Ratiometric Manner Research Articles

Multicolor Gold Clusterzyme-Enabled Construction of Ratiometric Fluorescent Sensor Array for Visual Biosensing.

The simultaneous detection of multiple bioanalytes with similar structures has been a long-standing challenge for biological research and disease diagnosis. Bioreceptor-inspired sensor arrays provide an attractive and competitive solution for addressing this challenge, but applying this technique to biosensing in practical application scenarios is complicated by many factors such as the lack of robust probes, interference from the biological matrix, and difficulty for on-site analysis. In this work, by taking advantage of the intrinsic fluorescence and enzyme-mimic properties of gold nanoclusters (AuNCs), we report the design of an innovative ratiometric sensor array toward enhanced fluorescent visual biosensing. With biologically important phosphates as an example, we show that the present fluorescent clusterzyme-based ratiometric sensor array could effectively discriminate and detect eight types of phosphates. In particular, AuNCs with three different emission colors (blue, green, and red) and good peroxidase-mimic properties were employed as the sensing units, and the presence of phosphates affected both the intrinsic fluorescence and the enzymatic activity of these AuNCs, yielding distinct optical responses in a ratiometric manner. Moreover, a portable sensor array was established by further integrating these fluorescent clusterzymes into a hydrogel matrix, which could visually identify different phosphates based on their distinct fluorescence color changes. Consequently, the point-of-care diagnosis of urinary tract infections at different levels was achieved by analyzing urinary microbial ATP via the present strategy. This study illustrates the great potential of clusterzyme-based fluorescent sensor arrays as a promising biosensing platform for disease diagnosis.

A near-infrared ratiometric fluorescent probe for the sensing and imaging of sulfur dioxide derivatives in living systems

As a reactive sulfur species, sulfur dioxide (SO2) and its derivatives play crucial role in various physiological processes, which can maintain redox homeostasis at normal levels and lead to the occurrence of many diseases at abnormal levels. So, the development of a suitable fluorescent probe is a crucial step in advancing our understanding of the role of SO2 derivatives in living organisms. Herein, we developed a near-infrared fluorescent probe (SP) based on the ICT mechanism to monitor SO2 derivatives in living organisms in a ratiometric manner. The probe SP exhibited excellent selectivity, good sensitivity, fast response rate (within 50 s), and low detection limit (1.79 µM). In addition, the cell experiment results suggested that the SP has been successfully employed for the real-time monitoring of endogenous and exogenous SO2 derivatives with negligible cytotoxicity. Moreover, SP was effective in detecting SO2 derivatives in mice.

FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Bifunctional ratiometric fluorescent membrane containing pyrene-based probe for visual monitoring and efficient removal of Al3+

A bifunctional fluorescent pyrene-based probe, PYR, was designed by incorporating pyridine and phenolic hydroxyl moieties into pyrene fluorophore, which exhibited ratiometric fluorescent response toward Al3+. PYR underwent unique monomer-excimer conversion in ratiometric manner through the formation of a 2:1 coordination complex and accomplished with fluorescent color variation from green to blue. PYR displayed excellent selectivity, high sensitivity (LOD = 0.33 μM), rapid response (<45 s), and good cytocompatibility to Al3+. Notably, a fluorescent cellulose membrane decorated with PYR was successfully developed and applied for accurately quantify of Al3+ in diverse actual samples, and high-efficiency removal (∼96%) of Al3+ from water systems, with sustainable recycling performance up to 10 times. Bifunctional membranes also played an important role in reducing the LOD of Al3+ to 16 nM through the preconcentration process. Therefore, this study is crucial to the development of environmentally sustainable strategy for monitoring and removing pollution.

Design, synthesis and application of dual-channel fluorescent probes for ratiometric detection of HClO and H2S based on phenothiazine coumarins

The ubiquity of diverse material entities in environmental matrices renders the deployment of unifunctional fluorescent indicators inadequate. Consequently, this study introduces a ratiometric dual-emission fluorescent sensor (Probe CP), synthesized by conjugating phenothiazine coumarin to hydroxycoumarin through a piperazine linker for concurrent detection of HClO and H2S. Upon interaction with HClO, the phenothiazine unit's sulfur atom undergoes oxidation to sulfoxide, facilitating a shift from red to green fluorescence in a ratiometric manner. Concurrently, at the opposite terminus of Probe CP, 2,4-dinitroanisole serves as the reactive moiety for H2S recognition; it restores the blue emission characteristic of 7-hydroxycoumarin while maintaining the red fluorescence emanating from phenothiazine coumarin as an internal standard for ratio-based assessment. Exhibiting elevated specificity and sensitivity coupled with minimal detection thresholds (0.0506 μM for HClO and 1.7292 μM for H2S) alongside rapid equilibration periods (3 min for HClO and half an hour for H2S), this sensor was efficaciously employed in cellular environments and within zebrafish models as well as imaging applications pertaining to alcohol-induced hepatic injury in murine subjects.

A disaggregation-driven BODPIY-based probe for ratiometric detection of G4 DNA

In conventional systems, fluorescent probes with planar structures usually experience the phenomenon of aggregation-induced quenching (ACQ) that limits their practical applications in bio-medical sensing. To confront such problem, an attractive way is to develop a disaggregation-induced emission (DIE) sensing strategy, where the quenched fluorescence is recovered via a disaggregation process. Currently most of the fluorescent probes designed based on DIE mechanism are signal off-on type with the fluorescent intensity changed at a single wavelength. On the other hand, ratiometric fluorescent probes have the advantage of having two distinct emission wavelengths, effectively avoiding environmental interference. The DIE probes with a ratiometric fluorescence response still poorly established. Herein, we report a BODIPY-based probe BODPA, where a 2,2′-dipicolylamine group was introduced to the meso-position of BODIPY core, for the selective ratiometric detection of G-quadruplex (G4) DNA. This probe in buffer solution formed aggregates with weak fluorescence at 508 nm. In the presence of G4 DNA, bright fluorescence was emitted at 518 nm through DIE mechanism. Taking advantage of the ratiometric manner, probe BODPA exhibited selectivity for G4 DNA over duplex DNA. The binding details between probe BODPA and G4 DNA were assessed by spectroscopic and molecular modeling methods, which suggested the ability of this probe could coordinate with G4 structure via end stacking and partial groove binding mode.

Real-time measurements of ATP dynamics via ATeams in Plasmodium falciparum reveal drug-class-specific response patterns.

Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.

Ratiometric pyrene-based fluorescent sensor for on-site monitoring of formaldehyde in foods and living cells

A ratiometric pyrene-based fluorescence sensor PN was developed for visual detection of formaldehyde (FA) by featuring homoallylamino group as specific recognition site. Sensor PN undergo a unique FA-triggered 2-aza-Cope rearrangement, which resulted in monomer-excimer conversion in a ratiometric manner accomplished with distinguishable fluorescent color change from blue to violet. Due to the advantage of high selectivity, fast response (< 100 s), low detection limit (18 μM), wide quantitative detection range (0.018–4 mM) and large ratio enhancement (up to 10-fold), PN has been successfully applied for detecting FA in diverse food samples. Notably, novel portable devices based on paper chip and alginate gel were constructed and realized visual quantitative detection of liquid and gaseous FA, as well as nondestructive monitoring volatile FA variation of real food samples during their shelf life, which were verified with the aid of chemometrics. Most importantly, sensor PN has been proven to be capable of fluorescence imaging of FA in living cells. Therefore, the impressive sensor PN is expected to provide advice and insights in the field of food safety and biological systems analysis.

Probing drug-mediated fluctuations of HClO levels in the endoplasmic reticulum by a ratiometric fluorescent probe with a large emission shift

Hypochlorous acid (HClO), one of the most important reactive oxygen species (ROS), is closely associated with endoplasmic reticulum (ER) stress, and therefore it is important to develop effective methods for detecting HClO in the ER. In this work, a ratiometric fluorescent probe (PTZC-ER) based on the phenothiazine-coumarin platform was constructed for HClO detection. In response to HClO, the fluorescence emission of the probe dramatically changes from 640 nm to 510 nm in seconds. Probe PTZC-ER exhibits outstanding selectivity and sensitivity (LOD = 11.8 nM) towards HClO. The probe can sense exogenous and endogenous HClO in a ratiometric manner, showing promising ER-targeting capability in the bioimaging experiments. Importantly, PTZC-ER can monitor fluctuations in HClO levels during drug-mediated oxidative stress.

A Photoacoustic Probe with Blood‐Brain Barrier Crossing Ability for Imaging Oxidative Stress Dynamics in the Mouse Brain

AbstractSpatiotemporal assessment of the oxidative stress dynamics in the brain is crucial for understanding the molecular mechanism underlying neurodegenerative diseases. However, existing oxidative stress probes have poor blood‐brain barrier permeability or poor penetration depth, making them unsuitable for brain imaging. Herein, we developed a photoacoustic probe that enables real‐time imaging of oxidative stress dynamics in the mouse brain. The probe not only responds to oxidative stress in a reversible and ratiometric manner, but it can also cross the blood‐brain barrier of the mouse brain. Notably, the probe displayed excellent photoacoustic imaging of oxidative stress dynamics in the brains of Parkinson's disease mouse models. In addition, we investigated the antioxidant properties of natural polyphenols in the brain of a Parkinson's disease mouse model using the probe as an imaging agent and suggested the potential of the probe for screening anti‐oxidative stress agents.

A Photoacoustic Probe with Blood-Brain Barrier Crossing Ability for Imaging Oxidative Stress Dynamics in the Mouse Brain.

Spatiotemporal assessment of the oxidative stress dynamics in the brain is crucial for understanding the molecular mechanism underlying neurodegenerative diseases. However, existing oxidative stress probes have poor blood-brain barrier permeability or poor penetration depth, making them unsuitable for brain imaging. Herein, we developed a photoacoustic probe that enables real-time imaging of oxidative stress dynamics in the mouse brain. The probe not only responds to oxidative stress in a reversible and ratiometric manner, but it can also cross the blood-brain barrier of the mouse brain. Notably, the probe displayed excellent photoacoustic imaging of oxidative stress dynamics in the brains of Parkinson's disease mouse models. In addition, we investigated the antioxidant properties of natural polyphenols in the brain of a Parkinson's disease mouse model using the probe as an imaging agent and suggested the potential of the probe for screening anti-oxidative stress agents.

FRET-based fluorescent probe with favorable water solubility for simultaneous detection of SO2 derivatives and viscosity

The intracellular viscosity is an important parameter of the microenvironment and SO2 is a vital gas signal molecule. At present, some dual-response fluorescence probes for simultaneous measurements of viscosity and SO2 derivatives (HSO3−/SO32−) possessed poor water solubility. In this work, we developed a water-soluble fluorescence probe CIJ (0.0864 g/100 mL of water at 20 °C) for simultaneous measurements of viscosity and SO2 derivatives. CIJ exhibited a sensitive fluorescence enhancement to environmental viscosity from 0.97 to 28.04 cP based on a twisted intramolecular charge transfer mechanism and was applied to effective measurement of viscosity in vitro and in vivo. CIJ could also respond to SO2 derivatives with a low detection limit (44 nM) and a fast response time (5 min) based on the nucleophilic addition reaction. Furthermore, CIJ was applied to monitor SO2 derivatives in ratiometric response manner in living cells.

A ratiometric radio-photoluminescence dosimeter based on a radical excimer for X-ray detection.

A novel radio-photoluminescence material featuring fluorochromic responses toward UV or X-ray irradiation has been obtained. Such a unique monomer- to excimer-based luminescence transition allows for dosimetry of ionizing radiation in a ratiometric manner. Rather than quenching the luminescence, the radiation-induced radical species of Th-105 boost the excimer emission, rendering it as a rare material possessing radical-excimers.

Development of an Fe2+ sensing system based on the inner filter effect between upconverting nanoparticles and ferrozine.

The ferrozine (FZ) assay is a vital oxidation state-specific colorimetric assay for the quantification of Fe2+ ions in environmental samples due to its sharp increase in absorbance at 562 nm upon addition of Fe2+. However, it has yet to be applied to corresponding fluoresence assays which typically offer higher sensitivites and lower detection limits. In this article we present for the first time its pairing with upconverting luminescent nanomaterials to enable detection of Fe2+via the inner filter effect using a low-power continuous wave diode laser (45 mW). Upon near infra-red excitation at 980 nm, the overlap of the upconversion emission of Er3+ at approximately 545 nm and the absorbance of the FZ:Fe2+ complex at 562 nm enabled measurement in the change of UCNP emission response as a function of Fe2+ concentration in a ratiometric manner. We first applied large, ultra-bright poly(acrylic acid) (PAA)-capped Gd2O2S:Yb3+,Er3+ UCNPs upconverting nanoparticles (UCNPs) for the detection of Fe2+ using FZ as the acceptor. The probe displayed good selectivity and sensitivity for Fe2+, with a low limit of detection (LoD) of 2.74 μM. Analogous results employing smaller (31 nm) PAA-capped hexagonal-phase NaYF4:Yb3+,Er3+ UCNPs synthesised in our lab were achieved, with a lower LoD towards Fe2+ of 1.43 μM. These results illustrate how the ratiometric nature of the system means it is applicable over a range of particle sizes, brightnesses and nanoparticle host matrices. Preliminary investigations also found the probes capable of detecting micromolar concentrations of Fe2+ in turbid solutions.

A ratiometric fluorescent probe based on phenothiazine and HBI motif for rapid detection of HClO and its applications

A deep-red emissive fluorescence probe PT-HBI with phenothiazine and 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazole-5(4H)-one (HBI) motif was designed and synthesized for rapid detection of HClO in a fine ratiometric manner. PT-HBI exhibited remarkable ratiometric fluorescence response to HClO within 50 s, achieving a large Stokes shift (120 nm), high sensitivity (LOD = 0.31 μM) and excellent selectivity. Among the tested analytes, only addition HClO to PT-HBI enables result from a significant fluorescence change from red emission (λmax = 660 nm) to yellow emission (λmax = 540 nm) with the ratio of F540/F660 displaying a 175-fold enhancement. In particular, PT-HBI was capable of monitoring exogenous HClO in Hela cells by ratiometric fluorescence imaging. Moreover, the probe PT-HBI has provided an extensive application for detection of HClO in real water samples and on paper-based sensor to detect HClO in different concentration.

Excitation-dependent ratiometric fluorescence response to mercury ion based on single hexagonal boron nitride quantum dots

Since a vast majority of mercury ion (Hg2+)-responsive fluorescence probes suffer from significant false positive interference in practical applications originating from their fluorescence quenching mechanism, it is still a challenge to develop a fluorescence enhancement sensor for its assay. In this work, single hexagonal boron nitride quantum dots (BNQDs)-based probe was presented for Hg2+ assay in a fluorescence enhancement-based ratiometric manner. When Hg2+ was present, the BNQDs exhibited a new fluorescence emission peak located at 560 nm under 280 nm excitation, while the original blue fluorescence of the probe at 461 nm was quenched, realizing a ratiometric fluorescence response to Hg2+. Furthermore, it is found that fluorescence enhancement at 560 nm was dramatically suppressed under 365 nm excitation, the mechanism behind this phenomenon has been explored by experiments and relevant theories. In addition to high selectivity and detection sensitivity with LOD of 0.7 nM, the prepared probe successfully demonstrated its accuracy in Hg2+ detection in environmental water samples. Moreover, the probe could be adopted for paper sensor design, and an accurate and reliable cell phone-based portable platform was demonstrated for Hg2+ assay with LOD of 1.9 nM, suggesting its potential in point-of-care detection application.

A NIR ratiometric fluorescent biosensor for sensitive detection and imaging of α‐L‐fucosidase in living cells and HCC tumor‐bearing mice

AbstractDetection and imaging of α‐L‐fucosidase (AFU) is of great value to understand its roles in hepatocellular carcinoma (HCC) and tumor early diagnosis, but ideal assays are still lacking. Herein, a near‐infrared (NIR) fluorescent biosensor (α‐Fuc‐DCM) was elaborately designed and synthesized for rapid and ratiometric detection of AFU activity in cells and HCC tumor mouse models. In the presence of AFU, this biosensor shows an enhancement in NIR emission in a ratiometric manner, which significantly improves the detection accuracy with the limit of detection as low as 4.8 mU/mL. Taking advantage of these merits, the activity of AFU in lysosomes could be visualized using ratiometric and NIR dual modality in living cells. Furthermore, its remarkable application for monitoring of endogenous AFU activity in HCC tumor‐bearing mouse model is also demonstrated with bright fluorescence signal, which indicated that the biosensor could clearly monitor the liver tumor in the early stage. Importantly, the α‐Fuc‐DCM probe can be utilized to detect the AFU in serum from HCC patients. This strategy offers a promising biosensor system for early diagnosis of HCC and studying the roles of AFU in cancers.

A versatile cellulose nanocrystal‑carbon dots architecture: Preparation and environmental/biological applications

In this work, a versatile cellulose nanocrystal‑carbon dots (CNC-CDs) architecture is prepared and applied in environmental remediation and biological sensing. The citric acid (CA) is employed to extract CNCs and in-situ synthesize blue-emissive CDs. The carboxyl-rich surface of CNC-CDs enables the chelation of Hg2+ and fluorescence quenching of CDs, realizing the detection and adsorption of Hg2+ with a film comprised of the architecture. The limit of detection (LOD) is measured as 12.8 nM with satisfying retention efficiency. The versatility of CNC-CDs is demonstrated by the fabrication of a fluorescence resonance energy transfer (FRET) sensor using m-tetrakis(4-sulfonatophenyl)porphine (TSPP) as FRET acceptor and Fe3+ receptor. Fluorescence of the sensor is responsive to Fe3+ in a ratiometric manner, which significantly contributes to the visualized and sensitive Fe3+ detection (6.9 nM). In combination with excellent biocompatibility, the FRET sensor is capable of imaging intracellular Fe3+.

A benzothiazole-based ratiometric fluorescent probe with large Stokes shifts for quantitative profiling of sulfite in real samples and living cells

Rapid, selective, and quantitative profiling of SO32− is a prerequisite for investigating its effect on different physiopathological processes. To address this challenge, our team prepared a ratiometric fluorescent probe HBTC-S, based on 4-(benzothiazol-2-yl)-3-hydroxybenzaldehyde for specific detection of SO32− under a single-wavelength excitation. The solution of probe HBTC-S itself exhibited orange fluorescence (λem = 614 nm), and became green fluorescent (λem = 471 nm) after the addition of SO32−. The vitro experiments suggested that the probe displayed high sensitivity, excellent selectivity, and a low detection limit (0.15 μM). Moreover, probe HBTC-S was successfully applied for qualitative and quantitative monitoring of SO32− in realistic samples and living HeLa cells in a ratiometric manner.

Role of Azole Drugs in Promoting Fungal Cell Autophagy Revealed by an NIR Fluorescence-Based Theranostic Probe.

Autophagy, a widespread degradation system in eukaryotes, plays an important role in maintaining the homeostasis of the cellular environment and the recycling of substances. Optical probes for the tracking of autophagy can be used as an effective tool not only to visualize the autophagy process but also to study autophagy-targeted drugs. Various molecule probes for autophagy of cancer cells emerge but are very limited for that of fungal cells, resulting in the lack of research on antifungal drugs targeting autophagy. To address this issue, we report an azole NIR fluorescence-based theranostic probe AF-1 with antifungal activity that is sensitive to autophagy-associated pH. The unique design of this probe lies in the introduction of both the pH-sensitive fluorophore with a detection range matching the pH range of the autophagy process and the conserved core structural fragment of azole drugs, providing a strategy to investigate the relationship between antifungal drug action and autophagy. As such, AF-1 exhibited excellent spectral properties and was found to target and induce the autophagy of the fungal cell membrane while maintaining moderate antifungal activity. Of note, using this theranostic probe as both a dye and drug, the autophagy process of fungi was visualized in a ratiometric manner, revealing the role of azole antifungal drugs in promoting autophagy to induce fungal cell apoptosis.