Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates. The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

DSFworld: A flexible and precise tool to analyze differential scanning fluorimetry data.

Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from which Tma is calculated vary widely in shape and complexity. However, the tools available for calculating Tma are only compatible with the simplest of DSF curves, hindering many otherwise straightforward applications of the technology. To overcome this limitation, we designed new mathematical models for Tma calculation that accommodate common forms of variation in DSF curves, including the number of transitions, the presence of high initial signal, and temperature-dependent signal decay. When tested these models against DSFbase, an open-source database of 6235 raw, real-life DSF curves, these models outperformed the existing standard approaches of sigmoid fitting and maximum of the first derivative. To make these models accessible, we created an open-source software and website, DSFworld (https://gestwickilab.shinyapps.io/dsfworld/). In addition to these improved fitting capabilities, DSFworld also includes features that overcome the practical limitations of many analysis workflows, including automatic reformatting of raw data exported from common qPCR instruments, labeling of data based on experimental variables, and flexible interactive plotting. We hope that DSFworld will enable more streamlined and accurate calculation of Tma values for DSF experiments.

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.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Simple enzyme based fluorimetric biosensor for urea in human biofluids.

As an important biomarker for renal related diseases, detection of urea is playing a vital role in human biofluids on clinical diagnosis concern. In this work, a synthetic salicyaldehyde based imine fluorophore was synthesized using sonication method and conjugated with urease which was used as fluorescent biosensor for the detection of urea in serum samples. This enzyme based biosensor has shown a good selectivity and sensitivity towards urea with the linear range from 2 to 80 mM and the detection limit of 73 µM. The sensing response obtain is highly agreeing with existing analytical technique for urea detection which strongly recommends this biosensor for clinical application.

A dual-mode fluorometric/colorimetric sensor for sulfadimethoxine detection based on Prussian blue nanoparticles and carbon dots.

A dual-mode (colorimetric/fluorescence) nanoenzyme-linked immunosorbent assay (NLISA) was developed based on Au-Cu nanocubes generating Prussian blue nanoparticles (PBNPs). It is expected that this method can be used to detect the residues of sulfonamides in the field, and solve the problem of long analysis time and high cost of the traditional method. Sulfadimethoxine (SDM) was selected as the proof-of-concept target analyte. The Au-Cu nanocubes were linked to the aptamer by amide interaction, and the Au-Cu nanocubes, SDM and antibody were immobilized on a 96-well plate using the sandwich method. The assay generates PBNPs by oxidising the Cu shells on the Au-Cu nanocubes in the presence of hydrochloric acid, Fe3+ and K3[Fe (CN)6]. In this process, the copper shell undergoes oxidation to Cu2+ and subsequently Cu2 + further quenches the fluorescence of the carbon point. PBNPs exhibit peroxidase-like activity, oxidising 3,3',5,5'-tetramethylbenzidine (TMB) to OX-TMB in the presence of H2O2, which alters the colorimetric signal. The dual-mode signals are directly proportional to the sulfadimethoxine concentration within the range 10- 3~10- 7 mg/mL. The limit of detection (LOD) of the assay is 0.023 ng/mL and 0.071 ng/mL for the fluorescent signal and the colorimetric signal, respectively. Moreover, the assay was successfully applied to determine sulfadimethoxine in silver carp, shrimp, and lamb samples with satisfactory results.

A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin.

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.

Ultrasensitive NIR fluorometric assay for inorganic pyrophosphatase detection via Cu2+-PPi interaction using bimetallic Au-Ag nanoclusters.

BACKGROUND: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis. RESULT: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu2+) ions. The off state of LA@AuAg_Cu2+ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu2+. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu2+ ions, and again quenched off state was retrieved due to interaction of free Cu2+ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples. SIGNIFICANCE: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.

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.

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.

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).

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.

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.

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.

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.

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.

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.