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Passports are the number one form of individual identification when crossing borders resulting in counterfeit or modified passports being used to allow illegal travel. Here we report on the use of Diamond Dye to detect cells deposited by touch onto parts of the photograph page of a passport. The passport page was divided into a grid of 11 rectangles of approximately equal dimensions. Thumbprints were deposited at 10 of the 11 squares, with the 11th left blank as a negative control. The location and number of deposited cells were determined and recorded. Cellular material was removed from each square separately, using a moistened swab or a tapelift. Tapelifting was better suited for the substrate resulting in an increase in DNA recovered. The highest total quantity of DNA isolated from the 10 samples post extraction was 0.162 ng. Short tandem repeat data were obtained from all 10 samples using the GlobalFilerTM PCR Amplification Kit. The DNA profiles were single source or two-person mixtures and the donor was always the major contributor. These data show the potential to visualize cellular material deposited by touch and then generate STR data from an item such as a passport after only brief physical contact. [ABSTRACT FROM AUTHOR]

Nucleic acids (deoxyribonucleic acid – DNA and ribonucleic acid – RNA) are essential components of all living organisms, with DNA encoding genetic information and RNA facilitating vital biological processes. The detection of nucleic acids having a specific sequence is crucial for identifying organisms and diagnosing genetic diseases. Because surface-enhanced Raman spectroscopy (SERS) is considered as one of the most promising analytical methods that offers important benefits such as short analysis time and exceptional sensitivity compared to other techniques, many groups are trying to apply SERS for nucleic acid detection. This review discusses how SERS spectroscopy can be used for DNA/RNA detection. Beginning with an overview of SERS theory, we delve into various SERS DNA/RNA sensors, including those based on a direct analysis of the SERS spectra of nucleic acids, and many types of sensors based on a selective hybridisation of probe and target nucleic acids. We describe how various types of sensors with increased sensitivity and reliability have evolved (from the first SERS DNA/RNA sensors described in the literature to recently developed ones). Challenges and future directions in SERS sensor development for nucleic acid detection and determination are also discussed. This comprehensive review aims to help researchers understand the field's nuances, and to foster advancements in the use of SERS spectroscopy in the medical sector. [ABSTRACT FROM AUTHOR]

Gravimetric resonant-inspired biosensors have attracted increasing attention in industrial and point-of-care applications, enabling label-free detection of biomarkers such as DNA and antibodies. Capacitive micromachined ultrasonic transducers (CMUTs) are promising tools for developing miniaturized high-performance biosensing complementary metal–oxide–silicon (CMOS) platforms. However, their operability is limited by inefficient functionalization, aggregation, crosstalk in the buffer, and the requirement for an external high-voltage (HV) power supply. In this study, we aimed to propose a CMUTs-based resonant biosensor integrated with a CMOS front–end interface coupled with ethylene–glycol alkanethiols to detect single-stranded DNA oligonucleotides with large specificity. The topography of the functionalized surface was characterized by energy-dispersive X-ray microanalysis. Improved selectivity for on-chip hybridization was demonstrated by comparing complementary and non-complementary single-stranded DNA oligonucleotides using fluorescence imaging technology. The sensor array was further characterized using a five-element lumped equivalent model. The 4 mm2 application-specific integrated circuit chip was designed and developed through 0.18 μm HV bipolar-CMOS-double diffused metal–oxide–silicon (DMOS) technology (BCD) to generate on-chip 20 V HV boosting and to track feedback frequency under a standard 1.8 V supply, with a total power consumption of 3.8 mW in a continuous mode. The measured results indicated a detection sensitivity of 7.943 × 10−3 μmol∙L−1∙Hz−1 over a concentration range of 1 to 100 μmol∙L−1. In conclusion, the label-free biosensing of DNA under dry conditions was successfully demonstrated using a microfabricated CMUT array with a 2 MHz frequency on CMOS electronics with an internal HV supplier. Moreover, ethylene–glycol alkanethiols successfully deposited self-assembled monolayers on aluminum electrodes, which has never been attempted thus far on CMUTs, to enhance the selectivity of bio-functionalization. The findings of this study indicate the possibility of full-on-chip DNA biosensing with CMUTs.

Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful technique for the detection and analysis of biomolecules due to its high sensitivity and selectivity. In recent years, SERS-based sensors have received significant attention for the detection of deoxyribonucleic acid (DNA) molecules, offering promising applications in fields such as medical diagnostics, forensic analysis, and environmental monitoring. This paper provides a concise overview of the principles, advancements, and potential of SERS-based sensors for DNA detection. First, the fundamental principles of SERS are introduced, highlighting its ability to enhance the Raman scattering signal by several orders of magnitude through the interaction between target molecules with metallic nanostructures. Then, the fabrication technologies of SERS substrates tailored for DNA detection are reviewed. The performances of SERS substrates previously reported for DNA detection are compared and analyzed in terms of the limit of detection (LOD) and enhancement factor (EF) in detail, with respect to the technical parameters of Raman spectroscopy (e.g., laser wavelength and power). Additionally, strategies for functionalizing the sensor surfaces with DNA-specific capture probes or aptamers are outlined. The collected data can be of help in selecting and optimizing the most suitable fabrication technology considering nucleotide sensing applications with Raman spectroscopy. [ABSTRACT FROM AUTHOR]

ABSTRACT Sporothrix sp. is a thermally dimorphic genus of fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases. Here, we adapted and evaluated a recently described real-time PCR assay targeting the β-tubulin gene to rapidly detect and differentiate two related species, S. schenckii and S. brasiliensis. The assay was tested with 55 S. brasiliensis, 19 S. schenckii, and 85 isolates from other clinically relevant fungi, and showed 100% concordance with reference identification methods. The assay showed high analytical sensitivity with a limit of detection of 1 pg of DNA per microliter of sample for both targets. The assay was further evaluated with 11 fresh and 17 formalin-fixed, paraffin-embedded (FFPE) tissues. This multiplex real-time PCR assay successfully detected the DNA from both S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this assay performs well and could be a helpful molecular tool to support rapid species identification in cultures and primary specimens.IMPORTANCEHaving available molecular tools to identify and differentiate closely related species will allow clinical, veterinarians, and public health labs to provide diagnostic results with accuracy and short turnaround time for the routine and outbreak response activities.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile. [ABSTRACT FROM AUTHOR]

Solid-state nanopores have become a prominent tool in the field of single-molecule detection. Conventional solid-state nanopores are thick, which affects the spatial resolution of the detection results. Graphene is the thinnest 2D material and has the highest spatial detection resolution. In this study, a graphene membrane chip was fabricated by combining a MEMS process with a 2D material wet transfer process. Raman spectroscopy was used to assess the quality of graphene after the transfer. The mechanism behind the influence of the processing dose and residence time of the helium ion beam on the processed pore size was investigated. Subsequently, graphene nanopores with diameters less than 10 nm were fabricated via helium ion microscopy. DNA was detected using a 5.8 nm graphene nanopore chip, and the appearance of double-peak signals on the surface of 20 mer DNA was successfully detected. These results serve as a valuable reference for nanopore fabrication using 2D material for DNA analysis. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNPs) exhibit improved optical and spectral properties compared to bulk materials, making them suitable for the detection of DNA, RNA, antigens, and antibodies. Here, we describe a simple, selective, and rapid non-cross linking detection assay, using approx. 35 nm spherical Au nanoprobes, for a common mutation occurring in exon 19 of the epidermal growth factor receptor (EGFR), associated with non-small-cell lung cancer cells. AuNPs were synthesized based on the seed-mediated growth method and functionalized with a specific 16 bp thiolated oligonucleotide using a pH-assisted method. Both AuNPs and Au nanoprobes proved to be highly stable and monodisperse through ultraviolet-visible spectrophotometry, dynamic light scattering (DLS), and electrophoretic light scattering (ELS). Our results indicate a detection limit of 1.5 µg mL−1 using a 0.15 nmol dm−3 Au nanoprobe concentration. In conclusion, this work presents an effective possibility for a straightforward, fast, and inexpensive alternative for the detection of DNA sequences related to lung cancer, leading to a potential platform for early diagnosis of lung cancer patients. [ABSTRACT FROM AUTHOR]

Precise DNA quantification and nuclear imaging are pivotal for clinical testing, pathological diagnosis, and drug development. The detection and localization of mitochondrial DNA serve as crucial indicators of cellular health. We introduce a novel conjugated oligoelectrolyte (COE) molecule, COE-S3, featuring a planar backbone composed of three benzene rings and terminal side chains. This unique amphiphilic structure endows COE-S3 with exceptional water solubility, a high quantum yield of 0.79, and a significant fluorescence Stokes shift (λex = 366 nm, λem = 476 nm), alongside a specific fluorescence response to DNA. The fluorescence intensity correlates proportionally with DNA concentration. COE-S3 interacts with double-stranded DNA (dsDNA) through an intercalation binding mode, exhibiting a binding constant (K) of 1.32 × 106 M−1. Its amphiphilic nature and strong DNA affinity facilitate its localization within mitochondria in living cells and nuclei in apoptotic cells. Remarkably, within 30 min of COE-S3 staining, cell vitality can be discerned through real-time nuclear fluorescence imaging of apoptotic cells. COE-S3's high DNA selectivity enables quantitative intracellular DNA analysis, providing insights into cell proliferation, differentiation, and growth. Our findings underscore COE-S3, with its strategically designed, shortened planar backbone, as a promising intercalative probe for DNA quantification and nuclear imaging. [ABSTRACT FROM AUTHOR]

Context. Wildlife trade is a prominent pathway for invasive species introductions into novel environments. Deliberate or accidental release of exotic pets can result in the establishment of alien populations, with damaging impacts for native species and environmental assets. This process is well documented for reptiles globally and is of considerable biosecurity concern in Australia. Boa constrictor is one species at high risk of establishment in Australia, and has insufficient biosecurity detection and post-border control capacity. Aims. We aimed to develop rapid DNA-based presumptive testing capacity for detecting B. constrictor, with appropriate sensitivity and specificity to operate in a trace DNA biosecurity context. Methods. Loop Mediated Isothermal Amplification (LAMP) is an emerging biosecurity tool that provides highly specific, sensitive, low-resource methods for detection of trace DNA in the absence of physical evidence. We developed colourimetric and fluorescent LAMP assays targeting the mitochondrial DNA control region of B. constrictor. We tested and validated these assays against synthetic DNA fragments, as well as DNA extracted from: (1) vouchered museum B. constrictor tissue; (2) shed B. constrictor skin samples; (3) a range of non-target species to test specificity; and (4) trace DNA recovered from glass tanks post B. constrictor presence. Key results. We successfully detected synthetic target DNA down to 1 fg and genomic B. constrictor DNA from tissue and shed skins down to <10 pg in under 30 minutes with our fluorescence-based LAMP assay. Additionally, we were able to detect B. constrictor trace DNA following 24 h of presence utilising a traditional laboratory-based DNA extraction method (approximately 180 min) and a rapid lysis step (approximately 8 min). Conclusions. Both colourimetric and fluorescent assays show promise for the specific detection of B.constrictor in biosecurity contexts, including post-border enforcement and compliance checksin the domestic illicit wildlife trade. Implications. Our findings greatly strengthen the ongoing development of biosecurity tools for trace DNA detection of commonly traded and trafficked species (i.e. reptiles) in wildlife enforcement contexts, advancing both preparedness and surveillance. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful technique for the detection and analysis of biomolecules due to its high sensitivity and selectivity. In recent years, SERS-based sensors have received significant attention for the detection of deoxyribonucleic acid (DNA) molecules, offering promising applications in fields such as medical diagnostics, forensic analysis, and environmental monitoring. This paper provides a concise overview of the principles, advancements, and potential of SERS-based sensors for DNA detection. First, the fundamental principles of SERS are introduced, highlighting its ability to enhance the Raman scattering signal by several orders of magnitude through the interaction between target molecules with metallic nanostructures. Then, the fabrication technologies of SERS substrates tailored for DNA detection are reviewed. The performances of SERS substrates previously reported for DNA detection are compared and analyzed in terms of the limit of detection (LOD) and enhancement factor (EF) in detail, with respect to the technical parameters of Raman spectroscopy (e.g., laser wavelength and power). Additionally, strategies for functionalizing the sensor surfaces with DNA-specific capture probes or aptamers are outlined. The collected data can be of help in selecting and optimizing the most suitable fabrication technology considering nucleotide sensing applications with Raman spectroscopy.

Due to their distinctive optical potential, controlled plasmonic features, control over size and shape, and ability to manipulate their localized surface plasmon resonance (LSPR) and their response to their surroundings, silver nanoparticle attracts special attention in the fields of the biosensor. Silver (Ag) is an appealing tool in many domains, including diagnosis, medicine delivery, environment, electronics, and as an antibacterial agent. Ag nanoparticle applications are ubiquitous today in the fields of biosensors. Numerous studies have demonstrated the effectiveness of using Ag nanoparticles to improve the specific detection of clinical indicators. The development of nanotechnology has greatly benefited the field of biosensors since nanomaterials have a high surface-to-volume ratio that enables them to operate with greater sensitivity and effectiveness. Utilizing interdisciplinary study in biology, chemistry, and material science, recent advancements in nanotechnology encouraged the manufacture of nanomaterials in a variety of shapes and sizes. In the context of disease diagnosis, this review highlighted the use of Ag nanoparticles in biosensors to detect small molecules to bigger entire cells. This paper also discusses how to make a glucose biosensor more sensitive. It describes how the detection performance of biomolecules including DNA, protein, tiny molecules, and complete cells can be improved. For future applications in the field of diagnosis, this review helps in developing an overall understanding of the clinical importance of Ag nanoparticles. These biosensors can be implemented in the field of diagnosis to speed up the response time. [ABSTRACT FROM AUTHOR]

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays. [ABSTRACT FROM AUTHOR]

Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36-43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25 °C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen’s two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

Despite actions to reduce the prevalence of antibiotic resistance, antibiotic resistance remains a major threat in the treatment and control of infections. Therefore, the development of rapid diagnostic methods plays a significant role in the detection of antibiotic resistance genes and the management of hospital infections. This study aimed to design and develop a gold nanoparticle colorimetric probe-based biosensor was developed for faster and more accurate detection of VIM-2 and IMP-1 Metallo-Beta-Lactamases (MBLs) genes in different clinical samples. After identifying 248 clinical isolates of bacteria with standard biochemical methods and evaluation of antibiotic resistance and identifying MBL-producing strains, PCR method was carried out for detecting VIM-2 and IMP-1 genes as a gold standard. Synthesis of AuNPs were done by citrate reduction method and AuNP -IMP-1 biosensor was used for detecting IMP-1 gene, after functionalization of thiol modified oligonucleotides. AuNP biosensor and IMP-1 PCR were compared in terms of detection indices. Method PCR was defined as the gold standard. Bacteria examined, 87 isolates were resistant to carbapenems. Out of the 87 carbapenem-resistant isolates, 85 (34.2%) were phenotypically positive for MBLs. Also, 7 isolates had IMP-1 gene, but none of them carried VIM-2 gene. The AuNP biosensor had 100% sensitivity, specificity, PPV and NPV, respectively. The final detection limit of IMP-1 genomic DNA (LOD) by PCR and AuNP-IMP-1 biosensor technique was 0.1 fg/μl (2.5 fg/25 reactions) and 0.001 fg/μl (0.025 fg/25 reaction), respectively. According to the promising results of Diagnostic indices, AuNP biosensor is a more efficient and accurate method for direct and indirect detection of antibiotic resistance genes in clinical samples.

To minimize the incorrect use of antibiotics, there is a great need for rapid and inexpensive tests to identify the pathogens that cause an infection. The gold standard of pathogen identification is based on the recognition of DNA sequences that are unique for a given pathogen. Here, we propose and test a strategy to develop simple, fast, and highly sensitive biosensors that make use of multivalency. Our approach uses DNA-functionalized polystyrene colloids that distinguish pathogens on the basis of the frequency of selected short DNA sequences in their genome. Importantly, our method uses entire genomes and does not require nucleic acid amplification. Polystyrene colloids grafted with specially designed surface DNA probes can bind cooperatively to frequently repeated sequences along the entire genome of the target bacteria, resulting in the formation of large and easily detectable colloidal aggregates. Our detection strategy allows "mix and read" detection of the target analyte; it is robust and highly sensitive over a wide concentration range covering, in the case of our test target genome Escherichia coli bl21-de3, 10 orders of magnitude from 101 to 1010 copies/mL. The sensitivity compares well with state-of-the-art sensing techniques and has excellent specificity against nontarget bacteria. When applied to real samples, the proposed technique shows an excellent recovery rate. Our detection strategy opens the way to developing a robust platform for pathogen detection in the fields of food safety, disease control, and environmental monitoring. [ABSTRACT FROM AUTHOR]

The accurate, rapid, and specific detection of DNA strands in solution is becoming increasingly important, especially in biomedical applications such as the trace detection of COVID-19 or cancer diagnosis. In this work we present the design, elaboration and characterization of an optofluidic sensor based on a polymer-based microresonator which shows a quick response time, a low detection limit and good sensitivity. The device is composed of a micro-racetrack waveguide vertically coupled to a bus waveguide and embedded within a microfluidic circuit. The spectral response of the microresonator, in air or immersed in deionised water, shows quality factors up to 72,900 and contrasts up to 0.9. The concentration of DNA strands in water is related to the spectral shift of the microresonator transmission function, as measured at the inflection points of resonance peaks in order to optimize the signal-over-noise ratio. After functionalization by a DNA probe strand on the surface of the microresonator, a specific and real time measurement of the complementary DNA strands in the solution is realized. Additionally, we have inferred the dissociation constant value of the binding equilibrium of the two complementary DNA strands and evidenced a sensitivity of 16.0 pm/µM and a detection limit of 121 nM. [ABSTRACT FROM AUTHOR]

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease. [ABSTRACT FROM AUTHOR]

Currently available DNA detection techniques frequently require compromises between simplicity, speed, accuracy, and cost. Here, we propose a simple, label-free, and cost-effective DNA detection platform developed at screen-printed carbon electrodes (SPCEs) modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs). The preparation of the detection platform involved a two-step electrochemical procedure based on GO reduction onto SPCEs followed by the electrochemical reduction of HAuCl4 to facilitate the post-grafting reaction with AuNPs. The final sensor was fabricated by the simple physical adsorption of a single-stranded DNA (ssDNA) probe onto a AuNPs–RGO/SPCE electrode. Each preparation step was confirmed by morphological and structural characterization using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy, respectively. Furthermore, the electrochemical properties of the modified electrodes have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the introduction of AuNPs onto RGO/SPCEs led to an enhancement in surface conductivity, a characteristic that favored an increased sensitivity in detection. The detection process relied on the change in the electrochemical signal induced by the binding of target DNA to the bioreceptor and was particularly monitored by the change in the charge transfer resistance of a [Fe(CN)6]4–/3– redox couple added in the test solution. [ABSTRACT FROM AUTHOR]

In paper-based microfluidics, the simplest devices are colorimetric, giving qualitative results. However, getting quantitative data can be quite a bit more difficult. Distance-based devices provide a user-friendly means of obtaining quantitative data without the need for any additional equipment, simply by using an included ruler or calibrated markings. This article details the development of a quantitative DNA detection device that utilizes the aggregation of polystyrene microspheres to affect the distance that microspheres wick through filter paper. The microspheres are conjugated to single-stranded DNA (ssDNA) oligomers that are partially complementary to a target strand and, in the presence of the target strand, form a three-strand complex, resulting in the formation of aggregates. The higher the concentration of the target strand, the larger the aggregate, and the shorter the distance wicked by the microspheres. This behavior was investigated across a wide range of target concentrations and under different incubation times to understand aggregate formation. The device was then used to successfully detect a target strand spiked in extracted plant DNA.

A novel integrated detection system that introduces a paper-chip-based molecular detection strategy into a polydimethylsiloxane (PDMS) microchip and temperature control system was developed for on-site colorimetric detection of DNA. For the paper chip-based detection strategy, a padlock probe DNA (PLP)-mediated rolling circle amplification (RCA) reaction for signal amplification and a radial flow assay according to the Au-probe labeling strategy for visualization were optimized and applied for DNA detection. In the PDMS chip, the reactions for ligation of target-dependent PLP, RCA, and labeling were performed one-step under isothermal temperature in a single chamber, and one drop of the final reaction solution was loaded onto the paper chip to form a radial colorimetric signal. To create an optimal analysis environment, not only the optimization of molecular reactions for DNA detection but also the chamber shape of the PDMS chip and temperature control system were successfully verified. Our results indicate that a detection limit of 14.7 nM of DNA was achieved, and non-specific DNAs with a single-base mismatch at the target DNA were selectively discriminated. This integrated detection system can be applied not only for single nucleotide polymorphism identification, but also for pathogen gene detection. The adoption of inexpensive paper and PDMS chips allows the fabrication of cost-effective detection systems. Moreover, it is very suitable for operation in various resource-limited locations by adopting a highly portable and user-friendly detection method that minimizes the use of large and expensive equipment. [ABSTRACT FROM AUTHOR]

Background and Aim: Antiviral therapy (AVT) is the mainstay of hepatitis B virus (HBV) management. We investigated whether AVT improves the outcomes of HBV‐related decompensated cirrhosis and undetectable HBV‐DNA. Methods: Between 2000 and 2017, treatment‐naïve patients with HBV‐related decompensated cirrhosis and undetectable HBV‐DNA were recruited from two tertiary hospitals. The endpoints included death and hepatocellular carcinoma (HCC). Results: A total of 429 patients were analyzed (50 and 379 patients in the AVT and non‐AVT groups, respectively). Patients in the AVT group were significantly younger and had higher alanine aminotransferase and alpha‐fetoprotein levels than those in the non‐AVT group (all P < 0.05). During follow‐up (median 49.6 months), 98 patients died and 105 developed HCC. The cumulative incidence rates of death (2.0%, 4.1%, and 6.4%, and 4.9%, 7.2%, and 10.2% at 6 months, 1 year, and 2 years, respectively) and HCC (8.6%, 15.8%, and 26.4% vs 1.6%, 7.7%, and 24.4% at 1, 2, and 5 years, respectively) were statistically comparable between the AVT and non‐AVT groups (all P > 0.05). Using Cox regression analysis, AVT was not significantly associated with death nor HCC (all P > 0.05). Similar results were observed after balancing baseline characteristics with inverse probability of treatment weighting. In the non‐AVT group, the cumulative incidence rates of HBV‐DNA detection at 6 months, 1 year, and 2 years were 2.0%, 3.1%, and 6.4%, respectively. Conclusions: Antiviral therapy did not attenuate the risk of death nor HCC in patients with HBV‐related decompensated cirrhosis and undetectable HBV‐DNA. [ABSTRACT FROM AUTHOR]

Solid-state nanopores have become a prominent tool in the field of single-molecule detection. Conventional solid-state nanopores are thick, which affects the spatial resolution of the detection results. Graphene is the thinnest 2D material and has the highest spatial detection resolution. In this study, a graphene membrane chip was fabricated by combining a MEMS process with a 2D material wet transfer process. Raman spectroscopy was used to assess the quality of graphene after the transfer. The mechanism behind the influence of the processing dose and residence time of the helium ion beam on the processed pore size was investigated. Subsequently, graphene nanopores with diameters less than 10 nm were fabricated via helium ion microscopy. DNA was detected using a 5.8 nm graphene nanopore chip, and the appearance of double-peak signals on the surface of 20 mer DNA was successfully detected. These results serve as a valuable reference for nanopore fabrication using 2D material for DNA analysis.

Gold nanoparticles (AuNPs) exhibit improved optical and spectral properties compared to bulk materials, making them suitable for the detection of DNA, RNA, antigens, and antibodies. Here, we describe a simple, selective, and rapid non-cross linking detection assay, using approx. 35 nm spherical Au nanoprobes, for a common mutation occurring in exon 19 of the epidermal growth factor receptor (EGFR), associated with non-small-cell lung cancer cells. AuNPs were synthesized based on the seed-mediated growth method and functionalized with a specific 16 bp thiolated oligonucleotide using a pH-assisted method. Both AuNPs and Au nanoprobes proved to be highly stable and monodisperse through ultraviolet-visible spectrophotometry, dynamic light scattering (DLS), and electrophoretic light scattering (ELS). Our results indicate a detection limit of 1.5 µg mL−1 using a 0.15 nmol dm−3 Au nanoprobe concentration. In conclusion, this work presents an effective possibility for a straightforward, fast, and inexpensive alternative for the detection of DNA sequences related to lung cancer, leading to a potential platform for early diagnosis of lung cancer patients.

Precise DNA quantification and nuclear imaging are pivotal for clinical testing, pathological diagnosis, and drug development. The detection and localization of mitochondrial DNA serve as crucial indicators of cellular health. We introduce a novel conjugated oligoelectrolyte (COE) molecule, COE-S3, featuring a planar backbone composed of three benzene rings and terminal side chains. This unique amphiphilic structure endows COE-S3 with exceptional water solubility, a high quantum yield of 0.79, and a significant fluorescence Stokes shift (λex = 366 nm, λem = 476 nm), alongside a specific fluorescence response to DNA. The fluorescence intensity correlates proportionally with DNA concentration. COE-S3 interacts with double-stranded DNA (dsDNA) through an intercalation binding mode, exhibiting a binding constant (K) of 1.32 × 106 M−1. Its amphiphilic nature and strong DNA affinity facilitate its localization within mitochondria in living cells and nuclei in apoptotic cells. Remarkably, within 30 min of COE-S3 staining, cell vitality can be discerned through real-time nuclear fluorescence imaging of apoptotic cells. COE-S3’s high DNA selectivity enables quantitative intracellular DNA analysis, providing insights into cell proliferation, differentiation, and growth. Our findings underscore COE-S3, with its strategically designed, shortened planar backbone, as a promising intercalative probe for DNA quantification and nuclear imaging.

Rapid, accurate, and portable on-site detection is critical in the face of public health emergencies. Infectious disease control and public health emergency policymaking can both be aided by effective and trustworthy point of care tests (POCT). A very promising POCT method appears to be the clustered regularly interspaced short palindromic repeats and associated protein (CRISPR/Cas)-based molecular diagnosis. For on-site detection, CRISPR/Cas-based detection can be combined with multiple signal sensing methods and integrated into smart devices. In this review, sensing methods for CRISPR/Cas-based diagnostics are introduced and the advanced strategies and recent advances in CRISPR/Cas-based POCT are reviewed. Finally, the future perspectives of CRISPR and POCT are summarized and prospected. [ABSTRACT FROM AUTHOR]

This communication aims at discussing strategies based on developments from nanotechnology focused on the next generation of sequencing (NGS). In this regard, it should be noted that even in the advanced current situation of many techniques and methods accompanied with developments of technology, there are still existing challenges and needs focused on real samples and low concentrations of genomic materials. The approaches discussed/described adopt spectroscopical techniques and new optical setups. PCR bases are introduced to understand the role of non-covalent interactions by discussing about Nobel prizes related to genomic material detection. The review also discusses colorimetric methods, polymeric transducers, fluorescence detection methods, enhanced plasmonic techniques such as metal-enhanced fluorescence (MEF), semiconductors, and developments in metamaterials. In addition, nano-optics, challenges linked to signal transductions, and how the limitations reported in each technique could be overcome are considered in real samples. Accordingly, this study shows developments where optical active nanoplatforms generate signal detection and transduction with enhanced performances and, in many cases, enhanced signaling from single double-stranded deoxyribonucleic acid (DNA) interactions. Future perspectives on miniaturized instrumentation, chips, and devices aimed at detecting genomic material are analyzed. However, the main concept in this report derives from gained insights into nanochemistry and nano-optics. Such concepts could be incorporated into other higher-sized substrates and experimental and optical setups. [ABSTRACT FROM AUTHOR]

Fluorescence enhancement, including metal-enhanced fluorescence (MEF), has been widely explored in the field of biosensor technology. Two- and three-dimensional (2D and 3D) MEF substrate approaches have been developed for biosensors to detect target biomarkers such as genes and proteins. However, several issues remain, not least poor reproducibility caused by limited recognition and diffusion of biomolecules among target molecules, biological probes, and MEF substrates in aqueous environments. Here, an Ag–ZnO nanocomplex as a 3D MEF substrate was developed in two steps: hydrothermal synthesis of ZnO nanowire (NW) as a building block of Ag nanoparticles (NPs), and photochemical deposition of AgNPs on the ZnO NW. Fluorescence enhancement on Ag–ZnO nanocomposites (NCs) compared with the glass substrate was 14.8-, 16.9-, 11.4-, and 14.2-fold in the presence of 10, 20, 40, and 60 base pair (bp) double-stranded (ds) DNA, respectively. The MEF effects on glass, ZnO NW, and Ag–ZnO NCs were analyzed by measuring the fluorescence lifetime of fluorescein (FAM)-labeled dsDNA, and the average lifetimes were 9.76, 9.34, and 7.86 ns, respectively. Ag–ZnO NCs showed a 50-fold higher sensitivity for the detection of dsDNA than Ag nanoisland film (AgNIF) as a 2D MEF substrate. Therefore, Ag–ZnO NCs may have potential as a 3D MEF substrate. The MEF phenomenon might involve broad-range interactions between the surface of the metal nanostructure on Ag–ZnO NCs and fluorophores in various scenarios. Our Ag–ZnO NCs could facilitate the development of sensitive MEF-based biosensors for DNA detection. [ABSTRACT FROM AUTHOR]

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans -cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans -cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications. [ABSTRACT FROM AUTHOR]

Abstract This review focuses on the most reliable and up-to-date methods for diagnosing trypanosomoses, a group of diseases of wild and domestic mammals, caused by trypanosomes, parasitic zooflagellate protozoans mainly transmitted by insects. In Africa, the Americas and Asia, these diseases, which in some cases affect humans, result in significant illness in animals and cause major economic losses in livestock. A number of pathogens are described in this review, including several Salivarian trypanosomes, such as Trypanosoma brucei sspp. (among which are the agents of sleeping sickness, the human African trypanosomiasis [HAT]), Trypanosoma congolense and Trypanosoma vivax (causing “Nagana” or animal African trypanosomosis [AAT]), Trypanosoma evansi (“Surra”) and Trypanosoma equiperdum (“Dourine”), and Trypanosoma cruzi, a Stercorarian trypanosome, etiological agent of the American trypanosomiasis (Chagas disease). Diagnostic methods for detecting zoonotic trypanosomes causing Chagas disease and HAT in animals, as well as a diagnostic method for detecting animal trypanosomes in humans (the so-called “atypical human infections by animal trypanosomes” [a-HT]), including T. evansi and Trypanosoma lewisi (a rat parasite), are also reviewed. Our goal is to present an integrated view of the various diagnostic methods and techniques, including those for: (i) parasite detection; (ii) DNA detection; and (iii) antibody detection. The discussion covers various other factors that need to be considered, such as the sensitivity and specificity of the various diagnostic methods, critical cross-reactions that may be expected among Trypanosomatidae, additional complementary information, such as clinical observations and epizootiological context, scale of study and logistic and cost constraints. The suitability of examining multiple specimens and samples using several techniques is discussed, as well as risks to technicians, in the context of specific geographical regions and settings. This overview also addresses the challenge of diagnosing mixed infections with different Trypanosoma species and/or kinetoplastid parasites. Improving and strengthening procedures for diagnosing animal trypanosomoses throughout the world will result in a better control of infections and will significantly impact on “One Health,” by advancing and preserving animal, human and environmental health. Graphical Abstract

Gold nanoparticles (AuNPs) are widely used in biomedical diagnostics due to their unique plasmonic properties, with larger AuNPs showing higher extinction coefficients for the plasmon band and, consequently, more intense colors than the more commonly used spherical 20 nm AuNPs. Other factors can be important in the performance of different‐sized AuNPs, including surface area, colloidal stability, and curvature effects. Here, the properties of spherical 20 nm AuNPs and 35 nm AuNPs functionalized with a specific thiol‐modified oligonucleotide–Au nanoprobes are compared when used on a colorimetric assay for the detection of a single nucleotide polymorphism related to lactose intolerance in humans. Successful functionalization of AuNPs is assessed by UV–vis spectroscopy, agarose gel electrophoresis, dynamic and electrophoretic light scattering, and nanoparticle tracking analysis. Statistical differences between Au‐nanoprobe DNA target groups are calculated using analysis of variance and a post hoc Tukey's test. These results show that both 35 and 20 nm Au nanoprobes have similar detection limits using a 0.15 nmol dm−3 nanoprobe concentration compared to 2.5 nmol dm−3. Interestingly, the use of 35 nm Au nanoprobes allows a reduction of 80% and 48% in the amount of gold and oligonucleotide used in the assay, respectively. [ABSTRACT FROM AUTHOR]

In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5′-TTATT-CCCCC-3′; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5′-TTATT-3′) used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin (stn) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems. [ABSTRACT FROM AUTHOR]

Creation of new two‐dimensional (2D) architectures has attracted significant attention in the field of self‐assembly for structural diversity and new functionalization. Although numerous 2D polymer nanosheets have been reported, 2D nanosheets with tubular channels have been unexplored. Herein, we describe a new strategy for the fabrication of stimulus‐responsive conjugated polymer 2D nanosheets with hollow cavities. Amphiphilic macrocyclic diacetylenes self‐assembled in an aqueous solution in a columnar manner to afford bilayered 2D nanosheets with intrinsically tubular nanochannels. UV‐induced polymerization resulted in the generation of blue‐colored tubular conjugated polydiacetylene 2D nanosheets. Immobilization of gold nanoparticles, fluorescence labeling with FRET phenomenon and colorimetric DNA sensing were demonstrated with these new 2D nanosheets. In addition, the free NH2 containing polymerized 2D nanosheet was utilized for conductivity behavior and grafting on graphene oxide (GO). [ABSTRACT FROM AUTHOR]

Porous silicon (PSi) layers are prepared on the single-crystal silicon surface by electrochemical etching, and PSi membranes (PSiMs) are fabricated by separating the PSi layers from the single-crystal silicon substrate using high-current pulses. PSiMs have better adsorption and response time than nonseparated PSi layers, making them more suitable for sensing applications. PSi microcavity (PSM) structures are widely used for the detection of biomolecules in biosensors because of their high transmission resonance peaks with narrow full-width at half-maximum (FWHM) in the reflection spectra. However, when compared to PSiMs, the permeability of the PSM on the silicon substrate is low, resulting in a significant reduction in its detection sensitivity. In this study, an efficient self-supported Bragg stacked microcavity (SBSM) structure was prepared for the detection of DNA biomolecules by combining the advantages of PSM and PSiMs. The redshift of the reflectance spectra obtained in the concentration range of 0.1– $0.5 ~\mu \text{M}$ shows a good linear relationship, with a detection limit of 2.2 nM. The detection sensitivity is improved by more than a factor of ten when compared to the PSM structure on the silicon substrate. The novel device allows DNA biomolecules to enter the porous layers more easily, especially the defective layer, thus improving its detection sensitivity. [ABSTRACT FROM AUTHOR]

Analytical verification of hazelnut in food supports risk-based approaches for proper allergen labeling to prevent unwanted allergic reactions or to quality control diagnostic or therapeutic allergen preparations for allergic subjects. We present the development and validation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of hazelnut by targeting the internal transcribed spacer 2 gene. The qualitative method requires neither sophisticated analytical equipment nor antibodies, allowing an easy-to-use application with no ethical concern related to the use of animals. It demonstrated a limit of detection at or below 10 mg/kg hazelnut in various food matrices, making it also suitable for verifying hazelnut at levels of clinically relevant eliciting doses. Validation against proficiency test samples and testing of applicability with commercial food items confirmed its usefulness in processed foods. The simplicity of the method, including visual colorimetric detection, combined with high specificity and sensitivity, represents an advancement over existing qualitative methods.

Sporothrix sp. is a thermally dimorphic genus of fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases. Here, we adapted and evaluated a recently described real-time PCR assay targeting the β-tubulin gene to rapidly detect and differentiate two related species, S. schenckii and S. brasiliensis . The assay was tested with 55 S . brasiliensis, 19 S . schenckii , and 85 isolates from other clinically relevant fungi, and showed 100% concordance with reference identification methods. The assay showed high analytical sensitivity with a limit of detection of 1 pg of DNA per microliter of sample for both targets. The assay was further evaluated with 11 fresh and 17 formalin-fixed, paraffin-embedded (FFPE) tissues. This multiplex real-time PCR assay successfully detected the DNA from both S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this assay performs well and could be a helpful molecular tool to support rapid species identification in cultures and primary specimens.IMPORTANCEHaving available molecular tools to identify and differentiate closely related species will allow clinical, veterinarians, and public health labs to provide diagnostic results with accuracy and short turnaround time for the routine and outbreak response activities.

The continued spread of antimicrobial resistance is a global problem. In fact, the WHO has declared antimicrobial resistance one of the top 10 global public health threats facing humanity. Molecular methods offer a faster means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular detection method that generates visible aggregates from DNA amplified products and functionalized magnetic nanoparticles. The amplification and detection procedure take less than 2 h. In this study we also investigated the factors behind the aggregation and confirmed that the size of the padlock probe is a relevant parameter for the amplification efficiency. We compared a 92 and a 69 nt long padlock probe and found the latter to perform over 4 times better than the 92 nt padlock probe. This effect was related to a higher quantity of amplification products produced by the shorter padlock probe. Consequently, the quantity of amplified DNA products affects the aggregation in a positive way, and thanks to it, a better assay sensitivity was achieved. Finally, using our developed method, we could detect as little as 1 amol of the antibiotic-resistance gene sul1 from a sample containing a multiresistance plasmid. We were also able to quantify the amount of plasmid DNA through absorbance spectroscopy and AC susceptometry. The presented results form the foundation of future development of an ultrasensitive and cheap detection approach that could be used in point of care settings.

The accurate, rapid, and specific detection of DNA strands in solution is becoming increasingly important, especially in biomedical applications such as the trace detection of COVID-19 or cancer diagnosis. In this work we present the design, elaboration and characterization of an optofluidic sensor based on a polymer-based microresonator which shows a quick response time, a low detection limit and good sensitivity. The device is composed of a micro-racetrack waveguide vertically coupled to a bus waveguide and embedded within a microfluidic circuit. The spectral response of the microresonator, in air or immersed in deionised water, shows quality factors up to 72,900 and contrasts up to 0.9. The concentration of DNA strands in water is related to the spectral shift of the microresonator transmission function, as measured at the inflection points of resonance peaks in order to optimize the signal-over-noise ratio. After functionalization by a DNA probe strand on the surface of the microresonator, a specific and real time measurement of the complementary DNA strands in the solution is realized. Additionally, we have inferred the dissociation constant value of the binding equilibrium of the two complementary DNA strands and evidenced a sensitivity of 16.0 pm/µM and a detection limit of 121 nM.

Currently available DNA detection techniques frequently require compromises between simplicity, speed, accuracy, and cost. Here, we propose a simple, label-free, and cost-effective DNA detection platform developed at screen-printed carbon electrodes (SPCEs) modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs). The preparation of the detection platform involved a two-step electrochemical procedure based on GO reduction onto SPCEs followed by the electrochemical reduction of HAuCl4 to facilitate the post-grafting reaction with AuNPs. The final sensor was fabricated by the simple physical adsorption of a single-stranded DNA (ssDNA) probe onto a AuNPs–RGO/SPCE electrode. Each preparation step was confirmed by morphological and structural characterization using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy, respectively. Furthermore, the electrochemical properties of the modified electrodes have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the introduction of AuNPs onto RGO/SPCEs led to an enhancement in surface conductivity, a characteristic that favored an increased sensitivity in detection. The detection process relied on the change in the electrochemical signal induced by the binding of target DNA to the bioreceptor and was particularly monitored by the change in the charge transfer resistance of a [Fe(CN)6]4–/3– redox couple added in the test solution.

Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo functions as a programmable DNA endonuclease by utilizing both short 5′‐phosphorylated and 5′‐hydroxylated single‐stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double‐stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago‐based DNA detection toolbox. [ABSTRACT FROM AUTHOR]

Rapid, simple, and inexpensive diagnostic point-of-care tests (POCTs) are essential for controlling infectious diseases in resource-limited settings. In this study, we developed a new detection system based on nanoparticle–DNA aggregation (STat aggregation of tagged DNA, STAT-DNA) to yield a visual change that can be easily detected by the naked eye. This simplified optical detection system was applied to detect hepatitis C virus (HCV). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers labeled with biotin and digoxigenin. Streptavidin-coated magnetic particles (1 μm) and anti-digoxigenin antibody-coated polystyrene particles (250–350 nm) were added to form aggregates. The limit of detection (LoD) and analytical specificity were analyzed. The STAT-DNA results were compared with those of the standard real-time PCR assay using serum samples from 54 patients with hepatitis C. We achieved visualization of amplified DNA with the naked eye by adding nanoparticles to the PCR mixture without employing centrifugal force, probe addition, incubation, or dilution. The LoD of STAT-DNA was at least 101 IU/mL. STAT-DNA did not show cross-reactivity with eight viral pathogens. The detection using STAT-DNA was consistent with that using standard real-time PCR. [ABSTRACT FROM AUTHOR]

Biosensors based on carbon nanotube (CNT) thin-film transistors (TFTs) have drawn intense interests for their exhibiting great potential in ultrasensitive and label-free DNA detection. However, the inevitable defects in the dielectric and channel of CNT-based TFTs may induce noisy or unreliable output signals. Here, we propose a low-temperature supercritical carbon dioxide (SCCO 2) fluid activation method to enhance the performance of transistor-based biosensors. An activation model of SCCO 2 treatment is proposed to elaborate that the SCCO 2 treatment can repair the dangling bonds of SiO 2 dielectric facilely and passivate the hydroxyl groups effectively. Furthermore, a cell-free DNA detection platform is built up based on all-carbon-nanotube TFTs (AC-TFTs) modified by the CW-Peptide probe. By adopting the SCCO 2 treatment, the response of AC-TFT biosensors to AKT2 gene related to triple-metastatic negative breast cancer can be enhanced by more than 114% with high uniformity, and a broad detection range of six orders of magnitude is yielded with a theoretical limit of detection of 42 fM. Additionally, the response has a linear correlation with the length of DNA targets. The nondestructive and eco-friendly SCCO 2 activation method provides a promising and universal strategy for achieving highly accurate and sensitive transistor-based biosensors in future clinical applications. [Display omitted] [ABSTRACT FROM AUTHOR]

A hollow core micro-bottle cavity (HC-MBC) biosensor with high quality (Q) factor for DNA detection with low detection limit has been proposed and demonstrated. Whispering gallery mode (WGM) spectra with Q factor >106 are excited through high-precision coupling of tapered fiber and HC-MBC. An improved surface functionalization method is used at 70 °C to ensure binding of probe single-stranded DNA onto the inner surface of the HC-MBC with high effectiveness and stability. Based on the principle of base complementary pairing, efficient detection of DNA with different concentrations and sequences are realized. The detection volume and limit of the proposed biosensor are ∼ nL and pM, respectively. Single-nucleotide mismatch recognition is also achieved. Benefiting from its label free, compact size, real-time and low detection limit, the proposed biosensor has broad application prospects in bioengineering and medical diagnosis. [ABSTRACT FROM AUTHOR]

In this paper, a novel platform for lab-in-fiber-based biosensors is studied. Hollow-core tube lattice fibers (HC-TLFs) are proposed as a label-free biosensor for the detection of DNA molecules. The particular light-guiding mechanism makes them a highly sensitive tool. Their transmission spectrum is featured by alternations of high and low transmittance at wavelength regions whose values depend on the thickness of the microstructured web composing the cladding around the hollow core. In order to achieve DNA detection by using these fibers, an internal chemical functionalization process of the fiber has been performed in five steps in order to link specific peptide nucleic acid (PNA) probes, then the functionalized fiber was used for a three-step assay. When a solution containing a particular DNA sequence is made to flow through the HC of the TLF in an 'optofluidic' format, a bio-layer is formed on the cladding surfaces causing a red-shift of the fiber transmission spectrum. By comparing the fiber transmission spectra before and after the flowing it is possible to identify the eventual formation of the layer and, therefore, the presence or not of a particular DNA sequence in the solution. [ABSTRACT FROM AUTHOR]