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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.

Non-covalent interactions between small molecules and nucleic acids are essential to many biological processes and the function of numerous therapeutic and diagnostic agents. These interactions are complex due to the structural diversity of DNA / RNA forms and the variety of possible binding modes. Factors such as nucleic acid topology, sequence, ionic conditions, and ligand aggregation further complicate analysis. Because no single technique can capture the full picture, a combination of spectroscopic and biochemical methods is required for accurate characterization. By combining working principles of four most common and available experimental methods with interpretative guidance and methodological details, this tutorial aspires to be a comprehensive reference for both novice and experienced researchers working in the field of small molecule-nucleic acid interactions. All discussed concepts apply equally to other types of DNA or RNA, and are generally relevant to most non-covalent ligand-biomolecule (including protein) interactions. Keywords: non-covalent binding to DNA, UV-Vis, fluorescence, thermal denaturation, circular dichroism, indicator displacement assay.

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 Zn 2+ -CQ affinity pair. Although the fluorescence enhancement effect of Zn 2+ 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 abstract Schematic illustration of the turn on-off fluorometric assay for for clioquinol (CQ) based on Zn 2+ -modulated sulfur quantum dots (SQDs).

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. A noninvasive and individual‐centered method is accomplished for monitoring uric acid (UA) in urine by executing indicator displacement assay, which successfully differentiates hyperuricemia patients from volunteers and targets a volunteer at high risk of hyperuricemia. To fit the family‐centered healthcare style, optical supramolecular sensing of UA is achieved by reading G value of images acquired with a portable smartphone.

Detection and quantification of trimethylamine -oxide (TMAO), a metabolite from gut microbial, is important for the disease diagnosis such as atherosclerosis, thrombosis and colorectal cancer. In this study, a novel method was established for the sensing and quantitative detection of TMAO via molecular recognition of guanidinium-modified calixarene from complex matrix. : Various macrocycles were tested for their abilities to serve as an artificial TMAO receptor. Using the optimized receptor, we developed an indicator displacement assay (IDA) for the facile fluorescence detection of TMAO. The quantification of TMAO was accomplished by the established calibration line after excluding the interference from the various interfering substances in artificial urine. : Among various macrocycles, water-soluble guanidinium-modified calix[5]arene (GC5A), which binds TMAO in submicromolar-level, was identified as the optimal artificial receptor for TMAO. With the aid of the GC5A•Fl (fluorescein) reporter pair, TMAO fluorescence \"switch-on\" sensing was achieved by IDA. The fluorescence intensity increased linearly with the elevated TMAO concentration. The detection was not significantly interfered by the various interfering substances. TMAO concentration in artificial urine was quantified using a calibration line with a detection limit of 28.88 ± 1.59 µM, within the biologically relevant low µM range. Furthermore, the GC5A•Fl reporter pair was successfully applied in analyzing human urine samples, by which a significant difference in fluorescence response was observed between the [normal + TMAO] and normal group. The proposed supramolecular approach provides a facile, low-cost and sensitive method for TMAO detection, which shows promise for tracking TMAO excretion in urine and studying chronic disease progression in humans.

Molecular containers are important tools to enhance the solubility and stability of bioactive compounds in water, and new macrocyclic scaffolds will expand the range of guests that can be encapsulated. α‐, β‐, and γ‐Cyclodextrin (CD), and derivatives thereof, are widely utilized for the complexation of active ingredients in product formulation, but large‐ring CDs, formed from more than eight glucose units, have been little explored due to a lack of availability. The recent development of a multi‐gram synthesis of δ‐CD (formed from nine glucopyranose units) means that this larger CD could now become a viable candidate for encapsulation of previously untargetable guests. Herein, it is reported that a bolaamphiphile with pyranine headgroups can act as a fluorescent probe for sensing of δ‐CD. Binding studies using fluorescence and NMR spectroscopy, as well as molecular dynamics simulations, reveal that partial unfolding of the bolaamphiphile upon binding to δ‐CD leads to fluorescence enhancement. It is showcased how this fluorescent probe can be implemented in an indicator displacement assay to detect host–guest interactions with δ‐CD. This proof‐of‐principle study paves the way forward toward high‐ throughput optical assays to screen libraries of bioactive guests and realize the potential of δ‐CD as a newly accessible molecular container. A molecular probe that partially unfolds and exhibits fluorescence enhancement upon binding to δ‐cyclodextrin (δ‐CD) is presented. Its implementation in a fluorescence indicator displacement assay to test the binding of various guest to δ‐CD is demonstrated.

Cellulose nanocrystals (CNCs) were successfully extracted and purified from cotton fabric waste via alkali treatment and acid hydrolysis. The resulting nanostructure was characterized using transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD), confirming the successful isolation of CNCs. A PV/Cu 2+ -CNCs/CNFs/PVA hydrogel was then synthesized through chemical crosslinking of polyvinyl alcohol (PVA), cellulose nanofibrils (CNFs), cellulose nanocrystals (CNCs), and borax. The incorporation of CNCs as a reinforcing agent significantly enhanced the hydrogel’s surface area and water absorption capacity, leading to a substantial improvement in the colorimetric sensitivity of the sensor. This hydrogel was employed as a non-invasive colorimetric sensor for urinary cysteine detection, utilizing an indicator-displacement assay (IDA) with pyrocatechol violet/copper ion (PV/Cu 2+ ) as the indicator. Upon exposure to cysteine, the sensor exhibited a distinct color change from greenish-blue to orange-red. The sensor demonstrated a linear detection range of 0–0.6 g/L and a limit of detection (LOD) of 0.017 g/L, effectively encompassing the clinical cut-off level for cysteine in human urine (0.25 g/L), relevant for Alzheimer’s disease indication. Eventually, this sensor was validated for cysteine determination in artificial urine samples, confirming its practical applicability. Graphical abstract

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)

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.

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.