DNA Capture Research Articles

Survival implications and radiographic predictors of prognosis in patients with IDH-mutant astrocytoma with CDKN2A/b homozygous deletion: An international, multi-centered study.

e14035 Background: Isocitrate Dehydrogenase (IDH) 1 or 2 mutations are associated with improved prognosis in glioma patients. Despite their less aggressive clinical course when compared to IDH-wild type gliomas, molecularly-defined IDH-mutant glioma subtypes with poor prognosis have been identified. Among these, CDKN2A/B homozygous deletion represents the strongest negative genetic predictor of prognosis yet identified in IDH-mutant astrocytoma. With the WHO 2021 classification recognizing CDKN2A/B homozygous deletion as sufficient for the designation of astrocytoma, IDH-mutant, WHO Grade 4, the translational implications and potential for therapies targeting CDKN2A/B homozygous deletion are an area of active investigation. We sought to further characterize overall (OS) and progression-free survival (PFS) of CDKN2A/B deleted patients within a large, multi-centered, international cohort and identify clinical characteristics that predict survival for patients with CDKN2A/B deletion. Methods: Patients with IDH-mutant glioma with CDKN2A/B homozygous deletion were retrospectively identified across four institutions (Massachusetts General Hospital, USA; University Hospital Heidelberg, Germany; University Hospital Dresden, Germany, and Foudan-Shanghai, China). IDH-mutation status was determined by immunohistochemistry or next-generation sequencing. CDKN2A/B status was determined by hybridized DNA capture or FISH. Results: We identified 75 patients with IDH-mutant astrocytoma with CDKN2A/B homozygous deletion. The cohort had a median follow-up time of 5.0 years, was 56% male, and had an average age of 38.8 years. Patients were treated with chemotherapy and radiation following initial surgery in 77% of cases. Preoperative contrast enhancement and T2 FLAIR mismatch were identified in 56 and 44% of cases, respectively. Compared to a cohort of 51 patients with CDKN2A/B intact, grade 4, IDH-mutant astrocytoma, those with CDKN2A/B-deleted tumors had significantly shorter median OS and PFS (OS: 4 vs 13.5 years, HR = 6.8, HR 95% CI = 3.1, 14.6, p < 0.0001; PFS: 2.8 vs 5.9 years, HR = 3.0, HR 95% CI = 1.7, 5.2, p = 0.0001). Within the CDKN2A/B deleted cohort, preoperative contrast enhancement predicted worse OS, but not PFS (OS: HR = 2.7, 95% CI = 1.1, 7.4, p = 0.04; PFS: HR = 1.17, 95% CI = 0.7, 3.7, p = 0.24). Conclusions: These data reinforce the strong negative prognostic impact of CDKN2A/B homozygous deletion on OS and PFS and identify a novel association between preoperative contrast enhancement and shorter OS in patients with CDKN2A/Bdeleted, IDH-mutant astrocytoma.

Cu NCs@MXene-luminescent Faraday cage and Cu Nanocone/Bi NPs array-based saliva exosome assay for asthma evaluation

In this work, a novel nano-sensing system with luminescent Faraday cage mode has been developed to detect miRNA-221-5p in clinic saliva exosomes to evaluate asthma. Firstly, copper nanoclusters (Cu NCs) were synthesized in situ on the defects in Ti3CN nanosheets based on the nanoconfinement effect. Due to the Electronic metal-support interactions (EMSI) between MXene and the anchored Cu NCs, Cu NCs@MXene displayed the strong luminescence performance, high electrochemical activity and good stability properties. Furthermore, MXene nanosheets with good conductivity and flexibility was used to expand the outer Helmholtz plane and improve the sensing ability. Meanwhile, Cu nanocone/Bi nanoparticles (NPs) arrays were constructed by step-by-step electrodeposition. Due to the surface plasmon coupling effect, the Cu nanocone/Bi NPs arrays can convert the electrochemiluminescence (ECL) signal of Cu NCs@MXene-Faraday cage into the directional emission with 13.4-fold enhancement. Finally, a thin phospholipid film and capture DNA were modified on the surface of the Cu nanocone/Bi NPs array as the sensing interface to detect miRNA-221-5p in saliva exosomes. The designed nano-sensing system had a detection range was 1.0 × 10-16 ∼ 1.0 × 10-8 mol/L with the detection limit of 34 aM. Finally, the biosensor has been successfully employed to evaluate asthma in the clinical analysis.

Fe-CP-based Catalytic Oxidation and Dissipative Self-Assembly of a Ferrocenyl Surfactant Applied in DNA Capture and Release

Fe-CP-based Catalytic Oxidation and Dissipative Self-Assembly of a Ferrocenyl Surfactant Applied in DNA Capture and Release

Lysis-Free Isolation and Direct Amplification of Pathogenic Bacterial DNA Using Diatom Frustules.

DNA has been implicated as an important biomarker for the diagnosis of bacterial infections. Herein, we developed a streamlined methodology that uses diatom frustules (DFs) to liberate and capture bacterial DNA and allows direct downstream amplification tests without any lysis, washing, or elution steps. Unlike most conventional DNA isolation methods that rely on cell lysis to release bacterial DNA, DFs can trigger the oxidative stress response of bacterial cells to promote bacterial membrane vesicle formation and DNA release by generating reactive oxygen species in aqueous solutions. Due to the hierarchical porous structure, DFs provided high DNA capture efficiency exceeding 80% over a wide range of DNA amounts from 10 pg to 10 ng, making only 10 μg DFs sufficient for each test. Since laborious liquid handling steps are not required, the entire DNA sample preparation process using DFs can be completed within 3 min. The diagnostic use of this DF-based methodology was illustrated, which showed that the DNA of the pathogenic bacteria in serum samples was isolated by DFs and directly detected using polymerase chain reaction (PCR) at concentrations as low as 102 CFU/mL, outperforming the most used approaches based on solid-phase DNA extraction. Furthermore, most of the bacterial cells were still alive after DNA isolation using DFs, providing the possibility of recycling samples for storage and further diagnosis. The proposed DF-based methodology is anticipated to simplify bacterial infection diagnosis and be broadly applied to various medical diagnoses and biological research.

A facile electrochemical aptasensor for chloramphenicol detection based on synergistically photosensitization enhanced by SYBR Green I and MoS2

This study reports the development of a photocatalytic electrochemical aptasensor for the purpose of detecting chloramphenicol (CAP) antibiotic residues in water by utilizing SYBR Green I (SG) and chemically exfoliated MoS2 (ce-MoS2) as synergistically signal-amplification platforms. The Au nanoparticles (AuNPs) were electrodeposited onto the surface of an indium tin oxide (ITO) electrode. After that, the thiolate-modified cDNA, also known as capture DNA, was combined with the aptamer. Subsequently, photosensitized SG molecules and ce-MoS2 nanomaterial were inserted into the groove of the resultant double-stranded DNA (dsDNA). The activation of the photocatalytic process upon exposure to light resulted in the generation of singlet oxygen. The singlet oxygen effectively split the dsDNA, resulting in significant enhancement in the current of [Fe(CN)6]3-/4-. When the CAP was present, both SG molecules and ce-MoS2 broke away from the dsDNA, which turned off the photosensitization response, leading to significant reduction in the current of [Fe(CN)6]3-/4-. Under the optimal conditions, the aptasensor exhibited a linear relationship between the current of [Fe(CN)6]3-/4- with logarithmic concentrations of CAP from 20 to 1000 nM, with a detection of limit (3σ) of 3.391 nM. The aptasensor also demonstrated good selectivity towards CAP in the presence of interfering antibiotics, such as tetracycline, streptomycin, levofloxacin, ciprofloxacin, and sulfadimethoxine. Additionally, the results obtained from the analysis of natural water samples using the proposed aptasensor were consistent with the findings acquired through the use of a liquid chromatograph-mass spectrometer. Therefore, with its simplicity and high selectivity, this aptasensor can potentially detect alternative antibiotics in environmental water samples by replacing the aptamers based on photosensitization.

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Integration of high-entropy nanozyme and hollow In2S3 nanotube heterostructures decorated with WO3 for ultrasensitive PEC aptasensing of highly toxic mycotoxin

As a highly toxic mycotoxin, patulin threatens human and animal health. Herein, a new signal amplification strategy was designed for photoelectrochemical (PEC) analysis of patulin based on nanotube-like In2S3/WO3 heterostructures with the assistance of PtRuFeCoW high-entropy alloy nanorods (HEANRs). Specifically, the prominent photoactivity of the In2S3/WO3 was investigated by UV−vis diffuse reflectance and photoluminescence spectroscopy, coupled by elaborating the photoelectron transport mechanism in detail. Simultaneously, the excellent peroxidase-like activity of the PtRuFeCoW HEANRs was certificated mainly by UV–vis absorption spectroscopy. On such foundation, the PtRuFeCoW HEANRs modified capture DNA was cast onto the In2S3/WO3 based photoanode for catalyzing 4-chloro-1-naphthol (4-CN) oxidation to form insoluble benz-4-chloro-hexadienone (4-CD) precipitate in the presence of H2O2, ultimately weakening the PEC currents. The resulting PEC sensor exhibited a wider detection range from 0.1 pg mL−1 to 500 ng mL−1 and a lower detection limit was down to 0.063 pg mL−1 (S/N = 3). The HEANRs nanozyme assisted PEC method holds great promise for ultrasensitive detection of environmental pollutants in practice.

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Selective Capture and Manipulation of DNA through Double Charged Nanopores.

In the past few decades, nanometer-scale pores have been employed as powerful tools for sensing biological molecules. Owing to its unique structure and properties, solid-state nanopores provide interesting opportunities for the development of DNA sequencing technology. Controlling DNA translocation in nanopores is an important means of improving the accuracy of sequencing. Here we present a proof of principle study of accelerating DNA captured across targeted graphene nanopores using surface charge density and find the intrinsic mechanism of the combination of electroosmotic flow induced by charges of nanopore and electrostatic attraction/repulsion between the nanopore and ssDNA. The theoretical study performed here provides a new means for controlling DNA transport dynamics and makes better and cheaper application of graphene in molecular sequencing.

MED23 pathogenic variant: genomic-phenotypic analysis.

The mediator complex subunit 23 (MED23) gene encodes a protein that acts as a tail module mediator complex, a multi-subunit co-activator involved in several cellular activities. MED23 has been shown to have substantial roles in myogenesis and other molecular mechanisms. The functions of MED23 in the neurological system remain unclear and the clinical phenotype is not thoroughly described. Whole exome sequencing was used to identify a novel mutation in the MED23 gene. DNA capture probes using next-generation sequencing-based copy number variation analysis with Illumina array were performed. The clinical, demographic, neuroimaging, and electrophysiological data of the patients were collected, and similarly, the data of all reported cases in the literature were extracted to compare findings. Screening a total of 9,662 articles, we identified 22 main regulatory processes for the MED23 gene, including suppressive activity for carcinogenic processes. MED23 is also involved in the brain's neurogenesis and functions. The identified cases mainly presented with intellectual disability (87.5%) and developmental delay (50%). Seizures were present in only 18.75% of the patients. Slow backgrounds and spike and sharp-wave complexes were reported on the electroencephalogram (EEG) of a few patients and delayed myelination, thin corpus callosum, and pontine hypoplasia on magnetic resonance imaging (MRI). The MED23 gene regulates several processes in which its understanding promotes considerable therapeutic potential for patients. It is crucial to consider genetic and laboratory testing, particularly when encountering potential carriers. Intellectual disability and developmental delay are the most notable clinical signs with heterogeneous features on EEG and MRI.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight

BackgroundThe Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes.ResultsTo provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate.ConclusionsFENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

Microsphere-Enhanced Fluorescence-Lightened Solid-Phase Hybridization Assay: The Strategy to Highly Selective Detection of Micro Ribonucleic Acids.

The detection of low-abundance microribonucleic acid (miRNA) frequently adopted nucleic acid sequence-based amplification detection, which was found to have poor selectivity for the nonspecific amplification of template-dependent ligation in enzyme-mediated cascade reactions. Here, a highly selective detection of miRNAs was developed that combined microsphere-enhanced fluorescence (MSEF) and solid-phase base-paired hybridization. The target miRNA could be accurately and quantitatively identified through the solid-phase hybridization assay on the surface of an optical microsphere, while the detected fluorescence signal could be physically amplified by MSEF. Hereinto, the optical microsphere acted as the fluorescence amplifier and whose surface supplied the space to carry out base-paired hybridization to recognize the target miRNA via the immobilized capture DNA sequence. The detected fluorescence signal of the single-base mismatched miRNA-21 sequence was just around 12% of that of the target miRNA-21 sequence in the measurement of model miRNA-21, while the limit of detection of miRNA-21 could be 1.0 fM. The developed detection of miRNA on an optical microsphere was demonstrated to be an excellent physically amplified method to selectively and sensitively detect the target miRNA and magnificently avoid the nonspecific amplification and false-positive results, which is expected to have wide applications in pathematology, pharmacology, clinic diagnosis, and on-site screening fields as well.

Smartphone-based device for rapid and single-step detection of piRNA–651 using anti-DNA:RNA hybrid antibody and enzymatic signal amplification

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs/piRs) are a class of small noncoding RNAs that play a crucial role in regulating various biological processes, including carcinogenesis. One specific piRNA, piR–651, has been reported to be overexpressed in both human blood serum and solid cancer tissues, that can be used a viable biomarker in cancer diagnosis. Early diagnosis of cancer can help reduce the burden of the disease and improve survival rates. In the present work, we report for the first time a smartphone-based colorimetric biosensor for highly sensitive and specific detection of piR–651 thanks to an enzymatic signal amplification, which yielded high colorimetric intensities. Indeed, a heteroduplex DNA:RNA was formed in the presence of piR–651 with the capture DNA probe immobilized on the magnetic beads for easy magnetic separation. Then, a HRP tethered to anti-DNA:RNA (S9.6) was used to reveal the DNA-RNA heteroduplex formed by catalyzing the oxidation of TMB substrate into colorimetric TMBox, which absorbs at 630 nm. The absorbance is positively proportional to the piR–651 concentrations. On the other hand, the colorimetric product of the assay can be photographed with a smartphone camera and analyzed using ImageJ software. Using a smartphone and under optimal conditions, the biosensor responded linearly to the logarithm of piRNA–651 from 8 fM to 100 pM with a detection limit of 2.3 fM and discriminates against other piRNAs. It was also successfully applied to the determination of piRNA–651 levels in spiked human serum.

Artificial nanochannels for highly selective detection of miRNA based on the HCR signal amplification

Artificial solid-state nanochannels have nanoscale spatial confinement and unique ion transport properties that have been widely used in biosensing, nanofluidic devices, and nucleic acid sequencing. However, most of the nanochannel sensors for miRNA detection are grounded on the complementary pairing of bases, lacking signal amplification, which limits its application in many fields. Herein, we proposed a signal amplification strategy for miRNA-182 detection based on introducing hybridization chain reaction (HCR) in artificial nanochannels to improve the detection sensitivity. In this method, the capture DNA (cDNA) was anchored in the nanochannel, and when the target miRNA appeared, the hairpin probes H1, H2, and initiator were introduced to trigger the HCR reaction to form long nucleic acid double strands, which significantly enhanced the negative charge and affected ion transport in nanochannel. The nanochannel sensor performed excellent selectivity to the target miRNA-182, and it can realize the detection of miRNA-182 with an ultra-low concentration of 1.65 aM, which can be further applied to the detection of miRNA-182 in actual biological samples. Moreover, the nanochannel system was proved by using the AND logic gate that cDNA, initiator, H1, H2, and miRNA-182 are indispensable for successfully triggering HCR. This work provides a simple and efficient signal amplification method for miRNA detection, which considerably expands the application of nanochannel sensors and holds great prospects in biomedical and clinical detection.

Deposition of high-quality, nanoscale SiO2 films and 3D structures

Silicon dioxide (SiO2) is ubiquitous in biomedical diagnostics and other applications as a capture medium for nucleic acids and proteins. Diagnostic devices have seen rapid miniaturisation in recent years, due to the increased demand for portable point-of-care diagnostics. However, there are increasing challenges with incorporating SiO2 nanostructures into diagnostic devices, due to the complexity of nanostructured SiO2 synthesis, often involving etching and chemical vapour deposition under high vacuum conditions.We report a novel and straightforward method for deposition of high-quality, nanoscale SiO2 films and 3D SiO2 structures using thermal decomposition of polydimethylsiloxane (PDMS), in a furnace at atmospheric pressure at 500 °C. This method allows individual nanometre controllability of conformal pinhole-free layers on a variety of materials and morphologies. The temperature ramp rate is a key factor in determining the SiO2 deposit morphology, with slower ramp rates leading to highly conformal 2D films and faster ones yielding 3D nanodentrite structures. For the 2D films, the film thickness, as determined by spectroscopic ellipsometry and confirmed by SEM data, is shown to correlate excellently with initial PDMS source material mass in the thickness range 0.8–18 nm. Fits to ellipsometry models confirm that the refractive index of the deposited film matches the expected value for SiO2, while electrical breakdown measurements confirm that the breakdown strength of the films is comparable to that of high-quality thermal oxides. Depositions on high aspect ratio ZnO nanostructures are shown to be highly conformal, leading to core-shell ZnO-SiO2 nanostructures whose shell thickness is in excellent agreement with the expected values from deposition on planar substrates. At faster ramp rates an abrupt morphological transition is seen to a deposit which displays a 3D nanodentrite morphology. The possibilities for applications of both morphologies (and core-shell combinations with other nanostructured materials) in biosensing and related areas are briefly discussed, and the DNA capture capabilities of each nanostructure are measured. The high aspect ratio nanodendrite structures allow for significant DNA capture within microfluidic devices in the presence of low DNA concentrations, with a maximum average capture efficiency of 43.4 % achieved in the presence of 10 ng/mL of DNA, which is an improvement by a factor of ∼ 3 over planar Si surfaces. Improvements by factors of >10 over planar surfaces were achieved at higher DNA concentrations of 100 and 1000 ng/mL.

Pulley Effect in the Capture of DNA Translocation through Solid-State Nanopores.

Nanopores are powerful single-molecule sensors for analyzing biomolecules such as DNA and proteins. Understanding the dynamics of DNA capture and translocation through nanopores is essential for optimizing their performance. In this study, we examine the effects of applied voltage and pore diameter on current blockage, translocation time, collision, and capture location by translocating λ-DNA through 5.7 and 16 nm solid-state nanopores. Ionic current changes are used to infer DNA conformations during translocation. We find that translocation time increases with pore diameter, which can be attributed to the decrease of the stall force. Linear and exponential decreases of collision frequency with voltage are observed in the 16 and 5.7 nm pores, respectively, indicating a free energy barrier in the small pore. Moreover, the results reveal a voltage-dependent bias in the capture location toward the DNA ends, which is explained by a "pulley effect" deforming the DNA as it approaches the pore. This study provides insights into the physics governing DNA capture and translocation, which can be useful for promoting single-file translocation to enhance nanopore sensing.

Development of surface-enhanced Raman scattering-sensing Method by combining novel Ag@Au core/shell nanoparticle-based SERS probe with hybridization chain reaction for high-sensitive detection of hepatitis C virus nucleic acid.

The ultrasensitive detection of hepatitis C virus (HCV) nucleic acid is crucial for the early diagnosis of hepatitis C. In this study, by combining Ag@Au core/shell nanoparticle (Ag@AuNP)-based surface-enhanced Raman scattering (SERS) tag with hybridization chain reaction (HCR), a novel SERS-sensing method was developed for the ultrasensitive detection of HCV nucleic acid. This SERS-sensing system comprised two different SERS tags, which were constructed by modifying Ag@AuNP with a Raman reporter molecule of 4-ethynylbezaldehyde, two different hairpin-structured HCR sequences (H1 or H2), and a detection plate prepared by immobilizinga capture DNA sequence onto the Ag@AuNP layer surface of the detection wells. When thetarget nucleic acid was present, the two SERS tags were captured on the surface of the Ag@AuNP-coated detection well to generate many "hot spots" through HCR, forming a strong SERS signal and realizing the ultrasensitive detection of thetarget HCV nucleic acid. The limit of detection of the SERS-sensing method for HCV nucleic acid was 0.47 fM, and the linear range was from 1 to 105 fM.

ACEK Biosensor for the Minute-Scale Quantification of Breast Cancer ctDNA.

Circulating tumor DNA (ctDNA) appears as a valuable liquid biopsy biomarker in the early diagnosis, treatment, and prognosis of cancer. Here, a biosensing method derived from the AC electrokinetics (ACEK) effect was constructed in this study for the simple, efficient, and rapid method of detection of ctDNA. In the proof-of-concept experiment, ctDNA from the PIK3CA E542K mutant in breast cancer was quantified by detecting a normalized capacitance change rate using a forked-finger gold electrode as the sensing electrode in combination with the ACEK effect. We compared two formats for the construction of the approach by employing varied immobilization strategies; one is to immobilize the DNA capture probe on the electrode surface by Au-S bonding, while the other immobilizes the probe on a self-assembled membrane on the electrode surface by amide bonding. Both formats demonstrated ultrafast detection speed by completing the ctDNA quantification within 1 min and a linear range of 10 fM-10 pM was observed. Meanwhile, the immobilization via the self-assembled membrane yielded improved stability, sensitivity, and specificity than its Au-S bonding counterpart. A detection limit of 1.94 fM was eventually achieved using the optimized approach. This research provides a label-free and minute-scale universal method for the detection of various malignant tumors. The ctDNA biosensors based on the ACEK effect improved according to the probe type or electrode structure and have potential applications in tumor drug efficacy prediction, drug resistance monitoring, screening of high-risk groups, differential diagnosis, monitoring of tiny residual lesions, and prognosis determination.

A SERS aptasensor based on HCR mediated signal conversion strategy for the rapid and reliable determination of Escherichia coli O157:H7

Rapid and reliable determination of pathogenic bacteria is crucial for food safety and public health. Surface-enhanced Raman spectroscopy (SERS) based aptasensors have attracted increasing attention in the determination of pathogenic bacteria due to their high specificity and sensitivity. However, the sensitivity and reliability of the SERS aptasensors for the determination of pathogens were hindered by the uneven distribution of SERS tags. To tackle this limitation, a SERS aptasensor based on a hybridization chain reaction (HCR) mediated signal conversion strategy was fabricated for the determination of E. coli O157:H7. In the presence of E. coli O157:H7, the blocked aptamer specifically binds with it and releases the blocker DNA. Then the blocker DNA hybridizes with the capture DNA on the SERS probe and initiates the HCR reaction, which further leads to the formation of double stranded DNA and the reduction of the SERS signal intensity of 4-mercaptobenzonitrile (4-MBN). Under the optimal conditions, the SERS signal intensity at 1073.9 cm−1 and the logarithmic value of the concentration of E. coli O157:H7 in the range of 8 × 102–8 × 107 CFU·mL−1 exhibited a good linear relationship, with a coefficient of determination of 0.92. The limit of detection for E. coli O157:H7 was 409 CFU·mL−1. Besides, this aptasensor exhibited satisfactory specificity for E. coli O157:H7. In addition, it showed excellent stability in real sample analysis with relative standard deviations (RSDs) below 3.76 % (n = 3). The established SERS aptasensor employed a HCR mediated signal conversion strategy and overcame the drawback of unevenly distribution of SERS tags in classical SERS aptasensors for the determination of pathogenic bacteria. This study offered a new reference for the rapid and reliable detection of pathogens with a portable Raman spectrometer.

Single-Molecule Analysis of SARS-CoV-2 Double-Stranded Polynucleotides Using Solid-State Nanopore with AI-Assisted Detection and Classification: Implications for Understanding Disease Severity.

This study utilized solid-state nanopores, combined with artificial intelligence (AI), to analyze the double-stranded polynucleotides encoding angiotensin-converting enzyme 2, receptor-binding domain, and N protein, important parts of SARS-CoV-2 infection. By examining ionic current signals during DNA translocation, we revealed the dynamic interactions and structural characteristics of these nucleotide sequences and also quantified their abundance. Nanopores of sizes 3 and 10 nm were efficiently fabricated and characterized, ensuring an optimal experimental approach. Our results showed a clear relationship between DNA capture rates and concentration, proving our method's effectiveness. Notably, longer DNA sequences had higher capture rates, suggesting their importance for potential disease marker analysis. The 3 nm nanopore demonstrated superior performance in our DNA analysis. Using dwell time measurements and excluded currents, we were able to distinguish the longer DNA fragments, paving the way for a DNA length-based analysis. Overall, our research underscores the potential of nanopore technology, enhanced with AI, in analyzing COVID-19-related DNA and its implications for understanding disease severity. This provides insight into innovative diagnostic and treatment strategies.