The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Comparative analysis of fixation techniques for signal detection in avian embryos.

The choice of fixation method significantly impacts tissue morphology and visualization of gene expression and proteins after in situ hybridization chain reaction (HCR) or immunohistochemistry (IHC), respectively. In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation techniques prior to HCR and IHC on chicken embryos. Our findings underscore the importance of optimizing fixation methods for accurate visualization and subsequent interpretation of HCR and IHC results, with implications for probe and antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei and neural tubes compared to PFA fixation. Additionally, TCA fixation altered the subcellular fluorescence signal intensity of various proteins, including transcription factors, cytoskeletal proteins, and cadherins. Notably, TCA fixation revealed protein signals in tissues that may be inaccessible with PFA fixation. In contrast, TCA fixation proved ineffective for mRNA visualization. These results highlight the need for optimization of fixation protocols depending on the target and model system, emphasizing the importance of methodological considerations in biological analyses.

An ultrasensitive electrochemical biosensor with dual-amplification mode and enzyme-deposited silver for detection of miR-205-5p.

An electrochemical biosensor based on dual-amplified nucleic acid mode and biocatalytic silver deposition was constructed using catalytic hairpin assembly-hybrid chain reaction (CHA-HCR). The electrochemical detection of silver on the electrode by linear sweep voltammetry (LSV) can be utilized to quantitatively measure miR-205-5p since the amount of silver deposited on the electrode is proportional to the target nucleic acid. The current response values exhibit strong linearity with the logarithm of miR-205-5p concentrations ranging from 0.1 pM to 10 µM, and the detection limit is 28 fM. A consistent trend was found in the results of the qRT-PCR and electrochemical biosensor techniques, which were employed to determine the total RNA recovered from cells, respectively. Moreover, the constructed sensor was used to assess miR-205-5p on various cell counts, and the outcomes demonstrated the excellent analytical efficiency of the proposed strategy. The recoveries ranged from 97.85% to 115.3% with RSDs of 2.251% to 4.869% in human serum samples. Our electrochemical biosensor for miR-205-5p detection exhibits good specificity, high sensitivity, repeatability, and stability. It is a potentially useful sensing platform for tumor diagnosis and tumor type identification in clinical settings.

CRISPR-based dual-aptamer proximity ligation coupled hybridization chain reaction for precise detection of tumor extracellular vesicles and cancer diagnosis.

Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (AptEpCAM, AptHER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 103-107 particles/µL, with a limit of detection (LOD) of 3.3 × 102 particles/µL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine.

Controlled Primary Amine-Enriched SG-Bonded Papain Surface: Synthesis, Characterization, and Extraction of Protonated Dichromate.

The single-step synthesis of nitro-derivatized SG using dimethyldichlorosilane in an aprotic solvent dichloromethane at 300 K is efficient and straightforward. Reduction and diazotization effectively functionalize the material for enzyme coupling at the O-carbon of the enzyme's tyrosine. The high extraction efficiency of protonated dichromate ions with a breakthrough capacity of 480 µmol·g-1 is notable. Eco-friendly elution using distilled water achieves a significant enrichment factor of 23.2. Excellent reusability (up to 900 cycles) and stable sorption efficiency (ζ ≥ 0.9) highlight the material's potential for practical applications and future research.

SERS biosensors based on catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification strategies for ultrasensitive microRNA-21 detection.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developed for the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.

Venom gland organogenesis in the common house spider.

Venom is a remarkable innovation found across the animal kingdom, yet the evolutionary origins of venom systems in various groups, including spiders, remain enigmatic. Here, we investigated the organogenesis of the venom apparatus in the common house spider, Parasteatoda tepidariorum. The venom apparatus consists of a pair of secretory glands, each connected to an opening at the fang tip by a duct that runs through the chelicerae. We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series. These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip, progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion. The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells. Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium, forkhead and sum-1 in the surrounding muscle layer, while Distal-less was predominantly expressed at the gland extremities. Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders, offering key insights into their evolution and development.

Integration of RCA-Based DNA Nanoscaffold with Target Triggered RNA-Cleaving DNAzyme for Sensitive Detection of miRNA21.

Cascaded amplification showed promising potential for detection of trace target miRNAs in molecular diagnosis and prevention of many diseases. In this study, miRNA21 was chosen as the target, and rolling circle amplification (RCA)-based DNA nanoscaffold was integrated with target triggered RNA-cleaving DNAzyme for sensitive detection of miRNA21. That is, the H1 probe was bound with the long-chain product of RCA to self-assemble into DNA nanoscaffold. Target miRNA21 triggered the hybridization chain reaction (HCR) located on the nanoscaffold, and led to rapid proximity of DNAzyme fragments modified at both ends of the H2 probe, which realized the cyclic cleavage of self-quenching substrate probe efficiently, and the fluorescence signal was restored. The results demonstrated that the proposed assay was sensitive, 0.76 pM of miRNA21 can be detected. The proposed assay was specific; only one-base mismatched miRNA21 can be effectively recognized, other nucleic acid sequence and the serum matrix did not cause any interference. The proposed assay was accurate; recoveries from 82.1 to 115.0% can be obtained in the spiked fetal bovine serum (FBS). The flexible and programmable characteristics of DNA nanoscaffold and DNAzyme provide a confident and robust strategy for more sensitive nucleic acid detection, and can be developed to be a universal sensing platform for detecting other miRNAs just needing modification on the corresponding sequence of H1 probe in HCR.

An electrochemical aptasensor for zearalenone detection based on the Co3O4/MoS2/Au nanocomposites and hybrid chain reaction.

An electrochemical aptasensor was used for the fast and sensitive detection of zearalenone (ZEN) based on the combination of Co3O4/MoS2/Au nanocomposites and the hybrid chain reaction (HCR). The glassy carbon electrode was coated with Co3O4/MoS2/Au nanomaterials to immobilize the ZEN-cDNA that had been bound with ZEN-Apt by the principle of base complementary pairing. In the absence of ZEN, the HCR could not be triggered because the ZEN-cDNA could not be exposed. After ZEN was added to the surface of the electrode, a complex structure was produced on the modified electrode by the combination of ZEN and ZEN-Apt. Therefore, the ZEN-cDNA can raise the HCR to produce the long-strand dsDNA structure. Due to the formation of dsDNA, the methylene blue (MB) could be inserted into the superstructure of branched DNA and the peak currents of the MB redox signal dramatically increased. So the concentration of ZEN could be detected by the change of signal intensity. Under optimized conditions, the developed electrochemical biosensing strategy showed an outstanding linear detection range of 1.0×10-10 mol/L to 1.0×10-6 mol/L, a low detection limit (LOD) of 8.5×10-11 mol/L with desirable selectivity and stability. Therefore, the fabricated platform possessed a great application potential in fields of food safety, medical detection, and drug analysis.

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

Development of SPQC sensor based on the specific recognition of TAL-effectors for locus-specific detection of 6-methyladenine in DNA.

N6-methyladenosine (6mA) plays a pivotal role in diverse biological processes, including cancer, bacterial toxin secretion, and bacterial drug resistance. However, to date there has not been a selective, sensitive, and simple method for quantitative detection of 6mA at single base resolution. Herein, we present a series piezoelectric quartz crystal (SPQC) sensor based on the specific recognition of transcription-activator-like effectors (TALEs) for locus-specific detection of 6mA. Detection sensitivity is enhanced through the use of a hybridization chain reaction (HCR) in conjunction with silver staining. The limit of detection (LOD) of the sensor was 0.63 pM and can distinguish single base mismatches. We demonstrate the applicability of the sensor platform by quantitating 6mA DNA at a specific site in biological matrix. The SPQC sensor presented herein offers a promising platform for in-depth study of cancer, bacterial toxin secretion, and bacterial drug resistance.

Risk and protective factors in risk assessment: Predicting inpatient aggression in adult males detained in a forensic mental health setting.

Structured clinical risk assessments represent a preferred means of assessing levels of aggression risk at different times and in different individuals. Increasing attention has been given to capturing protective factors, with sound risk assessment critical to high-secure forensic mental health care. The aim was to assess the predictive value of the HCR-20v3 for aggression risk and the long-term care pilot version of the SAPROF (the SAPROF-LC-pilot) in a high-secure forensic mental health inpatient population and to determine the incremental value of protective over risk factors. Participants were adult males detained in a high secure forensic mental health service, with a primary diagnosis of schizophrenia and/or personality disorder. The focus was on examining hospital based aggression (self- and other-directed) at two time points; up to 6 months (T1) and between 7 and 12 months (T2). The HCR-20V3 and SAPROF-LC-pilot demonstrated good predictive validity but with variability across subscales and aggression types/periods. Historical factors of the HCR-20V3 and External factors of the SAPROF-LC-pilot failed to predict, aside from a medium effect at T1 for verbal aggression and self-harm, for Historical factors. There was evidence for protective factors adding to prediction over risk factors alone, with the integration of protective and risk factors into a risk judgement particularly helpful in improving prediction accuracy. Protective factors contributed to risk estimates and particularly if integrated with risk factors. Combining risk and protective factors has clear predictive advantages, ensuring that protective factors are not supplementary but important to the aggression assessment process.

The predictive and prognostic value of risk factors in patients receiving hybrid coronary revascularization with postoperative pulmonary complications.

Background: The mortality rate of coronary artery disease ranks first in developed countries, and coronary revascularization therapy is an important cornerstone of its treatment. The postoperative pulmonary complications (PPCs) in patients receiving one-stop hybrid coronary revascularization (HCR) aggravate the dysfunction of multiple organs such as the heart and lungs, therefore increasing mortality. However, the risk factors are still unclear. The objective of this study was to explore the risk factors of PPCs after HCR surgery. Methods: In this study, the perioperative data of 311 patients undergoing HCR surgery were reviewed. All patients were divided into two groups according to whether the PPCs occurred. The baseline information and surgery-related indicators in preoperative laboratory examination, intraoperative fluid management, and anesthesia management were compared between the two groups. Results: Advanced age [odds ratio (OR): 1.065, 95% confidence interval (CI): 1.030-1.101, P<0.001], high body mass index (BMI; OR: 1.113, 95% CI: 1.011-1.225, P=0.02), history of percutaneous coronary intervention (PCI) surgery (OR: 2.831, 95% CI: 1.388-5.775, P=0.004), one-lung volume ventilation (OR: 3.804, 95% CI: 1.923-7.526, P<0.001), inhalation of high concentration oxygen (OR: 3.666, 95% CI: 1.719-7.815, P=0.001), the application of positive end-expiratory pressure (PEEP; OR: 2.567, 95% CI: 1.338-4.926, P=0.005), and long one-lung ventilation time (OR: 1.015, 95% CI: 1.006-1.023, P=0.001) may be risk factors for postoperative PPCs in patients undergoing one-stop coronary revascularization surgery. Using the above seven factors to jointly predict the risk of PPCs in patients undergoing one-stop coronary revascularization surgery, the receiver operating characteristic (ROC) curve showed an area under the curve (AUC) =0.873, 95% CI: 0.835-0.911, sensitivity: 84.81%, and specificity: 75.82%; the predictive model was shown to be effective. Conclusions: Patients undergoing HCR surgery with advanced age, high BMI, a history of PCI surgery, one-lung volume ventilation, inhalation of high concentration oxygen, use of PEEP, and prolonged single lung ventilation are more prone to PPCs.

Precision miRNA profiling: Electrochemiluminescence powered by CRISPR-Cas13a and hybridization chain reaction.

The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research.

N vacancy and self-enhancement induced high anodic electrochemiluminescence of 3D g-C3N4 for sensitive staphylococcus aureus analysis.

Here, 3D g-C3N4 with dense N vacancy in its 3D porous interconnected open-framework was synthesized, and the co-reactive 3-(dibutylamino)propylamine (DBAPA) was further covalently coupled onto the surface, resulting in a strong self-enhanced anodic electrochemiluminescence (ECL). Through introduction of high-density N vacancy, for the obtained 3D g-C3N4-NV, the band gap was broadened and the electrical conductivity was enhanced, realizing an obvious ECL improvement. Moreover, after the covalent binding of co-reactive DBAPA, the obtained 3D g-C3N4-NV-DBAPA exhibited a more intensive self-enhanced ECL signal due to the higher co-reaction efficiency originated from shorter electron transfer distance and lower energy loss. Based on the high initial signal of the proposed 3D g-C3N4-NV-DBAPA, a sensitive ECL biosensor with signal "on-off" was fabricated in assistance with multiple horizontal ordered hybridization chain reaction (HO-HCR). Through orderly fixing the reacted DNA chains on the Y-shape DNA structure on the electrode could effectively decrease diffusion process and improve the reaction efficiency of HCR process, resulting in the formation of numerous long horizontal double-strand DNA that could immobilize abundant ferrocene-doxorubicin (Fc-Dox) with ECL quenching effect. Meanwhile, compared to the traditional vertical HCR, the HO-HCR could make the quench reagent closer to the ECL emitter on the electrode surface and obtain a more effective quenching effect to enhance the sensing sensitivity. As a result, the proposed ECL biosensor archived the sensitive measurement of staphylococcus aureus with a detection limit of 10.3 aM.

An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 101-2 × 109 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.

A fluorescence-electrochemical dual-mode aptasensor based on novel DNA-dependent PBNFs@PtPd for highly selective and sensitive detection of procymidone through hybridization chain reaction.

Herein, a study for the first application of a hybridization chain reaction, a 1,8-naphthalimides-DNA (NDs) intercalator, and DNA-dependent Prussian blue nanoflowers@PtPd materials (PBNFs@PtPd) in the development of a fluorescence-electrochemical (FL-EC) aptasensor. This construction establishes an efficient sensing platform for the detection of procymidone (PCM). In the context of the described experiment, dual-mode detection is achieved through the generation of FL signals by an aptamer labeled with a Cy5 moiety and the formation of DPV signals by the modification of a thionine-appended 1,8-naphthalimide (Thi-NDs). In the presence of PCM, specific recognition occurs, followed by the utilization of magnetic separation technology to release DNA1 (S1) and aptamer-Cy5 (Apt-Cy5), subsequently introducing them onto both fluorescence and EC platforms. The presence of S1 effectively activates hybridization chain reaction (HCR) for the electrode surface, thereby significantly increasing the binding sites for Thi-NDs and consequently greatly amplifying the response signal of differential pulse voltammetry (DPV). The developed FL-EC dual-mode sensing platform demonstrates high sensitivity in the detection of PCM, with the detection limits of 0.173 µg·ml-1 (within the detection range of 500 pg·ml-1 to 500 ng·ml-1) and 0.074 ng·ml-1 (within the detection range of 100 pg·ml-1 to 100 ng·ml-1), respectively. The designed dual-mode sensor exhibits notable characteristics, including high selectivity, reproducibility, synergy, and reliable monitoring/capability for PCM in real samples.

"Two in one": A novel DNA cascade amplification strategy for trace detection of dual targets.

According to the characteristics of DNA programming, the cascaded nucleic acid amplification technology with larger output can overcome the problem of insufficient sensitivity of single nucleic acid amplification technology, and it combines the advantages of two or even multiple nucleic acid amplification technologies at the same time. In this work, a novel cascade signal amplification strategy with strand displacement amplification (SDA) and cascade hybridization chain reaction (HCR) was proposed for trace detection of hAAG and VEGF165. HAAG-induced SDA produced a large amount of S2 to open H2 on Polystyrene (PS) nanospheres, thereby triggering cascade HCR to form DNA dendritic nanostructures with rich fluorescence (FL) signal probes (565 nm). It could realize the amplification of FL signals for the detection of hAAG. Moreover, many doxorubicin (Dox) were loaded into the GC bases of DNA dendritic nanostructures, and its FL signal was effectively shielded. VEGF165 specifically bound to its aptamer to form G-quadruplex structures, which released Dox to produce a high FL signal (590 nm) for detection of VEGF165. This work developed a unique multifunctional DNA dendritic nanostructure fluorescence probe, and cleverly designed a new "On-off" switch strategy for sensitive trace detection of cancer markers.

High-Throughput RNA-HCR-FISH Detection of Endogenous Pre-mRNA Splice Variants.

RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Recent advances have increased RNA-FISH sensitivity, signal detection efficiency, and throughput. However, detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA-FISH fluorescence signals and due to the limited number of RNA-FISH probes per target. HiFENS (high-throughput FISH detection of endogenous pre-mRNA splicing isoforms) is a method that enables visualization and relative quantification of mRNA splice variants at single-cell resolution in an automated high-throughput manner. HiFENS incorporates HCR (hybridization chain reaction) signal amplification strategies to enhance the fluorescence signal generated by low abundance transcripts or a small number of FISH probes targeting short stretches of RNA, such as single exons. The technique offers a significant advance in high-throughput FISH-based RNA detection and provides a powerful tool that can be used as a readout in functional genomics screens to discover and dissect cellular pathways regulating gene expression and alternative pre-mRNA splicing events.

Enzyme-free sensitive SERS biosensor for the detection of thalassemia-associated microRNA-210 using a cascade dual-signal amplification strategy.

BACKGROUND: ß-thalassemia is a blood disorder caused by autosomal mutations. Gene modulation therapy to activate the γ-globin gene to induce fetal hemoglobin (HbF) synthesis has become a new option for the treatment of ß-thalassemia. MicroRNA-210 (miR-210) contributes to studying the mechanism regulating γ-globin gene expression and is a potential biomarker for rapid ß-thalassemia screening. Traditional miRNA detection methods perform well but necessitate complex and time-consuming miRNA sample processing. Therefore, the development of a sensitive, accurate, and simple miRNA level monitoring method is essential. RESULTS: We have developed a non-enzymatic surface-enhanced Raman scattering (SERS) biosensor utilizing a signal cascade amplification of catalytic hairpin assembly reaction (CHA) and proximity hybridization-induced hybridization chain reaction (HCR). Au@Ag NPs were used as the SERS substrate, and methylene blue (MB)- modified DNA hairpins were used as the SERS tags. The SERS assay involved two stages: implementing the CHA-HCR cascade signal amplification strategy and conducting SERS measurements on the resulting product. The HCR was started by the products of target-triggered CHA, which formed lengthy nicked double-stranded DNA (dsDNA) on the Au@Ag NPs surface to which numerous SERS tags were attached, leading to a significant increase in the SERS signal intensity. High specificity and sensitivity for miR-210 detection was achieved by monitoring MB SERS intensity changes. The suggested SERS biosensor has a low detection limit of 5.13 fM and is capable of detecting miR-210 at concentration between 10 fM and 1.0 nM. SIGNIFICANCE: The biosensor can detect miR-210 levels in the erythrocytes of ß-thalassemia patients, enabling rapid screening for ß-thalassemia and suggesting a novel approach for investigating the regulation mechanism of miR-210 on γ-globin gene expression. In the meantime, this innovative technique has the potential to detect additional miRNAs and to become an important tool for the early diagnosis of diseases and for biomedical research.