Hybridization Chain Reaction Research Articles

Conductivity-Regulated Bipolar Electrochemiluminescence Sensing Platform for Indicator-Free Homogeneous Bioassay.

Electrochemiluminescence (ECL) sensors have been widely developed because of their high sensitivity and low background. However, most of them suffered from tedious probe modification on the electrode and cross-interferences within the sensing and reporting reactions. The bipolar electrode based ECL (BPE-ECL) can effectively eliminate interference by physically separating the sensing and reporting cells, but there is still a need for exogenous electroactive indicators to transduce the variations between two poles of a BPE. Herein, based on the discovery that conductivity can be regulated in aqueous medium by homogeneous bioreaction, we showed a novel BPE-ECL sensing platform that combined the conductivity-based biosensing technology with ECL reporting system for the first time. Compared to many short nucleic acids, the target induced a hybridization chain reaction to produce the long nucleic acid aggregates, resulting in a conductivity decrease of the sensing cell and finally reducing the ECL response in the reporting cell. The BPE-ECL platform has already been applied to detect microRNA-21 for a demonstration. This innovative system not only separates the target sensing and reporting reactions but also avoids the use of electrochemical indicators for measurement. The BPE-ECL biosensing platform can be developed to detect different targets by changing the probe used.

Synergistic powering of DNA walker movement by endogenous dual enzymes for constructing dual-mode biosensors

To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01–500 U mL−1, presenting a good linearity in the concentration range of 0.01–10 U mL−1, with a detection limit of 0.01 U mL−1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01–50 U mL−1 and reducing the detection limit to 0.002 U mL−1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.

Dual-modal biosensor for mercuric ion detection based on Cu2O@Cu2S/D-TA COF heterojunction with excellent catalase-like, electrochemical and photoelectrochemical properties

In this study, a dual-mode biosensor based on the heterojunction of Cu2O@Cu2S/D-TA COF was constructed for ultra-sensitive detection of Hg2+ using both photoelectrochemical and electrochemical approaches. Briefly, a 2D ultra-thin covalent organic framework film (D-TA COF film) with excellent photoelectrochemical signals was prepared on ITO surfaces through an in situ growth method. Subsequently, the probe H1 was immobilized onto the biosensor via Au–S bonds. In the presence of Hg2+, the formation of T-Hg2+-T complexes triggered hybridization chain reactions (HCR), leading to the attachment of abundant Cu2O@Cu2S probes onto the biosensor. As a p-type semiconductor, Cu2O@Cu2S could form a heterojunction with the underlying D-TA COF films. Meanwhile, it exhibited catalase-like activity, and the O2 produced by its catalytic decomposition of H2O2 can interact with the D-TA COF films, thus achieving double amplification of the photocurrent signal. Benefiting from the excellent and inherent Cu2+/Cu+ redox pairs of Cu2O@Cu2S, satisfactory differential pulse voltammetry (DPV) signals were obtained. As expected, the dual-mode biosensor was realized with wider linear ranges and low detection limits. Additionally, the analytical performance for Hg2+ in real water samples was excellent. Briefly, this suggested approach offers a facile and highly efficient modality for monitoring heavy metal ions in aquatic environments.

Expression of novel androgen receptors in three GnRH neuron subtypes in the cichlid brain.

In teleosts, GnRH1 neurons stand at the apex of the Hypothalamo-Pituitary-Gonadal (HPG) axis, which is responsible for the production of sex steroids by the gonads (notably, androgens). To exert their actions, androgens need to bind to their specific receptors, called androgen receptors (ARs). Due to a teleost-specific whole genome duplication, A. burtoni possess two AR paralogs (ARα and ARβ) that are encoded by two different genes, ar1 and ar2, respectively. In A. burtoni, males stratify along dominance hierarchies, in which an individuals' social status determines its physiology and behavior. GnRH1 neurons have been strongly linked with dominance and circulating androgen levels. Similarly, GnRH3 neurons are implicated in the display of male specific behaviors. Some studies have shown that these GnRH neurons are responsive to fluctuations in circulating androgens levels, suggesting a link between GnRH neurons and ARs. While female A. burtoni do not naturally form a social hierarchy, their reproductive state is positively correlated to androgen levels and GnRH1 neuron size. Although there are reports related to the expression of ar genes in GnRH neurons in cichlid species, the expression of each ar gene remains inconclusive due to technical limitations. Here, we used immunohistochemistry, in situ hybridization chain reaction (HCR), and spatial transcriptomics to investigate ar1 and ar2 expression specifically in GnRH neurons. We find that all GnRH1 neurons intensely express ar1 but only a few of them express ar2, suggesting the presence of genetically-distinct GnRH1 subtypes. Very few ar1 and ar2 transcripts were found in GnRH2 neurons. GnRH3 neurons were found to express both ar genes. The presence of distinct ar genes within GnRH neuron subtypes, most clearly observed for GnRH1 neurons, suggests differential control of these neurons by androgenic signaling. These findings provide valuable insight for future studies aimed at disentangling the androgenic control of GnRH neuron plasticity and reproductive plasticity across teleosts.

An ultra-sensitive electrochemical biosensor for circulating tumor DNA utilizing dual enzyme-assisted target recycle and hybridization chain reaction amplification strategies

Circulating tumor DNA (ctDNA) has emerged as a pivotal biomarker for cancers, offering a non-invasive means of detection through liquid biopsies. However, the current methodologies for ctDNA detection often struggle with sensitivity, especially when confronted with ultra-low concentrations. To address this challenge, we have engineered a remarkably sensitive electrochemical biosensor that can detect low concentration of ctDNA. This breakthrough is achieved by integrating a dual enzyme-assisted amplification mechanism with hybridization chain reaction (HCR). In the initial phase, the presence of ctDNA triggers the unfolding of the H2 hairpin structure, resulting in a segment of double-stranded DNA. The subsequent introduction of Klenow (3′→5′ exo-) and nicking endonuclease (Nb.BbvCI) enzymes leads to the abundant production of extended DNA (EXTDNA). This step critically enhances the initial amplification of the target DNA, ensuring superior specificity and selectivity thanks to the synergistic enzymatic activity. EXTDNA efficiently unfolds the H1 hairpin structure immobilized on the gold electrode, which acts as the initiation point for the HCR. Further amplification of the trace target DNA is achieved through an additional H3 and H4 hybrid-mediated HCR. Finally, the differential pulse voltammetry signal of methylene blue is increased significantly. Our results reveal that the developed electrochemical biosensor displays an exceptional linear correlation with ctDNA across concentrations ranging from 10 fM to 20 pM, with an unprecedented detection limit of 2.3 fM (3 s/k). This innovative approach shows immense potential for the ultrasensitive detection of ctDNA, promising broad applications in early cancer detection and monitoring.

A portability self-powered sensor facilitates sensitive Cd2+ detection: Dual mechanism and three quantitative mode

As a common heavy metal contaminant, Cd2+ has adverse effects on food safety and consumer health. It is very important for human health to realize highly sensitive Cd2+ detection methods. The self-powered sensing system based on enzyme biofuel cells (EBFCs) does not need an external power supply, which can simplify the experimental equipment and has great application value in portable detection. Thus, the biosensor is innovatively integrated into the screen-printed electrode to construct a new type of portable sensor suitable for on-site and real-time Cd2+ detection. Hybridization chain reaction (HCR) combined with the Cd2+-dependent deoxyribose (DNAzyme) signal amplification strategy is used to enhance the detection sensitivity while specifically recognizing the Cd2+. Moreover, the self-powered sensor combines with smartphones to realize quantitative Cd2+ detection without other instruments and has the characteristic of Effectively improving the hazard detection technology is essential to ensure food safety. Portability, simplicity, and speed are suitable for real-time Cd2+ detection in the field. The dual mechanism and three quantitative modes combining colorimetric and two electrical signals output modes are adopted to realize the visualization and accurate detection. A series of research results confirm that this strategy is of great significance to strengthen the development of intelligent Cd2+ technology, expand the application of self-powered sensing technology, and improve the safety detection system.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip

BackgroundCircular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. ResultsHerein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SignificanceThis represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions.

Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.

Label-free and ultrasensitive detection of environmental lead ions based on spatially localized DNA nanomachines driven by hyperbranched hybridization chain reaction

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

HCR-Assisted RTF-EXPAR-Based Lateral Flow Analysis for Sensitive Detection of H1N1 Influenza Virus.

The H1N1 influenza virus is a significant pathogen responsible for seasonal influenza, and its frequent outbreaks pose substantial challenges to global public health. The present study successfully developed a lateral flow analysis platform that integrates reverse transcription-free exponential amplification reaction (RTF-EXPAR) and hybridization chain reaction (HCR) processes with functionalized quantum dots for the direct detection of H1N1 influenza virus RNA, eliminating the need for reverse transcription. The fluorescence signal on the band recorded with a smartphone can be utilized for the quantitative determination of the target. Interestingly, the dual signal amplification strategy exhibits high sensitivity with a remarkably low detection limit of 10 aM. Moreover, this platform exhibits excellent flexibility and universality, where the various pathogens can be determined by replacing the specific nucleic acid fragments in RTF-EXPAR. The aforementioned advantages reveal its huge potential in the early diagnosis of H1N1 influenza virus infection and developing point-of-care testing (POCT) equipment for nucleic acid analysis.

Catalytic hairpin-assembled and DNAzyme-driven DNA walker integrated hybridization chain reaction to construct a label-free electrochemical aptasensor for the detection of acetamiprid

As a new type of nicotine pesticide, the excessive residue of acetamiprid (ACE) in food and the environment seriously poses human health. Hence, this article developed an electrochemical aptasensor for detecting ACE based on a cascaded amplification circuit composed of catalytic hairpin-assembled and DNAzyme-driven DNA walker and hybridization chain reaction. Through the target triggered catalytic hairpin assembly (CHA), the free travelling strand of DNA walker and intact catalytic core of DNAzyme were formed, and with the assistance of Mg2+, DNAzyme was activated, driving the travelling strand to walk autonomously, repeatedly cutting hairpin DNA substrates on the electrode surface, and obtaining abundant DNA fragments. These fragments contained the initiators of hybridization chain reaction (HCR) could trigger HCR and produce the long double-strand DNA (dsDNA) for loading methylene blue (MB). Owing to the fact that Ce(Ⅲ, Ⅳ)-MOF material with electrocatalytic activity, the electrochemical reduction of MB could be effectively catalyzed, obtain significantly enhanced current signals, and ultimately achieve label-free and sensitive detection of ACE, with a detection limit of 16.38 fM (S/N = 3). Moreover, the aptasensor demonstrated excellent selectivity, stability, and reproducibility, and exhibited good reliability for detecting ACE in the vegetable soil and tomato samples, providing a new idea for the detection of pesticide residues in the fields of food and environment.

Aptasensors and Advancement in Molecular Recognition Technology

AbstractSynthetic oligonucleic acids known as aptamers exhibit remarkable selectivity and affinity for target recognition and binding. Selected via an iterative process known as “selective evolution of ligands by exponential enrichment” (SELEX), aptamers fold into defined 3D conformations to interact with their targets. The incorporation of aptamers as recognition elements has driven notable progress in biosensors, giving rise to the development of aptasensors. Here, the process of aptamer discovery and the development of various types of aptasensors are summarized. The fundamental design principles of aptasensors are elaborated along with the superiority of aptamers compared to antibodies. The various modes employed by aptasensors, such as structure‐switching design, hybridization chain reaction amplification, enzyme‐assisted recycling, and split aptamer design are examined. Further light is shed on the diverse landscape of aptasensors, their adaptability to different analytes aptasensors as well as their potential to propel advancements in modern biosensor technology. As a nucleic acids‐based biosensor platform, aptasensors poise to become a next generation of sensitive and cost‐effective technology to shape the future of molecular recognition in biosensing.

Topographic Organization of Glutamatergic and GABAergic Parvalbumin-Positive Neurons in the Lateral Habenula.

Parvalbumin-expressing (PV) neurons, classified by their expression of the calcium-binding protein parvalbumin, play crucial roles in the function and plasticity of the lateral habenular nucleus (LHb). This study aimed to deepen our understanding of the LHb by collecting information about the heterogeneity of LHb PV neurons in mice. To achieve this, we investigated the proportions of the transmitter machinery in LHb PV neurons, including GABAergic, glutamatergic, serotonergic, cholinergic, and dopaminergic neurotransmitter markers, using transcriptome analysis, mRNA in situ hybridization chain reaction, and immunohistochemistry. LHb PV neurons comprise three subsets: glutamatergic, GABAergic, and double-positive for glutamatergic and GABAergic machinery. By comparing the percentages of the subsets, we found that the LHb was topographically organized anteroposteriorly; the GABAergic and glutamatergic PV neurons were preferentially distributed in the anterior and posterior LHb, respectively, uncovering the anteroposterior topography of the LHb. In addition, we confirmed the mediolateral topography of lateral GABAergic PV neurons. These findings suggest that PV neurons play distinct roles in different parts of the LHb along the anteroposterior and mediolateral axes, facilitating the topographic function of the LHb. It would be interesting to determine whether their topography is differentially involved in various cognitive and motivational processes associated with the LHb, particularly the involvement of posterior glutamatergic PV neurons.

Highly conductive AuNPs/Co-MOF nanocomposites synergistic hybridization chain reaction enzyme-free electrochemical aptasensor for ultrasensitive detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is the most poisonous and carcinogenic pollutant among numerous mycotoxins, posing a serious risk to the health of humans. Herein, an electrochemical aptasensor was constructed for AFB1 detection using gold nanoparticles/cobalt-based metal–organic skeleton (AuNPs/Co-MOF) as a modifying material combined with an enzyme-free signal amplification strategy. AuNPs/Co-MOF have an extensive specific surface area and outstanding electrical conductivity, which allows for the loading of a large number of DNA strands and enhancement of the thionine (THi) redox signal, leading to a significant increase in signal response THi loaded bimetallic core–shell nanocomposites (Au@PtNPs) were synthesized as signal labels. Au@PtNPs have high catalytic activity towards THi while improving the efficiency of the loaded THi, which enables the electrochemical signal of THi to be amplified. In addition, the hybridization chain reaction (HCR) signal amplification strategies for enzyme-free amplification elicits a strong response in the electrochemical signal while ensuring sensor stability. The aptasensor has a detection range of 0.001–500 ng/mL and achieves the limit of detection (LOD) was 1.2 × 10-2 pg/mL and limit of quantification (LOQ) was 4.0 × 10-2 pg/mL under optimal experimental conditions. Therefore, applications for electrochemical aptasensors in the realm of food safety monitoring are quite promising.

LncRNA lnc-SPRR2G-2 contributes to keratinocyte hyperproliferation and inflammation in psoriasis by activating the STAT3 pathway and downregulating KHSRP

Psoriasis is a chronic inflammatory disease characterized by increased keratinocyte proliferation and local inflammation. Long noncoding RNAs (lncRNAs) play important regulatory roles in many immune-mediated diseases, including psoriasis. In this study, we aimed to investigate the role and mechanism of lnc-SPRR2G-2 (SPRR2G) in M5-treated psoriatic keratinocytes.Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction (qRT-PCR) showed that lnc-SPRR2G-2 was significantly upregulated in psoriasis tissues and psoriatic keratinocytes. In psoriatic keratinocytes, functional and molecular experiment analyses demonstrated that SPRR2G regulated proliferation, cell cycle and apoptosis, and induced the expression of S100 calcium binding protein A7 (S100A7), interleukin (IL)-1β, IL-8 and C-X-C motif chemokine ligand 10 (CXCL10). The function of SPRR2G in psoriasis is related to the STAT3 signaling pathway and can be inhibited by a STAT3 inhibitor. Moreover, KH-type splicing regulatory protein (KHSRP) was proved to be regulated by lnc-SPRR2G-2 and to control the mRNA decay of psoriasis-related cytokines (p < 0.05). In summary, we reported the functions of lnc-SPRR2G-2 and KHSRP in psoriasis. Our findings provide new insights for the further exploration of the pathogenesis and treatment of psoriasis.

DNA Nanocage-Assisted Size-Selective Recognition and Quantification toward Low-Mass Soluble β-Amyloid Oligomers.

Low-mass soluble β-amyloid peptide oligomers (LSAβOs) play a crucial role in the pathogenesis of Alzheimer's disease. However, these oligomers exhibit heterogeneity in terms of structure, stability, and stoichiometry, and their abundance in biofluids is low, making accurate identification challenging. In this study, we developed a DNA nanocage-assisted method for selective sizing and sensitive quantification of LSAβOs in serum. Using LSAβO less than 10 kDa (LSAβO10kD) and less than 30 kDa (LSAβO30kD) as models, the size-matching rules between DNA nanocages and LSAβOs were investigated, and two appropriate nanocages were selected for the detection of two LSAβOs, respectively. Both nanocages were functionalized by encapsulating oligomer's aptamer and a complementary sequence within their cavities. Once the LSAβO entered the corresponding nanocage cavity, the complementary sequence was released, triggering a hybridization chain reaction on an electrochemical sensing platform. The system achieved size-selective discrimination of LSAβO10kD with a linear range of 10-150 pM and LSAβO30kD with a linear range of 15-150 pM. Real sample testing confirmed the applicability of the method for blood-based diagnosis. The DNA nanocage-assisted electrochemical analysis platform provides an accurate, highly selective, and sensitive approach for oligomer analysis, which is significant for amyloid protein research and related disease diagnosis.

Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. We describedthe clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.

Au-Pt Coating Improved Catalytic Stability of Au@AuPt Nanoparticles for Pressure-Based Point-of-Care Detection of Escherichia coli O157:H7.

Point-of-care testing (POCT) technologies facilitate onsite detection of pathogens in minutes to hours. Among various POCT approaches, pressure-based sensors that utilize gas-generating reactions, particularly those catalyzed by nanozymes (e.g., platinum nanoparticles, PtNPs, or platinum-coated gold nanoparticles, and Au@PtNPs) have been shown to provide rapid and sensitive detection capabilities. The current study introduces Au-Pt alloy-coated gold nanoparticles (Au@AuPtNPs), an innovative nanozyme with enhanced catalytic activity and relatively high stability. For pathogen detection, Au@AuPtNPs are modified with H1 or H2 hairpin DNAs that can be triggered to undergo a hybridization chain reaction (HCR) that leads to their aggregation upon recognition by an initiator strand (Ini) with H1-/H2-complementary aptamers tethered to magnetic beads (MBs). Pathogen binding to the aptamer exposes Ini, which then binds Au@AuPtNPs and initiates a HCR, resulting in Au@AuPtNP aggregation on MBs. These Au@AuPtNP aggregates exhibit strong catalysis of O2 from the H2O2 substrate, which is measured by a pressure meter, enabling detection of Escherichia coli (E. coli) O157:H7 at concentrations as low as 3 CFU/mL with high specificity. Additionally, E. coli O157:H7 could also be detected in simulated water and tea samples. This method eliminates the need for costly, labor- and training-intensive instruments, supporting its further testing and validation for deployment as a rapid-response POCT application in the detection of bacterial contaminants.

Label-free, background-free detection of nucleic acid with immobilization-free heterogeneous biosensor and one-pot hybridization chain reaction amplification

Although immobilization-free and label-free electrochemical DNA (E-DNA) biosensors have engaged tremendous interest due to their superior properties, such as easy operation, time-saving and cost-saving, most of them are fabricated in homogeneous modes and usually produce high background current. In the present work, we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction (HCR) for one-pot nucleic acid detection with zero background. The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins, resulting in the generation of HCR products with multibranched arms, which can be captured onto the electrode via π-π stacking interactions between multibranched arms and reduced graphene oxide (rGO). Prior to the incubation process with an electrode, two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes. Thus, an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized. This strategy also presents additional merits of simplicity and cheap cost, since probe immobilization, signal tag labeling, and multiple incubation processes are avoided. Therefore, the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.