of baicalein and validated its function in baicalein‐induced apoptosis of HCC cells, which would be helpful to understand and use baicalein in HCC therapy. [ABSTRACT FROM AUTHOR]

A novel photoelectrochemical (PEC) sensor for the detection of microcystic toxins (MC-LR) was developed on the basis of signal-sensitive change strategy. NiO nanoarray as a basic photoactive material was grown directly on the ITO glass electrode via calcination after hydrothermal reaction, while dye N719 was used to sensitize the electrode for enhancing visible light absorption, and the first signal-on stage was obtained. In the meantime, p-type Cu2O was applied as the signal probe attached to probe DNA (DNA2) to improve the sensitivity, and the second "signal-on" stage appeared because of its synergistic effect with NiO nanoarrays. The PEC signal decreases after the target analyte MC-LR is modified on the electrode due to the stronger affinity between MC-LR and its complementary aptamer DNA; part of the Cu2O-DNA2 will dissociate from the electrode. This sensitive signal change strategy allows the detection limit of the MC-LR sensor to be as low as 1.7 pM, which offers an optional method for the sensitive and selective detection of other target molecules, with potential applications in environmental monitoring and toxin determination. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum (XP) is characterized by photosensitivity that causes pigmentary disorder and predisposition to skin cancers on sunlight-exposed areas due to DNA repair deficiency. Patients with XP group A (XP-A) develop freckle-like pigmented maculae and depigmented maculae within a year unless strict sun-protection is enforced. Although it is crucial to study pigment cells (melanocytes: MCs) as disease target cells, establishing MCs in primary cultures is challenging. Elucidation of the disease pathogenesis by comparison between MCs differentiated from XP-A induced pluripotent stem cells (iPSCs) and healthy control iPSCs on the response to UV irradiation. iPSCs were established from a XP-A fibroblasts and differentiated into MCs. Differences in gene expression profiles between XP-A-iPSC-derived melanocytes (XP-A-iMCs) and Healthy control iPSC-derived MCs (HC-iMCs) were analyzed 4 and 12 h after irradiation with 30 or 150 J/m2 of UV-B using microarray analysis. XP-A-iMCs expressed SOX10, MITF, and TYR, and showed melanin synthesis. Further, XP-A-iMCs showed reduced DNA repair ability. Gene expression profile between XP-A-iMCs and HC-iMCs revealed that, numerous gene probes that were specifically upregulated or downregulated in XP-A-iMCs after 150-J/m2 of UV-B irradiation did not return to basal levels. Of note that apoptotic pathways were highly upregulated at 150 J/m2 UV exposure in XP-A-iMCs, and cytokine-related pathways were upregulated even at 30 J/m2 UV exposure. We revealed for the first time that cytokine-related pathways were upregulated even at low-dose UV exposure in XP-A-iMCs. Disease-specific iPSCs are useful to elucidate the disease pathogenesis and develop treatment strategies of XP. • Induced pluripotent stem cells (iPSCs) were established from a XP-A patient. • Melanocytes(MCs) derived from XP-A-iPSCs and healthy control-iPSCs were analyzed. • Apoptotic pathways were highly upregulated at high-dose UV exposure in XP-A-iMCs. • Cytokine-related pathways were upregulated at low-dose UV exposure in XP-A-iMCs. • Disease-specific iPSCs are useful to elucidate the disease pathogenesis. [ABSTRACT FROM AUTHOR]

Simple Summary: Mitochondria are important organelles in the human body, and they act as a source of chemical energy for different cellular processes. Multiple copies of mitochondria are normally detected in cells, and their copy number has also been reported to be aberrated in cancers, including liver cancer. Target-panel sequencing is frequently used in oncology studies, but the optimal pooling ratio of nuclear and mitochondrial probes has remained elusive. Therefore, it is necessary to determine the physiological mitochondrial DNA copy number in non-liver cancer subjects, which can serve as a useful reference for deciding the desirable ratio of nuclear and mitochondrial probes for a less biased target enrichment of nuclear and mitochondrial genomes and hence their even representation. In addition, it can also be used as a physiological basis that can distinguish normal physiological variations from the pathological conditions in liver cancer. Mitochondria generate energy to support cells. They are important organelles that engage in key biological pathways. The dysfunction of mitochondria can be linked to hepatocarcinogenesis, which has been actively explored in recent years. To investigate the mitochondrial dysfunction caused by genetic variations, target-panel sequencing is a flexible and promising strategy. However, the copy number of mitochondria generally exceeds nuclear DNA, which raises a concern that uneven target enrichment of mitochondrial DNA (mtDNA) and nuclear DNA (ncDNA) in target-panel sequencing would lead to an undesirably biased representation of them. To resolve this issue, we evaluated the optimal pooling of mtDNA probes and ncDNA probes by a series of dilutions of mtDNA probes in both genomic DNA (gDNA) and cell-free DNA (cfDNA) samples. The evaluation was based on read count, average sequencing depth and coverage of targeted regions. We determined that an mtDNA:ncDNA probe ratio of around 1:10 would offer a good balance of sequencing performance and cost effectiveness. Moreover, we estimated the median physiological mtDNA:ncDNA copy ratio as 38.1 and 2.9 in cfDNA and gDNA samples of non-liver cancer subjects, respectively, whereas they were 20.0 and 2.1 in the liver cancer patients. Taken together, this study revealed the appropriate pooling strategy of mtDNA probes and ncDNA probes in target-panel sequencing and suggested the normal range of physiological variation of the mtDNA:ncDNA copy ratio in non-liver cancer individuals. This can serve as a useful reference for future target-panel sequencing investigations of the mitochondrial genome in liver cancer. [ABSTRACT FROM AUTHOR]

As an essential biomarker for diagnosing and treating various diseases, low-cost, quantitative detection methods for complementary DNA (cDNA) have received much attention. The surface plasmon resonance (SPR) sensing technique is an effective measurement scheme, but the ambient temperature and pH variations have a non-negligible impact. In this work, we developed a triple-probe SPR sensing system for detecting cDNA concentration, temperature, and pH. In order to satisfy the triple parameter measurements, we used a microstructured optical fiber as the sensing platform, silver and gold films as the excitation layer, and a MoS2 film as the modulation layer. First, we explore the modulation mechanism of SPR and the conditions for excitation of triple SPR and demonstrate that the carrier concentration is a crucial factor affecting the resonance wavelength. Then, the feasibility of the sensing system for triple-probing is theoretically analyzed. Finally, in the experiment, the optimal parameters of the sensor were determined, and the triple parameter detection was successfully realized. The experimental results show that the three probes can work independently, and the hybridized DNA probe can realize the selective detection of cDNA with a sensitivity of 0.249 nm/(nmol/l). The maximum sensitivity of the pH probe and the temperature probe are 51.5 nm/pH and 6.14 nm/°C. In addition, the experimental results show that the sensing probes have excellent reproducibility. This paper's innovation is using the fiber optic SPR effect to achieve quantitative detection for cDNA, temperature detection, and pH detection. Therefore, the sensor has a promising future in early diagnosis and biosensing. [ABSTRACT FROM AUTHOR]

DNA is fundamental for storing and transmitting genetic information. Analyzing DNA or RNA base sequences enables the identification of genetic disorders, monitoring gene expression, and detecting pathogens. Traditional detection techniques like polymerase chain reaction (PCR) and next-generation sequencing (NGS) have limitations, including complexity, high cost, and the need for advanced computational skills. Therefore, there is a significant demand for enzyme-free and amplification-free strategies for rapid, low-cost, and sensitive DNA detection. DNA biosensors, especially those utilizing plasmonic nanomaterials, offer a promising solution. This study introduces a novel DNA-functionalized waveguide-enhanced nanoplasmonic optofluidic biosensor using a nanogold-linked sorbent assay for enzyme-free and amplification-free DNA detection. Integrating plasmonic gold nanoparticles (AuNPs) with a glass planar waveguide (WG) and a microfluidic channel, fabricated through cost-effective, vacuum-free methods, the biosensor achieves specific detection of complementary target DNA sequences. Utilizing a sandwich architecture, AuNPs labeled with detection DNA probes enhance sensitivity by altering evanescent wave distribution and inducing plasmon resonance modes. The biosensor demonstrated exceptional performance in DNA detection, achieving a limit of detection (LOD) of 33.1 fg/mL (4.36 fM) with a rapid response time of approximately 8 min. This ultrasensitive, rapid, and cost-effective biosensor exhibits minimal background nonspecific adsorption, making it highly suitable for clinical applications and early disease diagnosis. The innovative design and fabrication processes offer significant advantages for mass production, presenting a viable tool for precise disease diagnostics and improved clinical outcomes. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are increasingly being considered essential diagnostic biomarkers and therapeutic targets for multiple diseases. In recent years, researchers have emphasized the need to develop probes that can harness extracellular miRNAs as input signals for disease diagnostics. In this study, we introduce a novel miRNA-responsive biosensor (miR-RBS) designed to achieve highly sensitive and specific detection of miRNAs, with a particular focus on targeted organ-specific visualization. The miR-RBS employs a Y-structured triple-stranded DNA probe (Y-TSDP) that exhibits a fluorescence-quenched state under normal physiological conditions. The probe switches to an activated state with fluorescence signals in the presence of high miRNA concentrations, enabling rapid and accurate disease reporting. Moreover, the miR-RBS probe had a modular design, with a fluorescence-labeled strand equipped with a functional module that facilitates specific binding to organs that express high levels of the target receptors. This allowed the customization of miRNA detection and cell targeting using aptameric anchors. In a drug-induced liver injury model, the results demonstrate that the miR-RBS probe effectively visualized miR-122 levels, suggesting it has good potential for disease diagnosis and organ-specific imaging. Together, this innovative biosensor provides a versatile tool for the early detection and monitoring of diseases through miRNA-based biomarkers. [ABSTRACT FROM AUTHOR]

Hand osteoarthritis (HOA), characterized by an earlier onset age and reduced susceptibility to mechanical stress compared with knee and hip osteoarthritis, is considered a suitable disease for identifying predictive biomarkers of osteoarthritis. In particular, DNA methylation variants, expected to contribute to HOA susceptibility, hold potential as osteoarthritis biomarkers. In this study, leukocyte DNA methylation patterns were analyzed in blood samples from patients with HOA, aiming to identify disease‐specific biomarkers for osteoarthritis. Using DNA methylation microarrays, we analyzed samples from three subjects with HOA and three age‐ and gender‐matched healthy individuals. For validation, pyrosequencing analysis was conducted using samples from 16 to 9 subjects with and without HOA, respectively. From 735,026 probes in the DNA methylation array, the Top 100 CpG sites associated with HOA, based on low adjusted P‐values, including those targeting bone morphogenetic protein 7 (BMP7), SBF2‐AS1, PLOD2, ICOS, and CSF1R were identified. Validation analysis revealed significantly higher methylation levels in the BMP7‐related site in the HOA group compared with the control group, even after adjusting for age, gender, and body mass index (p = 0.037). In contrast, no significant difference was observed in the other selected CpG sites between the HOA and control groups. This study highlights the significantly increased frequency of methylation at the specific BMP7 site in leukocytes of patients with HOA, suggesting its potential as a biomarker for HOA. Measurement of methylation levels at the CpG sites identified in this study offers a potential approach to prevent future osteoarthritis progression, providing valuable insights into disease management. [ABSTRACT FROM AUTHOR]

The ability to both sensitively and specifically assess the sequence composition of a nucleic acid strand is an ever-growing field. Designing a detection scheme that can perform this function when the sequence of the target being detected deviates significantly from the canonical sequence however is difficult in part because probe/primer design is based on established Watson-Crick base-pairing rules. We present here a robust and tunable toehold-based exchange probe that can detect a sequence with a variable number of SNPs of unknown identity by inserting a series of controlled, sequential mismatches into the protector seal of the toehold probe, in an effort to make the protector seal "sloppy". We show that the mismatch-tolerant system follows predicted behavior closely even with targets containing up to four mismatches that thermodynamically deviate from the canonical sequence by up to 15 kcal/mole. The system also performs faithfully regardless of the global mismatch position on either the protector seal or target. Lastly, we demonstrate the generalizability of the approach by testing the increasingly mismatch-tolerant protectors on HIV clinical samples to show that the system is capable of resolving multiple, iteratively mutated sequences derived from numerous HIV sub-populations with remarkable precision. [ABSTRACT FROM AUTHOR]

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella. [ABSTRACT FROM AUTHOR]

Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site. [ABSTRACT FROM AUTHOR]

Background: This study aims to prioritize genes potentially involved in multifactorial or causal relationships with gout. Methods: Using the Summary Data-based Mendelian Randomization (SMR) approach, this research analyzed expression quantitative trait loci (eQTL) data from blood and renal tissues and genome-wide association study (GWAS) data related to gout. It sought to identify genetic loci potentially involved in gout. Heterogeneity testing was conducted with the HEIDI test, and results were adjusted for the False Discovery Rate (FDR). Blood cis-eQTL data were sourced from the eQTLGen Consortium's summary-level data, and renal tissue data came from the V8 release of the GTEx eQTL summary data. Gout GWAS data was sourced from the FinnGen Documentation of the R10 release. Result: SMR analysis identified 14 gene probes in the eQTLGen blood summarylevel data significantly associated with gout. The top five ranked genes are: ENSG00000169231 (labeled THBS3, PSMR = 4.16 x 10--13), ENSG00000231064 (labeled THBS3-AS1, PSMR = 1.88 x 10--8), ENSG00000163463 (labeled KRTCAP2, PSMR = 3.88 x 10--6), ENSG00000172977 (labeled KAT5, PSMR = 1.70 x 10--5), and ENSG00000161395 (labeled PGAP3, PSMR = 3.24 x 10--5). Notably, increased expression of KRTCAP2 and PGAP3 is associated with an increased risk of gout, whereas increased expression of THBS3, THBS3-AS1, and KAT5 is associated with a reduced gout risk. No significant gene associations with gout were observed in renal tissue, likely due to the limited sample size of kidney tissue. Conclusion: Our findings have highlighted several genes potentially involved in the pathogenesis of gout. These results offer valuable insights into the mechanisms of gout and identify potential therapeutic targets for its treatment. [ABSTRACT FROM AUTHOR]

Anti-cancer therapy is crucial in cancer prevention and anti-cancer, and thus, highly sensitive methods for detecting cancer biomarkers are essential for cancer early diagnosis. Herein, an electrochemical aptamer biosensor based on the CRISPR-Cas12a system was constructed for the detection of cancer tumor biomarker MUC1 mucin. The sensitivity was significantly prompted by enzyme-catalyzed signal amplification, and the selectivity was improved by the dual recognition of the aptamer to MUC1 and crRNA-Cas12a system to the aptamer. Glucose oxidase (GOD) was loaded on the surface of magnetic Fe3O4@Au (MGNP) via probe single-stranded DNA (pDNA) with the terminal modification of mercapto (–SH) to form GOD-pDNA/MGNP. The corresponding aptamer of MUC1 (MUC1 Apt) binds to its complementary ssDNA (cDNA) to form the activator Apt/cDNA, which is specifically recognized by crRNA-Cas12a and excites the trans-cleavage function of Cas12a, thus in turn trans-cleaves pDNA and detaches GOD from the magnetic particles. The magnetic beads were separated and transferred into a glucose solution, and the oxidation current of H2O2 produced by the catalytic reaction of GOD was measured on a Pt-modified magnetically-controlled glassy carbon electrode, resulting in an indirect determination of MUC1. The current change was linear with the logarithm of MUC1 concentration in the range from 1.0 × 10−17 g mL−1 to 1.0 × 10−10 g mL−1. The detection limit was as low as 7.01 × 10−18 g mL−1. The method was applied for the detection of MUC1 in medical samples. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are formed in plant cells continuously. When ROS production exceeds the antioxidant capacity of the cells, oxidative stress develops which causes damage of cell components and may even lead to the induction of programmed cell death (PCD). The levels of ROS production increase upon abiotic stress, but also during pathogen attack in response to elicitors, and upon application of toxic compounds such as synthetic herbicides or natural phytotoxins. The commercial value of many synthetic herbicides is based on weed death as result of oxidative stress, and for a number of them, the site and the mechanism of ROS production have been characterized. This review summarizes the current knowledge on ROS production in plants subjected to different groups of synthetic herbicides and natural phytotoxins. We suggest that the use of ROS-specific fluorescent probes and of ROS-specific marker genes can provide important information on the mechanism of action of these toxins. Furthermore, we propose that, apart from oxidative damage, elicitation of ROS-induced PCD is emerging as one of the important processes underlying the action of herbicides and phytotoxins. [Display omitted] • The main reactive oxygen species (ROS) formed immediately after application of a phytotoxin indicate its mode of action. • ROS-specific marker genes and fluorescent probes can help to identify the major ROS induced in weeds by phytotoxins. • ROS induced in plants by natural phytotoxins can initiate programmed cell death (PCD). • Future herbicides with reduced toxicity for humans and animals should be based on induction of plant-specific PCD. [ABSTRACT FROM AUTHOR]

A CHA-based fluorescent DNA tetrahedral probe (FDTp) has been designed to detect the microRNAs miR-21 and miR-155 sensitively and specifically in living cells. The design consisted of functional elements (H1, H2, and Protector) connected to a DNA tetrahedron modified with two pairs of fluorophores and quenching groups. In the presence of miR-21, the chain displacement effect was triggered and Cy3 fluorescence was emitted. In the presence of miR-155, the signal of the catalytic hairpin assembly (CHA) between H1 and H2 on FDTp was amplified, making the fluorescence of FAM sensitive to miR-155. Using this method, the detection limit for miR-155 was 5 pM. The FDTp successfully imaged miR-21 and miR-155 in living cells and distinguished a variety of cell lines based on their expression levels of miR-21 and miR-155. The detection and imaging of dual targets in this design ensured the accuracy of tumor diagnosis and provided a new method for early tumor diagnosis. [ABSTRACT FROM AUTHOR]

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Dexamethasone (Dex) is a widely used glucocorticoid in medical practice, with applications ranging from allergies and inflammation to cerebral edema and shock. Despite its therapeutic benefits, Dex is classified as a prohibited substance for athletes due to its potential performance-enhancing effects. Consequently, there is a critical need for a convenient and rapid detection platform to enable prompt and accurate testing of this drug. In this study, we propose a label-free Förster Resonance Energy Transfer (FRET) aptasensor platform for Dex detection utilizing conjugated polymers (CPs), cationic conjugated polymers (CCPs), and gene finder probes (GFs). The system operates by exploiting the electrostatic interactions between positively charged CCPs and negatively charged DNA, facilitating sensitive and specific Dex detection. The label-free FRET aptasensor platform demonstrated robust performance in detecting Dex, exhibiting high selectivity and sensitivity. The system effectively distinguished Dex from interfering molecules and achieved stable detection across a range of concentrations in a commonly used sports drink matrix. Overall, the label-free FRET Dex detection system offers a simple, cost-effective, and highly sensitive approach for detecting Dex in diverse sample matrices. Its simplicity and effectiveness make it a promising tool for anti-doping efforts and other applications requiring rapid and accurate Dex detection. [ABSTRACT FROM AUTHOR]

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5′ end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection. [ABSTRACT FROM AUTHOR]

The detection of specific intracellular microRNAs could be potentially helpful in understanding the underlying mechanisms of cancer metastasis and invasion. MiRNAs are usually present in lower expression levels, especially in early stage of cancer. Here, we proposed a "one‐to‐more" amplification strategy for miRNA imaging, by virtue of DNA strand displacements with dual‐amplification. This approach involves leveraging high‐abundance endogenous mRNA as fuel strand to drive cascade reactions between DNA strands for amplification, enabling the monitoring of low‐abundance intracellular microRNA155. Notably, in comparison to the traditional "one‐to‐one" signal triggering mode, our "one‐to‐more" amplification strategy led to a remarkable 11.8‐fold increase in fluorescence signal. Our approach not only demonstrates a high sensitivity and specificity in detecting miR155, but also allows for discrimination of miR155 expression levels in different cell lines. With the advantages of intracellular signal amplification and reduced background signal, this approach holds substantial potential in the early diagnosis of cancer. [ABSTRACT FROM AUTHOR]

Background: Differentially methylated imprint control regions (ICRs) regulate the monoallelic expression of imprinted genes. Their epigenetic dysregulation by environmental exposures throughout life results in the formation of common chronic diseases. Unfortunately, existing Infinium methylation arrays lack the ability to profile these regions adequately. Whole genome bisulfite sequencing (WGBS) is the unique method able to profile the ICRs. However, it is very expensive and it requires not only a high coverage, but it is also computationally intensive to assess these regions. Findings: To address this deficiency, we developed a custom methylation array containing 22,819 probes. Among them, 10,438 are CG probes targeting unique CpG sites, with 9,757 probes successfully mapping to 1,088 out of the 1,488 candidate ICRs recently described. To assess the performance of the array, we created matched samples processed with the Human Imprintome array and WGBS, which is the current standard method for assessing the methylation of the Human Imprintome. We compared the methylation levels from the shared CpG sites, and obtained a mean R2 = 0.569. We also created matched samples processed with the Human Imprintome array and the Infinium Methylation EPIC v2 array, and obtained a mean R2 = 0.796. Furthermore, replication experiments demonstrated high reliability (ICC: 0.799–0.945). Conclusions: Our custom array will be useful for replicable and accurate assessment, mechanistic insight, and targeted investigation of ICRs. This tool should accelerate the discovery of ICRs associated with a wide range of diseases and exposures, and advance our understanding of genomic imprinting and its relevance in development and disease formation throughout the life course. [ABSTRACT FROM AUTHOR]

It is important to monitor miRNA-200c-3p as it can potentially serve as an important biomarker for respiratory diseases including COVID-19 and cancer. Despite the importance of microRNA monitoring, there are few previous studies for miRNA-200c-3p monitoring, and the application of hydroxyapatite nanoparticles (HaNP) in miRNA biosensors is quite limited. This study aims to fill this gap by utilizing the advantageous properties of HaNPs to develop a powerful strategy to detect microRNA-200c-3p. First, HaNPs were modified on the surface of pencil graphite electrodes (PGEs). Subsequently, hybridization between a phosphate-labeled miRNA-200c-3p-specific DNA probe and its complementary RNA target was carried out in the solution phase. The DNA-RNA hybrid forms were immobilized on the surface of the HaNP-PGEs and the impedimetric measurements were performed. The changes at the charge transfer resistance value (Rct) were evaluated in terms of the hybridization and optimization of the experimental conditions. The detection limits (DLs) were calculated as 0.12 µg/mL (16.19 nM) in phosphate buffer solution (PBS, pH 7.40) and 0.31 µg/mL (41.82 nM) in synthetic plasma. The selectivity of the developed biosensor was tested against miRNA-200c-5p and miRNA-141-3p. The results promise a significant improvement in public health in terms of a leap forward in the early diagnosis of many serious diseases. [ABSTRACT FROM AUTHOR]

A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered. [ABSTRACT FROM AUTHOR]

CRISPR/Cas system has been widely applied in the assay of disease-related nucleic acids. However, it is still challenging to use CRISPR/Cas system to detect multiple nucleic acids at the same time. Herein, we combined the preponderance of DNA logic circuit, label-free, and CRISPR/Cas technology to construct a label-free "AND" logical gate for multiple microRNAs detection with high specificity and sensitivity. With the simultaneous input of miRNA-155 and miRNA-141, the logic gate starts, and the activation chain of Cas12a is destroyed; thus, the activity is inhibited and the fluorescence of the signal probe ssDNA-AgNCs is turned on. The detection limit of this method for simultaneous quantitative detection of double target is 84 fmol/L (S/N = 3). In this "AND" logic gate, it is only necessary for the design of a simple DNA hairpin probe, which is inexpensive and easy, and since this method involves only one signal output, the data processing is very simple. What is more important, in this strategy two types of microRNAs can be monitored simultaneously by only using CRISPR/Cas12a and a type of crRNA, which offers a new design concept for the exploitation of single CRISPR/Cas system for multiple nucleic acid assays. [ABSTRACT FROM AUTHOR]

A fluorescence biosensor for determination of aflatoxin B1 (AFB1) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB1 by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1–100 ng/mL with a determination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheat samples. The stability, accuracy, and specificity of the method demonstrated the applicability for real sample analysis. [ABSTRACT FROM AUTHOR]

A competitive-type photoelectrochemical (PEC) aptasensor coupled with a novel Au@Cd:SnO2/SnS2 nanocomposite was designed for the detection of 17β-estradiol (E2) in microfluidic devices. The designed Au@Cd:SnO2/SnS2 nanocomposites exhibit high photoelectrochemical activity owing to the good matching of cascade band-edge and the efficient separation of photo-generated e−/h+ pairs derived from the Cd-doped defects in the energy level. The Au@Cd:SnO2/SnS2 nanocomposites were loaded into carbon paste electrodes (CPEs) to immobilize complementary DNA (cDNA) and estradiol aptamer probe DNA (E2-Apt), forming a double-strand DNA structure on the CPE surface. As the target E2 interacts with the double-strand DNA, E2-Apt is sensitively released from the CPE, subsequently increasing the photocurrent intensity due to the reduced steric hindrance of the electrode surface. The competitive-type sensing mechanism, combined with high PEC activity of the Au@Cd:SnO2/SnS2 nanocomposites, contributed to the rapid and sensitive detection of E2 in a "signal on" manner. Under the optimized conditions, the PEC aptasensor exhibited a linear range from 1.0 × 10−13 mol L−1 to 3.2 × 10−6 mol L−1 and a detection limit of 1.2 × 10−14 mol L−1 (S/N = 3). Moreover, the integration of microfluidic device with smartphone controlled portable electrochemical workstation enables the on-site detection of E2. The small sample volume (10 µL) and short analysis time (40 min) demonstrated the great potential of this strategy for E2 detection in rat serum and river water. With these advantages, the PEC aptasensor can be utilized for point-of-care testing (POCT) in both clinical and environmental applications. [ABSTRACT FROM AUTHOR]

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement. [ABSTRACT FROM AUTHOR]