The detection of antibiotics is crucial for safeguarding the environment, ensuring food safety, and promoting human health. However, developing a rapid, convenient, low-cost, and sensitive method for antibiotic detection presents significant challenges. Herein, an aptamer-free biosensor was successfully constructed using upconversion nanoparticles (UCNPs) coated with silk fibroin (SF), based on Förster resonance energy transfer (FRET) and the charge-transfer effect, for detecting roxithromycin (RXM). A synergistic FRET efficiency was achieved by utilizing alizarin red and RXM complexes as energy acceptors, with UCNP as the energy donor, and immobilizing an ultrathin SF protein corona within 10 nm. The biosensor detects RXM in deionized water with high sensitivity primarily through monolayer adsorption, with a detection range of 1.0 nM-141.6 nM and a detection limit as low as 0.68 nM. The performance of this biosensor was compared with the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for detecting antibiotics in river water separately and a strong correlation between the two methods was observed. The biosensor exhibited long-term stability in aqueous solutions (up to 60 d) with no attenuation of fluorescence intensity. Furthermore, the biosensor\'s applicability extended to the highly sensitive detection of other antibiotics, such as azithromycin. This study introduces a low-cost, eco-friendly, and highly sensitive method for antibiotic detection, with broad potential for future applications in environmental, healthcare, and food-related fields.

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A \"linear\" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.

Investigating the structural integrity of carriers in transit from ocular surface to ocular posterior segment is essential for an efficient topical drug delivery system. In this study, dual-carrier hydroxypropyl-β-cyclodextrin complex@Liposome (HPCD@Lip) nanocomposites were developed for the efficient delivery of dexamethasone. Förster Resonance Energy Transfer with near-infrared I fluorescent dyes and in vivo imaging system were used to investigate the structural integrity of HPCD@Lip nanocomposites after crossing Human conjunctival epithelial cells (HConEpiC) monolayer and in ocular tissues. The structural integrity of inner HPCD complexes was monitored for the first time. The results suggested that 23.1 ± 6.4 % of nanocomposites and 41.2 ± 4.3 % of HPCD complexes could cross HConEpiC monolayer with an intact structure at 1 h. 15.3 ± 8.4 % of intact nanocomposites could reach at least sclera and 22.9 ± 1.2 % of intact HPCD complexes could reach choroid-retina after 60 min in vivo, which showed that the dual-carrier drug delivery system could successfully deliver intact cyclodextrin complexes to ocular posterior segment. In conclusion, in vivo assessment of structural integrity of nanocarriers is greatly significant for guiding the rational design, higher drug delivery efficiency and clinical transformation for topical drug delivery system to the posterior segment of the eye.

For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non-radiatively, and are promising alternatives to fluorescent FRET acceptors. Small-molecule (dark) quenchers have been widely used as acceptors in FRET-based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small-molecule quenchers that are used in FRET-based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore-quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Hypoxia, induced by inadequate oxygen supply, is a key indication of various major illnesses, which necessitates the need to develop new nanoprobes capable of sensing hypoxia environments for the targeted system monitoring and drug delivery. Herein, we report a hypoxia-responsive, periodic mesoporous organosilica (PMO) nanocarrier for repairing hypoxia damage. β-cyclodextrin (β-CD) capped azobenzene functionalization on the PMO surface could be effectively cleaved by azoreductase under a hypoxia environment. Moreover, the nanosystem is equipped with fluorescence resonance energy transfer (FRET) pair (tetrastyrene derivative (TPE) covalently attached to the PMO framework as the donor and Rhodamine B (RhB) in the mesopores as the receptor) for intracellular visualization and tracking of drug release in real-time. The design of intelligent nanocarriers capable of simultaneous reporting and treating of hypoxia conditions highlights a great potential in the biomedical domain.

Membrane proteins have key functions in signal transduction, transport, and metabolism. Therefore, deciphering the interactions between membrane proteins provides crucial information on signal transduction and the spatiotemporal organization of protein complexes. However, detecting the interactions and behaviors of membrane proteins in their native environments remains difficult. Förster resonance energy transfer (FRET) is a powerful tool for quantifying the dynamic interactions and assembly of membrane proteins without disrupting their local environment, supplying nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we briefly introduce the basic principles of FRET and assess the current state of progress in the development of new FRET techniques (such as FRET-FLIM, Homo-FRET and smFRET) for the analysis of plant membrane proteins. We also describe the various FRET-based biosensors used to quantify the homeostasis of signaling molecules and the active state of kinases. Furthermore, we summarize recent applications of these advanced FRET sensors in probing membrane protein interactions, stoichiometry, and protein clustering, which have shed light on the complex biological functions of membrane proteins in living plant cells.

Herein, we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles (CNPs) modified by acid oxidation. The fluorescence of the fluorescein-labelled peptide was quenched by CNPs. The sensor reacted with trypsin to cleave the peptide, resulting in the release of the dye moiety and a substantial increase in fluorescence intensity, which was dose- and time-dependent, and trypsin could be quantified accordingly. Correspondingly, the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity, with a detection limit of 0.7 μg/mL. The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range, suitable for point-of-care testing. Furthermore, the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.

MiRNA-150, a gene regulator that has been revealed to be abnormal expression in non-small cell lung cancer (NSCLC), can be regarded as a serum indicator for diagnosis and monitoring of NSCLC. Herein, a new sort of nanoprobe, termed allosteric spherical nanoprobe, was first developed to sense miRNA-150. Compared with conventional hairpin, this new nanoprobe possesses more enrichment capacity and reaction cross section. Structurally, it consists of magnetic nanoparticles and dual-hairpin. In the absence of miRNA-150, the spherical nanoprobes form hairpin structure through DNA self-assembly, which could promote the Förster resonance energy transfer (FRET) of fluorophore (FAM) and quencher (BHQ1) nearby. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active \"Y\" structure which separated fluorophore and quencher to yield \"signal on\" fluorescence. In the manner of multipoint fluorescence detection, the target-bound allosteric spherical nanoprobe could provide high detection sensitivity with a linear range of 100 fM to 10 nM and a detection limit of 38 fM. More importantly, the proposed method can distinguish the expression of serum miRNA-150 among NSCLC patients and healthy people. Finally, we hoped that the potential bioanalytical application of this nanoprobe strategy will pave the way for point-of-care testing (POCT).

Nanocrystals (NCs) have been introduced for use in pulmonary delivery in recent decades. Although the deposition and bioavailability have been extensively studied, little is known about the biofate, which influences the drug release and absorption process of NCs. In this study, we fabricated three different sized curcumin NCs by adjusting the parameters of mill machine using a wet milling method and studied the size effect on pulmonary absorption. The small nanocrystals (NC-S, 246.16 ± 21.98 nm) exhibited a faster dissolution rate and higher diffusion percentage in vitro compared with middle (NC-M, 535.26 ± 50.33 nm) and large nanocrystals (NC-L, 1089.53 ± 194.34 nm). Multiple particle tracking experiments revealed that NC-S had larger mean squared displacement during diffusion in simulated mucus of 0.5% hydroxyethyl cellulose solution. Moreover, enhanced cellular uptake and transport efficiency were achieved by NC-S in Calu-3 cells and an air-liquid interface culturing model. NCs were mainly absorbed in the dissolved drug form, as assessed by using the Förster resonance energy transfer (FRET) technique. In vivo lung retention and distribution revealed that few smaller sized nanocrystals were retained in the lung after intratracheal administration. The pharmacokinetic study showed that the AUC(0-t) values of small sized nanocrystals were 1.75- and 3.32-fold greater than NC-M and NC-L, respectively. In conclusion, this study demonstrated that smaller sized nanocrystals were more easily absorbed into the blood system by increasing the dissolution rate.

Extensive studies on nanomedicines have been conducted for drug delivery and disease diagnosis (especially for cancer therapy). However, the intracellular and in vivo biofate of nanomedicines, which is significantly associated with their clinical therapeutic effect, is poorly understood at present. This is because of the technical challenges to quantify the disassembly and behaviour of nanomedicines. As a fluorescence- and distance-based approach, the Förster Resonance Energy Transfer (FRET) technique is very successful to study the interaction of nanomedicines with biological systems. In this review, principles on how to select a FRET pair and construct FRET-based nanomedicines have been described first, followed by their application to study structural integrity, biodistribution, disassembly kinetics, and elimination of nanomedicines at intracellular and in vivo levels, especially with drug nanocarriers including polymeric micelles, polymeric nanoparticles, and lipid-based nanoparticles. FRET is a powerful tool to reveal changes and interaction of nanoparticles after delivery, which will be very useful to guide future developments of nanomedicine.