FRAME: flap endonuclease 1-engineered PAM module for precise and sensitive modulation of CRISPR/Cas12a trans-cleavage activity.

CRISPR/Cas12a system, renowned for its precise recognition and efficient nucleic acid cleavage capabilities, has demonstrated remarkable performance in molecular diagnostics and biosensing. However, the reported Cas12a activity regulation methods often involved intricate CRISPR RNA (crRNA) structural adjustments or costly chemical modifications, which limited their applications. Here, we demonstrated a unique enzyme activity engineering strategy using flap endonuclease 1 (FEN1) to regulate the accessibility of the protospacer adjacent motif (PAM) module in the double-stranded DNA activator (FRAME). By identifying the three-base overlapping structure between the target inputs and substrate, FEN1 selectively cleaved and released the 5'-flap containing the 'TTTN' sequence, which triggered the secondary cleavage of FEN1 while forming a nicked PAM, ultimately achieving the sensitive switching of Cas12a's activity. The FRAME strategy exemplified the 'two birds with one stone' principle, as it not only precisely programmed Cas12a's activity but also simultaneously triggered isothermal cyclic amplification. Moreover, the FRAME strategy was applied to construct a sensing platform for detecting myeloperoxidase and miR-155, which demonstrated high sensitivity and specificity. Importantly, it proved its versatility in detecting multiple targets using a single crRNA without redesign. Collectively, the FRAME strategy opens up a novel avenue for modulating Cas12a's activity, promising immense potential in the realm of medical diagnostics.

A metal-organic framework and quantum dot-based ratiometric fluorescent probe for the detection of formaldehyde in food.

A green and sensitive ratio fluorescence strategy was proposed for the detection of formaldehyde (FA) in food based on a kind of metal-organic frameworks (MOFs), MIL-53(Fe)-NO2, and nitrogen-doped Ti3C2 MXene quantum dots (N-Ti3C2 MQDs) with a blue fluorescence at 450 nm. As a type of MOFs with oxidase-like activity, MIL-53(Fe)-NO2 can catalyze o-phenylenediamine (OPD) into yellow fluorescent product 2,3-diaminophenazine (DAP) with a fluorescent emission at 560 nm. DAP has the ability to suppress the blue light of N-Ti3C2 MQDs due to inner filter effect (IFE). Nevertheless, Schiff base reaction can occur between FA and OPD, inhibiting DAP production. This results in a weakening of the IFE which reverses the original fluorescence color and intensity of DAP and N-Ti3C2 MQDs. So, the ratio of fluorescence intensity detected at respective 450 nm and 560 nm was designed as the readout signal to detect FA in food. The linear range of FA detection was 1-200 µM, with a limit of detection of 0.49 µM. The method developed was successfully used to detect FA in food with satisfactory results. It indicates that MIL-53(Fe)-NO2, OPD, and N-Ti3C2 MQDs (MON) system constructed by integrating the mimics enzyme, enzyme substrate, and fluorescent quantum dots has potential application for FA detection in practical samples.

[Opening experiments: synthesis, purification, and characterization of polyethyleneimine-modified carbon dots].

Open experiments are an effective means of cultivating top-notch innovative talents. Based on student interests, research hotspots and our laboratory conditions, an experimental scheme was designed. In this experiment, polyethyleneimine modified carbon dots (PEI-CDs) were prepared via a one-step hydrothermal method using citric acid (CA) as the carbon source and PEI as the surface passivator. First, CA and PEI were completely dissolved in 0.1 mol/L HCl and transferred into an autoclave. The autoclave was heated to 130 ℃ for 2 h. PEI-CDs solution was obtained. After cooling to room temperature, the solution was concentrated to 2 mL by rotary evaporation. Finally, the PEI-CDs were precipitated, washed with ethanol, and dried under vacuum at 70 ℃ for 12 h. The obtained PEI-CDs were characterized by fluorescence spectrophotometry, absorption spectrophotometry, infrared spectrometry, and transmission electron microscopy. The results indicated that anhydrous-ethanol precipitation is a simple, rapid, economical, and green purification method. The as-prepared PEI-CDs had unique properties, such as good water solubility, high luminescence, uniform particle sizes, and good stability. Through this open experiment, students can not only master the operation of large-scale instruments but also enhance their interest in scientific research.

DNA Robots for CRISPR/Cas12a Activity Management and Universal Platforms for Biosensing.

The CRISPR/Cas12a system is a revolutionary genome editing technique that is widely employed in biosensing and molecular diagnostics. However, there are few reports on precisely managing the trans-cleavage activity of Cas12a by simple modification since the traditional methods to manage Cas12a often require difficult and rigorous regulation of core components. Hence, we developed a novel CRISPR/Cas12a regulatory mechanism, named DNA Robots for Enzyme Activity Management (DREAM), by introducing two simple DNA robots, apurinic/apyrimidinic site (AP site) or nick on target activator. First, we revealed the mechanism of how the DREAM strategy precisely regulated Cas12a through different binding affinities. Second, the DREAM strategy was found to improve the selectivity of Cas12a for identifying base mismatch. Third, a modular biosensor for base excision repair enzymes based on the DREAM strategy was developed by utilizing diversified generation ways of DNA robots, and a multi-signal output platform such as fluorescence, colorimetry, and visual lateral flow strip was constructed. Furthermore, we extended logic sensing circuits to overcome the barrier that Cas12a could not detect simultaneously in a single tube. Overall, the DREAM strategy not only provided new prospects for programmable Cas12a biosensing systems but also enabled portable, specific, and humanized detection with great potential for molecular diagnostics.

Self-Assembled DNA Nanospheres Driven by Carbon Dots for MicroRNAs Imaging in Tumor via Logic Circuit.

DNA nanostructures with diverse biological functions have made significant advancements in biomedical applications. However, a universal strategy for the efficient production of DNA nanostructures is still lacking. In this work, a facile and mild method is presented for self-assembling polyethylenimine-modified carbon dots (PEI-CDs) and DNA into nanospheres called CANs at room temperature. This makes CANs universally applicable to multiple biological applications involving various types of DNA. Due to the ultra-small size and strong cationic charge of PEI-CDs, CANs exhibit a dense structure with high loading capacity for encapsulated DNA while providing excellent stability by protecting DNA from enzymatic hydrolysis. Additionally, Mg2+ is incorporated into CANs to form Mg@CANs which enriches the performance of CANs and enables subsequent biological imaging applications by providing exogenous Mg2+. Especially, a DNAzyme logic gate system that contains AND and OR Mg@CANs is constructed and successfully delivered to tumor cells in vitro and in vivo. They can be specifically activated by endogenic human apurinic/apyrimidinic endonuclease 1 and recognize the expression levels of miRNA-21 and miRNA-155 at tumor sites by logic biocomputing. A versatile pattern for delivery of diverse DNA and flexible logic circuits for multiple miRNAs imaging are developed.

Fluorometric Assay of Tyrosinase and Atrazine Based on the Use of Carbon Dots and the Inhibition of Tyrosinase Activity.

Sensitive and convenient strategy of tyrosinase (TYR) and its inhibitor atrazine is in pressing demand for essential research as well as pragmatic application. In this work, an exquisite label-free fluorometric assay with high sensitivity, convenience and efficiency was described for detecting TYR and the herbicide atrazine on the basis of fluorescent nitrogen-doped carbon dots (CDs). The CDs were prepared via one-pot hydrothermal reaction starting from citric acid and diethylenetriamine. TYR catalyzed the oxidation of dopamine to dopaquinone derivative which could quench the fluorescence of CDs through a fluorescence resonance energy transfer (FRET) process. Thus, a sensitive and selective quantitative evaluation of TYR can be constructed on the basis of the relationship between the fluorescence of CDs and TYR activity. Atrazine, a typical inhibitor of TYR, inhibited the catalytic activity of TYR, leading to the reduced dopaquinone and the fluorescence was retained. The strategy covered a broad linear range of 0.1-150 U/mL and 4.0-80.0 nM for TYR and atrazine respectively with a low detection limit of 0.02 U/mL and 2.4 nM/mL. It is also demonstrated that the assay can be applied to detect TYR and atrazine in spiked complex real samples, which provides infinite potential in application of disease monitoring along with environmental analysis.

CRISPR/Cas12a-based fluorescence aptasensor integrated with two-dimensional cobalt oxyhydroxide nanosheets for IFN-γ detection.

Cytokine storm (CS) is a risky immune overreaction accompanied by significant elevations of pro-inflammatory cytokines including interferon-γ (IFN-γ), interleukin and tumor necrosis factor. Sensitive detection of cytokine is conducive to studying CS progress and diagnosing infectious diseases. In this study, we developed a tandem system combining aptamer, strand displacement amplification (SDA), CRISPR/Cas12a, and cobalt oxyhydroxide nanosheets (termed Apt-SCN tandem system) as a signal-amplified platform for IFN-γ detection. Owing to the stronger affinity, target IFN-γ bound specifically to the aptamer from aptamer-complementary DNA (Apt-cDNA) duplex. The cDNA released from the Apt-cDNA duplex initiated SDA, resulting in the generation of double-stranded DNA products that could activate the trans-cleavage activity of CRISPR/Cas12a. The activated CRISPR/Cas12a further cleaved FAM-labeled single-stranded DNA probe, preventing it from adhering to the cobalt oxyhydroxide nanosheets and recovering the fluorescence signal. Sensitive fluorometric analysis of IFN-γ was successfully performed with detection limit as low as 0.37 nM. Unlike traditional protein analysis methods, Apt-SCN tandem system incorporates multiple signal amplification techniques and may also be applicable for other cytokines assay. This study was the initial study to utilize SDA and CRISPR/Cas12a to detect IFN-γ, showing great potential for cytokines clinical assay and CS prevention.

A dual-emission ratiometric fluorescence strategy with enzyme-based inhibition for organophosphorus pesticides determination.

A fast, eco-friendly and accurate ratiometric fluorescent strategy is presented for the determination of organophosphorus pesticides (OPs) using intrinsic dual-emission silica nanoparticles modified with Rhodamine 6G (SiNPs-Rho6G). SiNPs-Rho6G had intrinsic dual-emission at 410 and 550 nm. The substrate acetylcholine was catalyzed by acetylcholinesterase (AChE) to produce thiocholine (TCh). TCh triggered the specific reaction of Ellman's reagent 5, 5-dithiobis (2-nitrobenzoic acid) to obtain 5-thio-2-nitrobenzoic acid, which caused the decrease in fluorescence intensity of SiNPs-Rho6G at 410 nm by the inner filter effect, while the fluorescence intensity of SiNPs-Rho6G at 550 nm was not significantly changed. OPs caused the recovery of the fluorescence at 410 nm by inhibiting the activity of AChE. Thus, the quantitative detection of OPs could be achieved through utilizing the catalytic characteristic of AChE. The linear curve from 0.010 to 0.250 µg mL-1 with a detection limit of 7 ng mL-1 was obtained for the determination of chlorpyrifos (Cpf). The ratiometric probe was used to detect the spiked Cpf in environmental and food samples with good recoveries. Therefore, combined with the dual emission characteristics of SiNPs-Rho6G and the specificity of the enzyme, the ratio fluorescence sensing platform has potential application prospects in OPs determinations.

Novel dual-emission sulfur quantum dot sensing platform for quantitative monitoring of pesticide 2,4-dichlorophenoxyacetic acid.

In this work, a novel environment-friendly dual-emission Rhodamine B modified sulfur quantum dots (RhB-SQDs) sensing platform was established to economically monitor organochlorine pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) through regulating the activity of alkaline phosphatase (ALP). This dual emission RhB-SQDs exhibited excellent fluorescence and high photostability with emission wavelengths of 455 nm and 580 nm. ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate to p-nitrophenol, which quenched RhB-SQDs fluorescence at 455 nm due to the internal filtration effect, but had no effect the fluorescence intensity of RhB-SQDs at 580 nm. When 2,4-D was present, the activity of ALP was specifically inhibited and enzymatic reaction was interrupted, leading to the reduction of p-nitrophenol production, so the fluorescence of RhB-SQDs at 455 nm was restored. It demonstrated a good linear relationship between the concentration of 2,4-D and F455/F580 in the range of 0.050-0.500 µg mL-1, with a detection limit of 17.3 ng mL-1. The dual-emission fluorescent probe was successfully realized in the identification of 2,4-D in natural water samples and vegetables with the advantages of exceptional accuracy, immunity to interference, and selectivity. The platform offers a fresh look at pesticide monitoring and has the potential to prevent pesticide-related health issues.

Spherical Hydrogel Sensor Based on PB@Fe-COF@Au Nanoparticles with Triplet Peroxidase-like Activity and Multiple Capture Sites for Effective Detection of Organophosphorus Pesticides.

The residues of organophosphorus pesticides (OPs) have drawn worldwide increasing attention because of their potential fatal effects on human health and ecological systems. It is of great significance to develop an efficient and portable method for in-field detection of OPs. Herein, a novel core-shell nanocomposite of prussian blue@Fe-covalent organic framework@Au (PB@Fe-COF@Au) was constructed. Fe2+ and Fe3+ in PB nanoparticle (PBNP) cores, Fe-porphyrin in COF shells, and AuNPs grown on shells all acted as peroxidase-like catalytic active sites, enabling PB@Fe-COF@Au to possess triplet peroxidase-like activity. A colorimetric, affordable, sensitive, and selective strategy was designed to detect OPs. Compared with previous reports, this sensor realized a wider linear range for chlorpyrifos of 10-800 ng mL-1 with a relatively lower detection limit of 0.61 ng mL-1, which was attributed to the overlapping triple catalytic sites of PB@Fe-COF@Au and triple response sites to OPs. The assay was successfully employed to detect chlorpyrifos in food and environmental samples. Moreover, to meet the demand of in-field detection for OPs, a spherical hydrogel method based on PB@Fe-COF@Au with visual, portable, and equipment-free features was fabricated. This work provides a new pathway to design and apply effective nanozymes for on-site monitoring of pesticides.

Programmable CRISPR-Cas12a and self-recruiting crRNA assisted dual biosensing platform for simultaneous detection of lung cancer biomarkers hOGG1 and FEN1.

Human 8-oxoguanine DNA glycosylase (hOGG1) and flap endonuclease 1 (FEN1) are recognized as potential biomarkers in lung cancer investigations. Developing analytical platforms of simultaneously targeting hOGG1 and FEN1 with high selectivity, sensitivity, especially programmability and universality is highly valuable for clinical research. Herein, we established a signal-amplified platform for simultaneously detecting hOGG1 and FEN1 on the basis of cleavage-induced ligation of DNA dumbbell probes, rolling circle transcription (RCT) and CRISPR-Cas12a. A hOGG1 cleavable site and FEN1 cleavable flap were dexterously designed at the 5' end of DNA flapped dumbbell probes (FDP) for hOGG1 and FEN1. After cleavage, the resulting nick sites with juxtaposition of 5' phosphate and 3' hydroxyl terminus could be linked to closed DNA dumbbell probes (CDP) by DNA ligase. The CDP served as a template for RCT, producing plentiful crRNA repeats to activate the trans-cleavage activity of CRISPR-Cas12a which could cleave fluorophores (TAMRA and FAM) and quenchers (BHQ2 and BHQ1) double-labeled ssDNA reporters. Then, hOGG1 and FEN1 could be detected by the recovered fluorescence signal, allowing for the highly sensitive calculated detection limits of 0.0013 and 0.0052 U/mL, respectively. Additionally, this method made it possible to evaluate the inhibitory effects, even to measure hOGG1 and FEN1 activities at the single-cell level. This novel target enzyme-initiated, circles-transcription without promoters, real-time generation, and self-assembly features of FDP-RCT-Cas12a system suppressed nonspecific background remarkably and relieved rigorous requirement of protospacer adjacent motif site. Hence, the universality of FDP-RCT-Cas12a system toward various disease-related non-nucleic acid targets which are tested without using aptamers was extremely improved.

Glucose and pH responsive fluorescence detection system based on simple synthesis of silicon-coated perovskite quantum dots.

Perovskite quantum dots (PQDs) are extremely unstable in ambient air due to their inherent structural instability, which limits the wide application of PQDs. In this work, silicon-coated CsPbBr3 PQDs (CsPbBr3@SiO2) was synthesized via a simple method. The SiO2 coating effectively isolated PQDs from water and oxygen in the environment, which were the main elements that destroyed the structure stability of PQDs. The synthesized CsPbBr3@SiO2 can be stored in water for more than 2 months and posessed wonderful dispersibility in aqueous solution. The fluorescence intensity remained unchanged within 7 days and only decreased by 11.9 % within 2 months. We found that CsPbBr3@SiO2 was extremely sensitive to environmental pH, and the fluorescence intensity decreased with the reduction of pH. In addition, an excellent linear relationship with pH value in the range of 1.0 âˆ¼ 5.0 was achieved. As we all known that glucose can be catalyzed by glucose oxidase to produce gluconic acid and hydrogen peroxide, in which a good deal of protons were produced and the pH was gradually lowered. Since CsPbBr3@SiO2 was stable to water and oxygen, and sensitive to ambient pH, we applied CsPbBr3@SiO2 to the detection of glucose. CsPbBr3@SiO2 showed fantastic selectivity and sensitivity to glucose, and the detection limit can even reach 18.5 µM. Furthermore, CsPbBr3@SiO2 was successfully applied to the detection of glucose in the human serum with satisfactory performance.

Thermal Stress Induces Metabolic Responses in Juvenile Qingtian Paddy Field Carp Cyprinus carpio var qingtianensis.

Extreme fluctuations in water temperature lead to significant economic losses for the aquaculture industry. Cyprinus carpio var qingtianensis (locally called Qingtian paddy field carp), is a local variety commonly found in Zhejiang province, China. Unlike traditional aquaculture environments, the water temperature range between day and night in the rice field environment is much larger, and the high temperature in summer may exceed the growth threshold of fish because there is no manual intervention; therefore, the study of how the Qingtian paddy field carp (PF carp) adapts to high-temperature conditions can shed light how the species adapt to the rice field environment. To investigate the molecular mechanisms of this fish under thermal stress, the liver metabolomics of Qiangtian paddy field carp (PF carp) were analyzed. In this study, metabolomics was used to examine the metabolic reaction of PF carp (102 days old, 104.69 ± 3.08 g in weight, 14.65 ± 0.46 cm in length) at water temperatures of 28 °C (control group, CG), 34 °C (experimental group (EG) 34), and 38 °C (EG38). The results show that 175 expression profile metabolites (DEMs), including 115 upregulated and 60 downregulated metabolites, were found in the CG vs. EG34. A total of 354 DEMs were inspected in CG vs. EG38, with 85 metabolites downregulated and 269 metabolites upregulated. According to the pathway enrichment study, various pathways were altered by thermal stress, including those of lipid, amino-acid, and carbohydrate metabolism. Our study presents a potential metabolic profile for PF carp under thermal stress. It also demonstrates how the host responds to thermal stress on a metabolic and molecular level.

Metal-organic frameworks-assisted nonenzymatic cascade amplification multiplexed strategy for sensing acute myocardial infarction related microRNAs.

Amplification strategies for multiple microRNAs (miRNAs) detection are pivotal for acute myocardial infarction (AMI). Herein, we rationally developed a metal-organic frameworks-assisted nonenzymatic cascade amplification strategy for simultaneous quantification of three AMI-related miRNAs (miR-21, miR-499 and miR-133a). The fluorescence of the elaborately designed DNA molecular beacons with the respective modification of FAM, TAMRA and Cy5 in the terminal was quenched by a metal-organic framework named Fe-MIL-88. When targets miRNA appeared, they hybridized with the corresponding DNA molecular beacons, and the catalyzed hairpin assembly (CHA) reaction would be triggered, producing "Y" shaped three-branched duplex nanostructure with the targets released, and initiating subsequent another cycle. The "Y" shaped nanostructures could not be adsorbed onto the surface of Fe-MIL-88 due to the weaker affinity between Fe-MIL-88 and "Y" shaped nanostructures. Therefore, the fluorescence of "Y" shaped nanostructures could not be quenched by Fe-MIL-88. In this way, three AMI-related miRNAs were simultaneously detected in the respective ranges of 0.05-30 nM, 0.08-30 nM and 0.1-20 nM with respective limits of detection down to 13, 25 and 40 pM. Furthermore, the method was successfully employed to determine three AMI-related miRNAs in human serum. The strategy offered great opportunity for ultrasensitive detecting multiple AMI-related miRNAs and substantially improving the accuracy of clinical early AMI diagnosis.

A ratiometric lanthanide-free fluorescent probe based on two-dimensional metal-organic frameworks and carbon dots for the determination of anthrax biomarker.

A lanthanide-free fluorescent probe has been constructed for the first time based on two-dimensional metal-organic frameworks (2D MOFs) and carbon dots (CDs) for ratiometric determination of dipicolinic acid (DPA), the biomarker of Bacillus anthracis. The fluorescence intensity at 659 nm increased due to the release of organic ligands TCPP resulting from the selective interaction between DPA and Zn2+ of 2D MOFs. CDs provided a reference signal at 445 nm which was almost unaffected, realizing self-calibration DPA sensing. F659/F445 versus the concentration of DPA shows good linear relationships in the range 0.01-0.2 µM and 0.2-10 µM under 390-nm excitation, with a detection limit of 7 nM. The ratiometric probe was prepared from 2D lanthanide-free MOFs so that the drawbacks of lanthanide-based probes were overcome. The proposed sensing system was successfully applied to the determination of DPA in spiked biological samples. These results suggest that a novel, simple, and selective strategy of determining DPA with 2D lanthanide-free MOFs is implemented. Graphical abstract Zn-TCPP nanosheets and a blue carbon dots (b-CDs) are synthesized to construct the ratiometric probe, which can exhibit fluorescence at 445and 659 nm with 390-nm excitation. Dipicolinic acid (DPA) can deprive the junction ions of Zn-TCPP nanosheets, triggering the collapse ofZn-TCPP nanosheets. The fluorescence at 659 nm is enhanced due to the release of TCPP, while the peak of b-CDs at 445 nm is almost not affected. Thus, the fluorescence intensity ratio (F659/F445) can serve as the response signal for sensitive DPA sensing.

A novel profuse color card for convenient visual determination of iodide in human urine based on catalytic oxidation reaction.

In this work, we reported a novel and convenient profuse color card for naked eye determination of iodide (I-) in urine using chromogenic substrate 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). I- catalyzed the oxidation of ABTS by peroxyacetic acid causing ABTS to yield cyan product ABTS+ with a new absorption peak at 730 nm. The addition of rose-red dye rhodamine B (RhB) changes the overall color of the solution from pink to purple and finally to blue, which makes the solution multicolor and easy to distinguish. A good linear relationship for I- was obtained ranging from 10.0 to 500.0 µg/L with the detection limit of 9.2 µg/L. Importantly, the sensor can semi-quantitatively estimate the concentration of I- in human urine with naked eye through the standard color card and assess the deficiency or excess of iodine in human body. The proposed profuse color card opens up a new colorimetric method for the rapid, simple and reliable determination of I- in clinic, and has promising applications in developing assay kit for the clinical diagnosis of I- in urine.

Swietenine extracted from Swietenia relieves myocardial hypertrophy induced by isoprenaline in mice.

As a traditional plant medicine in tropical areas, Swietenia macrophylla seeds are usually applied for some chronic diseases, including hypertension, diabetes, and so on. Few studies have been carried out to identify the effective elements in seed extract and their indications. In this study, we first investigated the functions of the swietenine, an extract from S. macrophylla seeds, using a model of myocardial hypertrophy induced by isoprenaline (ISO). At cellular level, H9c2 cell hypertrophy was also established through the treatment with ISO. The cardiac pathological remodeling was evaluated by echocardiography and histological analysis. Western blot and RT-qPCR were used to detect the expression of possible hypertrophy-promoting genes. Here, our results indicated that swietenine remarkably attenuated ISO-induced myocardial hypertrophy in vivo and in vitro. Moreover, Akt phosphorylation, ANP and BNP mRNA expression were efficiently decreased. Based on these findings, we concluded that swietenine might be a promising anti-hypertrophic agent against cardiac hypertrophy.

A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Dual-Emissive Fluorescent Sensor Based on Functionalized Quantum Dots for the Simultaneous Determination of Nitric Oxide and Hydrogen Sulfide.

A simple, direct fluorescent sensor was developed to simultaneously determine nitric oxide and hydrogen sulfide based on 4-(((3-aminonaphthalen-2-yl)amino)methyl)benzoic acid (DAN-1)-functionalized CdTe/CdS/ZnS quantum dots (QDs@DAN-1). In this sensor, DAN-1 could specifically recognize nitric oxide and yield highly fluorescent naphtho triazole (DAN-1-T). Meanwhile, the fluorescence intensity of the QDs could be quenched by hydrogen sulfide. The QDs and DAN-1-T could be simultaneously excited at 365 nm, and their maximum emission wavelengths were 635 and 440 nm, respectively. Nitric oxide and hydrogen sulfide were simultaneously determined by monitoring two different fluorescence signals. The limits of determination for nitric oxide and hydrogen sulfide were 0.051 and 0.13 µM, respectively. The QDs@DAN-1 sensor was also applied to determine nitric oxide and hydrogen sulfide in human plasma. This sensor may provide a new strategy for investigating the relationship between nitric oxide and hydrogen sulfide and elucidating their roles in related physiological and pathophysiological processes at the same time.

A redox modulated ratiometric fluorometric method based on the use of dual-color carbon dots for determination of the activity of enzymes participating in ascorbic acid-related reactions.

A turn-on ratiometric fluorescent assay is described for the determination of the activity of enzymes participating in ascorbic acid-forming reactions. Blue-emitting carbon dots (bCDs; with excitation/emission wavelength at 380/450 nm) serve as fluorescent indicator. Their fluorescence is reduced by Fe3+ ions via an inner filter effect. Yellow-emitting CDs (yCDs; with excitation/emission wavelength at 380/550 nm) serve as internal reference because their fluorescence is insensitive to Fe3+. Upon exposure to ascorbic acid (AA), Fe3+ is reduced to Fe2+. Hence, the fluorescence of the bCDs is restored. Thus, enzymes participating in AA-related reactions such as α-glucosidase (α-Glu) and alkaline phosphatase (ALP) can be determined. α-Glu activity was quantified in the range from 0.13 to 6.70 U mL-1, and ALP activity was determined between 2.0 and 130 U L-1. Endowed with excellent sensitivity, selectivity and low background signals, the method may also be used to screen the inhibitors of α-Glu and ALP. Graphical abstractSchematic representation of a redox modulated ratiometric fluorometric method based on the use of dual-color carbon dots for determination of the activity of enzymes participating in ascorbic acid-related reactions. Blue-emitting carbon dots (bCDs) serve as fluorescent indicator while yellow-emitting CDs (yCDs) serve as internal reference.