Fluorescence Recovery Research Articles

AND-Gate Logic Förster Resonance Energy Transfer/Magnetic Resonance Tuning Nanoprobe for Programmable Antitumor Immunity Imaging.

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.

Design of a Duplex-to-Complex Structure-Switching Approach for the Homogeneous Determination of Marine Biotoxins in Water

In this report, we describe a fluorescent assay for the detection of six marine toxins in water. The mechanism of detection is based on a duplex-to-complex structure-switching approach. The six aptamers specific to the targeted cyanotoxins were conjugated to a fluorescent dye, carboxyfluorescein (FAM). In parallel, complementary DNA (cDNA) sequences specific to each aptamer were conjugated to a fluorescence quencher BHQ1. In the absence of the target, an aptamer–cDNA duplex structure is formed, and the fluorescence is quenched. By adding the toxin, the aptamer tends to bind to its target and releases the cDNA. The fluorescence intensity is consequently restored after the formation of the complex aptamer–toxin, where the fluorescence recovery is directly correlated with the analyte concentration. Based on this principle, a highly sensitive detection of the six marine toxins was achieved, with the limits of detection of 0.15, 0.06, 0.075, 0.027, 0.041, and 0.026 nM for microcystin-LR, anatoxin-α, saxitoxin, cylindrospermopsin, okadaic acid, and brevetoxin, respectively. Moreover, each aptameric assay showed a very good selectivity towards the other five marine toxins. Finally, the developed technique was applied for the detection of the six toxins in spiked water samples with excellent recoveries.

Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.

Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

Aptamer-based sensitive fluorescence β-lactoglobulin food allergen bioassay via dual and cyclic bidirectional strand displacement amplifications.

β-Lactoglobulin (β-Lg) is a prevalent allergenic protein found in most dairy products, which poses great food safety risks for individuals with allergies, especially for infants. Sensitive and effective detection methods for such allergens are essential to reduce the risk of allergies in daily life. Herein, a fluorescent aptamer bioassay based on a dual and cyclic bidirectional strand displacement means is developed for thesensitive detection of β-Lg in infant rice porridge and milk. The aptamer in the duplex DNA probe binds β-Lg to release the assistance strand to further hybridize with two hairpins, which triggers the initiation of two cyclic amplification cycles through the polymerization, displacement, and nicking of the strands under the action of DNA polymerase and endonuclease restriction enzymes. The amplification cycles lead to the unfolding of many fluorescently quenched signal probes to exhibit substantially enhanced fluorescence recovery for detecting β-Lg. The assay can realize detection of β-Lg in concentrations as low as 4.41pM within the range of 0.01 to 10nM. Furthermore, our sensing method has the capability to discriminate β-Lg from other proteins with high selectivity, resulting in low levels of β-Lg detection in rice porridge and milk samples, demonstrating promising potentials of the developed sensing method for monitoring various food allergens.

Detection of C-reactive protein using a label-free NIR fluorescent aptasensor with a large Stokes shift based on an AIEE anthracene derivative

BackgroundC-reactive protein (CRP), one of the classic biomarkers of inflammation, is closely related to infectious inflammation, cardiovascular disease, cancer, and other diseases. Therefore, timely and accurate detection of CRP in human blood is crucial for the discovery, diagnosis, and treatment of the aforementioned diseases. Herein, a novel label-free NIR fluorescence aptasensor with a large Stokes shift based on an AIEE anthracene derivative B and a molybdenum disulfide (MoS2) platform was developed and used for the high sensitivity and specificity detection of CRP. ResultsCompound B could emit near-infrared (NIR) fluorescence with a large Stokes shift (190 nm). Notably, this compound could bind with the aptamer of CRP (CRP-Apt) through electrostatic attraction to form a B/CRP-Apt complex, generating an aggregation-induced emission enhancement effect and enhancing the fluorescent intensity of B. B/CRP-Apt could be adsorbed on the surface of MoS2 with the addition of MoS2 to its solution, and the fluorescence of Compound B was quenched. CRP was then added to the above solution. CRP-Apt had a substantially higher affinity for CRP than MoS2. Therefore, B/CRP-Apt detached from the surface of MoS2 and bound to CRP, thereby restoring the fluorescence of B. Experimental results showed a good linear relationship between the fluorescent recovery intensity of B and the concentration of CRP in the concentration range of 0.3–70 ng mL−1, with a limit of detection as low as 0.1 ng mL−1. Significance and NoveltyThe aptasensor integrates the advantages of high sensitivity of NIR fluorescence, high specificity of aptamers, good water-solubility and AIEE effect of Compound B. And it could be applied to the determination of CRP in human serum samples, while most of the reported methods can only determine CRP in spiked human serum samples.

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.

Conjugated Polymers Based on Carbazole and Tridentate Ligands as the "On-Off-On" Fluorescent Probes for Detection of Ni (II) Ion and Lysine.

Two novel conjugated polymers (polymer 1 and polymer 2) containing trisheterocyclic systemsand carbazole as the copolymerization unit were synthesized by the Suzuki coupling reaction and characterized using NMR spectroscopy and other methods. 4'-(3,5-Dibromophenyl)-2,2':6',2''-terpyridine and 2,2'-(4-(3,5-dibromophenyl)pyridine-2,6-diyl)dithiazole were used as the recognizing units of the two polymers respectively. The polymers show blue-violet fluorescence when dissolved in THF. The ability of the polymers to identify anions and metal ions was investigated by fluorescence sensing tests. It was found that I- not only quenched the fluorescence but also undergone some redshift. Ni (II) efficiently quenched the fluorescence of the polymers, and polymer 2 recognized Ni2+ with higher specificity. UV-visible absorption titration experiments showed that Ni2+ formed complexes with the polymers. In addition, the formation of complexes between Ni2+ and polymers were used for the detection of amino acids, and it was found that lysine could regenerate the fluorescence of [polymer 1-Ni2+] and [polymer 2-Ni2+] with 99% fluorescence recovery.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of Gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Stepwise virus assembly in the cell nucleus revealed by spatiotemporal click chemistry of DNA replication.

Biomolecular assemblies are fundamental to life and viral disease. The spatiotemporal coordination of viral replication and assembly is largely unknown. Here, we developed a dual-color click chemistry procedure for imaging adenovirus DNA (vDNA) replication in the cell nucleus. Late- but not early-replicated vDNA was packaged into virions. Early-replicated vDNA segregated from the viral replication compartment (VRC). Single object tracking, superresolution microscopy, fluorescence recovery after photobleaching, and correlative light-electron microscopy revealed a stepwise assembly program involving vDNA and capsid intermediates. Depending on replication and the scaffolding protein 52K, late-replicated vDNA with rapidly exchanging green fluorescent protein-tagged capsid linchpin protein V and incomplete virions emerged from the VRC periphery. These nanogel-like puncta exhibited restricted movements and were located with the capsid proteins hexon, VI, and virions in the nuclear periphery, suggestive of sites for virion formation. Our findings identify VRC dynamics and assembly intermediates, essential for stepwise productive adenovirus morphogenesis.

A fluorescent sensor for rapid and quantitative aquatic bacteria detection based on bacterial reactive oxygen species using Ag@carbon dots composites.

A novel fluorescent sensor based on silver nanoparticle-carbon dot composites (Ag@CDs) has been developed for the rapid and quantitative detection of aquatic bacteria. The sensor operates on the principle of plasmon-enhanced resonance energy transfer, where the fluorescence of CDs is quenched by Ag nanoparticles and restored upon bacterial interaction due to the generation of reactive oxygen species. The Ag@CDs exhibit a linear response to bacterial concentration over the range 7 × 104 ~ 4 × 107 CFU·mL-1, with a low detection limit of 4 × 104 CFU·mL-1. The fluorescence recovery is rapid, reaching maximum intensity within 5 min. The method demonstrates high selectivity, with minimal interference from common ions and compounds found in municipal and industrial wastewater. The Ag@CDs-based 96-well plate assay for quantitative measurement of bacteria was developed. The assay's performance was further validated through the analysis of real water samples, showing a recovery of 94.0 ~ 102% for domestic wastewater and 97.6 ~ 106% for industrial wastewater. Also, Ag@CDs-based test strips assay for semi-quantitation were developed for rapid in-field aquatic bacteria detection. Ag@CDs can be conveniently integrated into 96-well plates and test strips, providing rapid on-site aquatic bacteria testing.

Novel metal–organic framework biosensing platform for detection of COVID-19 RNA

The latest pandemic resulting from the novel SARS-CoV-2 coronavirus has significantly affected public health, the worldwide economy, and social life. Metal organic frameworks (MOFs) are currently being implemented in biosensors for rapid and accurate detection of viruses thanks to their exceptional properties. This research aims to develop a Zeolitic Imidazolate Framework-8 (ZIF-8) based fluorescent biosensor for facile and rapid COVID-19 RNA sequence detection. ZIF-8 was characterized using several tests, such as FT-IR, TGA, and PXRD, to examine the MOF’s crystalline structure and thermal stability. The results demonstrated high crystallinity and thermal stability up to a temperature of 550 °C. The experimental study showed that ZIF-8 is an excellent fluorescence quencher, with 78.39% quenching efficiency. Analyzing the adsorption mechanism of probe DNA into ZIF-8 revealed that they can form electrostatic and π–π stacking interactions, forming a P-DNA@ZIF-8 complex and that PET is more dominant than FRET in the quenching mechanism. This ZIF-8 biosensing platform showed high sensitivity towards COVID-19 RNA with an ultra-low limit of detection of 6.24 pM, a rapid detection time of 8 min, and high selectivity to COVID-19 RNA. Indeed, ZIF-8 experienced much lower fluorescence recovery when tested on two mismatched RNAs. The experimental results show the potential use of ZIF-8 as a novel biosensor for a rapid and sensitive COVID-19 diagnosis.

Human mesenchymal stem/stromal cell-derived extracellular vesicle transport in meniscus fibrocartilage.

Extracellular vesicles (EVs) derived from endometrial-derived mesenchymal stem/stromal cells (eMSC) play a crucial role in tissue repair due to their immunomodulatory and reparative properties. Given these properties, eMSC EVs may offer potential benefits for meniscal repair. The meniscus, being partly vascularized, relies on diffusivity for solute trafficking. This study focuses on EVs transport properties characterization within fibrocartilage that remains unknown. Specifically, EVs were isolated from Crude and CD146+ eMSC populations. Green fluorescence-labeled EVs transport properties were investigated in three structurally distinct layers (core, femoral, and tibial surfaces) of porcine meniscus. Diffusivity was measured via custom fluorescence recovery after photobleaching (FRAP) technique. Light spectrometry was used to determine EVs solubility. Both Crude and CD146+ eMSC EVs exhibited high purity (>90% CD63CD9 marker expression) and an average diffusivity of 10.924 (±4.065) µm²/s. Importantly, no significant difference was observed between Crude and CD146+ eMSC EV diffusivity on the meniscal layer (p > 0.05). The mean partitioning coefficient was 0.2118 (±0.1321), with Crude EVs demonstrating significantly higher solubility than CD146+ EVs (p < 0.05). In conclusion, this study underscores the potential of both Crude and CD146+ eMSC EVs to traverse all layers of the meniscus, supporting their capacity to enhance delivery of orthobiologics for cartilaginous tissue healing.

Crowder Chain Length Variability and Excluded Volume Effect on the Phase Separation Behavior of Mucin.

Phase separation within cellular membranes, a critical process underpinning diverse cellular functions, is significantly influenced by transmembrane proteins. Therefore, elucidating the behavior of a transmembrane protein in its phase-separated state is of utmost importance. Our study explores mucin behavior in the cellular milieu, aiming to determine the role of crowder chain length and excluded volume in phase separation. Confocal microscopy images demonstrate the strong partitioning of mucin into the condensed phase influenced by hydrophobic and electrostatic interactions. Fluorescence recovery after photobleaching analysis revealed increased mobility in the presence of shorter chain length crowders, indicating the dynamic behavior of protein within condensed phases. Excluded volume calculation using the theoretical model emphasizes its importance in mucin phase separation under crowded conditions. Our findings underscore the ability of mucin to phase-separate under crowded conditions, highlighting the crucial role of excluded volume and enhancing our understanding of its involvement in cancer progression.

A colorimetric fluorescent probe for reversible detection of HSO3−/H2O2 and effective discrimination of HSO3−/ClO− and its application in food and bioimaging

In view of the significant role of reactive sulfur species (RSS) and reactive oxygen species (ROS) in maintaining the redox homeostasis of organisms, we proposed a colorimetric fluorescent probe (HTN) for reversible detection of HSO3−/H2O2 and effective discrimination of HSO3−/ClO−. C = C is the active site for the Michael addition of HSO3− and the oxidation of ClO−. When HTN interacts with HSO3− and ClO−, it exhibits fluorescence quenching. The addition of oxidizing H2O2 to the system can restore the conjugate structure of the addition product of HSO3− (HTN-HSO3−) and the fluorescence recovery, but it cannot restore the structure of the oxidation product of ClO− (HTN-ClO−). By studying the change of the reversibility/non-reversibility of the probe structure with the addition of H2O2, the purpose of reversible detection of HSO3−/H2O2 and distinguishing HSO3−/ClO− is achieved. In addition, HTN can not only be used as a fluorescent ink to detect HSO3− on the test paper, but also has excellent detection effect on HSO3− and ClO− in real food samples and water samples. Meantime, HTN has good biocompatibility and can target mitochondria to achieve reversible detection of HSO3−/H2O2 and effective discrimination of HSO3−/ClO− in living cells. Therefore, HTN has great potential as a molecular tool for studying redox homeostasis in the interaction network of complex living systems.

Diffusion and Exchange Kinetics of Microparticle Formulations by Spatial Fourier Transform Fluorescence Recovery after Photobleaching with Patterned Illumination.

The mechanism of active pharmaceutical ingredient (API) mobility during release in microparticle formulation was investigated using periodically structured illumination combined with spatial Fourier transform fluorescence recovery after photobleaching (FT-FRAP). FT-FRAP applies structured photobleaching across a given field of view, allowing for the monitoring of molecular mobility through the analysis of recovery patterns in the FT domain. Encoding molecular mobility in the FT domain offers several advantages, including improved signal-to-noise ratio, simplified mathematical calculations, reduced sampling requirements, compatibility with multiphoton microscopy for imaging API molecules within the formulations, and the ability to distinguish between exchange and diffusion processes. To prepare microparticles for FT-FRAP analysis, a homogeneous mixture of dipyridamole and pH-independent methyl methacrylate polymer (Eudragit RS and RL) was processed using laminar jet breakup induced by vibration in a frequency-driven encapsulator. The encapsulated microparticles were characterized based on particle size distribution, encapsulation efficiency, batch size, and morphology. Utilizing FT-FRAP, the internal diffusion and exchange molecular mobility within RL and RS microparticles were discriminated and quantified. Theoretical modeling of exchange- and diffusion-controlled release revealed that both RL and RS microparticles exhibited similar exchange decay rates, but RL displayed a significantly higher diffusion coefficient. This difference in diffusion within RL and RS microparticles was correlated with their macroscopic dissolution performance.

Characterization of a Charged Biomimetic Lipid Membrane for Unique Antifouling Effects against Clinically Relevant Matrices in Biosensing.

Clinically relevant matrices such as human blood and serum can cause substantial interference in biosensing measurements, severely compromising the effectiveness of the sensors. We report the characterization of a positively charged lipid membrane that has demonstrated unique features to suppress the nonspecific signal for antifouling effects by using SPR, fluorescence recovery after photobleaching (FRAP), and MALDI-TOF-MS. The ethylphosphocholine (EPC) lipid membrane proved to be exceptionally effective at reducing irreversible interactions from human serum on a Protein A surface. The membrane formation conditions and their effects on membrane fluidity and mobility were characterized for understanding the antifouling functions when various capture molecules were immobilized. Specifically, EPC lipid membranes on a Protein A substrate appear to exhibit a strong interaction, likely through the electrostatic effect with the negatively charged proteins that resulted in a stable hydration layer. The strong interaction also limited lipid mobility, contributing to a robust, protective interface that remained undamaged in undiluted serum. Tailoring a surface with antifouling lipid membranes allows for a range of biosensing applications in highly complex biological media.

Microcapsules with condensates and liposomes composite membranes fabricated via soy protein simple coacervation

Coacervation is a common phenomenon in plant proteins which however has yet been well recognized. This study systematically investigated the properties of soy glycinin coacervates, explored the wetting behavior between the condensates and oil droplets in order to obtain microcapsules with core-shell structure, and furthermore figured out the strategy to stabilize the condensate membranes. Fluidity of glycinin coacervates was characterized by viscosity and fluorescence recovery after photo bleaching which was highly dependent on solution parameters and intriguingly increased with prolonged time due to protein structure reorganization. Complete engulfment of oil droplets by coacervates leading to core-shell structure formation was achieved at salinity≥0.1M NaCl and pH≥7 above room temperature. Core-shell structure could be preserved by membranization using liposome coating as it prevented the coalescence between coacervates. Condensate membranes were further hardened by a combination of physical and enzymatic crosslinking using calcium ions and transglutaminase respectively which enabled the microcapsules to survive in the gastric environment. This study is important as it paves way for fabricating well designed microcapsules based on the simple coacervation of plant proteins which are biocompatible and widely abundant.

Two new Cd(II)-based coordination polymers derived from flexible linker: Highly sensitive and selective “turn off-on” fluorescent sensors for ferric and dichromate ions

Coordination polymers (CPs) are peculiar multidimensional materials that find utility as luminescent sensors for ions and molecules existing in wastewater discharge. Herein, two new Cd(II)-based CPs with formula [Cd(L)(phen)]·0.5H2O (1) and [Cd(L)(2,2'-bipy)] (2) (H2L = 3,3'-((1,2-phenylenebis(methylene))bis(oxy))dibenzoic acid, phen = 1,10-phenanthroline and 2,2'-bipy = 2,2'-bipyridine) have been synthesized and characterized. The single crystal X-ray analysis for 1 reveals that, Cd(II)-based centre adopts an octahedral geometry and is coordinated to four carboxyl O atoms from three L2- ligands and two N atoms from phen molecules. While, in CP 2, the Cd(II) centre is seven coordinated, which is completed by five carboxylate O atoms from two distinct L2− ligands and two N atoms from the 2,2-bipy ligand. Both 1 and 2 have been used as turn-off fluorescence sensor for selective and sensitive detection of Fe3+ and Cr2O72-. The CP 1 exhibited superior selectivity and sensitivity than 2 with limit of detection (LOD) 1.732×10-5 M and 5.996×10-6 M against Fe3+ and Cr2O72- respectively. Interestingly, the emissive behaviour of 1 get restored on adding 2-hydroxybenzoic acid (2-hyd acid) (turn on) due to the competitive formation of 2-hyd acid @Fe3+ or 2-hyd acid@Cr2O72- and concomitant release of 1. The fluorescence recovery selectivity of 2-hyd acid over other antioxidants has been attributed to the synergistic effect of high EHOMO value and a small space steric hindrance. The sensing properties of the MOF have been addressed with the help of Hirshfeld surface and density functional theory analyses.

Highly sensitive β-amyloid1–42 oligomer aptamer-based assay via dual cyclic amplifications of three-way catalytic hairpin assembly and palindrome polymerase reaction

β-amyloid1–42 oligomer (AβO1–42), a neurotoxic protein, is considered the key factor in the progression of Alzheimer's disease (AD). And, high sensitivity detection of AβO1–42 is of considerable importance for the early diagnosis and deterioration prevention of AD. We present here an ultrasensitive fluorescent AβO1–42 detection approach based on aptamer recognition probes and a new dual cyclic signal amplification strategy by combining three-way catalytic hairpin assembly (3W-CHA) with palindromic sequence-based polymerase amplification (PBPA). Once the aptamers bind to target AβO1–42 molecules, ssDNA sequences are released to initiate 3W-CHA between three hairpins to unfold the signal hairpins and to form Y-shaped DNA structures (Y-DNAs). Subsequently, the assistance ssDNAs bind the Y-DNAs to expose the palindromic sequences to yield Y-DNA dimers, which lead to cyclic PBPA reaction with presence of polymerase to further unfold more signal hairpins for yielding considerably magnified fluorescence recovery, enabling sensitive assay of AβO1–42 down to 2.4 pM within a dynamic range of 5 pM∼50 nM. Furthermore, this strategy has been validated for interrogation of low AβO1–42 levels in artificial cerebrospinal fluid (CSF), suggesting its promising potential for application in early diagnosis and prevention of AD.

High-Sensitivity Enzyme-Free Fluorescence Probe Based on CRISPR/Cas13 and the Isothermal Amplification Strategy for Axl Sensing.

Axl is an important receptor tyrosine protein kinase that plays a key role in the development and progression of various diseases, such as cancer and inflammation. Developing a highly sensitive Axl detection method can help improve accuracy, better address-specific clinical needs, and guide personalized treatment. In this study, a CHA-CRISPR/Cas13 fluorescence probe was established using Axl-specific aptamers as a mediator to displace the polynucleotide chain (TA). Through TA construction, an entropy-driven nucleotide catalytic hairpin assembly system was created to cyclically release RNA that activates clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13 activity, triggering its cleavage activity. The activated CRISPR/Cas13 system cleaves the reporter labeled with BHQ1 and FAM at both ends, leading to the recovery of FAM fluorescence. Based on the optimization design using the free energy (△G) and secondary structure software simulation results of the nucleic acid sequence, the fluorescence intensity of the probe is proportional to the concentration of Axl. Results showed a good linear relationship between fluorescence intensity increment and log CAxl (CAxl in the range of 3.33-667 pM, r = 0.9907). The probe exhibited ultrahigh sensitivity with a detection limit of 0.84 pM. It was successfully applied in the detection of human serum samples, showing a higher Axl level in cervical cancer patients compared to breast cancer patients. The probe was also successfully applied in the imaging of various tumor cells, consistent with serum detection results. In conclusion, this probe represents an effective new method for detecting Axl, demonstrating outstanding specificity and sensitivity. It provides technological support for tumor diagnosis and shows the potential for detecting circulating tumor cells in blood through cell imaging.