Fluorescence Turn-on Assay Research Articles

Preparation of Conjugated Polymer Dots as a Fluorescence Turn-On Assay for Bovine Serum Albumin by Interaction with Graphene Oxide

Fluorescent conjugated polymers with various monomer compositions were synthesized via Suzuki coupling polymerization and spherically-shaped conjugated polymer dots (CPdot) with uniform size were prepared via conventional reprecipitation technique. Chemically modified graphene oxide (GO) was prepared to use as a component of sensor platform for protein detection. GO showed an excellent interaction with CPdot on its surface via hydrophobic interaction, which, in turn, induced the quenching of the fluorescence of CPdot. Upon exposure to bovine serum albumin (BSA), the quenched fluorescence was recovered, resulting from the release of CPdot from the complex with GO, as BSA adsorbed preferentially on the GO surface.

Sensitive turn-on fluorescence assay of methimazole based on the fluorescence resonance energy transfer between acridine orange and silver nanoparticles

A new method for highly sensitive detection of methimazole was developed based on the fluorescence resonance energy transfer (FRET) between acridine orange (AO) and silver nanoparticles (AgNPs), in which AO acts as a donor and AgNP as an acceptor. The fluorescence of AO molecules was severely quenched when they were attached to the surface of AgNP by electrostatic attraction. Upon the addition of methimazole, AO molecules were detached from NPs because of strong adsorption of methimazole on the surface of AgNPs. Under optimum conditions, the enhanced fluorescence intensity displayed a linear relationship with the concentration of methimazole in the range of 8.0×10−9–3.75×10−7M, with a limit of detection of 5.5nM. The method was applied for the determination of therapeutic levels of methimazole in human plasma samples with satisfactory results.

A versatile biosensing system for DNA-related enzyme activity assay via the synthesis of silver nanoclusters using enzymatically-generated DNA as template

In the present day, oligonucleotide-encapsulated silver clusters (DNA–AgNCs) have been widely applied into bio-analysis as a signal producer. Herein, we developed a novel method to synthesize DNA–AgNCs encapsulated by long-chain cytosine (C)-rich DNA. Such DNA was polymerized in a template-free way by terminal deoxynucleotidyl transferase (TdT). We demonstrated that TdT-polymerized long chain C-rich DNA can serve as an excellent template for AgNCs synthesis. Based on this novel synthesis strategy, we developed a label-free and turn-on fluorescence assay to detect TdT activity with ultralow limit of detection (LOD) of 0.0318U and ultrahigh signal to background (S/B) of 46.7. Furthermore, our proposed method was extended to a versatile biosensing strategy for turn-on nucleases activity assay based on the enzyme-activated TdT polymerization. Two nucleases, EcoRI and ExoIII as model of endonuclease and exonuclease, respectively, have been detected with high selectivity and competitive low LOD of 0.0629U and 0.00867U, respectively. Our work demonstrates the feasibility of TdT polymerization-based DNA–AgNCs synthesis strategy as a versatile and potent biosensing platform to detect the activity of DNA-related enzymes.

Efficient fluorescence resonance energy transfer between oppositely charged CdTe quantum dots and gold nanoparticles for turn-on fluorescence detection of glyphosate

We designed a turn-on fluorescence assay for glyphosate based on the fluorescence resonance energy transfer (FRET) between negatively charged CdTe quantum dots capped with thioglycolic acid (TGA-CdTe-QDs) and positively charged gold nanoparticles stabilized with cysteamine (CS-AuNPs). Oppositely charged TGA-CdTe-QDs and CS-AuNPs can form FRET donor–acceptor assemblies due to electrostatic interactions, which effectively quench the fluorescence intensity of TGA-CdTe-QDs. The presence of glyphosate could induce the aggregation of CS-AuNPs through electrostatic interactions, resulting in the fluorescence recovery of the quenched QDs. This FRET-based method has been successfully utilized to detect glyphosate in apples with satisfactory results. The detection limit for glyphosate was 9.8ng/kg (3σ), with the linear range of 0.02–2.0μg/kg. The attractive sensitivity was obtained due to the efficient FRET and the superior fluorescence properties of QDs. The proposed method is a promising approach for rapid screening of glyphosate in real samples.

A DNA-scaffolded silver nanocluster/Cu²⁺ ensemble as a turn-on fluorescent probe for histidine.

A new type of rapid, sensitive, and selective fluorescence turn-on assay was developed for detection of histidine using a DNA-scaffolded silver nanocluster/Cu(2+) ensemble (DNA-AgNC/Cu(2+)). Cu(2+) was first bound to nucleic acids of the DNA-AgNCs forming a DNA-AgNC/Cu(2+) ensemble and then liberated into solution via the highly specific interaction between histidine and Cu(2+) in the presence of other amino acids. The fluorescence of DNA-AgNCs was greatly quenched with the addition of Cu(2+), and then the DNA-AgNC/Cu(2+) ensemble exhibited marvelous fluorescent enhancement in the presence of histidine, which showed the possibility for constructing a turn-on chemosensor of histidine. Compared to other methods, this approach promises high sensitivity, simplicity in design, convenient operation, and minimized use of organic solvents. The ultra-high selectivity demonstrated the feasibility of the assay for detecting histidine in sophisticated physical environments. The fitting range of the proposed assay is from 0 to 100 μM, with a detection limit of 1.4 μM (S/N = 3) in artificial urine samples. The protocol was evaluated by analysis of artificial urine samples with good recoveries and showed great potential for practical applications.

DNA-Templated Silver Nanoclusters for Fluorescence Turn-on Assay of Acetylcholinesterase Activity

We have developed a fluorescence turn-on assay using DNA-templated silver nanoclusters (Ag NCs) (i.e., 12 polycytosine-templated silver nanoclusters, dC12-Ag NCs), which is amenable to rapid, ultrasensitive assay of acetylcholinesterase (AChE). The detection mechanism is based on the concept, that is, AChE hydrolyzes the acetylthiocholine (ATCh) chloride to produce thiocholine (TCh). Subsequently, TCh sensitively and rapidly reacts with dC12-Ag NCs via Ag-S bond forming and enhances the fluorescence of dC12-Ag NCs. Using dC12-Ag NCs, detection of TCh has a linear concentration range of 2.0 nM to 16.0 nM and a detection limit of 0.3 nM. Due to the sensitive and rapid fluorescence turn-on response of dC12-Ag NCs to TCh, AChE with activity as low as 0.5 × 10(-4) U/mL (signal/noise = 3) can be analyzed with a dynamic range of 0.1 to 1.25 × 10(-3) U/mL. The promising application of the proposed method in AChE inhibitor screening was demonstrated. AChE concentrations were determined in human blood red cell (RBC) membranes from clinical specimens using dC12-Ag NCs, and the quantitative results were validated with Ellman's method. Aside from the ease of manufacture, reduction of matrix effect, and low background noise, the continuous detection format and detection sensitivity can open up to wider applications to AChE activity assay in neurobiology, toxicology, and pharmacology, among other fields.

A fluorescence turn-on method for real-time monitoring of protease activity based on the electron transfer between a fluorophore labeled oligonucleotide and cytochrome c

A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1nM trypsin and the IC50 values are 3.23μgmL−1 and 0.303μgmL−1 for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.

Inner filter effect of gold nanoparticles on the fluorescence of rare-earth phosphate nanocrystals and its application for determination of biological aminothiols

A simple, sensitive fluorescent method for detecting biological aminothiols has been developed based on the inner filter effect principle that utilizes CePO4:Tb3+ luminescent nanoparticles as the donor and gold nanoparticles (AuNPs) as the energy receptor. Stable, water-soluble and well-dispersible CePO4:Tb3+ nanoparticles with low photobleaching features were synthesized conveniently by a facile solvothermal method. At the same time, AuNPs with a high extinction coefficient are expected to be capable of functioning as powerful receptor. Based on the complementary overlap between the emission spectrum of CePO4:Tb3+ nanoparticles and the absorption spectrum of Au NPs, an inner filter effect system was constructed. In the presence of aminothiols (such as cysteine), AuNPs interacted with the aminothiols, thereby inducing the aggregation of AuNPs, which induced the fluorescence recovery. In the present work, we developed a turn-on fluorescent assay for the determination of biological aminothiols. Under the optimum conditions, the linear concentration ranges were 1.0×10−7–2.0×10−6M for cysteine, 5.0×10−8–5.0×10−7M for glutathione and 8.0×10−8–1.0×10−6M for homocysteine, respectively. The method is successfully applied to the quantification of biological aminothiols in synthetic samples.

A new fluorometric turn-on assay for alkaline phosphatase and inhibitor screening based on aggregation and deaggregation of tetraphenylethylene molecules

Based on the consideration that compound 1 with a -PO3H2 group can be hydrolyzed into compound 3 which shows low solubility in aqueous solutions and thus aggregation can occur easily, a new fluorescence turn-on assay has been constructed for alkaline phosphatase (ALP) with compound 1. ALP at concentrations as low as 18 mUmL(-1) can be assayed with compound 1. Moreover, compound 1 has been successfully applied for ALP assay in living cells. Also, compound 1 is useful for screening inhibitors of ALP.

A real-time fluorescence turn-on assay for trypsin based on a conjugated polyelectrolyte.

We report a continuous and sensitive fluorescence turn-on assay for trypsin by using an anionic conjugated polyelectrolyte (PPE-CO2H) and a cationic peptide substrate labelled with p-nitroaniline (RxG-pNA). Applications of the assay in trypsin activity study and high-throughput screening of protease inhibitors were demonstrated.

Tyrosine-functionalized CuInS2 quantum dots as a fluorescence probe for the determination of biothiols, histidine and threonine

A novel, rapid and highly sensitive fluorescence turn-on assay for the detection of biothiols (glutathione, and L-cysteine), histidine and threonine was developed. Water-soluble CuInS2 ternary quantum dots (QDs) capped by mercaptopropionic acid (MPA) were directly synthesized in aqueous solution, and then functionalized using tyrosine molecules to form tyrosine-functionalized CuInS2 QDs (T-CuInS2 QDs). The fluorescence of T-CuInS2 QDs would decrease in the presence of Cu(2+) due to the coordination effect of phenolic hydroxyls of the tyrosine molecules. Subsequently, the addition of biothiols (glutathione and L-cysteine), histidine or threonine could turn on the fluorescence of the T-CuInS2 QDs-Cu(2+) system due to their strong affinity for Cu(2+). The proposed method was simple in design and fast in operation, and it was applied for the detection of glutathione, L-cysteine, histidine, and threonine in human serum samples with satisfactory results.

Real‐Time Fluorescence Turn‐On Detection of Alkaline Phosphatase Activity with a Novel Perylene Probe

A tetracationic perylene probe (probe 1) was designed and synthesized. Probe 1 was used for the real-time fluorescence turn-on assay of alkaline phosphatase (ALP) activity and inhibitor screening. Probe 1 monomer fluorescence could be very efficiently quenched by ATP through the formation of an ATP/probe 1 complex. ALP triggered the degradation of ATP, the breakdown of the ATP/probe 1 complex, and the recovery of the probe 1 monomer fluorescence. In the presence of an ALP inhibitor, a decrease in fluorescence recovery was observed.

Continuous and Sensitive Acid Phosphatase Assay Based on a Conjugated Polyelectrolyte

We report a novel continuous and sensitive fluorescence turn-on assay for ACPs, which consists of a cationic conjugated polyelectrolyte (PPE4+) and a commonly used phosphatase substrate p-nitrophenyl phosphate (pNPP). The kinetics of the ACP catalyzed hydrolysis of the substrate pNPP was monitored by the fluorescence change of PPE4+ and corresponding kinetic parameters were derived to be consistent with the literature reports. The applications of PPE4+/pNPP-based ACP assay in high-throughput screening of ACP inhibitors and detection of prostatic acid phosphotase (PAP) in vitro were demonstrated.

Label-free fluorescence turn on detection of tiopronin with tunable dynamic range based on the ensemble of Alizarin Red S/copper ion

In this study, a new type of rapid, label-free fluorescence turn-on assay for detection of tiopronin using Alizarin Red S (ARS)/copper ion ensemble is developed. ARS is high fluorescence in BR buffer solution. But, the fluorescence of ARS can be significantly quenched by copper ions due to ground-state complexation. However, in the presence of tiopronin, copper ions were released from the ARS and thus restored the fluorescence of ARS. The assay has several important features. First, the system is simple in design, fast in operation and is more convenient and promising than other methods. Second, the proposed assay eliminated the separation process and sophisticated instrumentations. Third, the detection process can be seen with the naked eye and can be easily adapted to automated high-throughput screening. At last, the assay has high sensitivity and selectivity for tiopronin and the detection limit is 0.8ng/mL which is lower than or at least comparable to the previous methods. Moreover, the dynamic range of the sensor can be tuned simply by adjusting the concentration of copper ions. Importantly, the protocol offers high selectivity for the determination of tiopronin in pharmaceutical tablets, injection and biological samples with satisfactory results. Thus, the assay shows great potential applications in the fields of pharmaceuticals and clinical analysis.

Alizarin red S/copper ion-based ensemble for fluorescence turn on detection of glutathione with tunable dynamic range

In this study, a new type of rapid, high sensitive and selective fluorescence turn-on assay for detection of glutathione using an Alizarin Red S/copper ion ensemble is developed. This assay is based on the highly specific interaction between the glutathione and the copper ions and the strong fluorescence Alizarin Red S probe in a competition assay format. The system is simple in design, fast in operation and is more convenient and promising than other methods. The novel strategy eliminated the separation process, chemical modifications, and sophisticated instrumentations. The detection and discrimination process can be seen with the naked eye and can be easily adapted to automated high-throughput screening. The assay has high sensitivity and selectivity for glutathione. The detection limit is 2.3nM, it is lower than or at least comparable to previous methods. The dynamic range of the sensor can be tuned simply by adjusting the concentration of copper ions. Importantly, the protocol offers high selectivity for the determination of glutathione among amino acids found in proteins, as well as in serum samples. The assay shows great potential for practical application as a disease-associated biomarker and it will be needed to satisfy the great demand of amino acid determination in the fields such as biochemistry, pharmaceuticals, and clinical analysis.

Binding-Induced Fluorescence Turn-On Assay Using Aptamer-Functionalized Silver Nanocluster DNA Probes

We present here a binding-induced fluorescence turn-on assay for protein detection. Key features of this assay include affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters (Ag NCs) using guanine-rich DNA sequences. In an example of an assay for human α-thrombin, two aptamers (Apt15 and Apt29) were used and were modified by including additional sequence elements. A 12-nucleotide (nt) sequence was used to link the first aptamer with a nanocluster nucleation sequence at the 5'-end. The second aptamer was linked through a complementary sequence (12-nt) to a G-rich overhang at the 3'-end. Binding of the two aptamer probes to the target protein initiates hybridization between the complementary linker sequences attached to each aptamer and thereby bring the end of the G-rich overhang to close proximity to Ag NCs, resulting in a significant fluorescence enhancement. With this approach, a detection limit of 1 nM and a linear dynamic range of 5 nM-2 μM were achieved for human α-thrombin. This fluorescence assay is performed in a single tube, and it does not require washing or separation steps. The principle of the binding-induced DNA hybridization and fluorescence enhancement of Ag NCs can be extended to other homogeneous assay applications provided that two appropriate probes are available to bind with the same target molecule.

Templated Synthesis of Glycoluril Hexamer and Monofunctionalized Cucurbit[6]uril Derivatives

We report that the p-xylylenediammonium ion (11) acts as a template in the cucurbit[n]uril forming reaction that biases the reaction toward the production of methylene bridged glycoluril hexamer (6C) and bis-nor-seco-CB[10]. Hexamer 6C is readily available on the gram scale by a one step synthetic procedure that avoids chromatography. Hexamer 6C undergoes macrocylization with (substituted) phthalaldehydes 12, 14, 15, and 18-in 9 M H(2)SO(4) or concd HCl at room temperature to deliver monofunctionalized CB[6] derivatives 13, 16, 17, and 19-that are poised for further functionalization reactions. The kinetics of the macrocyclization reaction between hexamer and formaldehyde or phthalaldehyde depends on the presence and identity of ammonium ions as templates. p-Xylylenediammonium ion (11) which barely fits inside CB[6] sized cavities acts as a negative template which slows down transformation of 6C and paraformaldehyde into CB[6]. In contrast, 11 and hexanediammonium ion (20) act as a positive template that promotes the macrocyclization reaction between 6C and 12 to deliver (±)-21 as a key intermediate along the mechanistic pathway to CB[6] derivatives. Naphthalene-CB[6] derivative 19 which contains both fluorophore and ureidyl C═O metal-ion (e.g., Eu(3+)) binding sites forms the basis for a fluorescence turn-on assay for suitable ammonium ions (e.g., hexanediammonium ion and histamine).

Simple and sensitive fluorescence sensor for detection of potassium ion in the presence of high concentration of sodium ion using berberine–G-quadruplex complex as sensing element

In this work, a novel potassium ion (K+) sensor is presented using berberine–G-quadruplex complex as a fluorescent probe. This sensor is based on the K+that can induce the G-rich DNA to form G-quadruplex conformation. The G-quadruplex can bind berberine to form berberine–G-quadruplex complex, resulting in remarkable enhancement of fluorescence emission of the berberine–G-quadruplex system. In the presence of 800mM sodium ion (Na+), the fluorescence of the berberine–G-quadruplex complex increased linearly with increasing K+ concentration in the range of 0.005–1.0mM. The turn-on fluorescent assay is simple, inexpensive, and highly sensitive. We observed that Na+ in 10,000-fold molar excess does not interfere. The molecular mechanisms which produce enhanced fluorescence of berberine were discussed.

A New Fluorescence Turn-on Assay for Trypsin and Inhibitor Screening Based on Graphene Oxide

In this paper, we describe a new continuous fluorescence turn-on method for trypsin assay and inhibitor screening in situ. This assay is designed based on the following assumptions: (1) It is expected that the fluorescein-labeled peptide composed of six arginine residues (Arg(6)-FAM) with positive charges will interact with the negatively charged edge of water-soluble graphene oxide (GO) because of electrostatic interactions to form a GO/Arg(6)-FAM complex. As a result, the fluorescence of fluorescein will be quenched because of the energy transfer from fluorescein to GO. (2) Arg(6)-FAM can be hydrolyzed into small fragments in the presence of trypsin, and accordingly, the GO/Arg(6)-FAM complex will be dissociated, gradually leading to fluorescence recovery for the solution. In this way, the trypsin activity can be easily assayed with the ensemble of Arg(6)-FAM and GO. Additionally, the ensemble can be employed for screening of the inhibitors of trypsin.

Modulating DNA-templated silver nanoclusters for fluorescence turn-on detection of thiol compounds

Based on the fact that the fluorescence response pattern of a silver nanocluster to a specific analyte is highly dependent on the nature of the DNA template, we develop a novel fluorescence turn-on assay for thiol compounds with high specificity and sensitivity by modulating DNA-templated silver nanoclusters.