Rapid and sensitive detection of large yellow croaker iridovirus by real-time RPA and RPA-LFD.

Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/µL, while the detection limit of RPA-LFD was 101 copies/µL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.

A universal fluorescence biosensor based on rolling circle amplification and locking probe for DNA detection.

A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.

Turn-on Coumarin Precursor: From Hydrazine Sensor to Covalent Inhibition and Fluorescence Detection of Rabbit Muscle Aldolase.

Hydrazine, a highly toxic compound, demands sensitive and selective detection methods. Building upon our previous studies with pre-coumarin OFF-ON sensors for fluoride anions, we extended our strategy to hydrazine sensing by adapting phenol protecting groups (propionate, levulinate, and γ-bromobutanoate) to our pre-coumarin scaffold. These probes reacted with hydrazine, yielding a fluorescent signal with low micromolar limits of detection. Mechanistic studies revealed that hydrazine deprotection may be outperformed by a retro-Knoevenagel reaction, where hydrazine acts as a nucleophile and a base yielding a fluorescent diimide compound (6,6'-((1E,1'E)-hydrazine-1,2diylidenebis(methaneylylidene))bis(3(diethylamino)phenol, 7). Additionally, our pre-coumarins unexpectedly reacted with primary amines, generating a fluorescent signal corresponding to phenol deprotection followed by cyclization and coumarin formation. The potential of compound 3 as a theranostic Turn-On coumarin precursor was also explored. We propose that its reaction with ALDOA produced a γ-lactam, blocking the catalytic nucleophilic amine in the enzyme's binding site. The cleavage of the ester group in compound 3 induced the formation of fluorescent coumarin 4. This fluorescent signal was proportional to ALDOA concentration, demonstrating the potential of compound 3 for future theranostic studies in vivo.

Modified glove removal technique to prevent hand contamination in routine phlebotomy.

The World Health Organization and the Centers for Disease Control and Prevention (CDC) have established guidelines recommending the performance of hand hygiene routines for healthcare workers following glove removal. However, the completion of frequent hygiene routines can cause allergic and adverse skin reactions. This double-blind, randomized study aimed to address this concern by developing and evaluating a modified glove removal technique that minimizes contamination risk during routine phlebotomy procedures. Furthermore, this study used fluorescent detection to compare the frequency of contamination associated with the CDC-recommended technique and the modified technique using fluorescent detection. One hundred healthcare personnel were enrolled and divided into two groups: one group followed the CDC technique, while the other group implemented the modified technique. Participants received instructional videos and practiced under supervision. They subsequently performed blood collection using a simulation arm covered with fluorescent cream as a contamination marker. After removing gloves, hand contamination was assessed under a black light. The median time required for glove removal in the modified group was four seconds longer than that in the group that followed the CDC technique (p < 0.001). Contamination was observed in 2% (1/50) of subjects using the CDC-recommended technique, while no contamination was detected with the modified technique (p ≥ 0.05). Both the group that followed the CDC technique and the group that used modified glove removal techniques demonstrated the potential to prevent contamination during phlebotomy, thereby reducing the need for hand hygiene and the occurrence of contamination and adverse skin reactions. These findings prompt further exploration into whether proper glove removal can reduce the frequency of completing a hand hygiene routine after each glove removal, specifically within the context of phlebotomy. However, it is essential to note that hand hygiene following glove removal is still recommended to prevent contamination. Further research is warranted to validate these findings.

Nanomaterials for fluorescent detection of vitamin B2: A review.

Vitamin B2 plays vital roles in maintaining human health. It is of tremendous significance to construct sensitive sensors of VB2. In this review, we first briefly presented the sensing mechanisms of fluorescent nanomaterials for sensing VB2. Subsequently, the advances of nanomaterials for fluorescent determination of VB2 were highlighted. And sensing performance of traditional approaches and fluorescent nanosensors was further compared. In last section, the challenges and perspectives concerning the topic were discussed.

Nitrogen and Sulfur Co-doped Carbon Dots as a Turn-Off Fluorescence Probe for the Detection of Cerium and Iron.

Carbon dots has becoming one of the most promising fluorescence sensors to determine the trace level of heavy metals in environments because of their advantages in optical properties, response time, and convenient operation procedures. Herein, a novel nitrogen and sulfur co-doped carbon dots (NS-CDs) were prepared though microwave assisted approach using DL-malic acid and allyl thiourea for the first time. Due to the existence of nitrogen and sulfur, the as-prepared NS-CDs exhibited bright blue fluorescence at 430 nm upon 330 nm excitation, with a fluorescence quantum yield of 19.8%. The sensitivity study of NS-CDs against metal ions and organic molecules has approved that the fluorescence could be further quenched by Ce4+ and Fe3+ ions, with the same linear detection ranges varying from 10 to 90 µM. The limits of detection (LOD) were determined as low as 0.75 µM and 0.67 µM for Ce4+ and Fe3+ ions, respectively. The possible quenching mechanism is explained by inner filter effect and static quenching mechanism for Ce4+ ions, while the quenching effect caused by Fe3+ ions is attributed to the inner filter effect, static and dynamic quenching mechanisms. Additionally, the developed sensor was used for the detection of Ce4+ and Fe3+ ions in tap water with satisfactory recoveries. Finally, the designed NS-CDs sensor possesses good biocompatibility against MA104 cells, suggesting the sensor can be potentially applied to detect Ce4+ and Fe3+ ions in environment and biological systems.

Facile one-pot synthesis of fluorescent aminoclay and the applications for fluorescence sensing Cr2 O7 2.

Here, we developed a facile one-pot strategy for the fabrication of fluorescent aminoclay (F-AC) through in situ solvothermal treatment of 3-aminopropyltrimethoxysilane, MgCl2 , and sodium ascorbate at 180°C for 6 h. The obtained F-AC exhibited blue emission, good water solubility, and satisfactory photostability. It was observed that Cr2 O7 2- could selectively quench the fluorescence of F-AC through the inner filter effect and static quenching process. As a result, a novel fluorescent F-AC-based nanosensor was constructed with good linearity in the range 0.1-75 µM. The nanosensor was successfully applied in real water samples with satisfactory results. This work not only provides a novel nanosensor for Cr2 O7 2- , but also highlights the F-AC's promising applications in wider fields due to the versatility and simplicity of the preparation strategy.

Interfacial reactivity-modulated fluorescent metal-organic frameworks for sensitive detection of interferon-γ towards tuberculosis diagnosis.

A new aptamer-based method has been developed for interferon-γ (IFN-γ) detection by utilizing interface reactivity-modulated fluorescent metal-organic frameworks (MOFs). Specifically, the binding of IFN-γ to its aptamer decreases the interface reactivity between the biotin-labeled aptamer and the streptavidin-functionalized magnetic beads by generating significant steric effects. As a result, several biotin-labeled aptamers escape from the enrichment of magnetic beads and remain in the supernatant, which subsequently undergo the terminal deoxynucleotidyl transferase-catalyzed polymerization elongation. Along with the elongation, pyrophosphate is continuously produced as the by-product, triggering the decomposition of fluorescent MOFs to generate a remarkable fluorescent response with the excitation/emission wavelength of 610 nm/685 nm. Experimental results show that the method enables the detection of IFN-γ in the range 0.06 fM to 6 pM with a detection limit of 0.057 fM. The method also displays high specificity and repeatability with an average relative standard deviation of 2.04%. Moreover, the method demonstrates satisfactory recoveries from 96.3 to 105.5% in serum samples and excellent utility in clinical blood samples. Therefore, this work may provide a valuable tool for IFN-γ detection and is expected to be of high potential in tuberculosis diagnosis in the future.

Sensitive Detection of Various Forms of Hydrogen Sulfide via Highly Selective Naphthalimide-Based Fluorescent Probe.

Hydrogen sulfide (H2S) is an important gasotransmitter, but only a few methods are available for real-time detection. Fluorescent probes are attractive tools for biological applications because of their high sensitivity, convenience, rapid implementation, noninvasive monitoring capability, and simplicity in fluorescent imaging of living cells and tissues. Herein, we report on a pro-fluorescent probe, NAP-Py-N3 based on naphthalimide derivative, which was found to show high selectivity toward H2S over various other analytes, including biothiols, making it feasible to detect H2S. After reaction with H2S, this probe showed rapid and significant turn-on green fluorescent enhancement at 553 nm (about 54-fold, k2 = 9.62 M-1s-1), high sensitivity (LOD: 15.5 nM), significant Stokes shift (118 nm), and it was found that the fluorescence quantum yield of fluorescence product can reach 0.36. Moreover, the probe has also been successfully applied to detect the gaseous H2S and to confirm the presence of H2S released from modern organic donors, which in recent years have been commonly used to investigate the role of H2S in biological systems. All the results indicate that this probe is excellent and highly valuable.

Ascorbic Acid-Caused Quenching Effect of Protein Clusteroluminescence Probe: The Fast Fluorescent Detection of Ascorbic Acid in Vegetables.

It is interesting and meaningful to explore fluorescent probes for novel rapid detection methods. In this study, we discovered a natural fluorescence probe, bovine serum albumin (BSA), for the assay of ascorbic acid (AA). Due to clusterization-triggered emission (CTE), BSA has the character of clusteroluminescence. AA shows an obvious fluorescence quenching effect on BSA, and the quenching effect increases with increasing concentrations of AA. After optimization, a method for the rapid detection of AA is established by the AA-caused fluorescence quenching effect. The fluorescence quenching effect reaches saturation after 5 min of incubation time and the fluorescence is stable within more than one hour, suggesting a rapid and stable fluorescence response. Moreover, the proposed assay method shows good selectivity and a wide linear range. To further study the mechanisms of AA-caused fluorescence quenching effect, some thermodynamic parameters are calculated. The main intermolecular force between BSA and AA is electrostatic, presumably leading to the inhibiting CTE process of BSA. This method also shows acceptable reliability for the real vegetable sample assay. In summary, this work will not only provide an assay strategy for AA, but also open an avenue for the application expansion of CTE effect of natural biomacromolecules.

Carbon dots based ratiometric fluorescent sensing platform for food safety.

Food safety has become a major global concern and the rapid detection of food nutritional ingredients and contaminants has aroused much more attention. Nanomaterials-based fluorescent sensing holds great potential in designing highly sensitive and selective detection strategies for food safety analysis. Carbon dots (CDs) possess tremendous prospects in fluorescent sensing food ingredients and contaminants due to their superior properties of chemical and photostability, highly fluorescence with tunability, and no/low-toxicity. Numerous endeavors are demanded to contribute to overcoming the challenge of lower sensitivity and selectivity of the sensors interfered by various components in intricate food matrices to ensure food safety and human health. Nanohybrid CDs based ratiometric fluorescent sensing with self-calibration is regarded as an efficient strategy for the CDs based sensors for the specific recognition of target analyte in the food matrices. This work is devoted to reviewing the development of nanohybrid CDs based ratiometric fluorescent sensing platform and the perspectives of the platform for food safety. The applications of nanohybrid CDs in sensing are summarized and the sensing mechanisms are briefly discussed.

MicroRNA detection in biologically relevant media using a split aptamer platform.

MicroRNA (miRNA)-based intercellular communication has been implicated in many functional and dysfunctional biological processes. This has raised interest in the potential use of miRNAs as biomarkers for diagnosis and prognosis. Though the list of clinically significant miRNA biomarkers is expanding, it remains challenging to adapt current chemical tools to investigate miRNAs in complex environments native to cells and tissues. We describe here a methodology for rapidly developing aptamer-based fluorescent biosensors that can specifically detect miRNAs in biologically relevant media (10-30% v/v), including medium collected from cultured HeLa cells, human serum, and human plasma. This methodology involves the semi-rational design of the hybridization between DNA oligonucleotides and the miRNA target to build a pool of potential aptamers, and the screening of this pool for high signal-to-background ratio and target specificity. The DNA oligonucleotides are readily available and require no chemical modification, rendering these chemical tools highly adaptable to any novel and niche miRNA target. Following this approach, we developed sensors that detect distinct oncogenic miRNA targets (miR-19b, miR-21, and miR-92a) at concentrations as low as 5 nM without amplification and are selective against single-nucleotide mutants. This work provides a systematic approach toward the development of miRNA biosensors that are easily accessible and can perform in biological environments with minimal sample handling.

A smartphone integrated ratiometric fluorescent sensor for point-of-care testing of fluoride ions.

In view of the high toxicity and widespread availability, it is of great importance to develop accurate, sensitive, and convenient assays for fluoride ion (F-) detection. Herein, a ratiometric fluorescent system is established for point-of-care testing (POCT) of F- with a smartphone as analyzer. The sensing system of calcein-QDs-Eu3+ contains two fluorescent probes of calcein (green emission) and ZnCdSe/ZnS QDs (red emission). The sensing system only presents red emission in that the calcein emission is quenched due to the combination between calcein and Eu3+. When F- is introduced, the fluorescence of calcein is recovered due to the stronger interaction between F- and Eu3+, which changes the emission from red to green. The ratiometric strategy offers an obvious fluorescence color change of the system, which eliminates interference and improves the detection accuracy. Specifically, the sensing system has excellent selectivity in that Eu3+ is more inclined to bind with F- rather than other anions. The developed assay was further used to prepare a test paper and hydrogel for POCT. To further improve the detection sensitivity and realize quantitative analysis, a smartphone installed with a color scan app is integrated as signal reader and analyzer, which is used for POCT of F- in real samples, showing great application potential in environmental protection and food safety evaluation.

Highly dispersive AuNCs/ChOx@ZIF-8/PEI nanocomplexes for fluorescent detection of cholesterol in human serum.

Gold nanoclusters (AuNCs) are widely used in the fluorescence detection of biomolecules in human serum due to their good fluorescence properties, low toxicity, and better biocompatibility. However, the weak fluorescence intensity of AuNCs limits the fluorescence detection of molecules within a wide concentration range. It is reported that coating AuNCs in ZIF-8 with adjustable pore size can effectively improve the fluorescence intensity of AuNCs and broaden the detection range. And the AuNCs wrapped in the gaps of ZIF-8 can prevent the fluorescence quenching caused by the aggregation of AuNCs. However, ZIF-8 has high crystallinity, poor dispersion, and easy deposition, which reduces the fluorescence stability of the detection system and affects the detection. Based on the above research, the highly hydrophilic polymer PEI was modified to the surface of ZIF-8, and a kind of nanocomposite material AuNCs/ChOx@ZIF-8/PEI was obtained by co-encapsulating AuNCs prepared with glutathione as a ligand and cholesterol oxidase (ChOx) into ZIF-8 modified with PEI. The composite material emits strong red light at 650 nm under the excitation of 395-nm light, and the system can sensitively detect cholesterol (Chol) in human serum. Compared with other materials, the PEI-modified composite has better solubility and stability, so the detection effect of Chol is better. Encapsulation of ChOx in the ZIF-8 shell can protect the enzyme and increase the local concentration of ChOx, thereby speeding up the reaction rate. Compared with free AuNCs/ChOx, the quenching rate of AuNCs/ChOx@ZIF-8/PEI system is doubled. Secondly, the addition of Fe2+ to the detection process results in higher quenching rate and detection sensitivity. The system can detect Chol in the concentration range 0.1-2.4 µM, with a detection limit of 0.073 µM. The determination is a fast and sensitive strategy. In addition, the practicability of this assay in the detection of Chol in human serum has been verified. Due to its selectivity and sensitivity, it has potential application value in clinical diagnosis.

Temperature-Triggered Structural Dynamics of Non-Coordinating Guest Moieties in a Fluorescent Actinide Polyrotaxane Framework.

We present here the synthesis of a novel fluorescent actinide polyrotaxane compound URCP1 through the utilization of an end-cutting pseudorotaxane precursor with only the cucurbit[6]uril (CB[6]) macrocyclic components acting as linking struts. The non-coordinating guest motif in the obtained polyrotaxane, with increased freedom and structural flexibility, can display intriguing temperature-triggered conformational variations inside the cavity of CB[6], which was clearly evidenced by crystallographic snapshots at different temperatures. Notably, this observation of temperature-triggered structural dynamics in URCP1 represents the first report of actinide polyrotaxane with such feature in solid-state. Moreover, URCP1 has a high photoluminescence quantum yield (PLQY) of 49.8 %, comparable to other luminescent uranyl compounds, and can work as a fluorescent probe to selectively detect Fe3+ over other eight competing cations in aqueous solution, with the limit of detection being as low as 4.4×10-3  ppm.

Development of a rapid and sensitive quantum dot nanobead-based double-antigen sandwich lateral flow immunoassay and its clinical performance for the detection of SARS-CoV-2 total antibodies.

Owing to the over-increasing demands in resisting and managing the coronavirus disease 2019 (COVID-19) pandemic, development of rapid, highly sensitive, accurate, and versatile tools for monitoring total antibody concentrations at the population level has been evolved as an urgent challenge on measuring the fatality rate, tracking the changes in incidence and prevalence, comprehending medical sequelae after recovery, as well as characterizing seroprevalence and vaccine coverage. To this end, herein we prepared highly luminescent quantum dot nanobeads (QBs) by embedding numerous quantum dots into polymer matrix, and then applied it as a signal-amplification label in lateral flow immunoassay (LFIA). After covalently linkage with the expressed recombinant SARS-CoV-2 spike protein (RSSP), the synthesized QBs were used to determine the total antibody levels in sera by virtue of a double-antigen sandwich immunoassay. Under the developed condition, the QB-LFIA can allow the rapid detection of SARS-CoV-2 total antibodies within 15 min with about one order of magnitude improvement in analytical sensitivity compared to conventional gold nanoparticle-based LFIA. In addition, the developed QB-LFIA performed well in clinical study in dynamic monitoring of serum antibody levels in the whole course of SARS-CoV-2 infection. In conclusion, we successfully developed a promising fluorescent immunological sensing tool for characterizing the host immune response to SARS-CoV-2 infection and confirming the acquired immunity to COVID-19 by evaluating the SRAS-CoV-2 total antibody level in the crowd.

Detecting Mercury (II) and Thiocyanate Using "Turn-on" Fluorescence of Graphene Quantum Dots.

In this work, 1.8 nm graphene quantum dots (GQDs), exhibiting bright blue fluorescence, were prepared using a bottom-up synthesis from citric acid. The fluorescence of the GQDs could be almost completely quenched (about 96%) by adding Hg2+. Quenching was far less efficient with other similar heavy metals, Tl+, Pb2+ and Bi3+. Fluorescence could be near quantitatively restored through the introduction of thiocyanate. This "turn-on" fluorescence can thus be used to detect both or either environmental and physiological contaminants mercury and thiocyanate and could prove useful for the development of simple point-of-care diagnostics in the future. Graphical Abstract.

A facile synthesis of multifunctional carbon dots as fluorescence 'turn on' and 'turn off' probes for selective detection of Al3+ and 2,4,6-trinitrophenol.

Carbon dots (CDs) have drawn increasing interests due to their unique optical properties and promising application in various fields. In this study, citric acid (CA) and 5-chloromethyl-8-hydroxyquinoline (LQ) were used to synthesize nitrogen-doped CDs as novel fluorescent probes using a one-step solvothermal route. The as-prepared CDs had strong blue-white fluorescence emission when excited at 405 nm wavelength with a quantum yield (QY) of 25%, behaving with high ion concentration stability. Water-soluble CDs with a 8-hydroxyquinoline structure on their surface could be used to detect Al3+ using a 'turn on' mechanism and trinitrophenol (TNP) using a 'turn off' mechanism with detection limits of 229 nM and 44.4 nM, respectively. Al3+ enhances the fluorescence of CDs by forming a coordination complex to generate a fluorescence synergistic role and limit CD nonradiative transition. TNP quenched the fluorescence with high selectivity and sensitivity, which was attributed to the inner filter effect and static quenching. These results indicated that these CDs with their unique 'turn on' and 'turn off' nature have potential application in the environmental protection field and in prevention of terrorist threats.

A fluorescent aptasensor for Staphylococcus aureus based on strand displacement amplification and self-assembled DNA hexagonal structure.

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) is designed, which takes advantage of strand displacement amplification (SDA) technology and unique self-assembled DNA hexagonal structure. In the presence of S. aureus, a partially complementary strand of S. aureus aptamer (cDNA) is competitively released from cDNA/aptamer duplex immobilized on magnetic beads due to the affinity of the aptamer for S. aureus. The addition of primer starts the SDA reaction. With the catalysis of Bsm DNA polymerase and Nb.bpu10I endonuclease, a large number of single-stranded DNA (ssDNA) is produced, which induces the opening of a hairpin probe and the subsequent self-assembly to form a hexagonal structure. The staining of the DNA hexagon with SYBR Green I excites the fluorescence signal, which is used for detection. The aptasensor exhibits a broad linear range from 7 to 7 × 107 CFU/mL, with a detection limit of 1.7 CFU/mL for S. aureus. The sensor shows negligible responses to other bacteria. Moreover, the aptasensor has been applied to detect S. aureus in milk samples, and the results demonstrate the general applicability of the sensor and its prospect in systematic detection of S. aureus in food safety control and medicine-related fields. Graphical abstract The presence of S. aureus can be converted to the formation of unique DNA hexagonal structure and subsequent fluorescent signal by the combination of SDA with self-assembly technology.

Photoinducible Oncometabolite Detection.

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.