Fluorescence Resonance Energy Transfer Research Articles

Probing G-Quadruplexes Conformational Dynamics and Nano-Mechanical Interactions at the Single Molecule Level: Techniques and Perspectives

The analysis of nucleic acid structures, topologies, nano-mechanics and interactions with ligands and other biomacromolecules (most notably proteins) at the single molecule level has become a fundamental topic in molecular biophysics over the last two decades. Techniques such as molecular tweezers, single-molecule fluorescence resonance energy transfer, and atomic force microscopy have enabled us to disclose an unprecedented insight into the mechanisms governing gene replication, transcription and regulation. In this minireview, we survey the main working principles and discuss technical caveats of the above techniques, using as a fil-rouge the history of their achievements in dissecting G-quadruplexes. The revised literature offers a clear example of the superior ability of single-molecule techniques with respect to ensemble techniques to unveil the structural and functional diversity of the several polymorphs corresponding to a single G-quadruplex folding sequence, thus shedding new light on the extreme complexity of these fascinating non-Watson–Crick structures.

FRET verification of crucial interaction sites in RhoA regulation mediated by RhoGDI

AbstractThe small GTPase Rho family are the major factors in mediating actin cytoskeleton dynamics. Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) serve as important negative regulators by complexing with inactive Rho into the cytoplasm. However, how these two molecules interact still needs experimental verification. Based on fluorescence resonance energy transfer (FRET) measurements, we would demonstrate crucial sites in RhoGDI and RhoA for this regulatory role. Cotransfection of RhoGDI markedly reduced RhoA or Cdc42 activity in airway smooth muscle (ASM) cells, while D185R-RhoGDI mutant reversed this decrease, indicating that RhoGDI Asp185 residue is essential for the molecular interaction. R68D-RhoA (mutation in the switch II region) resulted in a deficiency in RhoGDI regulation, while TV37/38NG-RhoA (in the switch I region) displayed low RhoA activity. Hence, the Arg68 site in RhoA is indispensable for regulation by RhoGDI, and Thr37Val38 site is important for maintaining RhoA activity. Additionally, microtubule but not actin cytoskeleton showed inhibitory role in RhoA activity, while the dissolution of either cytoskeleton did not change the regulatory role of RhoGDI. In checking the downstream effect, reduction of RhoA activity induced by PDGF stimulation or RhoGDI decreased cellular stress fibers. In this study, FRET visualization was applied to have experimentally demonstrated the interaction sites and crucial role of RhoGDI in regulating RhoA activity. Graphical Abstract

An Upconversion Fluorescent Resonant Energy Transfer Biosensor for the Detection of microRNA through DNA Hybridization

An Upconversion Fluorescent Resonant Energy Transfer Biosensor for the Detection of microRNA through DNA Hybridization

Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Abstract Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing ‘designer’ PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localization of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. To address this, we determined the structure of a dPPR bound to its target sequence and used two- and three-color single-molecule fluorescence resonance energy transfer to interrogate the mechanism of ssRNA binding to individual dPPRs in real time. We demonstrate that dPPRs are slower to bind longer ssRNA sequences (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR. Importantly, dPPR binds only to its target sequence (i.e. it does not associate with non-target ssRNA sequences) and does not ‘scan’ longer ssRNA oligonucleotides for the target sequence. The kinetic constraints imposed by random 3D diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus).

Early Diagnosis of Tumorigenesis via Ratiometric Carbon Dots with Deep-Red Emissive Fluorescence Based on NAD+ Dependence

The early diagnosis of tumorigenesis is crucial for clinical treatment, but the resolution and sensitivity of conventional short-wavelength biomarkers are not ideal because of the complicated interference in living tissue. Herein, a nicotinamide adenine dinucleotide (NAD+)-responsive probe with deep-red emissive ratiometric fluorescence was synthetized as a promising target for energy metabolism patterns during tumorigenesis. Interestingly, the solvents H3PO4 and 2,2′-dithiodibenzoic acid enhanced the red emission (640 and 680 nm) of o-phenylenediamine-based carbon dots (CDs), leading to the formation of a nanoscale graphite-like skeleton covered with -P=O, -CONH-, -COOH and -NH2 on their surfaces. Meanwhile, this method exhibited high sensitivity to the discriminating target NAD+, with a detection limit of 63 μM due to the inner filter effect and fluorescence resonance energy transfer process between NAD+ and CDs, which is superior to the reported capillary electrophoresis and liquid chromatographic detection methods (the reported detection limit was about 0.2 mM) in complex biological samples and even cancer cells. Encouragingly, NAD+ significantly promoted nucleus-targeting fluorescence and cell migration compared to GSH and pH stimulation, which were gradually eliminated in human hepatocellular carcinoma (HepG2) cells after 2-deoxy-d-Glucose inhibited the glycolytic phenotype. The proposed method holds great potential for the temporal and spatial resolution of NAD+-dependent tumor diagnosis in complex living systems.

Engineering modular and tunable single-molecule sensors by decoupling sensing from signal output.

Biosensors play key roles in medical research and diagnostics. However, the development of biosensors for new biomolecular targets of interest often involves tedious optimization steps to ensure a high signal response at the analyte concentration of interest. Here we show a modular nanosensor platform that facilitates these steps by offering ways to decouple and independently tune the signal output as well as the response window. Our approach utilizes a dynamic DNA origami nanostructure to engineer a high optical signal response based on fluorescence resonance energy transfer. We demonstrate mechanisms to tune the sensor's response window, specificity and cooperativity as well as highlight the modularity of the proposed platform by extending it to different biomolecular targets including more complex sensing schemes. This versatile nanosensor platform offers a promising starting point for the rapid development of biosensors with tailored properties.

Quantitative Examination and Mechanistic Insights of Polymer Chain Conformation Confined in Nanopores by Time-Resolved Fluorescence Resonance Energy Transfer.

The conformational studies of polymers confined at the nanoscale remain challenging and controversial due to the limitations of characterization techniques. In this study, we utilized the high sensitivity of time-resolved fluorescence resonance energy transfer (trFRET) and a site-specific dye-labeling strategy to characterize the conformation of polymer chains confined in anodic aluminum oxide (AAO) nanopores. This strategy introduced a fluorescent donor (carbazole) and acceptor (anthracene) at the center of poly(butyl methacrylate) (PBMA) chains grown by atom transfer radical polymerization (ATRP). By quantitatively analyzing fluorescence decay through the Förster mechanism and the Drake-Klafter-Levitz (DKL) formalism, we can determine both the energy transfer efficiency and the spatial distribution of the dyes. This analysis revealed that the PBMA chains, with a molecular weight of 40 kDa, maintained their bulk-like conformation even when confined within nanopores as small as 10 nm in diameter. This study is the first to demonstrate the use of trFRET for investigating chain conformation in confined polymer systems, which can be generalized to other polymer types and polymer topologies in different confined geometries.

Towards the Development of an Optical Biosensor for the Detection of Human Blood for Forensic Analysis

Blood is a common biological fluid in forensic investigations, offering significant evidential value. Currently employed presumptive blood tests often lack specificity and are sample destructive, which can compromise downstream analysis. Within this study, the development of an optical biosensor for detecting human red blood cells (RBCs) has been explored to address such limitations. Aptamer-based biosensors, termed aptasensors, offer a promising alternative due to their high specificity and affinity for target analytes. Aptamers are short, single-stranded DNA or RNA sequences that form stable three-dimensional structures, allowing them to bind to specific targets selectively. A nanoflare design has been employed within this work, consisting of a quenching gold nanoparticle (AuNP), DNA aptamer sequences, and complementary fluorophore-labelled flares operating through a fluorescence resonance energy transfer (FRET) mechanism. In the presence of RBCs, the aptamer–flare complex is disrupted, restoring fluorescence and indicating the presence of blood. Two aptamers, N1 and BB1, with a demonstrated binding affinity to RBCs, were selected for inclusion within the nanoflare. This study aimed to optimise three features of the design: aptamer conjugation to AuNPs, aptamer hybridisation to complementary flares, and flare displacement in the presence of RBCs. Fluorescence restoration was achieved with both the N1 and BB1 nanoflares, demonstrating the potential for a functional biosensor to be utilised within the forensic workflow. It is hoped that introducing such an aptasensor could enhance the forensic workflow. This aptasensor could replace current tests with a specific and sensitive reagent that can be used for real-time detection, improving the standard of forensic blood analysis.

Beating the Odds with Binuclear N‐Heterocyclic Carbene Metallacycles: A Practical and Efficient Full‐Color Tunable Fluorescence System

AbstractAchieving full‐color emission with just two emitters presents a significant challenge. Two N‐heterocyclic carbene metallacycles (NHC‐M; M ═ Ag, Au) featuring a tetraphenylethene core, combining covalent and coordination bonds are synthesized to restrict rotation within the NHC‐M‐BC (BC = bicyclic) metallacycles and greatly enhanced their quantum yields. The enhancement is accomplished by adjusting the CH3CN/H2O solvent mixture, allowing emission tuning from blue to green for NHC‐Ag‐BC. Further diversification of the emission spectrum, including access to high‐quality white light (CIE coordinates 0.33, 0.34), is facilitated through the addition of sulforhodamine B via fluorescence resonance energy transfer (FRET). The compatibility of NHC‐Ag‐BC with agarose gel extends its applicability to UV‐LEDs chromic coatings, as well as information encryption and anti‐counterfeiting materials. The results underscore the viability of dual‐fluorophore systems for achieving full‐color emission and highlight the potential for developing versatile, multi‐colored functional materials.

Purification, fibrinolytic activity and substrate binding of nattokinase from Bacillus subtilis: A rapid and sensitive detection for fibrinolytic activity of nattokinase

Nattokinase (NK, EC 3.4.21.62) is an alkaline serine protease secreted by B. subtilis, which has a strong fibrinolytic activity in vitro and in vivo. Here, we fermented NK using a B. subtilis strain and purified the protein, designed a peptide segment derived from fibrin as a substrate of NK. Based on fluorescence resonance energy transfer (FRET), we found that NK exhibits a high hydrolytic activity towards the peptide, with Km of 9.33 ± 1.28 μM, kcat of 0.20 ± 0.01 s−1, and kcat/Km of 21,436.23 s−1 M−1, demonstrating that the peptide is a substrate for NK. Furthermore, we investigated the binding of the substrate with NK by tryptophan fluorescence quenching, molecular docking and dynamics simulation. Fluorescence quenching showed that the substrate binds to NK mainly through hydrogen bonding and van der Waals forces, with dissociation constants KD of 13.73 and 21.24 μM at 25 and 35 °C, respectively. Molecular docking and dynamics analysis revealed that the substrate recognizes the active site of NK, and provides new information on the characteristics of the binding of the substrate with NK. Our study demonstrated a rapid and sensitive method for the quantitative measurement of fibrinolytic activity of NK based on the substrate, which is very reproducible and rapid with virtually complete results in approximately 20 min.

Synthesis, Characterization, Proteolytic Activity Inhibition, ADMET Prediction, and Molecular Docking Studies of Novel Indole Derivatives as Potential SARS-CoV-2 Protease Inhibitors

Both the main protease (Mpro) and papain protease (PLpro) are fundamental enzymes in SARS-CoV-2 life cycle. In this study, we have targeted these two enzymes using novel indole derivatives as antiviral agents. The synthesized compounds were thoroughly characterized by Fourier-transform infrared spectroscopy (FT-IR), UV-vis, 1H, 13C, and 2D nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), and melting point (m.p.). The optimized structure, reactivity, and stability of the synthesized compounds were calculated using Density Functional Theory (DFT) at the B3LYP/6-31G(d,p) level. Using in silico ADMET prediction along with molecular docking, we found that the synthesized compounds presented good docking scores and very low predicted inhibition constants (Pki) from low micromolar to nanomolar ranges. In particular, compounds 10 and 11 with respective Pki values of 1.68 μM and 387.71 nM for Mpro and 402.50 nM and 27.10 nM for PLpro. These two compounds are engaged in vast range of interactions with the active sites of both enzymes. Further in vitro investigations using fluorescence resonance energy transfer (FRET) assays demonstrated that compounds 10 and 11 inhibited Mpro proteolytic activity with approximate IC50 values of 20 µM and 10 µM, respectively. These findings suggest that these new indole derivatives could serve as promising candidates for the development of drugs against SARS-CoV-2 and potentially other future coronaviruses.

Alginate‑carbon dot nanocomposite: A green approach towards designing turn-on aptasenor for Candida albicans fungus

Candida albicans fungus, an opportunistic microorganism that becomes a pathogen in its host, can often cause superficial and severe infections in human body. In this work, a turn-on fluorescent aptasenor based on Alginate‑carbon dot nanocomposite has been designed through a green approach. Carbon dots (CDs) with average size of 4 ± 1 nm were synthesized hydrothermally using Philodendron bipinnatifidum plant extract as precursor. Then, mixture of carbon dot and alginate was cross-linked by Ca2+ ions to make Alginate-CD nanocomposite with negligible effect on fluorescence intensity of CDs (λex = 360 nm, λem = 400 nm). The fluorescence quantum yield and lifetime were found as 14.1 % and 3.15 ns, respectively. The nanocomposite was characterize using different spectroscopic (Photoluminescence, UV–visible, XPS, FT-IR) and microscopic (TEM and SEM) analytical techniques. An aptamer was used to selectively target (1 → 3)-β-D-glucan on the surface of the Candida albicans cell wall. It was found that the fluorescence energy resonance transfer between nanocomposite and aptamer quenches the fluorescence intensity of nanocomposite, making it suitable for designing turn-on fluorescent probe. It was found that the fluorescence could be recovered in the presence of Candida albicans fungus (in <30 min), owing to higher affinity between aptamer and (1 → 3)-β-D-glucan on the surface of Candida albicans fungus, where there were two linear ranges: low concentration of 50–200 cells/mL and high concentration of 1000–6000 cells/mL (R2 ≥ 0.989 for both linear ranges), with limit of detection of 40 cells/mL. The selectivity of the designed probe was examined against a mixture of microbial agents (Staphylococcus aureus and Escherichia coli) and Curdlan polysaccharide (1→3)-b-D-Glucan (L). The designed biosensor showed an excellent ability to directly detect Candida albicans fungus via a simple and fast (<30 min) approach. The results of this experimental study show that Alginate-CD-aptamer nanocomposite is a bright prospect in clinical applications and a suitable alternative to traditional methods of Candida albicans diagnosis.

Development of an assay to quantify tranexamic acid levels in plasma

Dysregulations during blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labelled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7% and 3.0%, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.

Characterizations of angiotensin-converting enzyme-2 (ACE2) peptidase activity

Angiotensin (Ang) II (1-8) is a potent vasoconstrictor known for its role in hypertension. Angiotensin-converting enzyme (ACE2) converts Ang II (1-8) to a vasodilator Ang (1-7) by removing the carboxy-terminal Phe. ACE2 more recently gained attention as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the coronavirus disease 2019 (COVID-19) pandemic. Given the pathophysiological importance of ACE2, the present study examined the mechanism of ACE2 catalytic activity by comparing the ability of angiotensin molecules of various lengths to compete with the artificial fluorogenic substrate. The Fluorimetric SensoLyte 390 ACE2 Activity Assay uses an Mca/Dnp fluorescence resonance energy transfer peptide as the substrate. Results showed that the natural substrate Ang II (1-8) competed with the fluorogenic substrate, reducing the fluorescence signals. Deletion of C-terminal Phe resulted in the loss of the ability to compete with the artificial substrate, as shown by the actions of Ang (1-7), Ang (2-7), and Ang (5-7). By contrast, the loss of N-terminal Asp potentiated the ability to compete with the substrate as seen by the action of Ang III (2-8). However, the loss of two amino acids (Asp-Arg) from the N-terminus reduced the ability to compete with the substrate as observed by the actions of Ang IV (3-8) and Ang (5-8). Ang I (1-10) and Ang (1-9) did not strongly compete with the substrate. Interestingly, shorter peptides Ang (1-5) and Ang (1-4) potentiated the ACE2 activity. These results suggest that Ang II and Ang III are the best natural substrates for ACE2.

A handheld fluorescent platform integrated with a Sm(III)-CdTe quantum dot-based ratiometric nanoprobe for point-of-use determination of phosphate.

Phosphate (Pi) is crucial for various physiological processes and aquatic environments, which emphasizes the need for a simple, on-site sensor to promptly detect Pi for human health and environmental conservation. In this study, we propose a dual-emission ratiometric fluorescence sensor for highly sensitive and visual Pi detection. The sensor employs samarium ions (Sm3+) as a core component, with cadmium telluride quantum dots (CdTe QDs) and ofloxacin (OFL) serving as signal carriers. The CdTe-Sm(III)-OFL nanoprobe emits a purple fluorescence resulting from the red fluorescence of CdTe QDs and the blue-green fluorescence of OFL. The fluorescence of OFL is quenched by Sm3+ through fluorescence resonance energy transfer (FRET). Upon Pi interaction, the FRET process is disrupted due to the competitive Pi-Sm3+ binding, which leads to the fluorescence recovery of OFL while the red fluorescence of CdTe remains steady. This enables the construction of a ratiometric fluorescent sensor for Pi detection, manifesting as a color change from purple to blue. The sensor demonstrated a linear response for Pi detection within the range of 0.1-75 μM, with a low detection limit of 17.0 nM. By utilizing the distinct fluorescence responses of various physiological phosphates and employing chemometrics, this innovative dual-emission sensor accurately distinguishes among different physiological phosphates. Furthermore, a portable lab-on-paper device based on CdTe-Sm(III)-OFL, coupled with a smartphone-integrated mini-device, is developed for swift Pi detection using an ordinary smartphone. Analytical performance validated on environmental and biological samples demonstrates the sensor's excellent robustness and adaptability. This study introduces a pioneering approach to fabricate ratiometric fluorescence sensors and customize portable, cost-effective mini-devices for precise target detection, thus opening avenues for advanced sensing strategies in various applications.

Effects of the restrictive cardiomyopathy-associated cardiac troponin I K178E mutation on cAMP signalling at the myofilament

Abstract Background Catecholaminergic stimulation of cardiac β-adrenergic receptors (β-ARs) and subsequent cAMP signalling control cardiac inotropy and lusitropy. cAMP signalling is known to be compartmentalised to optimise sympathetic control by producing heterogeneous cAMP signals and protein kinase A (PKA) activation at distinct subcellular sites. This signalling may be disrupted in diseases such as restrictive cardiomyopathy (RCM) characterised by diastolic dysfunction, which is associated with certain mutations including the K178E missense mutation in cardiac troponin I (TPNI). The impaired ventricular relaxation associated with this mutation is thought to arise from myofilament calcium hypersensitivity, which can be modulated by PKA-dependent TPNI phosphorylation. However, a detailed understanding of the pathological effects mediated by the K178E-TPNI mutation is lacking. Purpose This study aims to determine whether the K178E-TPNI mutation alters local cAMP/PKA signalling at TPNI, potentially affecting myofilament calcium sensitivity and cardiac myocyte relaxation. Methods Real-time imaging of cAMP was carried out using the Fluorescence Resonance Energy Transfer (FRET)-based sensor CUTie (cAMP Universal Tag for imaging experiments) [1] fused to wild type (WT)- or K178E-TPNI. The sensors were expressed by transfection in neonatal rat ventricular myocytes (NRVMs) and FRET-changes were recorded at the myofilament on application of the β-AR stimulant isoproterenol (ISO). We further assessed the involvement of cAMP hydrolysing phosphodiesterases (PDEs) in the regulation of cAMP levels selectively at TPNI. Results The peak FRET-change and FRET-change after 5mins on ISO application were significantly higher in NRVMs expressing the mutant compared to the WT sensor, as was the rate of cAMP accumulation. A strong trend showed that the rise in FRET following PDE4 inhibition was larger in NRVMs expressing the WT compared to the mutant sensor, suggesting the involvement of this isoform in degrading cAMP locally at TPNI and reduced PDE4 activity in the presence of the K178E mutation. Co-immunoprecipitation analysis in HEK293 cells demonstrated interaction between WT-TPNI and a PDE4D isoform, and a weaker interaction of this isoform with the K178E-TPNI mutant. Conclusions This study shows that the K178E-TPNI mutation attenuates the TPNI/PDE4D interaction and alters the magnitude and kinetics of the local cAMP response at the myofilament. This mechanistic insight may aid the development of therapeutics targeting the TPNI/PDE4D interaction to treat RCM-associated diastolic dysfunction.The TPNI-CUTie SensorFRET Response Trace

Evaluation of Selected Plant Phenolics via Beta-Secretase-1 Inhibition, Molecular Docking, and Gene Expression Related to Alzheimer’s Disease

Background: The goal of the current study was to investigate the inhibitory activity of six phenolic compounds, i.e., rosmarinic acid, gallic acid, oleuropein, epigallocatechin gallate (EGCG), 3-hydroxytyrosol, and quercetin, against β-site amyloid precursor protein cleaving enzyme-1 (BACE1), also known as β-secretase or memapsin 2, which is implicated in the pathogenesis of Alzheimer’s disease (AD). Methods and Results: The inhibitory potential against BACE1, molecular docking simulations, as well as neurotoxicity and the effect on the AD-related gene expression of the selected phenolics were tested. BACE1 inhibitory activity was carried out using the ELISA microplate assay via fluorescence resonance energy transfer (FRET) technology. Molecular docking experiments were performed in the human BACE1 active site (PDB code: 2WJO). Neurotoxicity of the compounds was carried out in SH-SY5Y, a human neuroblastoma cell line, by the Alamar Blue method. A gene expression analysis of the compounds on fourteen genes linked to AD was conducted using the real-time polymerase chain reaction (RT-PCR) method. Rosmarinic acid, EGCG, oleuropein, and quercetin (also used as the reference) were able to inhibit BACE1 with their respective IC50 values 4.06 ± 0.68, 1.62 ± 0.12, 9.87 ± 1.01, and 3.16 ± 0.30 mM. The inhibitory compounds were observed to occupy the non-catalytic site of the BACE1. However, hydrogen bonds were found to be present between rosmarinic acid and EGCG and aspartic amino acid D228 in the catalytic site. Oleuropein and quercetin effectively suppressed the expression of PSEN, APOE, and CLU, which are recognized to be linked to the pathogenesis of AD. Conclusions: The outcomes of the work bring quercetin, EGCG, and rosmarinic acid to the forefront as promising BACE1 inhibitors.

Development of a high-throughput screening system targeting the protein-protein interactions between PRL and CNNM

Phosphatase of regenerating liver (PRL) is an oncogenic protein that promotes tumor progression by directly binding to cyclin M (CNNM) membrane proteins and inhibiting their Mg2+ efflux activity. In this study, we have developed a high-throughput screening system to detect the interactions between PRL and CNNM proteins based on homogenous time-resolved fluorescence resonance energy transfer (HTR-FRET, HTRF). We optimized the tag sequences attached to the recombinant proteins of the CNNM4 CBS domains and PRL3 lacking the carboxyl terminal CAAX motif, and successfully detected the interaction by observing the FRET signal in the mixture of the tagged proteins and fluorophore-conjugated antibodies. Moreover, we performed compound library screening using this system and discovered several compounds that could efficiently inhibit the PRL-CNNM interaction. Characterization of one candidate compound revealed that it was relatively stable compared with thienopyridone, a known inhibitor of the PRL-CNNM interaction. The candidate compound can also inhibit PRL function in cells: suppression of CNNM-dependent Mg2+ efflux, and has sufficient in vitro drug metabolism and pharmacokinetic properties. Overall, these results demonstrate the effectiveness of this screening system for identifying novel inhibitors of the PRL-CNNM interaction, which could contribute to the development of novel anti-cancer drugs.

FRET-Based Dual-Color Carbon Dot Ratiometric Fluorescent Sensor Enables the Smartphone-Integrated Device for Noninvasive and Portable Diagnosis of Chronic Kidney Disease.

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.

Optical Biosensors for Detection of Cancer Biomarkers: Current and Future Perspectives.

Optical biosensors are emerging as a promising technique for the sensitive and accurate detection of cancer biomarkers, enabling significant advancements in the field of early diagnosis. This study elaborates on the latest developments in optical biosensors designed for detecting cancer biomarkers, highlighting their vital significance in early cancer diagnosis. When combined with targeted nanoparticles, the bio-fluids can help in the molecular stage diagnosis of cancer. This enhances the discrimination of disease from the normal subjects drastically. The optical sensor methods that are involved in the disease diagnosis and imaging of cancer taken for the present review are surface plasmon resonance, localized surface plasmon resonance, fluorescence resonance energy transfer, surface-enhanced Raman spectroscopy and colorimetric sensing. The article meticulously describes the specific biomarkers and analytes that optical biosensors target. Beyond elucidating the underlying principles and applications, this article furnishes an overview of recent breakthroughs and emerging trends in the field. This encompasses the evolution of innovative nanomaterials and nanostructures designed to augment sensitivity and the incorporation of microfluidics for facilitating point-of-care testing, thereby charting a course towards prospective advancements.