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Dual temperature- and photoresponsive polymer nanoparticles with sensitivity toward temperature variation through fluorescence resonance energy transfer (FRET)-assisted white light emission (WLE) were synthesized using distillation–precipitation polymerization. A fluorescence chemosensor of temperature, white light-emitting anticounterfeiting inks, and different products for security encryption of confidential documents were prepared on the basis of the FRET mechanism due to an efficient spectral overlap between the emission of the coumarin donor and the absorption of the rhodamine B (RhB) acceptor. At the 150 μL RhB doping, a strong WLE was observed as a consequence of the FRET mechanism between the two complementary colors. Upon increasing the temperature in the range of 25–85 °C, the efficient FRET distance was affected due to squeezing of the thermoresponsive polymer moieties in response to heating; thus, FRET was turned off. These stimulus-responsive fluorescent nanoparticles were finally used as temperature sensors and in security encryption applications by incorporating on/off WLE based on the FRET mechanism and the preparation of white light-emitting paper, silk, and ink through the incorporation of FRET species into paper pulp, impregnation of silk in the colloidal fluorescent nanoparticles, and adding poly-(ethylene glycol) and glycerol to the polymer colloids, respectively. [ABSTRACT FROM AUTHOR]

The creation of yolk–shell nanoparticles (YS NPs) with a movable core and periodic mesoporous organosilica (PMO) shell has been simplified using a " self-template" strategy. In this method, the knitting of the PMO shell and the formation of the interlayer cavity co-occur. This benefit contributes to the simplification of preparation processes and the realization of greater YS NP preparation efficiency with varied functional cores. By incorporating a silver nanocore into the YS NP (Ag-PMO-YS), a ratiometric Cu2+ fluorescence sensor is created by the "like dissolves like" principle driving the self-assembly of fluorescein derivatives and Cu2+ ligands within the surfactant micelles. Due to the coordination of Cu2+ and its ligands and their impact on the emission of fluorescein derivatives, which take part in the fluorescence resonance energy transfer (FRET) process with carbon dots (CDs), this sensor system demonstrates extraordinary detection sensitivity to Cu2+ (10–7 M). Additionally, the movable Ag nanocore's metal-enhanced fluorescence (MEF) effect strengthens the high detection sensitivity and stability. [ABSTRACT FROM AUTHOR]

Aggregation-induced emission (AIE) photosensitizers are promising for photodynamic therapy, yet their short excitation wavelengths present a limitation. In this study, we develop a series of organic photosensitizers with dual modulation capabilities based on excited-state intramolecular proton transfer (ESIPT) and AIE. Notably, we synthesize near-infrared (NIR)-excited photosensitive nanoparticles through a coassembly strategy utilizing upconversion nanoparticles (UCNPs) and amphiphilic polymers. The spectroscopic analysis and theoretical calculations elucidate the significant impact of additional or π-spacer groups on the conformational change and the energy barrier of the ESIPT process. An efficient Förster resonance energy transfer between the photosensitizer and UCNPs is achieved through the coassembly strategy. Both in vitro and in vivo experiments demonstrate the antitumor efficacy of these nanoparticles under NIR excitation. This work not only introduces a novel approach for simultaneously modulating AIE properties and the ESIPT process but also provides a robust solution for overcoming the excitation wavelength limitations of many organic photosensitizers.

Abnormal fluctuations in blood biomarker levels serve as critical indicators of the disease. However, detecting endogenous substances in whole blood using fluorescent probes is challenging due to its complex composition. This challenge primarily arises from two factors: the high autofluorescence of whole blood and the intrinsic fluorescence of the probe, both contributing to significant background fluorescence in the detection system. To overcome these obstacles, we introduced a donor-acceptor "one-to-many" FRET-based sensing strategy integrated with blood autofluorescence suppression to design a multifunctional fluorescent nanoprobe. The donor effectively suppresses blood autofluorescence through the inner filter effect and efficiently quenches donor fluorescence by adjusting the acceptor-to-donor ratio, achieving a "zero" background in whole blood detection. Leveraging this excellent background fluorescence quenching effect, we successfully detected endogenous ONOO - and ClO - levels in whole blood samples from mice with sepsis or hemolytic diseases. Furthermore, we monitored the changes in the ONOO - and ClO - levels throughout the disease course, revealing a positive correlation between the ONOO - and ClO - concentrations and disease severity. This innovative sensing strategy for achieving a "zero" background in whole blood detection provides valuable insights for designing fluorescent probes to directly detect endogenous substances in whole blood.

Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles remains challenging, limiting our understanding of the relationships between sequence, structure, and function. Combining single molecule Förster resonance energy transfer (smFRET) experiments with molecular dynamics simulations could provide experimentally grounded, all-atom models of a protein's structural ensemble. However, agreement between the two techniques is often insufficient to achieve this goal. Here, we explore whether accounting for important experimental details like averaging across structures sampled during a given smFRET measurement is responsible for this apparent discrepancy. We present an approach to account for this time-averaging by leveraging the kinetic information available from Markov state models of a protein's dynamics. This allows us to accurately assess which time scales are averaged during an experiment. We find this approach significantly improves agreement between simulations and experiments in proteins with varying degrees of dynamics, including the well-ordered protein T4 lysozyme, the partially disordered protein apolipoprotein E (ApoE), and a disordered amyloid protein (Aβ40). We find evidence for hidden states that are not apparent in smFRET experiments because of time averaging with other structures, akin to states in fast exchange in nuclear magnetic resonance, and evaluate different force fields. Finally, we show how remaining discrepancies between computations and experiments can be used to guide additional simulations and build structural models for states that were previously unaccounted for. We expect our approach will enable combining simulations and experiments to understand the link between sequence, structure, and function in many settings. Understanding protein dynamics is crucial for understanding protein function, yet few methodologies report on protein motion at an atomic level. Combining single molecule Förster resonance energy transfer (smFRET) experiments with computer simulations could provide atomistic models of protein ensembles which are grounded in experiments, however, there has been limited agreement between the two methods to date. Here, we present an algorithm to recapitulate smFRET experiments from molecular dynamics simulations. This approach significantly improves agreement between simulations and experiments for proteins across the ordered spectrum. Moreover, with this approach we can confidently create atomic models for states observed during smFRET experiments which were otherwise difficult to model due to high amounts of flexibility, disorder, or large deviations from crystal-like states.

We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units─a peptide and a peptide nucleic acid (PNA), fused together. The peptide presents the substrate of MMP2, while the PNA serves as a nucleic acid output for subsequent processing. This chemical translator was immobilized on micrometer magnetic beads as a physical support for an activity-based assay. We incorporated into our design a single-stranded DNA partially hybridized with the PNA sequence and bearing a region complementary to the RNA guide of CRISPR-Cas12a. The target-induced nuclease activity of Cas12a results in the degradation of FRET-labeled DNA reporters and amplified fluorescence signal, enabling the detection of MMP2 in the low picomolar range, showing a limit of detection of 72 pg/mL. This study provides new design principles for a broader applicability of CRISPR-Cas-based biosensing.

Simultaneous detection of different biomarkers related to the spatiotemporally dynamic immune events is of particular importance for the accurate evaluation of antitumor immune effects. Here, we have developed an AND-gate logic dual resonance energy transfer nanoprobe (named DRET) for dynamic monitoring of programmed CD8 + T cell activation and tumor cell apoptosis. Immunotherapy-induced granzyme B secretion from CD8 + T cells and the subsequent caspase-3 release from apoptotic tumor cells individually activate one of the tiers of the "AND-gate" logic DRET. The resulting fluorescence recovery and magnetic resonance T1 enhancement can be used for precise immunomodulatory drug screening, early efficacy prediction, and immune stratification. Particularly, not only "Responders" can be distinguished from "Non-responders", but also "Acquired resistance" can be identified from "Maintain responders", providing a novel approach to put forward the accurate evaluation of antitumor immunity.

Decorating a gold surface with molecular-level control over the positioning of DNA probes was demonstrated using a self-assembled monolayer (SAM) of wireframe DNA nanocube structures. The DNA nanocubes were specifically adsorbed and oriented using thiol-modified DNA on one face of the cube. The DNA nanocube SAM had a uniform coverage over the gold single crystal bead electrode with a separation of 20-30 nm measured by AFM. The face of the nanocube furthest from the gold surface was designed to hybridize with two different sequences of a 50 base single-stranded DNA probe that was modified with a fluorophore. The first 20 bases were hybridized with the DNA nanocube. One of a pair of FRET fluorophores was used for each probe strand. The dimensions of the nanocube controlled the relative spacing between these fluorophores. When the DNA probes were single-stranded, a FRET signal was observed. FRET decreased to background levels when a complementary DNA target was hybridized to either probe, resulting in a turn-off sensor with little cross-talk between the individual hybridization events. Hybridization isotherms for one target gave K A = 170 pM and a detection limit <50 pM. In addition, the DNA nanocube SAM was configured to be used as a turn-on NeutrAvidin sensor using biotinylated DNA targets hybridized to each probe resulting in an increase in FRET. We show that the wireframe DNA nanocube can be an effective scaffold for preparing biosensors with controlled separation between surface-bound probes facilitating precise sensor surface design and enabling a wide range of sensing modalities with more than one signal available for correlative confirmation of the target binding.

Sensing caspase-3 activity is essential for understanding the role of apoptosis in cancer dynamics, controlling therapeutic strategies, and improving patient care in cancer treatment. In this study, we demonstrate a highly sensitive recombinant human caspase-3 (rhC3) detection technique in biological fluids. This technique uses a copper nanocluster stabilized with bovine serum albumin (BSA-CuNCs) as a metal-based fluorescent biosensor, conjugated with anti-human caspase-3 (ahC3). To turn its fluorescence off, molybdenum disulfide nanosheets (MoS 2 NSs) are added; this partnership is termed ahC3@BSA-CuNCs/MoS 2 nanocouple. In the presence of rhC3, the energy transfer process is affected by strong ahC3/rhC3 interactions. When in close proximity, the rhC3 molecules cause detachment of the nanocluster from the MoS 2 NS surface by attracting the ahC3 component of the nanocluster. This increases the distance between the nanocluster and quencher with a consequent restoration of intensity. As the concentration of rhC3 increases, the fluorescence intensity of the system also increases. A proportional response is seen in the concentration between 0.1 and 1.3 ng/mL with a very low limit of detection of 2.75 pg/mL and a quantification limit of 8.60 pg/mL. A simple filter paper strip was made to visually identify the presence of rhC3 under UV light.

Cystatin C (Cys C), a crucial renal disease marker for chronic kidney disease (CKD), plays a vital role in early diagnosis and treatment guidance. However, most current methods for detecting Cys C rely on a single signal and find it difficult to perform noninvasive and portable diagnosis. Here, we developed a ratiometric fluorescent carbon dot (CD) detection system for point-of-care testing (POCT) of Cys C through fluorescence resonance energy transfer (FRET). The detection is based on the hydrolysis effect of papain on a bovine serum albumin (BSA) scaffold and the specific inhibitory effect of Cys C on papain, endowing high-resolution color variance. Moreover, a low-cost, portable, yet reliable smartphone-assisted miniaturized device for real-time quantitative POCT of Cys C has been developed with a limit of detection (LOD) as low as 0.4 μg/mL. This sensing platform can effectively differentiate patients from healthy volunteers, which facilitates self-screening for healthy individuals and home monitoring for CKD patients.

Amyloid β protein oligomer (AβO) has been found to be a crucial biomarker of Alzheimer's disease (AD). Exploring an accurate detection method of AβO is of great significance for early AD diagnosis and treatment. Herein, a fluorescent biosensor for AβO detection is designed. It integrates fluorescence resonance energy transfer (FRET)-based fluorescence detection with a poly-(dimethylsiloxane) (PDMS)-based microfluidic chip. The carboxyl fluorescein-modified AβO aptamers are employed as the fluorophore, and three-dimensional Fe3O4/MXene nanospheres are used as the fluorescence quencher for the first time. Moreover, the PDMS-based multichamber microfluidic chip serves as a fluorescence detection platform. The Fe3O4/MXene-based nanosphere-based biosensor shows an excellent linear relationship between the fluorescence intensity and the logarithm of the AβO concentration in the range of 0.10–200 nM. The detection limit is ∼0.05 nM at the ultralow sample volumes of ∼4.50 μL. These results strongly show that the fabricated high-performance biosensor can be widely used in smart healthcare including but not limited to medical diagnosis, health monitoring, and biological studies. [ABSTRACT FROM AUTHOR]

Cesium lead bromide quantum dot (CsPbBr3 QD) is emerging as a promising luminescent material for gamma ray detection. However, its intensive self-absorption traps the luminescent photons generated and diminishes the scintillation light yield. The self-absorption is magnified at high CsPbBr3 QD loading and large scintillator volume, which are required to effectively attenuate gamma photons. We report a liquid scintillator loaded with up to 40 wt % of CsPbBr3 QDs. Pyrromethene 580 (PM-580), a fluorescent dye, was co-dissolved in the solution as a fluorescence resonance energy-transfer acceptor to overcome the self-absorption of the QDs. The rapid energy transfer from the QDs to PM-580 also accelerates the scintillation decay kinetics. The scintillation light yield of the liquid containing 20 wt % QDs and 0.75 wt % PM-580 was 8800 photons/MeV. The decay lifetime is 24.3 ns, faster than most inorganic crystal scintillators. The light yield was 7300 photons/MeV at 40 wt % QD loading. Gamma pulse spectroscopy of the 40 wt % QD liquid produced the 662 keV gamma photopeak with a 27% energy resolution, demonstrating the potential of the CsPbBr3 QD loaded organic scintillators for spectroscopic gamma detection. [ABSTRACT FROM AUTHOR]

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters ( e.g. , ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain ( k cat / K M = 1.63 × 10 3 M -1 s -1 ) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide ( k I = 0.00325 s -1 ; t 1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

In situ visualization of microRNA (miRNA) in cancer cells and diseased tissues is essential for advancing our comprehension of the onset and progression of associated diseases. Two-photon (TP) imaging, as an imaging technology with high spatiotemporal resolution, deep tissue penetration, and accurate target quantification, has distinctive advantages over single-photon imaging and has attracted increasing attention. Extensive research has been conducted on two-photon dye-doped silica nanoparticles, which exhibit a large two-photon absorption (TPA) cross-section, high fluorescence quantum yield, and excellent biocompatibility. However, the low abundance of RNA in tumor cells leads to insufficient signal output. Based on functional nucleic acid, a catalyzed hairpin self-assembly (CHA) signal amplification strategy, which has simplicity, robustness, and nonenzymatic characteristics, can achieve the amplification of DNA or RNA signals. Here, a two-photon silica nanoamplifier (TP-SNA) utilizing TP dye-doped silica nanoparticles (SiNPs) and functional nucleic acid was constructed, employing triggering catalyzed hairpin self-assembly and fluorescence resonance energy transfer (FRET) for highly sensitive detection and precise TP imaging of endogenous miRNAs in tumor cells and tissues at varying depths. The TP-SNA demonstrated the capability to detect miR-203 with a detection limit of 33 pM. The maximum two-photon tissue penetration depth of the two-photon nanoamplifier was 210 μm. The two-photon nanoamplifier developed in this study makes full use of the advantages of accurate TP ratiometric bioimaging and the CHA signal amplification strategy, which shows good application value for future transformation into clinical diagnosis.

DNA polymerase β (Pol β) fills single nucleotide gaps during base excision repair. Deficiencies in Pol β can lead to increased mutagenesis and genomic instability in the cell, resulting in cancer. Our laboratory has previously shown that the I260 M somatic mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260 M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics and the nature and rates of conformational changes using steady-state fluorescence and Förster resonance energy transfer (FRET). Our data indicate that the I260 M mutation affects the fingers region of rat Pol β by creating a "collapsed" state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. I260 M is a temperature-sensitive mutator and binds nucleotides tighter than the WT protein, resulting in reduced fidelity compared to the WT. Additionally, we have generated a kinetic model of WT and I260 M using FRET and single turnover data, which demonstrates that I260 M precatalytic conformation changes differ compared to the WT as it is missing a precatalytic noncovalent step. Taken together, these results suggest that the collapsed state of I260 M may decrease its ability for nucleotide discrimination, illustrating the importance of the "fingers closing" conformational change for polymerase fidelity and accurate DNA synthesis.

O -GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme ( K D ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

Understanding the heterogeneity of molecular environments within cells is an outstanding challenge of great fundamental and technological interest. Cells are organized into specialized compartments, each with distinct functions. These compartments exhibit dynamic heterogeneity under high-resolution microscopy, which reflects fluctuations in molecular populations, concentrations, and spatial distributions. To enhance our comprehension of the spatial relationships among molecules within cells, it is crucial to analyze images of high-resolution microscopy by clustering individual pixels according to their visible spatial properties and their temporal evolution. Here, we evaluate the effectiveness of similarity metrics based on their ability to facilitate fast and accurate data analysis in time and space. We discuss the capability of these metrics to differentiate subcellular localization, kinetics, and structures of protein-RNA interactions in Forster resonance energy transfer (FRET) microscopy videos, illustrated by a practical example from recent literature. Our results suggest that using the correlation similarity metric to cluster pixels of high-resolution microscopy data should improve the analysis of high-dimensional microscopy data in a wide range of applications.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.

Much attention has been given to studying the translational diffusion of globular proteins, whereas the translational diffusion of intrinsically disordered proteins (IDPs) is less studied. In this study, we investigate the translational diffusion and how it is affected by the self-association of an IDP, κ-casein, using pulsed-field gradient nuclear magnetic resonance and time-resolved Förster resonance energy transfer. Using the analysis of the shape of diffusion attenuation and the concentration dependence of κ-casein diffusion coefficients and intermolecular interactions, we demonstrate that κ-casein exhibits continuous self-association. When the volume fraction of κ-casein is below 0.08, we observe that κ-casein self-association results in a macroscopic phase separation upon storage at 4 °C. At κ-casein volume fractions above 0.08, self-association leads to the formation of labile gel-like networks without subsequent macroscopic phase separation. Unlike α-casein, which shows a strong concentration dependence and extensive gel-like network formation, only one-third of κ-casein molecules participate in the gel network at a time, resulting in a more dynamic and less extensive structure. These findings highlight the unique association properties of κ-casein, contributing to a better understanding of its behavior under various conditions and its potential role in casein micelle formation.

Rapid proliferation and a faster rate of glycolysis in cancer cells often result in an elevated local temperature (40-43 °C) at the tumor site. Nanoparticles prepared from polymers with two lower critical solution temperatures (LCSTs) can be utilized to take advantage of this subtle temperature elevation to deliver anticancer drugs preferably to the cancer cells, thereby enhancing the overall therapeutic efficacy and reducing side effects. In this direction, we synthesized N -vinyl-2-pyrrolidone (NVP) and substituted NVP (sub-NVP: C 2 -NVP, C 4 -NVP)-based polymers with precisely controlled LCSTs by varying the ratio of NVP and sub-NVP. The first LCST (LCST1) was kept below 37 °C to promote self-assembly, drug loading, and structural stability in physiological conditions and the second LCST (LCST2) was in the range of 40-43 °C to ensure mild hyperthermia-induced drug release. Additionally, covalent attachment of tetraphenylethylene (TPE, AIEgen) resulted in aggregation-induced emission in thermoresponsive micellar nanoparticles in which TPE acted as a Förster Resonance Energy Transfer (FRET) pair with the loaded anticancer drug doxorubicin (DOX). Tracking of FRET-induced fluorescence recovery of TPE molecules was utilized to confirm the real-time thermoresponsive release of DOX from nanoparticles and eventual localization of TPE in the cytoplasm and DOX in the nucleus. In vitro cellular studies such as cytotoxicity, cellular uptake, and thermoresponsive drug release showed that the DOX-loaded polymeric nanoparticles were nontoxic to normal cells (HEK-293) but significantly more effective in cancer cells (MCF-7) at 40 °C. To our knowledge, this is the first report of preferential delivery of anticancer drugs only by exploiting the slightly elevated temperature of cancer cells.

Mitochondrial damage is implicated with a variety of pathological processes and is featured by the dramatically raised NADH level. To date, the detection and in situ imaging of NADH in living animals is still a challenge. Herein, we designed an upconversion luminescent nanoprobe for the in vivo imaging of NADH. The Nile blue (NB) with a strong absorption band at 500–700 nm can quench the red luminescence of the upconversion nanoparticle at 654 nm by the fluorescence resonance energy transfer effect. Under the presence of NADH, the NB was bleached, so that the fluorescence of the probe was "turned on". The nanoprobe achieved highly selective detection of NADH, and the in vivo monitoring of NADH was also demonstrated on living mice. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) have proven to be promising delivery vehicles for RNA-based vaccines and therapeutics, particularly in LNP formulations containing ionizable cationic lipids that undergo protonation/deprotonation in response to buffer pH changes. These nanoparticles are typically formulated using a rapid mixing technique at low pH, followed by a return to physiological pH that triggers LNP-LNP fusion. A detailed understanding of these dynamic processes is crucial to optimize the overall performance and efficiency of LNPs. However, knowledge gaps persist regarding how particle formation mechanisms impact drug loading and delivery functions. In this work, we employ single-molecule Convex Lens-induced Confinement (CLiC) microscopy in combination with Förster resonance energy transfer (FRET) measurements to study LNP fusion dynamics in relation to various formulation parameters, including lipid concentration, buffer conditions, drug loading ratio, PEG-lipid concentrations, and ionizable lipid selection. Our results reveal a strong correlation between the measured fusion dynamics and the formulation parameters used; these findings are consistent with DLS and Cryo-TEM-based assays. These measurements offer a cost-effective method for characterizing and screening potential drug candidates and can provide additional insights into their design, with opportunities for optimization.

A major impediment to the clinical translation of DNA tiling nanostructures is a technical bottleneck for the programmable assembly of DNA architectures with well-defined local geometry due to the inability to achieve both sufficient structural rigidity and a large framework. In this work, a Y-backbone was inserted into each face to construct a superlarge, sufficiently rigidified tetrahedral DNA nanostructure (called RDT) with extremely high efficiency. In RDT, the spatial size increased by 6.86-fold, and the structural rigidity was enhanced at least 4-fold, contributing to an ∼350-fold improvement in the resistance to nucleolytic degradation even without a protective coating. RDT can be mounted onto an artificial lipid-bilayer membrane with molecular-level precision and well-defined spatial orientation that can be validated using the fluorescence resonance energy transfer (FRET) assay. The spatial orientation of Y-shaped backbone-rigidified RDT is unachievable for conventional DNA polyhedrons and ensures a high level of precision in the geometric positioning of diverse biomolecules with an approximately homogeneous environment. In tests of RDT, surface-confined horseradish peroxidase (HRP) exhibited nearly 100% catalytic activity and targeting aptamer-immobilized gold nanoparticles showed 5.3-fold enhanced cellular internalization. Significantly, RDT exhibited a 27.5-fold enhanced structural stability in a bodily environment and did not induce detectable systemic toxicity.

Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with different head groups (imidazolium, morpholinium, and pyridinium) and alkyl chain lengths (ranging from decyl to tetradecyl). Analysis of the protein fluorescence spectra indicates noticeable changes in the secondary structure of HSA with varying concentrations of all SAILs tested. Helicity calculations based on the Fourier transform infrared (FTIR) data show that HSA becomes more organized at the micellar concentration of SAILs, leading to an increased protein activity at this level. Small-angle neutron scattering (SANS) data confirm the formation of a bead-necklace structure between the SAILs and HSA. Atomistic molecular dynamics (MD) simulation results identify several hotspots on the protein surface for interaction with SAIL, which results in the modulation of protein conformational fluctuation and stability. Furthermore, fluorescence resonance energy transfer (FRET) experiments with the intramolecular charge transfer (ICT) probe trans -ethyl p -(dimethylamino) cinnamate (EDAC) demonstrate that higher alkyl chain lengths and SAIL concentrations result in a significantly increased energy transfer efficiency. The findings of this study provide a detailed molecular-level understanding of how the protein structure and function are affected by the presence of SAILs, with potential implications for a wide range of applications involving protein-SAIL composite systems.

Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.

Herein, CsPbBr 3 perovskite quantum dots (CPB PQDs)@poly(methyl methacrylate) (PMMA) (CPB@PMMA) nanospheres were used as energy donors with high Förster resonance energy transfer (FRET) efficiency and exceptional biocompatibility for ultrasensitive dynamic imaging of tiny amounts of microRNAs in living cells. Impressively, compared with traditional homogeneous single QDs as energy donors, CPB@PMMA obtained by encapsulating numerous CPB PQDs into PMMA as energy donors could not only significantly increase the efficiency of FRET via improving the local concentration of CPB PQDs but also distinctly avoid the problem of cytotoxicity caused by divulged heavy metal ions entering living cells. Most importantly, in the presence of target miRNA-21, DNA dendrimer-like nanostructures labeled with 6-carboxy-tetramethylrhodamine (TAMRA) were generated by the exposed tether interhybridization of the Y-shape structure, which could wrap around the surface of CPB@PMMA nanospheres to remarkably bridge the distance of FRET and increase the opportunity for effective energy transfer, resulting in excellent precision and accuracy for ultrasensitive and dynamic imaging of miRNAs. As proof of concept, the proposed strategy exhibited ultrahigh sensitivity with a detection limit of 45.3 aM and distinctly distinguished drug-irritative miRNA concentration abnormalities with living cells. Hence, the proposed enzyme-free CPB@PMMA biosensor provides convincing evidence for supplying accurate information, which could be expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

The guanine-rich telomeric repeats can form G-quadruplexes (G4s) that alter the accessibility of the single-stranded telomeric overhang. In this study, we investigated the effects of Na + and K + on G4 folding and accessibility through cation introduction and exchange. We combined differential scanning calorimetry (DSC), circular dichroism (CD), and single molecule Förster resonance energy transfer (smFRET) to monitor the stability, conformational dynamics, and complementary strand binding accessibility of G4 formed by single-stranded telomeric DNA. Our data showed that G4 formed through heating and slow cooling in K + solution exhibited fewer conformational dynamics than G4 formed in Na + solution, which is consistent with the higher thermal stability of G4 in K + . Monitoring cation exchange with real time smFRET at room temperature shows that Na + and K + can replace each other in G4. When encountering high K + at room or body temperature, G4 undergoes a slow conformational rearrangement process which is mostly complete by 2 h. The slow conformational rearrangement ends with a stable G4 that is unable to be unfolded by a complementary strand. This study provides new insights into the accessibility of G4 forming sequences at different time points after introduction to a high K + environment in cells, which may affect how the nascent telomeric overhang interacts with proteins and telomerase.

The prevailing but not undisputed amyloid cascade hypothesis places the β-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3 , offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.

Ultraphotostable phosphorescent nanosensors have been designed for continuously sensing the lysosome pH over a long duration. The nanosensors exhibited excellent photostability, high accuracy, and capability to measure pH values during cell proliferation for up to 7 days. By arranging a metal-ligand complex of long phosphorescence lifetime and pH indicator in silica nanoparticles, we discover efficient Förster resonance energy transfer, which converts the pH-responsive UV-vis absorption signal of the pH indicator into a phosphorescent signal. Both the phosphorescent intensity and lifetime change at different pH values, and intracellular pH values can be accurately measured by our custom-built rapid phosphorescent lifetime imaging microscopy. The excellent photostability, high accuracy, and good biocompatibility prove that these nanosensors are a useful tool for tracing the fluctuation of pH values during endocytosis. The methodology can be easily adapted to design new nanosensors with different p K a or for different kinds of intracellular ions, as there are hundreds of pH and ion indicators readily available.

Probing the structural characteristics of biomolecular ions in the gas phase following native mass spectrometry (nMS) is of great interest, because noncovalent interactions, and thus native fold features, are believed to be largely retained upon desolvation. However, the conformation usually depends heavily on the charge state of the species investigated. In this study, we combine transition metal ion Förster resonance energy transfer (tmFRET) and ion mobility-mass spectrometry (IM-MS) with molecular dynamics (MD) simulations to interrogate the β-hairpin structure of GB1p in vacuo. Fluorescence lifetime values and collisional cross sections suggest an unfolding of the β-hairpin motif for higher charge states. MD simulations are consistent with experimental constraints, yet intriguingly provide an alternative structural interpretation: preservation of the β-hairpin is not only predicted for 2+ but also for 4+ charged species, which is unexpected given the substantial Coulomb repulsion for small secondary structure scaffolds.

DNA circuits, as a type of biochemical system, have the capability to synchronize the perception of molecular information with a chemical reaction response and directly process the molecular characteristic information in biological activities, making them a crucial area in molecular digital computing and smart bioanalytical applications. Instead of cascading logic gates, the traditional research approach achieves multiple logic operations which limits the scalability of DNA circuits and increases the development costs. Based on the interface reaction mechanism of Lambda exonuclease, the molecular perceptron proposed in this study, with the need for only adjusting weight and bias parameters to alter the corresponding logic expressions, enhances the versatility of the molecular circuits. We also establish a mathematical model and an improved heuristic algorithm for solving weights and bias parameters for arbitrary logic operations. The simulation and FRET experiment results of a series of logic operations demonstrate the universality of molecular perceptron. We hope the proposed molecular perceptron can introduce a new design paradigm for molecular circuits, fostering innovation and development in biomedical research related to biosensing, targeted therapy, and nanomachines.

Telomerase is an important biomarker for early diagnosis of cancers, but current telomerase assays usually rely on measuring the extension products of telomerase substrates, which increases the assay complexity. More evidence indicates that human telomerase RNA (hTR), as a core component of telomerase, is positively correlated with the telomerase activity. Herein, we demonstrate the development of a duplex-specific nuclease (DSN)-propelled 3D quantum dot (QD) nanoassembly with two-step Föster resonance energy transfer (FRET) for the one-step sensing of hTR in breast cancer cells and tissues. This assay involves only one hairpin probe modified with a Cy5 at the sixth base from the 5'-biotin end and a BHQ2 at the 3'-terminus, which integrates three functions of target recognition, target recycling amplification, and signal readout. The anchoring of the hairpin probe on the 605QD surface results in the formation of a 3D 605QD-Cy5-probe-BHQ2 nanoassembly in which two-step FRET occurs among the 605QD, Cy5, and BHQ2 quencher. Notably, the formation of 605QD-Cy5-probe-BHQ2 nanoassembly facilitates the reduction of background signal and the increase of signal-to-background ratio due to its dense, highly oriented nucleic acid shell-induced steric hindrance effect. This assay can achieve one-step and rapid detection of hTR with a detection limit of 2.10 fM, which is the simplest and most rapid hTR assay reported so far. Moreover, this assay can efficiently distinguish single-base mismatched sequences, and it can discriminate the hTR level between breast cancer patients and healthy donors with a high accuracy of 100%, with great prospects for early diagnosis of cancers.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL -1 ) with a limit of detection of 0.02 ng mL -1 . Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Intracellular cancer-related biomarker imaging strategy has been used for specific identification of cancer cells, which was of great importance to accurate cancer clinical diagnosis and prognosis studies. Localized DNA circuits with improved sensitivity showed great potential for intracellular biomarkers imaging. However, the ability of localized DNA circuits to specifically image cancer cells is limited by off-site signal leakage associated with a single-biomarker sensing strategy. Herein, we integrated the endogenous enzyme-powered strategy with logic-responsive and localized signal amplifying capability to construct a self-assembled endogenously AND logic DNA nanomachine (EDN) for highly specific cancer cell imaging. When the EDN encountered a cancer cell, the overexpressed DNA repairing enzyme apurinic/apyrimidinic endonuclease 1 (APE1) and miR-21 could synergistically activate a DNA circuit via cascaded localized toehold-mediated strand displacement (TMSD) reactions, resulting in amplified fluorescence resonance energy transfer (FRET) signal. In this strategy, both endogenous APE1 and miR-21, served as two "keys" to activate the AND logic operation in cancer cells to reduce off-tumor signal leakage. Such a multiplied molecular recognition/activation nanomachine as a powerful toolbox realized specific capture and reliable imaging of biomolecules in living cancer cells.

Alpha-synuclein (α-Syn) is a key protein of Parkinson's disease (PD). Oligomers formed by misfolding and aggregation of α-Syn can cause many pathological phenomena and aggravate the development of PD. Therefore, sensitive and accurate detection of oligomers is essential to understanding the pathology of PD and beneficial to screening and developing new drugs against PD. Here, we demonstrated a simple and sensitive method to detect the early aggregation of α-Syn via Förster resonance energy transfer (FRET) technology. We performed systematic investigations of the FRET sensitizations, efficiencies, and donor-to-acceptor distances during α-Syn aggregation, which was proved to be more sensitive to reflect small distance changes in the early stage of α-Syn aggregation, especially for α-Syn oligomers. The FRET assays were also applied to study the influence of Ser129 phosphorylation (pS129) on the aggregation rate of α-Syn. Our results showed that pS129 modification promotes α-Syn aggregation and enhances the ability of preformed fibrils to induce monomer aggregation. pS129 also increased the cytotoxicity of α-Syn. These results are of great significance for a better understanding of the pathological mechanisms of PD and future PD drug development.

Different phases of lyotropic liquid crystals (LLCs), made up of mesogen-like sodium dodecyl sulfate (SDS), mainly bestow different bulk viscosities. Along with this, the role of microviscosities of the individual LLC phases is of immense interest because a minute change in it due to guest incorporation can cause significant alteration in their property as a potential energy transfer scaffold. Recently, LLCs have been identified as plausible drug delivery agents for ocular treatments. In this direction, the present work illustrates photophysical modulations of an important laser dye as well as an ophthalmic medicine, coumarin 6 (C6), inside different LLC phases in an aqueous medium. C6 molecules spontaneously accumulate in water, leading to aggregation-caused quenching (ACQ) of fluorescence. However, the different phases of the LLCs prepared from SDS and water helped in disintegrating the C6 colonies to various extents depending upon the microviscosity. The heterogeneity in the LLC phases, in turn, could modulate the Förster resonance energy transfer (FRET) between C6 and the LLC incorporated with N-doped carbon nanoparticles (N-CNPs). The N-CNPs act as potential photosensitizers and generate singlet oxygen ( 1 O 2 ), a reactive oxygen species (ROS), to different extents. Microviscosities of the prepared LLCs were calculated by using fluorescence correlation spectroscopy (FCS). The different phases of the LLCs, viz., lamellar and hexagonal, with different microviscosities controlled the extent of C6 disaggregation and hence the FRET and the ROS generation. The results are encouraging since ROS generation has a significant role in the vision mechanism and PDT-based applications. LLC-based drug administration with potential FRET to control ROS generation may become handy in ophthalmology. The LLC phases used in this experiment not only served the purpose of drug delivery but also the photophysical events therein are compatible with the ocular environment.

The massive use of pyrethroid pesticides in agriculture has brought growing concerns about food safety due to their several harmful effects on human health, especially through the accumulation of the food chain. To date, most of the available analytical methods for pyrethroids still suffer from insufficient detection universality, complicated sample pretreatment, and detection processes, which severely limit their practical applications. Herein, a novel Förster resonance energy transfer (FRET)-assisted host-guest supramolecular nanoassembly is reported, for the first time, successfully realizing ratiometric fluorescent detection of pyrethroids in real samples through the indicator displacement assay (IDA) mechanism. This method is capable of detecting a broad spectrum of pyrethroids, including bifenthrin, cyfluthrin, cypermethrin, deltamethrin, etofenprox, fenvalerate, and permethrin, with ultrahigh detection sensitivity, great selectivity, high anti-interference ability, and, in particular, distinct emission color response from red to green. Such a large chromatic response makes this method available for fast and on-site detection of pyrethroids in real samples with the aid of several simple portable analytical apparatuses.

Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb → QD → AF647 → Cy5.5 (→ = ET step), which is detected in a time-gated modality, and QD → AF647 → Cy5.5, which is detected from direct excitation. The labeled DNA-displaying QD construct is then further assembled with a Ru II -modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.

Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-β (Αβ) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aβ complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αβ species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aβ-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αβ, which discloses the binding region and the inhibitor's orientation.

The efficiency of the enzyme-free toehold-mediated strand displacement (TMSD) technique is often insufficient to detect single-nucleotide polymorphism (SNP) that possesses only single base pair mismatch discrimination. Here, we report a novel dual base pair mismatch strategy enabling TMSD biosensing for SNP detection under enzyme-free conditions when coupled with catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET). The strategy is based on a competitive strand displacement reaction mechanism, affected by the thermodynamic stability originating from rationally designed dual base pair mismatch, for the specific recognition of mutant-type DNA. In particular, enzyme-free nucleic acid circuits, such as CHA, emerge as a powerful method for signal amplification. Eventually, the signal transduction of this proposed biosensor was determined by FRET between streptavidin-coated 605 nm emission quantum dots (605QDs, donor) and Cy5/biotin hybridization (acceptor, from CHA) when incubated with each other. The proposed biosensor displayed high sensitivity to the mutant target (MT) with a detection concentration down to 4.3 fM and led to high discrimination factors for all types of mismatches in multiple sequence contexts. As such, the application of this proposed biosensor to investigate mechanisms of the competitive strand displacement reaction further illustrates the versatility of our dual base pair mismatch strategy, which can be utilized for the creation of a new class of biosensors.

Photoluminescence (PL) imaging and bioanalysis with smartphone-based devices are of growing interest for point-of-care/point-of-need diagnostics. Strategies for maximizing sensitivity have been explored in this context, but color multiplexing has been very limited, with its maximum level unexplored. Here, we evaluated color multiplexing with smartphone-based PL imaging by using supra-nanoparticle assemblies of quantum dots (supra-QDs). These materials were prepared as composite colors that were tailored to the red-green-blue (RGB) color space of smartphone cameras by coassembling different ratios of R-, G-, and B-emitting QDs on a silica nanoparticle scaffold. The supra-QDs were characterized and used to label cell-sized objects that were measured under flow with a smartphone-based device. Each color followed an approximately linear trajectory in the RGB space, and training of support vector machine models enabled color classification with overall accuracies ≥87% for 10-color multiplexing and better accuracies for fewer colors. Most misclassification occurred at low signal levels, such that establishing a nonclassifiable zone near the origin of RGB color space improved the overall 10-color classification accuracy to ≥94%. Similar improvements in accuracy with greater retention of data were possible with a probabilistic rather than a radial threshold. Simulations that were parameterized by experimental data suggested that ≥14-color multiplexing with accuracies ≥90% should be possible with an optimized supra-QD color set. This study is an important foundation for advancing RGB color-based multiplexing for imaging and analyses with smartphone cameras and related charge-coupled device and CMOS color image sensor technologies.

Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GP FRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters but the local effects were considerably stronger (GP FRET ≫ GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby enhancing the depth of information obtained from the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures globally, imposing a substantial burden on patients and their families. The pathological role of peroxynitrite (ONOO - ), which can trigger oxidative stress, inflammation, and neuronal hyperexcitability, is critical in epilepsy. However, the development of reliable, in situ, and real-time optical imaging tools to detect ONOO - in the brain encounters some challenges related to the depth of tissue penetration, background interference, optical bleaching, and spectral overlapping. To address these limitations, we present Ir-CBM , a new one-photon and two-photon excitable and long-lived ratiometric luminescent probe designed specifically for precise detection of ONOO - in epilepsy-based on the Förster resonance energy transfer mechanism by combining an iridium(III) complex with an organic fluorophore. Ir-CBM possesses the advantages of rapid response, one-/two-photon excitation, and ratiometric luminescent imaging for monitoring the cellular levels of ONOO - and evaluating the effects of different therapeutic drugs on ONOO - in the brain of an epilepsy model rat. The development and utilization of Ir-CBM offer valuable insights into the design of ratiometric luminescent probes. Furthermore, Ir-CBM serves as a rapid imaging and screening tool for antiepileptic drugs, thereby accelerating the exploration of novel antiepileptic drug screening and improving preventive and therapeutic strategies in epilepsy research.

The label with a large Stokes shift and strong fluorescence properties could improve the sensitivity of the lateral flow immunoassay (LFIA). Herein, two aggregation-induced emission (AIE) luminogens with spectral overlap were encapsulated in polymers by using the microemulsion method as a label, and the construction of a fluorescence resonance energy transfer mode was further verified via theoretical calculation and spectral analysis. Satisfactorily, the doped AIE polymer microspheres (DAIEPMs) exhibited a large Stokes shift of 285 nm and a 10.8-fold fluorescence enhancement compared to those of the AIEPMs loaded with acceptors. Benefiting from the excellent optical performance, DAIEPMs were applied to the LFIA for sensitive detection of chlorothalonil, which is an organochlorine pesticide. The limit of detection of the proposed DAIEPMs-LFIA was 1.2 pg/mL, which was 4.8-fold and 11.6-fold lower than those of quantum dot bead LFIA and gold nanoparticle LFIA, respectively. This work provides a new strategy to improve the optical properties of fluorescent materials and construct a sensitive and reliable detection platform.

Optical imaging and photoacoustic (PA) imaging have become essential tools to investigate physiological or pathological processes at the molecular level in vivo . The detection of variations at the molecular level in vivo is particularly important owing to the rapid progression of diseases. However, most studies have mainly focused on plain qualitative molecular imaging and detection, which is characterized by the absence of a reference signal in one-channel responsive imaging. To overcome the limitation and quantitatively detect molecules in situ , this Account reviews the recent contributions of our group to the quantitative imaging field in the form of ratiometric optical and PA imaging in vivo in the second near-infrared window (NIR-II, 950-1700 nm).In this Account, we present recent advances that our group has made in ratiometric imaging probe design and biomedical applications by constructing probes based on ratiometric optical imaging and ratiometric PA imaging. First, we highlight the design strategies of ratiometric optical probes that were based on organic ratiometric molecular probes, radio-activated organic ratiometric probes, and hybrid organic-inorganic assembled ratiometric probes. Subsequently, the design strategies of the ratiometric NIR-II optical nanoprobes with activated bioluminescence resonance energy transfer (BRET), Förster resonance energy transfer (FRET), and nonradiative energy transfer (NRET) effects provide a reliable tool to achieve the ratiometric detection of endogenous signaling molecules and thereby apply it to the monitoring and evaluation of the efficacy of photodynamic therapy, radiotherapy, and immunotherapy to guide the treatment process. In addition, we systematically introduce the functional design principles of ratiometric PA imaging probes based on core-shell nanoprobes, core-satellite nanoprobes, and universal hybrid nanoprobes, where we have established that reference signal and sensing signal can be obtained from the random assortment of plasmonic components and organic semiconducting molecules using a phase separation strategy. On these insights, we discuss the rational and detailed biomedical applications of ratiometric PA imaging probes which include accurate quantitative detection of disease-related molecules in inflammation or tumors in real time. In these champion implementations of ratiometric PA imaging probes, different diagnostic modules have been linked through compound modification with activation characteristics (e.g., pH, redox, enzyme, hypoxia). Finally, we present the challenges and perspectives for ratiometric probes based on optical imaging and PA imaging for multitarget design and future clinical translation. We believe that the upcoming generations of ratiometric imaging probes would have promising potential applications in the precise diagnosis of diseases. Finally, this Account may stimulate innovative studies in the design of ratiometric imaging probes and exploration of their clinical applications.

Near-infrared (NIR)-responsive bioassays based on upconversion nanoparticle (UCNP) incorporating high-performance semiconductors have been developed by researchers, but most lack satisfactory ultrasensitivity for exceedingly trace amounts of target. Herein, for the first time, the CRISPR/Cas13a system is combined with cascade DNA circuits, fluorescent resonance energy transfer (FRET) effect, and luminescence-confined UCNPs-bonded CuInS 2 /ZnO p - n heterostructures-functionalized paper-working electrode to construct dual-signal-on paper-supported NIR-irradiated photoelectrochemical (PEC) (NIR-PEC) and upconversion luminescence (UCL) bioassay for high-sensitive quantification of miRNA-106a (miR-106a). By constructing an ideal FAM-labeled aminating molecular beacon (FAM-H2) model, a relatively good FRET ratio between the UCNP and FAM (≈85.3%) can be achieved. In the existence of miR-106a, the hairpin-structure FAM-H2 was unwound, bringing about the distance increase of UCNP and FAM and the restraint of FRET. Accordingly, both the NIR-PEC signal and the UCL intensity gradually recovered distinctly. Unlike conventional single-mode PEC sensors, with NIR excitation, the designed dual-mode sensing system could implement minimized misdiagnose assay and quantitative miR-106a determination with low detection limits, that is, 76.54 and 51.36 aM for NIR-PEC and UCL detection, respectively. This work not only broadens the horizon of application of the CRISPR/Cas13a strategy toward biosensing but also constructs a new structure of the UCNP-semiconductor in the exploration of efficient NIR-responsive tools and inspires the construction of a no-misdiagnosed and novel biosensor for dual-mode liquid biopsy.

Nucleic acids, as one kind of significant biomarker, have attracted tremendous attention and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and a donor donor-acceptor fluorescence resonance energy transfer ("DD-A" FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labeled probe as FRET donor), and H2 (TAMRA-labeled probe as FRET acceptor). First, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMB complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1-H2 duplexes were formed during the CHA process, inducing an obvious FRET signal. In contrast, CHA could not be triggered, and the FRET signal was weak, while target was absent. With the aid of magnetic separation and "DD-A" FRET, errors from background interference were effectively eliminated. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application. [ABSTRACT FROM AUTHOR]