Probe Design Research Articles

The Molecular Mechanism of Fluorescence Lifetime of Fluorescent Probes in Cell Membranes.

Fluorescence probes play crucial roles in unraveling the structure and dynamics of cell membranes including membrane fluidity, polarity, and lipid molecule ordering. The fluorescence lifetime of probes describes the average duration of time that a fluorescent molecule remains in an excited state before returning to the ground state, which is sensitive to environmental changes. However, the molecular mechanism and inherent properties to determine the fluorescence lifetimes remain unexplored and inadequately studied. Furthermore, the effects of the probe on the membrane are also unclear. In this study, we investigated the interactions between probes and lipids, as well as the structural properties of probes within the outer and inner membrane of Mycobacterium smegmatis (Msm) by combining molecular dynamics (MD) simulations, enhanced sampling methods, fluorescence lifetime imaging microscopy (FLIM), and time-correlated single photon counting (TCSPC). The results show that even though the probes have very little effect on the membrane lipids, different membrane environments significantly affect the fluorescence lifetime of the probes. The analysis based on the all-atom simulations shows a strong correlation between the probe's immersion depth within the membrane and its fluorescence lifetime. Specifically, probes buried in the membrane environment shielded from rapid water molecule collisions exhibit longer fluorescence lifetimes. The molecular basis of the fluorescence lifetime of probes in cell membranes revealed in this work would enhance the comprehension of fluorescence probes and facilitate the rational design of novel efficient probes.

Design and photophysical characterization of quasi-intrinsic fluorescent probes utilized in DNA sequencing

Design and photophysical characterization of quasi-intrinsic fluorescent probes utilized in DNA sequencing

Design and synthesis of piperine-based photoaffinity probes for revealing potential targets of piperine in neurological disease.

Piperine (PIP) has attracted extensive attention due to its diverse biological activities. In this study, we developed two photoaffinity probes PIP-1 and PIP-2, which are biologically safe and retain PIP's bioactivity, to investigate its protein targets in vivo. Using in situ labeling and cell imaging, we were able to effectively detect and visualize the drug targets of PIP with our probes. Additionally, a series of protein targets of PIP were fished using PIP-2 through proteome profiling, with further validation suggesting that TGFβ1 might be a potential target involved in PIP's effects on neurological diseases. These findings demonstrate that PIP-2 is a valuable tool for identifying the targets of PIP.

The rational design of ratiometric probe based on fluorescence carbon dots and its PVA film platform for the detection of Hg2+: tunable mechanism and the performance

Carbon dots (CDs) are recognized as sensitive probes for the detection of Hg2+ due to their remarkable fluorescent properties and ease of synthesis. However, drawbacks such as high costs, intricate synthesis, and non-portability hinder their broader applications. This study reported the synthesis of tunable multicolor CDs, including blue (B-CDs), green (G-CDs), and orange (O-CDs), by using hydrothermal methods while adjusting precursors and temperature. The observed fluorescence redshift from B-CDs to G-CDs or O-CDs arose from interactions among particle size, sp² conjugation, and surface oxidation, while the redshift from G-CDs to O-CDs was linked to surface fluorophores. Moreover, a ratiometric probe was developed by mixing B-CDs (response signal) and O-CDs (reference signal). Hg2+ significantly quenched B-CDs fluorescence due to static quenching from electron transfer. The probe demonstrated excellent stability and sensitivity, with limits of detection (LOD) of 4.8nM in the 0-0.6μM range and 9.2nM in a broader range (0-50μM). A portable smartphone-assisted detection platform was further developed by integrating the probe into polyvinyl alcohol (PVA) hydrogel. Both methods manifested the capability to accurately quantify Hg2+ in real water samples. This research provides innovative strategies for multicolor CDs and a sensing platform for rapid on-site Hg2+ detection.

68Ga labeled Olmutinib: Design, synthesis, and evaluation of a novel PET EGFR probe.

Radiolabeled tyrosine kinase inhibitors (TKIs) offer a promising approach for molecular imaging of EGFR-positive cancers. Despite the development of various EGFR small-molecule probes, none of the 68Ga-labeled small-molecule probes based on the chelator DOTA have shown tumor-specific uptake. To address this challenge, we selected Olmutinib, a third-generation EGFR covalent inhibitor, as a PET imaging tracer for EGFR-positive tumors. We synthesized the precursor DOTA-Olmutinib through a five-step process and subsequently radiolabeled it with 68Ga to prepare 68Ga-DOTA-Olmutinib. 68Ga-DOTA-Olmutinib displayed moderate lipophilicity (log P=0.85) and exhibited high stability in vitro and in vivo. Western blot analysis was used to detect the level of EGFR in multiple tumor cells. In cell uptake experiments, 68Ga-DOTA-Olmutinib exhibited enhanced uptake specifically in tumor cells with a higher level of EGFR supporting it as an EGFR-specific tracer. Additionally, PET/CT imaging with 68Ga-DOTA-Olmutinib showed significant tumor uptake at 60min with 4% ID/g post-injection, marking a breakthrough, though the uptake is not yet ideal. Overall, our results suggest that 68Ga-labeled Olmutinib holds promise as a potential PET tracer for detecting EGFR-positive cancers.

Reaction-Based SERS Probes for the Detection of Raman-Inactive Species.

Surface-enhanced Raman spectroscopy (SERS) has the advantages of high sensitivity, low water interference, narrow spectral peaks for multicomponent analysis, and rich molecular fingerprint information, presenting great potential to be a robust analytical technology. However, a key issue is the unavailability in directly detecting Raman-inactive species with a small Raman scattering cross-section. Current research has addressed this issue by using specific chemical reactions to induce significant characteristic changes in SERS signals, enabling the sensitive and selective detection of Raman-inactive species. This reaction-activated SERS sensing strategy provides a clever approach to the precise determination of Raman-inactive species. In this review, we have first summarized the design principles and types of reaction-based SERS probes. Furthermore, we have examined the enormous potential of reaction-based SERS probes in the detection of bioactive species, environmental pollutants, and food contaminants. Finally, we have discussed in depth the challenges and prospects of reaction-based SERS probes on stability, reliability, and intelligence. The review is aimed to inspire a more advanced design of reaction-based SERS probes, thus further facilitating their extensive applications in SERS analysis.

Enhancing Multi-Hole Pressure Probe Data Processing in Turbine Cascade Experiments Using Structural Risk Minimization Principle

Multi-hole pressure probes are crucial for turbomachinery flow measurements, yet conventional data processing methods often lack generalization for complex flows. This study introduces an innovative approach by integrating machine learning techniques with the structural risk minimization (SRM) principle, significantly enhancing the generalization capability of regression models. A comprehensive framework has been developed, combining SRM-based machine learning regression methods, such as ridge regression and kernel ridge regression, with hyperparameter optimization and S-fold cross-validation, to ensure robust model selection and accuracy. Validated using the McCormick function and applied to VKI-RG transonic turbine cascade measurements, the SRM-based methods demonstrated superior performance over traditional empirical risk minimization approaches, with lower error ratios and higher R2 values. Novel insights from SHAP analysis revealed subtle but significant differences in aerodynamic parameters, including a 0.63122° discrepancy in exit flow angle predictions, guiding the probe design and calibration strategies. This study presents a holistic workflow for improving multi-hole pressure probe measurements under high-subsonic conditions, representing a meaningful enhancement over traditional empirical methods and providing valuable references for practical applications.

The Effect of the Picture Exchange Communication System on Individuals with Autism Spectrum Disorder

Introduction: The aim of this study was to examine the effect of the Picture Exchange Communication System (PECS) protocol in teaching individuals with moderate/severe autism spectrum disorder (ASD) the ability to initiate spontaneous communication and request food. Method: In the study, a single-subject research model, multiple probe design with across-participant was used. The participants of the study were three boys diagnosed with moderate/severe ASD attending a special education practice school. The independent variable of the study was the first three phases of the PECS protocol, and the dependent variable was the child's ability to initiate spontaneous communication and request food. The study included baseline, intervention, generalization and follow-up sessions. Results: The first three phases of the PECS protocol were found to be effective in children with moderate/severe ASD in learning the skill of requesting food. The children were able to maintain the food-requesting skill they learned one and three weeks after the last intervention session and were able to generalize this skill to different environments and people. Teachers who participated in the study as communication partners expressed positive opinions about the study. Discussion: The effectiveness findings of the study are consistent with other studies in the literature that examined the effect of PECS on the acquisition of requesting skills by individuals with ASD. The findings were discussed in comparison with the related literature and suggestions were made for future research and practice.

Synthesis and characterization of a vitamin B6-tetrazole hydrazone as a fluorescence probe for selective detection of Cd2+ and Ga3+ ions

A simple selective fluorescence probe for cadmium(II) and gallium(III) ions was designed and prepared by a reaction of hydrazone formation between pyridoxal-5′-phosphate and 5-hydrazinyl-1H-tetrazole. The probe shows an enhanced blue fluorescence in the presence of Cd2+ ions and a yellow-green fluorescence in the presence of Ga3+ ions in aqueous DMSO. The conditional stability constants and stoichiometry of the Cd2+ and Ga3+ complexes were determined using the maximum likelihood method. Gallium(III) ions form two complexes of different structures with probe in solution. The DFT quantum chemical calculations were performed for obtaining molecular structures of probe 1 and its complexes with Ga3+ and Cd2+. Simulation of electronic absorption spectra and NMR spectra by TDDFT and GIAO methods allowed to determine the structures, which was obtained. The probe demonstrated a low detection limit of 0.19 μM for Cd2+ and 0.97 μM for Ga3+, making it suitable for water analysis. This research offers important insights into the design and enhancement of fluorescence probes for the selective detection of metal ions.

Redefining Molecular Probes for Monitoring Subcellular Environment: A Perspective.

The development of small-molecule fluorescent probes has revolutionized the monitoring of in vivo physicochemical parameters, offering unprecedented insights into biological processes. In this perspective, we critically examine recent advances and trends in the design and application of fluorescent probes for real-time in vivo monitoring of subcellular environments. Traditional concepts such as membrane potential, microviscosity, and micropolarity have been superseded by more biologically relevant parameters like membrane voltage, tension, and hydration, enhancing the accuracy of physiological assessments. This redefinition not only presents an evolved concept with broader applications in monitoring subcellular dynamics but also addresses the unmet needs of subcellular biology more effectively. We also highlight the limitations of commonly used probes in providing specific information about the redox environment, noting their nonspecificity to oxidants and the influence of various chemical interactions. These probes typically rely on free radical mechanisms and require metal catalysts to react with hydrogen peroxide. They include naphthalimide, fluorescein, BODIPY, rhodamine, cyanine cores to cover the UV-vis-near-infrared window. The motif of this perspective is to provide critical insights into trending fluorescent-based systems employed in real-time or in vivo physicochemical-responsive monitoring, thus aiming to inform and inspire further research in creating robust and efficient fluorescent probes for comprehensive in vivo monitoring applications.

Design and Validation of Specific Oligonucleotide Probes on Planar Magnetic Biosensors.

Planar DNA biosensors employ surface-tethered oligonucleotide probes to capture target molecules for diagnostic applications. To improve the sensitivity and specificity of biosensing, hybridization affinities should be enhanced, and cross-hybridization with off-targets must be minimized. To this end, assays can be designed using the thermodynamic properties of hybridization between probes and on-targets or off-targets based on Gibbs free energies and melting temperatures. However, the nature of heterogeneous hybridization between the probes on the surface and the targets in a solution imposes challenges in predicting precise hybridization affinities and the degree of cross-hybridization due to indeterminable thermodynamic penalties induced by the solid surface and its status. Herein, we suggest practical and convenient guidelines for designing oligonucleotide probes based on data obtained from planar magnetic biosensors and thermodynamic properties calculated by using easily accessible solution-phase prediction. The suggested requirements comprised Gibbs free energy ≥ -7.5 kcal mol-1 and melting temperature ≤10 °C below the hybridization temperature, and we validated for the absence of cross-hybridization. Additionally, the effects of secondary structures such as hairpins and homodimers were investigated for better oligonucleotide probe designs. We believe that these practical guidelines will assist researchers in developing planar magnetic biosensors with high sensitivity and specificity for the detection of new targets.

Novice-friendly probes for the gathering and analysis of requirements and subsequent design of software

Developing software, both in its entirety or some specific elements and components, poses a significant challenge. One of the main obstacles one may face during this process is conducting a thorough survey and subsequent analysis of requirements. This difficulty arises from stakeholders struggling to accurately articulate their needs, desires, and expectations. To address this issue, qualitative strategies such as interviews, surveys, work tables, brainstorming, observation, and user stories are commonly employed. However, mastering and utilising them effectively often takes years of experience. To simplify this process for novices (e.g., students, beginners, enthusiasts) in the field of computer science and related areas, inspired by participatory design guidelines, we devised a series of design probes that we call Mirrors. To explore their feasibility, we conducted two different interventions with students. In this document, we present these tools along with a methodology for their application. Additionally, we show the results obtained through their implementation. Lastly, we talk about their benefits and limitations, as well as our future work in order to consolidate their effectiveness.

In-situ nucleic acid amplification induced by DNA self-assembly for rapid and ultrasensitive detection of miRNA

BackgroundTo improve the sensitivity and specificity of nucleic acid detection, coupling two or more signal amplification systems is a feasible pattern, such as nucleic acid isothermal amplification coupling genome-editing technology, and cascaded DNA self-assembly circuits. And representative signal amplification strategies include loop-mediated isothermal amplification (LAMP), clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) systems, and catalyzed hairpin assembly (CHA). However, these detection strategies often require the enrichment of intermediate products, the replacement of reaction conditions and the design of multiple probes, which may seriously affect the reliability of detection results. ResultsHerein, we propose a novel nucleic acid detection system which is named as catalyzed hairpin assembly (CHA) coupled with embedded primer triggered isothermal amplification (CEA for short). DNA self-assembly probes in CEA contain a specially designed primer. When target nucleic acid (e.g., miRNA) initiates CHA reaction (the first signal amplification), the self-assembly product of CHA will expose a primer (named as embedded primer). The embedded primer will trigger a special nucleic acid isothermal amplification in situ, then generate plenty of double-stranded DNA products in 30 min with varying lengths (the second signal amplification). Compared to that of a typical CHA reaction, the sensitivity of CEA has increased by three orders of magnitude and the detection limit is as low as 0.228 fM. Besides, it has excellent detection performance in cancer and stem cell samples. SignificanceBy coupling embedded primer with DNA self-assembly system, a new nucleic acid detection system (CEA) with one-step operation and dual signal amplification has been successfully established. Compared with traditional dual signal amplification systems, CEA can not only significantly improve the reaction efficiency, but also greatly reduce the difficulty of detection system design and experimental operation.

Near-Infrared Bioimaging Using Two-photon Fluorescent Probes.

Near-infrared (NIR) bioimaging has emerged as a transformative technology in biomedical research. Among many fluorescent probes that are suitable for NIR imaging studies, two-photon absorption (TPA) ones represent a particularly promising category, because TPA fluorescent probes can overcome the inherent limitations of one-photon absorption (OPA) counterparts. By leveraging the unique properties of two-photon absorption, TPA fluorescent probes achieve superior tissue penetration, significantly reduced photodamage, and enhanced spatial resolution. This perspective article delves into the fundamental principles, design strategies, and representative TPA probes for various imaging applications. In particular, a number of molecular fluorescent probes, ranging from organic, inorganic, and COF/MOF-based systems are highlighted to showcase the vast scope of possible TPA probe design and application scenarios. In addition, the employment of stimulated TPA probes that are responsive to different external factors, including pH, redox species, enzymes, and hypoxia, is also discussed. In the end, the future perspectives for the continuous advancement of TPA fluorescent probes in the NIR bioimaging field are presented. For instance, it is essential to transition from cellular to in vivo imaging studies to obtain more physiologically relevant insights. Additionally, the development of "dual-function" TPA probes for both disease diagnosis and therapeutic treatment is particularly promising.

Flecainide to Prevent Atrial Arrhythmia after Patent Foramen Ovale Closure, the AFLOAT Study: A Randomized Clinical Trial.

The real incidence of atrial arrhythmia (AA) after patent foramen ovale (PFO) closure and whether this complication can be prevented remain unknown. This study assessed if flecainide is effective to prevent AA during the first 3 months after PFO closure, and if 6 months treatment by flecainide is more effective than 3 months to prevent AA after PFO closure. AFLOAT is a prospective, multicentre, randomized, open-label, superiority trial with a blind evaluation of all the endpoints (PROBE design). Patients were randomized in a 1:1:1 ratio after PFO closure to receive flecainide (150 mg once a day in a sustained-release (SR) dose) for 3 months, flecainide (150 mg od SR dose) for 6 months, or no additional treatment (standard-of-care) for 6 months. The primary endpoint was the percentage of patients with at least one episode AA (≥30s) recorded within 3 months after PFO closure on long-term monitoring with an insertable cardiac monitor (ICM). The secondary endpoint was the percentage of patients with at least one episode of AA (≥30s) recorded with ICM during the 3-6 months period after PFO closure. 186 patients were included (mean age 54 years, male 68.8%) and AA (≥30s) occurred in 53 (28.5%) patients during the 6-month follow-up; 86.8% of these AA events occurred in the first month after PFO closure. The primary outcome occurred in 33/123 (26.8%) and 16/63 (25.4%) patients receiving flecainide for at least 3 months or standard of care, respectively [Risk Difference (RD) 1.4%; 95% confidence interval (CI) -12.9% to 13.8%, NS]. The secondary endpoint occurred in 3/60 (5.0%), 4/63 (6.3%), and 5/63 (7.9%) patients receiving flecainide 6 months, 3 months or standard of care, respectively [RD -2.9%; 95% CI -12.7% to 6.9%, and RD -1.6%; 95% CI -11.8% to 8.6%, respectively]. In the first 6 months following successful PFO closure, AA (≥30s) occurred in 28.5% of cases, mostly in the first month after the procedure. Flecainide did not prevent AA after PFO closure.

Design, Synthesis and Crystal Structure of a Novel Fluorescence Probe for Zn2+ Based on Pyrano[3,2-c] Carbazole.

Zinc is a trace element, which plays an important role in many biological processes. The deficiency of zinc will lead to many diseases. Thus, it is of great significance to develop fast and efficient quantitative detection technology for zinc ions. In this study, a novel fluorescence probe FP2 was designed for Zn2+ quantification based on pyrano[3,2-c] carbazole. The structure of FP2 was characterized by 1HNMR, 13CNMR, HRMS, and X-ray diffraction. In the HEPES buffer solution, FP2 is responsive to Zn2+ and greatly enhanced. The pH value and reaction time were investigated, and the optimum reaction conditions were determined as follows: the pH was 7~9 and the reaction time was longer than 24 min. Under the optimized conditions, the concentration of FP2 and Zn2+ showed a good linear relationship in the range of 0~10 μM, and the LOD was 0.0065 μmol/L. In addition, through the 1H NMR titration experiment, density functional theory calculation, and the job plot of FP2 with Zn2+ in the HEPES buffer solution, the binding mode of FP2 and Zn2+ was explained. Finally, the method of flame atomic absorption spectrometry (FAAS) and FP2 were used to detect the content of Zn2+ in the water extract of tea. The results showed that the FP2 method is more accurate than the FAAS method, which shows that the method described in this work could be used to detect the content of Zn2+ in practical samples and verify the practicability of this method.

Probe set selection for targeted spatial transcriptomics

Targeted spatial transcriptomic methods capture the topology of cell types and states in tissues at single-cell and subcellular resolution by measuring the expression of a predefined set of genes. The selection of an optimal set of probed genes is crucial for capturing the spatial signals present in a tissue. This requires selecting the most informative, yet minimal, set of genes to profile (gene set selection) for which it is possible to build probes (probe design). However, current selections often rely on marker genes, precluding them from detecting continuous spatial signals or new states. We present Spapros, an end-to-end probe set selection pipeline that optimizes both gene set specificity for cell type identification and within-cell type expression variation to resolve spatially distinct populations while considering prior knowledge as well as probe design and expression constraints. We evaluated Spapros and show that it outperforms other selection approaches in both cell type recovery and recovering expression variation beyond cell types. Furthermore, we used Spapros to design a single-cell resolution in situ hybridization on tissues (SCRINSHOT) experiment of adult lung tissue to demonstrate how probes selected with Spapros identify cell types of interest and detect spatial variation even within cell types.

Eudaemonia-Focused Design as Intentional Praxis: An Inclusive Approach to Realising Flourishing.

In this paper, the authors consider a novel eudaemonic approach to spatial design, proposed by Mikus et al., and examine how researchers and practitioners can co-design with participants to achieve a flourishing interpretation of Aristotle's concept of eudaemonia (i.e., being one's best self). The approach was tested in doctoral research conducted during 2021 at a time when many areas were under lockdown, necessitating a virtual approach. The research engaged nine older adults, aged 65-80 and living alone in Australia, and nine design professionals via creative methods including interviews, cultural probes, and co-design workshops. Combining these methods in concert with respectful engagement was found to produce not only the aimed-for principles to guide eudaemonic home design but also unintended yet beneficial consequences resulting from meaningful inclusive praxis. Here, the authors reflect on the importance of recognising eudaemonic design as a means of careful methodological praxis to precipitate promising participant outcomes, ranging from individual intrinsic motivation to collective mutual inspiration, to enhance the research experience, and to promote flourishing.

Rational Design of a Tandem Activatable Fluorescent Probe for Differential Diagnosis and Therapeutic Assessment of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a formidable disease, distinguished by its high aggressiveness and dismal outcomes. Although leucine aminopeptidase (LAP) has been widely employed as a biomarker in biological imaging of HCC, it is still susceptible to interference from false-positive signals activated in injured liver tissues. In this study, based on the significant difference of GSH levels in alcohol-damaged liver tissues and tumor tissues, a dual-tandem activatable probe (PCLT) was designed for differential diagnosis and treatment guidance of HCC by near-infrared fluorescence (NIRF) imaging. This probe comprised a dual-locked hemicyanine dye decorated with a tetraethylene glycol chain and dual-recognition unit of glutathione (GSH) and LAP, which could be sequentially cleaved by GSH and LAP to restore its NIRF signal. PCLT excellently discriminated orthotopic HCC from ALI far earlier (7 days) than histological analysis (28 days) and exhibited higher specificity toward early orthotopic HCC than the single-locked probe (PCL). In addition, PCLT is capable of accurately delineating the tumor contour, assisting in surgical resection of HCC tumors under fluorescence visualization, and noninvasively assessing the antitumor effect of HCC chemotherapy during ferroptosis, thereby presenting promising clinical implications for clinical diagnosis and therapy of HCC.

Dual-Locked Chemiluminescent Probe Enables Precise Imaging and Timely Diagnosis of Colitis via Chymotrypsin/Vanin-1 Cascade Activation.

The development of precise diagnosis and the discovery of individualized drugs go together to provide effective therapy against inflammatory bowel disease (IBD). The exploitation of the unique imaging advantages of chemiluminescent probes represents a pivotal strategy for achieving this goal. Nevertheless, the dual-locked strategy, which is believed to enhance precision, is rarely employed in the design of chemiluminescent probes. A novel dual-locked chemiluminescent probe, BPan-CL, was designed based on IBD candidate biomarkers chymotrypsin (CHT) and vanin-1. BPan-CL exhibited specific reactivity and chemiluminescence response when subjected to simultaneous stimulation of CHT and vanin-1, with a signal-to-noise ratio superior to that of the fluorescent probe with the same dual-locked mode. In both live cell and IBD mice imaging, BPan-CL demonstrated superior sensitivity compared to its single-locked counterpart, Pan-CL. In contrast to Pan-CL, BPan-CL was able to more accurately identify IBD and healthy mice by in vivo imaging and allowed for early prediction of IBD using a noninvasive fecal test. BPan-CL has identified CHT and vanin-1 as valuable combinatorial biomarkers for accurate and early IBD diagnosis. This strategy has significant potential for use in biomedical imaging and future individualized therapies.