Recent Advances in Gold Nanocluster-Based Biosensing and Therapy: A Review.

Gold nanoclusters (Au NCs) with bright emission and unique chemical reactivity characters have been widely applied for optical sensing and imaging. With a combination of surface modifications, effective therapeutic treatments of tumors are realized. In this review, we summarize the recently adopted biosensing and therapy events based on Au NCs. Homogeneous and fluorometric biosensing systems toward various targets, including ions, small molecules, reactive oxygen species, biomacromolecules, cancer cells, and bacteria, in vitro and in vivo, are presented by turn-off, turn-on, and ratiometric tactics. The therapy applications are concluded in three aspects: photodynamic therapy, photothermal therapy, and as a drug carrier. The basic mechanisms and performances of these systems are introduced. Finally, this review highlights the challenges and future trend of Au NC-based biosensing and therapy systems.

Fluorimetric Detection of Insulin Misfolding by Probes Derived from Functionalized Fluorene Frameworks.

A group of functionalized fluorene derivatives that are structurally similar to the cellular prion protein ligand N,N'-(methylenedi-4,1-phenylene)bis [2-(1-pyrrolidinyl)acetamide] (GN8) have been synthesized. These compounds show remarkable native fluorescence due to the fluorene ring. The substituents introduced at positions 2 and 7 of the fluorene moiety are sufficiently flexible to accommodate the beta-conformational folding that develops in amyloidogenic proteins. Changes in the native fluorescence of these fluorene derivatives provide evidence of transformations in the amyloidogenic aggregation processes of insulin. The increase observed in the fluorescence intensity of the sensors in the presence of native insulin or amyloid aggregates suggest their potential use as fluorescence probes for detecting abnormal conformations; therefore, the compounds can be proposed for use as "turn-on" fluorescence sensors. Protein-sensor dissociation constants are in the 5-10 µM range and an intermolecular charge transfer process between the protein and the sensors can be successfully exploited for the sensitive detection of abnormal insulin conformations. The values obtained for the Stern-Volmer quenching constant for compound 4 as a consequence of the sensor-protein interaction are comparable to those obtained for the reference compound GN8. Fluorene derivatives showed good performance in scavenging reactive oxygen species (ROS), and they show antioxidant capacity according to the FRAP and DPPH assays.

Open Meter Duo: Low-Cost Instrument for Fluorimetric Determination of Cholinesterase Activity.

Environmental screening is essential due to the increased occurrence of harmful substances in the environment. Open Meter Duo (OMD) is an open-source field photo/fluorimeter that uses an RGB diode that imitates a color according to the selected wavelength and uses a UV LED from the security kit diode as an excitation light source. The prepared PCB shield with a 3D-printed aperture was connected to Arduino UNO R4 WiFi. This system was used for the fluorescent detection of cholinesterase activity with the indoxyl acetate method. Carbofuran-a toxic pesticide-and donepezil-a drug used to treat Alzheimer's disease-were tested as model inhibitors of cholinesterase activity. The limit of detection of indoxyl acetate was 11.6 µmol/L, and the IC50 values of the inhibitors were evaluated. This system is optimized for wireless use in field analysis with added cloud support and power source. The time of analysis was 5 min for the fluorimetric assay and 20 min for the optional photometric assay. The time of field operation was approximately 4 h of continuous measurement. This system is ready to be used as a cheap and easy control platform for portable use in drug control and point-of-care testing.

ß-Glucuronidase-triggered reaction for fluorometric and colorimetric dual-mode assay based on the in situ formation of silicon nanoparticles.

BACKGROUND: ß-Glucuronidase (GUS) is considered as a promising biomarker for primary cancer. Thus, the reliable detection of GUS has great practical significance in the discovery and diagnosis of cancer. Compared with traditional organic probes, silicon nanoparticles (Si NPs) have emerged as robust optical nanomaterials due to their facile preparation, superior photobleaching resistance and excellent biocompatibility. However, most nanomaterials-based methods only output a single signal which is easily influenced by external factors in complex systems. Hence, developing nanomaterial-based multi-signal optical assays for highly sensitive GUS determination is still urgently desired. RESULTS: In this study, we developed a simple and efficient one-step method for the in situ preparation of yellow color and yellow-green fluorescent Si NPs. This was achieved by combining 3-[2-(2-aminoethylamino) ethylamino] propyl-trimethoxysilane with p-aminophenol (AP) in an aqueous solution. The obtained Si NPs showed yellow-green fluorescence at 535 nm when excited at 380 nm, while also exhibiting an absorption peak at a wavelength of 490 nm. Taking inspiration from the easy synthesis step regulated by AP, which is generated through the hydrolysis of 4-aminophenyl ß-D-glucuronide catalyzed by GUS, we constructed a direct fluorometric and colorimetric dual-mode method to measure GUS activity. The developed fluorometric and colorimetric sensing platform showed high sensitivity and accuracy with detection limits for GUS determination as low as 0.0093 and 0.081 U/L, respectively. SIGNIFICANCE: This study provides a facile dual-mode fluorometric and colorimetric approach for determination of GUS activity based on novel Si NPs for the first time. This designed sensing approach was successfully employed for the quantification of GUS in human serum samples and screening of GUS inhibitors, indicating the feasibility and potential applications in clinical cancer diagnosis and anti-cancer drug discovery.

Mercury (II) sensing using a simple turn-on fluorescent graphene oxide based aptasensor in serum and water samples.

A simple, highly sensitive, and selective fluorometric aptasensing platform based on aptamer and graphene oxide (GO) is proposed for the determination of mercury (II) ion (Hg2+). In the designed assay, two aptamer probes, a carboxy-fluorescein (FAM) labeled aptamer (aptamer A) and its complementary (aptamer B) with partial complement containing several mismatches and GO as the quencher were used. In the absence of Hg2+, both A and B aptamers were adsorbed on the surface of GO by π-π-stacking, leading to fluorescence quenching of FAM due to fluorescence resonance energy transfer (FRET). Upon exposure to Hg2+, the A and B aptamer strands bind Hg2+ and form T-Hg2+-T complexes, leading to the formation of a stable double-stranded aptamer. The double-stranded aptamer is detached from the GO surface, resulting in the recovery of FAM fluorescence. The fluorescence intensity (FI) of the developed sensor was correlated with the Hg2+ concentration under optimized experimental conditions in two wide linear ranges, even in the presence of 10 divalent cations as interferences. The linear ranges were obtained from 200.0 to 900.0 fM and 5.0 to 33.0 pM, a limit of detection (LOD) of 106.0 fM, and a limit of quantification (LOQ) of 321.3 fM. The concentration of Hg2+ was determined in five real samples containing three water and two serum samples, using spiking and standard addition methods and the results were compared with the spiked amounts and atomic absorption (AAS) as standard method respectively, with acceptable recoveries. Furthermore, in the standard addition method, to overcome the effects of matrix influence of real samples in quantitative predictions, the excitation-emission matrix (EEM) data for samples was simultaneously analyzed by multivariate curve resolution with alternating least squares (MCR-ALS) as a second-order standard addition method (SOSAM).

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Highly efficient dual-mode detection of AFB1 based on the inner filter effect: Donor-acceptor selection and application.

BACKGROUND: The utilization of inner filter effect (IFE) brings more opportunities for construction of fluorescence immunoassays but remains a great challenge, especially how to select best donor in the face of extensive fluorescent nanomaterials. Aflatoxin B1 possesses high toxicity among mycotoxins and is frequently found in agricultural products that may significantly threaten to human health. Therefore, with the help of signal transduction mechanism of IFE to develop a convenient and sensitive approach for AFB1 detection is of great significance in ensuring food safety. RESULTS: Herein, the classical alkaline phosphatase (ALP) catalyzes hydrolysis of p-nitrophenylphosphate to produce p-nitrophenol (PNP) was employed as a model reaction, which intends to explore tunable multicolor fluorescence of gold nanoclusters (AuNCs) for matching PNP to maximize IFE efficiency. The luminescent green-emitting AuNCs were selected as an optimal donor in terms of excellent spectral overlap, high photoluminescence, and adequate system adaptability, thus achieving a 22-fold increase in sensitivity improvement compared to colorimetric method for ALP detection. The fluorescence quenching mechanism between PNP and AuNCs was validated as IFE by studying ultraviolet absorption, zeta potentials and fluorescence lifetime. In light of this, we integrated a highly specific antibody-antigen recognition system, efficient enzymatic reaction and excellent optical characteristics of AuNCs to develop dual-mode immunoassay for AFB1 monitoring. The sensitivity of fluorometric immunoassay was lower to 0.06 ng/mL, which obtained a 3.5-fold improvement compared to "gold standard" ELISA. Their practicability and applicability were confirmed in the tap water, corn, wheat and peanuts samples. SIGNIFICANCE: This work provides an easy-to-understand screening procedure to select optimal donor-acceptor pairs in IFE analysis. Furthermore, we expect that integration of IFE-based signal conversion strategy into mature immunoassay not only extends the signal types, simplifies signal amplification steps, and reduces the false-positive/false-negative rates, but also provides a simple, convenient, and versatile strategy for monitoring of trace other contaminants.

Green and inventive fluorescence approach for levodropropizine determination in human plasma.

A green, rapid and sensitive fluorimetric method to quantify levodropropizine (LVP) in human plasma was exploited for the first time. The proposed method adopts LVP's intrinsic fluorescence in distilled water at a detecting emission of 345 nm following excitation at 240 nm. LVP displayed linearity across concentrations ranging from 50 to 1000 ng mL-1, with a detection limit of 0.77 ng mL-1 and a quantification limit of 2.33 ng mL-1. Thorough validation confirmed its reliability, successfully determining LVP in tablets with an average recovery of 98.64 ± 1.07 %. Furthermore, the method's applicability extended to estimate the studied drug in spiked human plasma with excellent obtained percentage recoveries (98.68 ± 1.28-100.14 ± 1.23).

Fluorescence-Based Protein Stability Monitoring-A Review.

Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.

Combination of gold nanoclusters and silicon quantum dots for ratiometric fluorometry: One system, two mechanisms.

A ratiometric fluorometry based on silicon quantum dots (SiQDs) and gold nanoclusters (AuNCs) is constructed for detecting activity of butyrylcholinesterase (BChE) in human serum. By using thiobutyrylcholine iodide (BTCh) as the substrate of BChE-catalyzed hydrolysis reaction, variation of fluorescence emission from AuNCs is employed as an indicator of BChE activity since one of the hydrolysis products, thiocholine (TCh), would influence the aggregation state of AuNCs and consequently led to the change of fluorescence quantum efficiency of AuNCs. It is interesting that there are two mechanisms working for the fluorescence emission of aggregated AuNCs: aggregation-induced emission enhancement (AIEE) and aggregation-caused quenching (ACQ) with the presence of TCh at very low and higher concentration levels, respectively. Although both of these mechanisms can be utilized for sensing BChE, their opposite influence on the fluorescence emission of aggregated AuNCs should be worthy of attention, especially in the process of developing fluorescence methods for detecting trace targets by using AuNCs. In order to eliminate the fluctuation of fluorophotometer, SiQDs is chosen as the fluorophore to develop by ratiometric fluorescence methods in this work. Additionally, obvious aggregation of AuNCs induces significant decrease of inner filter effect (IFE) on the fluorescence emitted from SiQDs, while mild aggregation of AuNCs demonstrates little IFE. The linear ranges for detecting activity of BChE are 0.004 - 0.05 U/L and 0.5 - 20 U/L by ratiometric fluorometry based on the AIEE and ACQ, respectively. The very different responses originated from AIEE and ACQ of AuNCs would respectively make their own contributions to the determination of BChE activities at very low or high levels, which facilitate the developments of enhanced or quenched fluorescence methods. However, the detection of BChE activities at medium levels might suffer from the combination of AIEE and ACQ with ambiguous fractions. Therefore, it must be careful during the processes of developing and applying fluorescence methods based on the AIEE and ACQ of AuNCs, as well as the process of evaluating their analytical performance.

Fluorometric and colorimetric dual-mode sensing of α-glucosidase based on aggregation-induced emission enhancement of AuNCs.

Herein, a fluorometric and colorimetric dual-mode assay platform used for α-glucosidase (α-Glu) activity sensing based on aggregation-induced emission enhancement (AIEE) of AuNCs was developed for the first time. The quantum yield (QY) and fluorescence lifetime of AuNCs were successfully ameliorated by Ce3+-triggered AIEE (Ce@AuNCs). Subsequently, on the basis of the inner filter effect (IFE) and dynamic quenching effect (DQE) between 2,6-dichlorophenolindophenol (DCIP) and Ce@AuNCs as well as the reduction of DCIP by ascorbic acid (AA) generated from α-Glu-catalyzed hydrolysis of L-ascorbic acid-2-O-α-D-glucopyranosyl (AA2G), the marriage of fluorometric and colorimetric modes applied for α-Glu activity monitoring was achieved. Besides, the feasibility of this dual-mode sensing system was confirmed by the assays versus potential interfering substances and in real samples. In particular, this system was further applied to evaluate natural α-Glu inhibitors (AGIs) including luteolin, apigenin, and hesperidin. Overall, the multi-mode optical sensor newly designed here has the potential for the accurate discovery of natural anti-diabetes drugs and the therapy of diabetes.

Multicolor emissive carbon dot-based fluorometric analysis platform for rapid quantification and discrimination of nitroimidazole antibiotic residues.

The development of efficient, rapid, portable, and accurate analysis of veterinary drug residues in food matrices is in great demand for food safety assessment. Here, we have developed a smartphone-integrated platform for fluorometric quantification of metronidazole (MNZ) residues and constructed a sensor array for discrimination of different nitroimidazole antibiotics (NIIMs). Multicolor CDs (B-CDs, C-CDs, Y-CDs, and R-CD) were prepared and showed different fluorescence response to MNZ. The fluorescence of C-CDs was quenched Because of the inner filter effect (IFE) between the C-CDs and MNZ, while that of R-CDs was enhanced due to the passivation of surface defects by MNZ. Based on the response pattern, the fluorometric quantification of MNZ based on the fluorescence images of C-CD + R-CD system (R/G values) was achieved with a low detection limit of 0.45 µM. By designing a smartphone-integrated platform, the analysis can be completed within 20 min. In addition, a fluorescence sensor array based C-CDs and R-CDs was also developed. The unique fingerprint of each NIIMs was obtained by linear discriminant analysis (LDA) of the response patterns, indicating an effective discrimination of five NIIMs. Moreover, the platform was used for quantification of MNZ in food samples and the recoveries were within 84.0-106.3 % with relative standard deviations 1.2-10.2 %. Therefore, the proposed method shows great potential as a universal platform for rapid detection of veterinary drug residues.

Ratiometric fluorometric assay triggered by alkaline phosphatase: Proof-of-concept toward a split-type biosensing strategy for DNA detection.

Herein, a sensitive ratiometric and split-type fluorescent sensing platform has been constructed for DNA detection based on one signal precursor and two fluorescent signal indicators. In this assay, o-phenylenediamine (OPD) was selected as the signal precursor. On one hand, Cu2+ can oxidize OPD to produce 2, 3-diaminophenazine (DAP), which with an emission peak at 555 nm. On the other hand, ascorbic acid (AA) could react with Cu2+ to generate dehydroascorbic acid (DHAA), which could further react with OPD to form 3-(1, 2-dihydroxy ethyl)furo[3, 4-b]quinoxalin-1 (3H)-on (DFQ) with a strong emission peak at 420 nm. As a result, the formation of DAP was inhibited, and leading to the decrease of fluorescence intensity at 555 nm. Alkaline phosphatase (ALP) could catalyze the substrate l-ascorbic acid-2-phosphate (AA2P) to produce AA in situ. Inspired by the successful use of ALP as a biocatalytic marker in bioassay, a split-type ratiometric fluorescent assay has been designed for DNA detection by using H1N1 DNA as the target model. It was realized for ratiometric fluorescent determination of H1N1 in a linear ranging from 50 pM to 1.5 nM with a limit of detection of 10 pM. The novel strategy could reduce the mutual interferences between the biomolecular recognition system and the fluorescence signal conversion system, which improving the accuracy of detection and effectively reducing the background signal. Furthermore, the strategy provided a promising platform for biomarkers detection in the fields of ratiometric fluorescent biosensors and bioanalysis.

Dopamine-based selective spectrophotometry p-aminosalicylic acid assay by hydrolyzate-triggered formation of azamonardine-like products.

BACKGROUND: The selective recognition of drugs and its metabolism or decomposition products is significant to drug development and drug resistance research. Fluorescence-based techniques provide satisfying sensitivity by target-triggered chemical reaction. However, the interference from the matrix or additives usually restricts the specific detection. It is highly desirable to explore specific chemical reactions for achieving selective perception of these species. RESULTS: We report a specific m-aminophenol (MAP)-dopamine (DA) reaction, which generates highly fluorescent azamonardine-like products. Based on this reaction, fluorometric and indirect detection of p-aminosalicylic acid (typical antituberculosis drug, PAS) can be realized using the DA-based probe with high sensitivity. The acid induces the decarboxylation of PAS and produces MAP, which reacts with DA and generates fluorescent azamonardine-like products. The practical application of the proposed method is validated by the accurate PAS analysis in urine samples and Pasinazid tablets. Interestingly, none of additives in the Pasinazid tablets contribute comparable fluorescence variation. SIGNIFICANCE: This work discovers a new MAP-DA reaction for the first time, it not only explores sensitive PAS drug detection probe, but also demonstrates the feasibility of the development of novel drug analysis platform by recognizing decomposition product with specific reaction. Thus, new avenues for the exploration of simple and rapid spectrophotometric probes toward various drug analytes with high specify and sensitivity based on this tactic might be possible in analytical and drug-related fields.

Development of a fluorometric and colorimetric dual-mode sensing platform for acid phosphatase assay based on Fe3+ functionalized CuInS2/ZnS quantum dots.

BACKGROUND: The spectral dual-mode response towards analyte has been attracted much attention, benefiting from the higher detection accuracy of such strategy in comparison to single signal readout. However, the currently reported dual-mode sensors for acid phosphatase (ACP) activity are still limited, and most of them more or less exist some deficiencies, such as complicated construction procedure, high-cost, poor biocompatibility, aggregation-caused quenching and limited emission capacity. RESULTS: Herein, we employed Fe3+ functionalized CuInS2/ZnS quantum dots (CIS/ZnS QDs) as nanosensor to develop a novel fluorometric and colorimetric dual-mode assay for ACP activity, combing with ACP-triggered hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA). The Fe3+ binding to CIS/ZnS QDs can be reduced into Fe2+ during the determination, resulting in the dramatically weakened photoinduced electron transfer (PET) effect and the disappearance of competition absorption. Thus, a highly sensitive ACP assay in the range of 0.22-12.5 U L-1 through fluorescence "turn-on" mode has been achieved with a detection of limit (LOD) of 0.064 U L-1. Meanwhile, the ACP activity can also be quantified by spectrophotometry based on the chromogenic reaction of the formed Fe2+ with 1,10-phenanthroline (Phen). Moreover, the designed nanosensor with good biocompatibility was successfully applied to image and monitor the ACP levels in living cells. SIGNIFICANCE: We believe that the proposed method has remarkable advantages and potential application for ACP assay in terms of the high accuracy, simplicity, low cost, as well as its adequate sensitivity.

A Study of Small Molecule-Based Rhodamine-Derived Chemosensors and their Implications in Environmental and Biological Systems from 2012 to 2021: Latest Advancement and Future Prospects.

Rhodamine-based chemosensors have sparked considerable interest in recent years due to their remarkable photophysical properties, which include high absorption coefficients, exceptional quantum yields, improved photostability, and significant red shifts. This article presents an overview of the diverse fluorometric, and colorimetric sensors produced from rhodamine, as well as their applications in a wide range of fields. The ability of rhodamine-based chemosensors to detect a wide range of metal ions, including Hg+2, Al3+, Cr3+, Cu2+, Fe3+, Fe2+, Cd2+, Sn4+, Zn2+, and Pb2+, is one of their major advantages. Other applications of these sensors include dual analytes, multianalytes, and relay recognition of dual analytes. Rhodamine-based probes can also detect noble metal ions such as Au3+, Ag+, and Pt2+. They have been used to detect pH, biological species, reactive oxygen and nitrogen species, anions, and nerve agents in addition to metal ions. The probes have been engineered to undergo colorimetric or fluorometric changes upon binding to specific analytes, rendering them highly selective and sensitive by ring-opening via different mechanisms such as Photoinduced Electron Transfer (PET), Chelation Enhanced Fluorescence (CHEF), Intramolecular Charge Transfer (ICT), and Fluorescence Resonance Energy Transfer (FRET). For improved sensing performance, light-harvesting dendritic systems based on rhodamine conjugates has also been explored for enhanced sensing performance. These dendritic arrangements permit the incorporation of numerous rhodamine units, resulting in an improvement in signal amplification and sensitivity. The probes have been utilised extensively for imaging biological samples, including imaging of living cells, and for environmental research. Moreover, they have been combined into logic gates for the construction of molecular computing systems. The usage of rhodamine-based chemosensors has created significant potential in a range of disciplines, including biological and environmental sensing as well as logic gate applications. This study focuses on the work published between 2012 and 2021 and emphasises the enormous research and development potential of these probes.

Fluorimetric Cell Analysis with 9-Aryl-Substituted Berberine Derivatives as DNA-Targeting Fluorescent Probes.

DNA-sensitive fluorescent light-up probes based on berberine are presented. This biogenic fluorophore was chosen as central unit to use its potential biocompatibility and its DNA-binding properties. To provide predictable fluorescence quenching in aqueous solution and a fluorescence light-up effect upon DNA binding, aryl substituents were attached at the 9-position by Suzuki-Miyaura coupling reactions. The 9-arylberberine derivatives have a very low fluorescence quantum yield (Φfl =<0.02), which is caused by the radiationless deactivation of the excited state by torsional relaxation about the biaryl axis. In addition, these berberine derivatives intercalate into DNA with high affinity (Kb =2.0-22×104  M-1 ). Except for the nitrophenyl- and hydroxyphenyl-substituted derivatives, all tested compounds exhibited a pronounced fluorescence light-up effect upon association with DNA, because the deactivation of the excited-state by torsional relaxation is suppressed in the DNA binding site. Most notably, it was shown exemplarily with the 9-(4-methoxyphenyl)- and the 9-(6-methoxynaphthyl)-substituted derivatives that these properties are suited for fluorimetric cell analysis. In particular, these probes generated distinct staining patterns in eukaryotic cells (NIH 3T3 mouse fibroblasts), which enabled the identification of nuclear substructures, most likely heterochromatin or nucleoli, respectively.

A near-infrared fluorescent probe for rapid and on-site detection of sulfur dioxide derivative in biological, food and environmental systems.

Emission of toxic gaseous sulfur dioxide (SO2) and its derivative bisulfite (HSO3-) from various industrial applications, like food processing, transportation, and the coking process, has raised substantial concerns regarding environmental quality and public health. The probes for specific and sensitive detection of SO2 derivatives plays an essential role in their regulation, and ultimately mitigating their environmental and health implications, but the one that can detect SO2 derivatives onsite by end users remains limited. Herein, we report a new near-infrared fluorescence probe (SL) for rapid and onsite detection of SO2 derivative, HSO3- in industrial wastewater, food samples and for sensing its interaction with biological organisms. The SL is developed through coupling of quinolinium and coumarin moiety through an electron deficit CC bond that can specifically react with HSO3- via a Michael addition. By recording the blue shift of absorption and emission spectra, SL can sensitively detect HSO3- (limit of detection, 38 nM) in aqueous solution within 40 s SL is biocompatible, can be used for evaluating toxicity of SO2 derivatives in living organisms. The preparation of SL-stained test paper allows the colorimetric/fluorometric analysis for quantification of HSO3- onsite in food, river and coking wastewater samples using a smartphone. The successful development of SL not only provides a new tool to investigate HSO3- in biological, food and environmental systems, but also potentially promotes the application of fluorescence technique for rapid and onsite analysis of real-world samples by end users.

A sensitive fluorometric-colorimetric dual-mode intelligent sensing platform for the detection of formaldehyde.

Excess formaldehyde (FA) is a strong carcinogen, so the development of a rapid visualized and portable formaldehyde detection platform is of great research importance. A multi-color fluorescence sensing system constituted of model compound (NAHN) and red-emitting InP/ZnS QDs was constructed herein, which can simultaneously realize fluorometric-colorimetric dual-mode sensing when exposed to FA environment. Its preparation process was simplified, the detection process was green, and the limits of detection (LOD) were 0.623 µM and 0.791 µM, respectively. The high recoveries of FA in actual water samples indicated that the sensor had broad application prospects. The prepared fluorescent film can be utilized for rapid visual simulation analysis of FA on the surface of various fruits and vegetables. In addition, a serial logic gate was designed to quickly semi-quantitatively assess FA concentration, which promoted the realization of on-site intelligent evaluation of FA.

Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry.

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the "dark" nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.