Fluorescence Signal Research Articles

A clinicopathological and molecular series of five TFEB-altered renal cell carcinoma (RCC) cases: highlighting an aggressive subset of TFEB-rearranged RCC concomitant with TFEB amplification/gene copy number gains.

The classification of TFEB-altered renal cell carcinoma (RCC) has been revised to include TFEB-rearranged RCC and TFEB-amplified RCC in the 2022 World Health Organization (WHO) Classification of Tumors of the Urinary System. Given the wide spectrum of TFEB-altered RCC in terms of morphology and clinical behavior, an accurate diagnosis is challenging yet crucial, particularly in aggressive cases. Moreover, the concurrence of TFEB gene rearrangement and amplification/gene copy number (GCN) gains was also observed, but there was limited knowledge of these cases. We presented three TFEB-rearranged RCC cases, one TFEB-amplified RCC case, and one case of concomitant TFEB-rearranged and -amplified RCC, comparing the similarities and differences among these three subgroups. Furthermore, we summarized the clinicopathological and molecular features of TFEB-rearranged RCC concomitant with TFEB amplification/GCN gains from the literature and the present study. TFEB-altered RCCs exhibit significant heterogeneity in morphology and clinical behavior while displaying similar immunohistochemical profiles, including positive staining for Melan-A, PAX8, and CD117, and negative staining for CK7. A typical biphasic "rosette-like" morphology has been observed in a proportion of TFEB-rearranged RCC concomitant with TFEB amplification/GCN gains, which has been noted in TFEB-rearranged RCC, but not in cases with only TFEB amplification. Notably, TFEB-rearranged RCCs concomitant with TFEB amplification/GCN gains tend to be aggressive, in contrast to the often indolent nature of TFEB-rearranged cases, irrespective of the extent of TFEB gene copy increase. Therefore, a TFEB FISH assay is essential for unclassified RCC cases that exhibit melanocytic marker expression, and fluorescent signals should be counted and interpreted acurrately.

Reversible addition-fragmentation chain transfer enhanced aggregation signal-on fluorescence detection of alkaline phosphatase.

The instability of the signal intensity of fluorescent biosensors and the false signals have been significant factors affecting the performance of biosensors. Herein, a novel signaling system is devised through the application of reversible addition-fragmentation chain transfer (RAFT) polymerization with monomers containing the tetraphenylethylene (TPE) groups. TPE exhibits an aggregation-induced emission (AIE) phenomenon in certain solvents, mainly due to the blockage of the rotation of its four benzene rings, which also exist in the aggregated state. With this property, a series of molecules are modified based on click chemistry for RAFT polymerization using Fe3O4 magnetic beads as the carriers, and stable aggregated luminescent TPE polymers are formed on the surface of magnetic beads to realize the transformation of fluorescence signal from "0" to "1". In addition, the fluorescence signal demonstrates a positive correlation with alkaline phosphatase (ALP) activity, which can be quantified by measuring the fluorescence intensity. The biosensor exhibits high sensitivity and good linearity in the range of 0.1-5 U/L, with a LOD of 0.079 U/L. Furthermore, the designed strategy demonstrated satisfactory performance in the quantitative determination of ALP activity in serum samples, indicating that the signaling system developed by combining RAFT polymerization and AIE molecules has an important application in the field of fluorescent biosensors.

Visualizing the human olfactory projection and ancillary structures in a 3D reconstruction

Visualizing in 3D the histological microanatomy of the human olfactory projection from the olfactory mucosa in the nasal cavity to the olfactory bulbs in the cranial cavity necessitates a workflow for handling a great many sections. Here, we assembled a 3D reconstruction of a 7.45 cm3en-bloc specimen extracted from an embalmed human cadaver. A series of 10 µm coronal sections was stained with quadruple fluorescence histology and scanned in four channels. A trained anatomist manually segmented six structures of interest in a subset of the sections to generate the ground truth. Six convolutional neural networks were then trained for automatic segmentation of these structures in 1234 sections. A high-performance computing solution was engineered to register the sections based on the fluorescence signal and segmented structures. The resulting 3D visualization offers several novel didactic opportunities of interactive exploration and virtual manipulation. By extrapolating manual counts of OSNs in a subset of sections to the calculated volume of the envelope of the entire olfactory epithelium, we computed a total of ~2.7 million OSNs in the specimen. Such empirically derived information helps assess the extent to which the organizational principles of the human olfactory projection may differ from those in mice.

Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques

Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques.

Locking-Fluorescence Signals Regulated CRISPR/Cas12a Biosensor Based on Metal-Organic Framework for Sensitive Detection of Salmonella typhimurium.

The efficient, sensitive, and rapid detection of Salmonella typhimurium (S. typhimurium) in food and food products is important to ensure food safety and health. This study developed a fluorescence biosensing assay that integrated recombinase-aided amplification (RAA) and CRISPR/Cas12a with a zeolitic imidazolate framework-8@fluorescein sodium (ZIF-8@FLS) nanocomposite for the sensitive detection of S. typhimurium. In this approach, using RAA as a preamplification module, CRISPR/Cas12a-AChE as a target recognition and dual-enzyme cascade amplification module, and the prepared ZIF-8@FLS with high porosity and rapid pH responsiveness as a fluorescence signal explosive amplification module, the RAA-CRISPR/Cas12a-ZIF-8@FLS biosensor was constructed. Under optimal conditions, it exhibited an excellent linear relationship for S. Typhimurium, with a sensitive detection limit as low as 1.3 × 102 CFU/mL and could complete sample detection within 2 h relying on the RAA and ZIF-8@FLS explosive fluorescence rapid response, demonstrating its significant advantages in specificity, sensitivity, and reliability in food-borne pathogens detection.

Tracking the uptake of labelled host-derived extracellular vesicles by the human fungal pathogen Aspergillus fumigatus

Abstract Extracellular vesicles (EVs) have gained attention as facilitators of intercellular as well as interkingdom communication during host-microbe interactions. Recently we showed that upon infection, host polymorphonuclear leukocytes produce antifungal EVs targeting the clinically important fungal pathogen Aspergillus fumigatus; however, the small size of EVs (< 1 µm) complicates their functional analysis. Here, we employed a more tractable, reporter-based system to label host alveolar epithelial cell-derived EVs and enabled their visualisation during in vitro A. fumigatus interaction. Fusion of EV marker proteins (CD63, CD9, and CD81) with a Nanoluciferase (NLuc) and a green fluorescent protein (GFP) facilitated their relative quantification by luminescence and visualization by a fluorescence signal. The use of an NLuc fused with a GFP is advantageous as it allows for quantification and visualisation of EVs simultaneously without additional external manipulation and to distinguish subpopulations of EVs. Using this system, visualisation and tracking of EVs was possible using confocal laser scanning microscopy and advanced imaging analysis. These experiments revealed the propensity of host cell-derived EVs to associate with the fungal cell wall and ultimately colocalize with the cell membrane of A. fumigatus hyphae in large numbers. In conclusion, we have created a series of tools to better define the complex interplay of host-derived EVs with microbial pathogens.

Multispatially Localized DNA Walker Coupling Covalent Organic Framework for Dual-Mode Detection of Nucleocapsid Protein Using the Walking-Recycling-Conversion Strategy.

DNA walkers have emerged as a powerful tool in bioanalysis; however, many existing approaches are still restricted by low reaction kinetics and inaccurate single-mode detection. Herein, a fluorescence (FL) and electrochemical (EC) dual-mode biosensor was proposed based on a multispatially localized DNA walker (m-DNA walker) coupling covalent organic framework (COF) using the walking-recycling-conversion strategy. Specifically, the functionalized COF not only served as a three-dimensional nanocarrier but also acted as an effective quencher of the walking tracks. In the presence of the target, the activated m-DNA walker moved fast along the numerous quenching tracks, leading to the cleavage of Cy3-H1 and the recovery of the FL signal. To further improve the detection sensitivity, the Cy3-H1 fragments' recycling process was implemented with the generation of a large amount of S1 and S2, which caused the assembly of DNA-Fe3+-polydopamine network amplifiers on the electrode. The rapid electrochemical conversion was introduced to convert DNA-Fe3+-polydopamine into electroactive Prussian Blue, providing a significant EC signal output. Using nucleocapsid protein (N-protein) as the model target, the designed biosensing platform produced a FL/EC dual-mode readout with the detection limits of 65.0 fg/mL for FL mode and 2.3 fg/mL for EC mode, which could eliminate the interference from different reactive pathways and improve the detection accuracy, holding potential application in early disease diagnosis and treatment.

Visual aptasensor based on PtNPs/g-C3N4 and DNase I for the field detection of acetamiprid.

A visual aptasensor based on the combination of graphitic carbon nitride loaded with platinum nanoparticles (PtNPs/g-C3N4) and deoxyribonuclease I (DNase I) was developed for detecting acetamiprid. The prepared PtNPs/g-C3N4 exhibited excellent peroxidase (POD)-like activity, demonstrating the capacity to oxidize the colourless o-phenylenediamine (OPD) to produce the coloured product diaminophenazine (DAP) in the presence of hydrogen peroxide (H2O2). The DNA aptamer of acetamiprid can adsorb onto the surface of PtNPs/g-C3N4, thereby inhibiting its POD activity. The binding of acetamiprid to its aptamer results in the aptamer desorbing from the surface of PtNPs/g-C3N4 and subsequent digestion by DNase I. Ascribed to the synergistic effect of these factors, the aptasensor can achieve the rapid on-site detection of acetamiprid based on the acetamiprid-induced the colour change of the solution just with the smartphone. Furthermore, due to the remarkable fluorescence signal of DAP at 570nm, the aptasensor under fluorescence mode enables highly sensitive and quantitative detection of acetamiprid with a linear range of 1 ng/mL to 4µg/mL and a detection limit as low as 0.31 ng/mL.Theaptasensor has successfully realized the detection of acetamiprid in river water and cucumber samples, offering a novel perspective for the rapid assessment of environmental pollution and food safety risks associated with acetamiprid residues.

Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales

The worldwide spread of carbapenemase-producing Enterobacterales (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (blaKPC and blaNDM) within 50 min, including 10 min for DNA extraction and 30–40 min for RCCS reaction (a 20 min RPA reaction with a 10–20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting blaKPC and blaNDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of Klebsiella pneumoniae and Escherichia coli; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.

Superconducting nanowire single-photon detector enhanced near-infrared II portable confocal microscopy for tissue imaging with indocyanine green.

In this Letter a novel, to our knowledge, approach for near-infrared (NIR) fluorescence portable confocal microscopy is introduced, aiming to enhance fluorescence imaging of biological samples in the NIR-II window. By integrating a superconducting nanowire single-photon detector (SNSPD) into a confocal microscopy, we have significantly leveraged the detection efficiency of the NIR-II fluorescence signal from indocyanine green (ICG), an FDA-approved dye known for its NIR-II fluorescence capabilities. The SNSPD, characterized by its extremely low dark count rate and optimized NIR system detection efficiency, enables the excitation of ICG with 1 mW and the capture of low-light fluorescence signals from deep regions (up to 512 µm). Consequently, our technique was able to produce high-resolution images of bio samples with a superior signal-to-noise ratio, making a substantial advancement in the field of fluorescence microscopy and offering a promising opportunity for future clinical study.

A powerful and versatile new fixation protocol for immunostaining and in situ hybridization that preserves delicate tissues

BackgroundUnderstanding how genes function to heal wounds and restore lost tissue is essential for studying regeneration. Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression patterns of genes in different biological systems. Yet, existing methods to permeabilize samples for WISH can damage or destroy fragile regenerating tissues, thereby preventing such experiments.ResultsHere, we describe a new protocol for in situ hybridization (ISH) and immunostaining in the highly regenerative planarian Schmidtea mediterranea. This new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both the assays and prevents degradation of the epidermis and regeneration blastema. The NAFA protocol achieves this without the use of proteinase K digestion which likely leads to better preservation of antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Furthermore, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Additionally, the tissue fixation protocol was easily adapted for regenerating killifish tail fin, which yielded better ISH signal with minimal background.ConclusionsThus, the NAFA protocol robustly preserves the delicate wounded tissues while also facilitating probe and antibody penetration into internal tissues. Furthermore, the fixation protocol is compatible for WISH on regenerating teleost fins suggesting that it will be a valuable technique for studying the processes of wounding response and regeneration in multiple species.

Unprecedented Photoinduced‐Electron‐Transfer Probe with a Turn‐ON Chemiluminescence Mode‐of‐Action

PeT‐based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence‐based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT‐based turn‐on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT‐based turn‐on mechanism. The probe consists of a phenoxy‐1,2‐dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole‐dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal‐to‐noise ratio (over 5000‐fold). The PeT‐dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc‐DBCO and Max‐DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT‐based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT‐quenching pathways.

Unprecedented Photoinduced-Electron-Transfer Probe with a Turn-ON Chemiluminescence Mode-of-Action.

PeT-based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence-based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT-based turn-on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT-based turn-on mechanism. The probe consists of a phenoxy-1,2-dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole-dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal-to-noise ratio (over 5000-fold). The PeT-dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc-DBCO and Max-DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT-based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT-quenching pathways.

A d10-Cd Cluster Containing Sandwich-Type Arsenotungstate Exhibiting Fluorescent Recognition of Carcinogenic Dye in Methanol.

A d10-Cd cluster containing sandwich-type arsenotungstate [C3H12N2]6[Cd4Cl2(B-α-AsW9O34)2] was synthesized and its structure characterized through elemental analyses, X-ray powder diffraction (XRPD), IR spectroscopy, X-ray photoelectron spectroscopy (XPS), and single-crystal X-ray diffraction. The X-ray analysis revealed that the molecular unit of the compound consists of a captivating tetra-Cd-substituted sandwich-type polyoxoanion, accompanied by six elegantly protonated 1,2-diaminopropane as counter ions. The further novelty of the tetranuclear cadmium cluster lies in its occupied chlorine atom sites. This makes it highly susceptible to coordinate reactions with nitrogen on polycyclic aromatic hydrocarbons, thereby exhibiting different fluorescent signals that facilitate the identification and detection of these carcinogenic substances in methanol.

Dual-mode, signal-amplified DNA biosensor for label-free, reliable assay of gestational diabetes mellitus-related miRNA (miR-135a)

miR-135a is highly expressed in patients with gestational diabetes mellitus, and its target genes are also involved in insulin signaling pathway, so it is one biomarker for gestational diabetes mellitus. Herein we designed a dual-mode DNA biosensor for reliable assay of miR-135a based on the fluorescence and colorimetric signals. Several experiments were carried out to demonstrate the assay feasibility and mechanism for this dual-mode DNA biosensor. With optimum parameters, this proposed dual-mode biosensor has been realized sensitive and quantitative assay of miR-135a. For the fluorescence and colorimetric signals, the working ranges are 0.56-61 and 8.3-74nM, while limits of detection are 0.18 and 3.7nM respectively. This dual-mode strategy allows two independent signals for miR-135a assay, so it can verify each other to show more accurate results with good fidelity. Furthermore, there is a good selectivity in the biosensor for target miR-135a over other nucleotide variants, as well as good anti-interference ability in complex samples. This dual-mode DNA biosensor provides a new approach for miR-135a assay and miRNA expression profiling in gestational diabetes mellitus.

Mechanoresponsive self-reinforcement composite hydrogels with triple-network structures

Composite hydrogels featuring multiple-network structures hold immense potential owing to their superior mechanical attributes and exceptional capacity for dissipating energy. Nonetheless, many multiple-network hydrogels lack mechanoresponsive self-reinforcement capabilities, rendering them susceptible to enduring structural fractures. Hence, it exists a critical need to engineer composite hydrogels with both multiple-network structures and mechanoresponsive self-reinforcement abilities. In this study, we devised a triple-network (TN) hydrogel employing poly (2-acrylamido-2-methylpropane sulfonic acid) sodium salt (PNaAMPS) as the first network, which can generate mechanical radicals upon fracture. While polyvinyl alcohol (PVA) and carboxymethyl cellulose (CMC)-Al3+ served as the secondary and tertiary networks, respectively, so that to optimize its mechanical properties effectively. Upon breakage, the fragmented PNaAMPS chains could act as radical initiators, catalyzing the polymerization of the N-isopropyl acrylamide (NIPAM) monomers within the TN hydrogels to form PNIPAM chains. Furthermore, through successive network disruptions and the infusion of NIPAM monomers, the mechanical strength of the triple-network gel could be significantly enhanced. Furthermore, we evaluated the extent of mechanoresponsive self-reinforcement using the fluorochrome 8-Anilino-1-naphthalene sulfonic acid (ANS) as a fluorescent probe. This probe enabled the quantification of the PNIPAM production visually, which provided valuable feedback of the mechanical strength self-reinforcement levels by the fluorescence signals. Our approach set the stage for the development of mechanoresponsive composite hydrogels with fluorescence feedback capabilities for self-reinforcement assessment.

Development of an assay to quantify tranexamic acid levels in plasma

Dysregulations during blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labelled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7% and 3.0%, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.

Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR/Cas12a technology

Fasciola hepatica is a foodborne zoonotic parasite causing significant economic losses and impacting human and livestock health in resource-limited regions. We developed a rapid, reliable, and sensitive detection method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a, allowing visualization with the naked eye or a fluorescence reader. Multiple visual methods were used to analyze the assay results. Fluorescence signals were collected using a fluorescence reader or observed under UV or blue light. Lateral flow strips (LFS) were used for visual detection. Among seven primer pairs and three CRISPR RNA (crRNA) screened, F1/R1 and crRNA3 were optimal. The Cas12a reaction buffer was optimized with 50 mM Tris-HCl and 80 mM NaCl, with an RPA reaction time of 20 min. The assay showed high specificity and excellent sensitivity for F. hepatica, detecting 0.122 copies/μL with fluorescence and 8.6 copies/μL with LFS. Testing of 143 sheep and 43 human fecal samples showed 98.39 % consistency with qPCR and nested PCR, with prevalence rates of 52.45 % and 18.6 % in sheep and humans, respectively. Our assay offers substantial potential for point-of-care testing in resource-limited areas, addressing the need for rapid and accurate diagnosis of F. hepatica.

Enzyme-Assisted Fluorescence Biosensor Based on Circular Single-Stranded DNA Without Group Modification for MicroRNA Detection

Fluorescent biosensor, which has the characteristics of high sensitivity, specificity, and low cost, can be directly detected in physiological fluids such as blood and serum. Therefore, the development of fluorescence sensor platforms for miRNA detection has a positive effect on the prevention and treatment of various diseases. In this paper, miR-34a was selected as a biological indicator of Alzheimer’s disease (AD). We designed a circular single-stranded DNA (CSSD) biosensor, which uses two unmodified single-stranded DNA (ssDNA) with complementary ends, DNAa and DNAb, to form CSSD by DNA sequence pairing to improve thermal stability and achieve signal amplification. At the same time, CSSD can react with miR-34a, and then the DNA of the DNA–RNA chain is hydrolyzed by duplex-specific nuclease (DSN enzyme). Finally, miR-34a is released to partake in the subsequent step, thus realizing cycle amplification. By evaluating the change in fluorescence signal under the optimized conditions, we discovered that this approach exhibits impressive sensitivity, with a detection threshold reaching as low as 0.36 nM. This surpasses the performance of numerous preceding miRNA detection biosensors. Furthermore, the system displays excellent detection capabilities even in intricate settings like serum, showcasing a strong ability to differentiate and choose effectively. In summary, this is a signal-off fluorescent biosensor, which realizes the purpose of double amplification of biosensor signal by using CSSD and enzyme assistance so that it can be used as a valuable tool for early diagnosis of diseases.

Microwave enhanced carbon dots synthesis from eggshell membrane: Versatile applications in heavy metal ion sensing, strain free detection of fingerprints, UV shielding, food packing and anti-counterfeiting

In this study, a one-step microwave irradiation process combined with a sodium hydroxide (NaOH) solution allowed for the quick and easy synthesis of carbon dots (CDs) from eggshell membrane (ESM) ashes. The resultant CDs demonstrated exceptional selectivity for Fe3+ and excellent fluorescence (FL) with a quantum yield (QY) of 15.3%. Utilizing synthetic CDs, a sensitive nanoprobe is utilized to identify free Fe3+ within the 0-350μM (R2 = 0.9976) range, with a 0.281μM limit of detection (LoD). Additionally, the CDs were shown to be useful for intracellular Fe3+ detection applications because of their low cytotoxicity and biocompatibility. Investigations using real samples at 420 and 500nm excitation wavelengths showed promising results in terms of real-world application. In order to transform molecular data into FL signal outputs, a multi-input logic gate is also constructed. In the UV-A (93%), UV-B (88%), UV-C (98%) and HEBL (79%) regions, blue colour-emitting CDs had the maximum UV blockage, but pure polyvinyl alcohol (PVA) absorbed less than 22–30% of the light in all UV regions. The integration of CDs into the polymer improved the thermal characteristics of the PVA film. Additionally, testing of in vitro cell viability demonstrated that the CDs, when embedded in the PVA, did not cause cytotoxicity to the Neuroblastoma cell line (SH-SY-5Y). As a paradigm for perishable commodities, the effects of CDs and PVA were assessed on the shelf life of tomatoes. When fresh tomatoes were coated with CDs@PVA and PVA, weight and moisture loss were effectively decreased, according to the results of the long-term monitoring study. Throughout almost 34 days at room temperature, it also dramatically reduced the growth of fungi and prevented spoiling, all the while maintaining the fruits original colour and appearance. The CDs FL under 365nm UV light excitation makes them well-suited for creating fluorescent inks to deter counterfeiting. Additionally, the study explored the CDs exceptional FL properties for use as a luminescent fingerprint powder, aiding in the detection of latent fingerprints (LFPs) on various surfaces. The findings from this study highlight the potential of cost-effective and safe CDs for a range of applications, including non-cytotoxic UV blocking, active packaging, Fe3+ sensing, fingerprint detection, anti-counterfeiting (AC) measures and flexible nanocomposite (NC) films.