Water-Soluble Triazolium Covalent Cages for ATP Sensing.

Water-soluble organic cages are attractive targets for their molecular recognition and sensing features of biologically relevant molecules. Here, we have successfully designed and synthesized a pair of water-soluble cationic cages employing click reaction as the fundamental step followed by the N-methylation of the triazole rings. The rigid and shape-persistent 3D hydrophobic cavity, positively charged surface, H-bonding triazolium rings, and excellent water solubility empower both cages to exhibit a superior affinity and selectivity for binding with adenosine-5'-triphosphate (ATP) compared to cyclophanes and other macrocyclic receptors. Both cage molecules (PCC⋅Cl and BCC⋅Cl) can bind a highly emissive dye HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) to form non-fluorescent complexes. The addition of ATP resulted in the stronger cage⊂ATP complexes with the retention of HPTS emission upon its displacement. The resultant indicator-displacement assay system can efficiently sense and quantify ATP in nanomolar detection limits in buffer solutions and human serum matrix. Spectroscopic and theoretical studies revealed the synergistic effect of π⋅⋅⋅π stacking interaction between the aromatic moiety of the cationic cages and the adenine moiety of ATP, as well as the electrostatic and hydrogen bonding interaction between the phosphate anion of ATP and triazole protons of cages, played the pivotal roles in the sensing process.

High-throughput screening of α-chiral-primary amines to determine yield and enantiomeric excess.

A novel screening protocol was developed using a combination of a fluorescent indicator displacement assay and a circular dichroism (CD) active Fe(II) complex to determine concentration and enantiomeric excess (ee) of α-chiral amines, respectively. The analyte concentration is quantified with a pre-formed non-fluorescent imine, where transimination with the chiral amine results in displacement of the fluorophore 2-naphthylamine. After discerning the concentration of amine via fluorescence in a wellplate reader, the analyte is then incorporated into a three-component octahedral Fe(II) assembly for ee determination using an EKKO CD plate-reader. With these two assays, both the ee and yield of asymmetric transformations of 192 samples could be determined with acceptable errors in under fifteen minutes (not counting the preparation time). This combined speed and accuracy provides an attractive solution to overcoming analytical bottlenecks when creating α-chiral amines.

Supramolecular Bioimaging through Signal Amplification by Combining Indicator Displacement Assay with Förster Resonance Energy Transfer.

Fluorescent chemosensors are powerful imaging tools in the fields of life sciences and engineering. Based on the principle of supramolecular chemistry, indicator displacement assay (IDA) provides an alternative approach for constructing and optimizing chemosensors, which has the advantages of simplicity, tunability, and modularity. However, the application of IDA in bioimaging continues to face a series of challenges, including interfering signals, background noise, and inconsistent spatial location. Accordingly, we herein report a supramolecular bioimaging strategy of Förster resonance energy transfer (FRET)-assisted IDA by employing macrocyclic amphiphiles as the operating platform. By merging FRET with IDA, the limitations of IDA in bioimaging were addressed. As a proof of concept, the study achieved mitochondria-targeted imaging of adenosine triphosphate in live cells with signal amplification. This study opens a non-covalent avenue for bioimaging with advancements in tunability, generality, and simplicity, apart from the covalent approach.

Fluorescent nanoprobe array based on carbon nanodots for qualitative and quantitative determination of biogenic polyamine.

A nanoprobe array based on fluorescent nitrogen-rich carbon dots (N-CDs) and Ag+ was constructed for simultaneous qualitative and quantitative determination of seven kinds of biogenic polyamines (BAs), including tryptamine (Try), histamine (His), putrescine (Put), cadaverine (Cad), spermine (Spm), spermidine (Spd), and agmatine (Agm). Ag+ can specifically bind to the N-CDs and quench the fluorescence of the N-CDs through a static mechanism. BAs further statically quench the fluorescence of the N-CD@Ag+ composite by bridging two Ag+ centers of the N-CD@Ag+. The nanoprobe array was constructed based on the differential fluorescence response arising from the differential binding affinity of various BAs. BAs can be differentiated and analyzed by the nanoprobe array within the concentration range 0.5-500 µM. The preliminary diluted and artificially spiked commercial human serum was utilized to simulate the serum environment for assessing the performance of the nanoprobe array in real samples. The N-CD@Ag+ system can recognize BAs with 100% accuracy in simulated human serum samples. The quantitative determination of BAs - no matter in a one-component system or a three-component system - was also realized by using the N-CD@Ag+ system even in the simulated serum environment. The recovery rates from spiked serum samples were higher 99%, and the relative standard deviation (RSD) was less than 3%. Based on the excellent multi-BA determination performance, a BA-related disease model about cerebral ischemia was constructed. Healthy cases as well as mild, moderate, and severe cerebral ischemia cases can be well identified from the disease model based on the N-CD@Ag+ nanoprobe array. Schematic representation of fluorescent nanoprobe array constructed by carbon nanodots (N-CDs) and Ag+ for qualitative and quantitative analyses of biogenic polyamines (BAs) and diagnosis of cerebral ischemia (CI) through linear discriminant analysis (LDA) and support vector machine (SVM).

Fluorescence Detection of Deoxyadenosine in Cordyceps spp. by Indicator Displacement Assay.

A rapid, sensitive and reliable indicator displacement assay (IDA) for specific detection of 2'- and 3'-deoxyadenosine (2'-dAde and 3'-dAde), the latter is also known as cordycepin, was established. The formation of inclusion complex between protonated acridine orange (AOH+) and cucurbit[7]uril (CB7) resulted in the hypochromic shift of fluorescent emission from 530 nm to 512 nm. Addition of cordycepin to the highly fluorescent AOH+/CB7 complex resulted in a unique tripartite AOH+/CB7/dAde complex with diminished fluorescence, and such reduction in emission intensity serves as the basis for our novel sensing system. The detection limits were 11 and 82 µM for 2'- and 3'-deoxyadenosine, respectively. The proposed method also demonstrated high selectivity toward 2'- and 3'-deoxyadenosine, owing to the inability of other deoxynucleosides, nucleosides and nucleotides commonly found in Cordyceps spp. to displace the AOH+ from the AOH+/CB7 complex, which was confirmed by isothermal titration calorimetry (ITC), UV-Visible and proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our method was successfully implemented in the analysis of cordycepin in commercially available Ophiocordyceps and Cordyceps supplements, providing a novel and effective tool for quality assessment of these precious fungi with several health benefits.

Catalytic Chemosensing Assay for Selective Detection of Methyl Parathion Organophosphate Pesticide.

Herein, a catalytic chemosensing assay (CCA), based on a bimetallic complex, [RuII (bpy)2 (CN)2 ]2 (CuI I)2 (bpy=2,2'-bipyridine), is described. This complex integrates a task-specific catalyst (CuI -catalyst) and a signaling unit ([RuII (bpy)2 (CN)2 ]) to specifically hydrolyze methyl parathion, a highly toxic organophosphate (OP) pesticide. The bimetallic complex catalyzed the hydrolysis of the phosphate ester to generate o,o-dimethyl thiophosphate (DTP) anion and 4-nitrophenolate. Intrinsically, 4-nitrophenolate absorbed UV/Vis light at λmax =400 nm, creating the first level of the chemosensing signal. DTP interacted with the original complex to displace the chromophore, [RuII (bpy)2 (CN)2 ], which was monitored by spectrofluorometry; this was classified as the second level of chemosensing signal. By integrating both spectroscopic and spectrofluorometric signals with a simple AND logic gate, only methyl parathion was able to provide a positive response. Other aromatic and aliphatic OP pesticides (diazinon, fenthion, meviphos, terbufos, and phosalone) and 4-nitrophenyl acetate provided negative responses. Furthermore, owing to the metal-catalyzed hydrolysis of methyl parathion, the CCA system led to the detoxification of the pesticide. The CCA system also demonstrated its catalytic chemosensing properties in the detection of methyl parathion in real samples, including tap water, river water, and underground water.

A novel dye-based colorimetric chemosensors for sequential detection of Cu2+ and cysteine in aqueous solution.

An IDAs based chemosensing ensembles for sensitive and selective sequential detection of Cu2+ and cysteine (Cys) in 100% aqueous solution was designed on the basis of the complex formation between 4-(2-Pyridylazo)resorcinol (PAR). In the first step, PAR was used for colorimetric detection of Cu2+ in aqueous solution by the obvious color change. The detection limit (31.0 nmol L-1) for Cu2+ much lower than the guideline (31.5 µmol L-1) of WHO in drinking water. In the second step the produced ensemble (PAR-Cu2+), sensitively and selectively detected a low concentration of Cys via indicator displacement assay system. The detection limit for Cys was determined to be 72 nmol L-1. The colorimetric detection operation is low-cost using PAR and copper ion and has a simple operation without any further modifications. Any enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentations are also not required in this experiment. It could find applications for the detection of analytes in environmental, biological samples based on these results, dual logic gates (IMPLICATION and INHIBIT) were obtained by controlling the chemical inputs.

Using zinc ion-enhanced fluorescence of sulfur quantum dots to improve the detection of the zinc(II)-binding antifungal drug clioquinol.

A turn on-off fluorometric assay for clioquinol (CQ) is described here. It is based on modulation of the fluorescence of sulfur quantum dots (SQDs; best measured at excitation/emission wavelengths of 360/426 nm) by using the Zn2+-CQ affinity pair. Although the fluorescence enhancement effect of Zn2+ on SQDs was not obvious, a good quenching modulation effect was observed in the presence of CQ. This resulted in a linear analytical range that is increased by two orders of magnitude (from 0.024 µM to 0.24 µM, and 0.62 µM to 30 µM), with a detection limit (3 s) of 0.015 µM. The selectivity of the method is also improved. Graphical abstractSchematic illustration of the turn on-off fluorometric assay for for clioquinol (CQ) based on Zn2+-modulated sulfur quantum dots (SQDs).

Fluorescent Trimethylated Naphthyridine Derivative with an Aminoalkyl Side Chain as the Tightest Non-aminoglycoside Ligand for the Bacterial A-site RNA.

The bacterial ribosomal decoding region of the aminoacyl-tRNA site (A-site) is one of the most validated target RNAs for antibiotic agents. Although natural aminoglycosides are well-characterized A-site binding ligands, high off-target effects and the growing emergence of bacterial resistance against aminoglycosides limit their clinical use. To circumvent these concerns with the aminoglycoside family, non-aminoglycoside A-site binding ligands have great potential as novel antibiotics against bacterial infections. This work describes a new class of small heterocyclic ligands based on the 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) structure for the bacterial (Escherichia coli) A-site. ATMND possessing an aminoethyl side chain is found to strongly and selectively bind to the internal loop of the A-site (Kd =0.44 µm; pH 7.0, I=0.06 m, 5 °C). Significantly, this ligand shows the tightest binding reported to date among non-aminoglycoside ligands. The binding study based on the thermodynamics and molecular modelling reveals key molecular interactions of ATMND-C2 -NH2 for high affinity to the A-site. This ligand is also demonstrated to be applicable to the fluorescence indicator displacement assay for assessing ligand/A-site interactions.

A Highly Selective and Strong Anti-Interference Host-Guest Complex as Fluorescent Probe for Detection of Amantadine by Indicator Displacement Assay.

Amantadine (AMA) and its derivatives are illicit veterinary drugs that are hard to detect at very low concentrations. Developing a fast, simple and highly sensitive method for the detection of AMA is highly in demand. Here, we designed an anthracyclic compound (ABAM) that binds to a cucurbit[7]uril (CB[7]) host with a high association constant of up to 8.7 × 108 M−1. The host-guest complex was then used as a fluorescent probe for the detection of AMA. Competition by AMA for occupying the cavity of CB[7] allows ABAM to release from the CB[7]-ABAM complex, causing significant fluorescence quenching of ABAM (indicator displacement assay, IDA). The linear range of the method is from 0.000188 to 0.375 µg/mL, and the detection limit can be as low as 6.5 × 10−5 µg/mL (0.35 nM). Most importantly, due to the high binding affinity between CB[7] and ABAM, this fluorescence host-guest system shows great anti-interference capacity. Thus, we are able to accurately determine the concentration of AMA in various samples, including pharmaceutical formulations.

Development of a New Colorimetric Chemosensor for Selective Determination of Urinary and Vegetable Oxalate Concentration Through an Indicator Displacement Assay (IDA) in Aqueous Media.

The paper proposes a method that exhibits operational simplicity for the indirect spectrophotometric determination of oxalate ion. We developed Reactive Blue 4 (RB4) as a sensor by complexation with copper ion as a simple, inexpensive yet selective colorimetric chemosensing ensemble for the recognition of oxalate over other available competitive analytes via indicator displacement assay (IDA) in both solution (aqueous medium) and solid state (paper-based experiment). The addition of oxalate to RB4-Cu2+ complex changed the colour from sky blue to dark blue due to the regeneration of RB4 by the chelation of oxalate as the competitive analyte with Cu2+. The absorbance band increase is linear with oxalate concentration from 1.76 to 49.4 µmol/L with a detection limit of 0.62 µmol/L. This measurement mode did not show any influence of interferences (available anions and ascorbic acid). This approach eliminated the need for the separation stages, enzymatic multiple-step reactions, sample preparation, organic solvent mixture, chemical modifications and equipment developed to a high degree of complexity. The oxalate determination gave results in different real samples such as urine, mushroom and spinach, which demonstrated the applicability of the existing method. Furthermore, this colorimetric system can serve as IMPLICATION molecular logic gate using Cu2+ and oxalate (C2O4 2-) as inputs and UV-Vis absorbance signal as the output with potential monitoring applications.

Strong binding and fluorescence sensing of bisphosphonates by guanidinium-modified calix[5]arene.

Based on the indicator displacement assay (IDA) approach, we herein report the fluorescence "switch-on" sensing and quantitative detection of bisphosphonates (BPs), a class of drugs extensively used in the treatment of patients with various skeletal diseases. Guanidinium-modified calix[5]arene (GC5A) affords strong binding on the micromolar to nanomolar level towards BPs dominantly via multiple salt bridge interactions, which was evaluated by fluorescence competitive titrations. Fluorescent IDA enables the highly sensitive and label-free detection of BPs in buffer solution, and more importantly, in artificial urine. Calibration lines were therefore set up in untreated artificial urine, allowing for quantifying the concentrations of BPs in the biologically relevant low range.

Analyzing urinary hippuric acid as a metabolic health biomarker through a supramolecular architecture.

The prevalence of metabolic disorders has been found to increase concomitantly with alternations in habitual diet and lifestyle, indicating the importance of metabolic health monitoring for early warning of high-risk status and suggesting effective intervention strategies. Hippuric acid (HA), as one of the most abundant metabolites from the gut microbiota, holds potential as a regulator of metabolic health. Accordingly, it is imperative to establish an efficient, sensitive, and affordable method for large-scale population monitoring, revealing the association between HA level and metabolic disorders. Upon systematic screening of macrocycle•dye reporter pair, a supramolecular architecture (guanidinomethyl-modified calix[5]arene, GMC5A) was employed to sense urinary HA by employing fluorescein (Fl), whose complexation behavior was demonstrated by theoretical calculations, accomplishing quantification of HA in urine from 249 volunteers in the range of 0.10 mM and 10.93 mM. Excitedly, by restricted cubic spline, urinary HA concentration was found to have a significantly negative correlation with the risk of metabolic disorders when it exceeded 0.76 mM, suggesting the importance of dietary habits, especially the consumption of fruits, coffee, and tea, which was unveiled from a simple questionnaire survey. In this study, we accomplished a high throughput and sensitive detection of urinary HA based on supramolecular sensing with the GMC5A•Fl reporter pair, which sheds light on the rapid quantification of urinary HA as an indicator of metabolic health status and early intervention by balancing the daily diet.

Enzymatic colorimetric method for turn-on determination of l-lactic acid through indicator displacement assay.

l-Lactic acid is a natural α-hydroxy carboxylic acid and is commonly used as an addictive. Quantitation of l-lactic acid is indispensable in food and cosmetic industries. An enzymatic colorimetric method was developed for the determination of l-lactic acid by competitive indicator displacement assay. Boric acid inhibited the colorimetric reaction of l-3,4-dihydroxyphenylalanine (l-DOPA) catalyzed by tyrosinase. l-Lactic acid competitively displaced and released l-DOPA bound with boric acid to serve as substrate, and thus restored the tyrosinase activity. Recovery of color reaction could be spectrophotometrically determined at 475 nm and was proportional to the amount of l-lactic acid. A calibration curve between l-lactic acid concentration and recovery of absorbance were built. The concentration range of the l-lactic acid was 0.25-2.25 mM. The limit of detection (LOD) and the limit of quantification (LOQ) for l-lactic acid was estimated to be 0.05 mM and 0.16 mM, respectively. The method achieved turn-on and visual sensing with good precision, accuracy, specificity, and robustness. The assay method exhibited a promising prospect to determine the content of l-lactic acid in foods and cosmetics.

Fluorescence "turn-on" sensing for five PDE5 inhibitors in functional food based on bimetallic nanoclusters.

Some phosphodiesterase type-5 (PDE5) inhibitors are active ingredients of prescription drugs that are widely used in the treatment of erectile dysfunction (ED). Recently, a large number of substances with this activity have been developed. Illegal addition of PDE5 inhibitors to foods could lead to cardiovascular diseases and even death, which poses a serious threat to food safety, therefore an on-site rapid screening method is urgently needed. Herein, a host functionalized bimetallic nanoclusters, CD/Au Ag NCs, were synthesized through self-assembly of 6-Aza-2-thiothymine gold nanoclusters (ATT-Au NCs), Arginine silver nanoclusters (Arg-Ag NCs) and carboxymethyl ß-cyclodextrin (ß-CMCD). The introduction of Rhodamine 6G (R6G) could quench the fluorescence of CD/Au Ag NCs based on the inner filter effect (IFE) and fluorescence resonance energy transfer effect (FRET). Importantly, it was discovered that several PDE5 inhibitors exhibited a higher binding affinity to ß-CMCD and could displace R6G binding with CD cavity, which disrupted the fluorescence quenching effects and resulted in the fluorescence recovery of CD/Au Ag NCs. This fluorescence turn-on signal could be utilized for the detection of PDE5 inhibitors. At present, emerging PDE5 inhibitor analogues pose a great challenge to food safety due to their unknown efficacy and safety. The proposed method holds the advantages of high sensitivity, simple probe synthesis, easy operation, and simultaneous detection of multiple PDE5 inhibitors. Meanwhile, the successful application in functional food sample demonstrated its high application potential in multiple PDE5 inhibitors screening.

Cucurbit[n]uril-based fluorescent indicator-displacement assays for sensing organic compounds.

The widespread conversion of synthetic receptors into luminescent sensors has been achieved via the use of fluorescent-indicator displacement assays (F-IDAs). Due to their rigid structures and efficient binding affinities, cucurbit[n]urils, combined with a variety of fluorescent guests, have gained extensive utilization in fluorescent-indicator displacement assays for sensing non-fluorescent or weakly fluorescent organic compounds (analytes) in a selective and specific manner. This mini-review summarizes recent advances in the design of cucurbit[n]uril-based fluorescent-indicator displacement assays and discusses the current challenges and future prospects in this area.

Graphene oxide-alginate hydrogel-based indicator displacement assay integrated with diaper for non-invasive Alzheimer's disease screening.

Pyrocatechol violet/copper ion-graphene oxide/alginate (PV/Cu2+-GO/Alg) hydrogel was fabricated and applied as a colorimetric sensor for monitoring urinary cysteine via an indicator-displacement assay (IDA) and Cu2+-cysteine affinity pair. The hydrogel-based sensor was formed by Ca2+ cations cross-linked PV/Cu2+-GO/Alg. The morphologies of hydrogel were characterized by field-emission scanning electron microscopy with energy-dispersive X-ray spectroscopy and Fourier-transform Raman spectroscopy. Incorporating GO into the hydrogel improved its uniformity of porosity, large surface area, and compressive strength, leading to amplified colorimetric signals of the hydrogel sensor. Under optimal conditions, this sensor offered a linear range of 0.0-0.5 g/L with a detection limit of 0.05 g/L for cysteine without interfering effects in urine. Furthermore, this hydrogel-based sensor was applied for urinary cysteine detection and validated with laser desorption ionization mass spectrometry. This platform could be used to determine cysteine at its cutoff (0.25 g/L) in human urine, which was distinguishable between normal and abnormal individuals, to evaluate an early stage of Alzheimer's disease. Eventually, this system was integrated with diapers for a wearable cysteine sensor.

Noninvasive and Individual-Centered Monitoring of Uric Acid for Precaution of Hyperuricemia via Optical Supramolecular Sensing.

Characterized by an excessively increased uric acid (UA) level in serum, hyperuricemia induces gout and also poses a great threat to renal and cardiovascular systems. It is urgent and meaningful to perform early warning by noninvasive diagnosis, thus conducing to blockage of disease aggravation. Here, guanidinocalix[5]arene (GC5A) is successfully identified from the self-built macrocyclic library to specifically monitor UA from urine by the indicator displacement assay. UA is strongly bound to GC5A at micromolar-level, while simultaneously excluding fluorescein (Fl) from the GC5A·Fl complex in the "switch-on" mode. This method successfully differentiates patients with hyperuricemia from volunteers except for those with kidney dysfunction and targets a volunteer at high risk of hyperuricemia. In order to meet the trend from hospital-centered to individual-centered testing, visual detection of UA is studied through a smartphone equipped with a color-scanning feature, whose adaptability and feasibility are demonstrated in sensing UA from authentic urine, leading to a promising method in family-centered healthcare style. A high-throughput and visual detection method is provided here for alarming hyperuricemic by noninvasive diagnosis.

Azocalixarene-Based Supramolecular System for the Detection of Paraquat via an Improved Indicator Displacement Assay.

In view of the lethal toxicity of paraquat (PQ) on human health, herein, a simple indicator displacement assay (IDA) based on an azo-modified calixarene host (azoCX[4]) and a fluorophore guest (p-DPD) were elaborately constructed for PQ detection in environmental water samples and plant surfaces. The fluorescent signal of p-DPD in the probe can be quenched by azoCX[4] through a photon-induced electron transfer process and recovered upon the addition of PQ within 10 s. The detection range of the p-DPD@azoCX[4] probe was calculated to be 0.35-8 µM in the Tris-HCl buffer solutions (pH = 7.4). Moreover, this probe exhibited excellent detection selectivity toward PQ over five herbicides (glyphosate, bispyribac, atrazine, ametryn, and bensulfuron methyl), together with anti-interference abilities in the presence of inorganic ions (K+, Na+, Zn2+, Ni2+, Li+, F-, Cl-, Br-, CO32-, HCO3-, and NO3-) and amino acids (Asp, Arg, Glu, Ala, and Cys). Particularly, the probe was successfully used to detect PQ in real water samples with acceptable accuracy and showed potential applications for on-site detection with paper-based test strips and on the leaf surface. We believe that this simplified IDA-based probe provided an effective detecting tool for PQ, and the design strategy would guide the further development of new IDA sensing systems.

The Detection of Food Additives Using a Fluorescence Indicator Based on 6- p-Toluidinylnaphthalence-2-sulfonate and Cationic Pillar[6]arene.

The current study investigated host-guest complexation in 6-p-toluidinylnaphthalene-2-sulfonate (TNS), a fluorescence probe used to investigate hydrophobic regions that contain the water-soluble cationic pillar[6]arene (CP6). After complexation with CP6, the fluorescence intensity of TNS was significantly increased. The decreases in the fluorescence intensity of the TNS•CP6 complex when phenolic food-additives are added have been used in indicator displacement assays to detect food additives in the water.