Ratiometric Properties Research Articles

Ratiometric Fluorescent pH Sensing with Carbon Dots: Fluorescence Mapping across pH Levels for Potential Underwater Applications.

Ocean acidification has become a major climate change concern requiring continuous observation. Additionally, in the industry, pH surveillance is of great importance. Consequently, there is a pressing demand to develop robust and inexpensive pH sensors. Ratiometric fluorescence pH sensing stands out as a promising concept. The application of carbon dots in fluorescent sensing presents a compelling avenue for the advancement of pH-sensing solutions. This potential is underpinned by the affordability of carbon dots, their straightforward manufacturing process, low toxicity, and minimal susceptibility to photobleaching. Thus, investigating novel carbon dots is essential to identify optimal pH-sensitive candidates. In this study, five carbon dots were synthesized through a simple solvothermal treatment, and their fluorescence was examined as a function of pH within the range of 5-9, across an excitation range of 200-550 nm and an emission range of 250-750 nm. The resulting optical features showed that all five carbon dots exhibited pH sensitivity in both the UV and visible regions. One type of carbon dot, synthesized from m-phenylenediamine, displayed ratiometric properties at four excitation wavelengths, with the best results observed when excited in the visible spectrum at 475 nm. Indeed, these carbon dots exhibited good linearity over pH values of 6-9 in aqueous Carmody buffer solution by calculating the ratio of the green emission band at 525 nm to the orange one at 630 nm (I525nm/I630nm), demonstrating highly suitable properties for ratiometric sensing.

A near-infrared fluorescent ratiometric probe with large Stokes shift for multi-mode sensing of Pb2+ and bioimaging

Pb2+ is a heavy metal ion pollutant that poses a serious threat to human health and ecosystems. The conventional methods for detecting Pb2+ have several limitations. In this study, we introduce a novel fluorescent probe that enables the detection of Pb2+ in the near-infrared region, free from interference from other common ions. A unique characteristic of this probe is its ability to rapidly and accurately identify Pb2+ through ratiometric measurements accompanied by a large Stokes shift of 201 nm. The limit of detection achieved by probe was remarkably low, surpassing the standards set by the World Health Organization, and outperforming previously reported probes. To the best of our knowledge, this is the first organic small-molecule fluorescent probe with both near-infrared emission and ratiometric properties for the detection of Pb2+. We present a triple-mode sensing platform constructed using a probe that allows for the sensitive and selective recognition of Pb2+ in common food items. Furthermore, we successfully conducted high-quality fluorescence imaging of Pb2+ in various samples from common edible plants, HeLa cells, Caenorhabditis elegans, and mice. Importantly, the probe-Pb2+ complex exhibited tumour-targeting capabilities. Overall, this study presents a novel approach for the development of fluorescent probes for Pb2+ detection.

Effective Azo-functionalized metal-organic framework colorimetric and ratiometric sensor for detection of heavy metals in aqueous solution by the naked eye

In this work, azo-functionalized zirconium metal organic framework grafted on carboxymethlated cellulose membrane (UiO-66-Azo-Im@CCM) was synthesized by solvothermal method. The developed sensor was systematically characterized and their sensitivity, selectivity and ratiometric properties were investigated. The effect of pH on the sensing performance of the fabricated sensor was also studied. The findings show that developed sensor exhibited excellent sensitivity, selectivity and ratiometric property toward copper, cobalt and nickel heavy metal ions. The calculated limit of detection (LOD) these heavy metals ions were lower than the world health organization permissible concentration that reveals its potential candidacy in monitoring these heavy metals. This work confirms that the developed UiO-66-Azo-Im@CCM can used as powerful tool for the monitoring of Cu2+, Ni2+, and Co2+ without need of the sophisticated instrument. Therefore, we anticipate that it will play its role in environmental remediation in general wastewater treatment in particular.

New High-Performance Fluorescent Dye Scaffolds: Applications for Bioimaging and Biosensing

AbstractFluorescence imaging in the shortwave infrared region (SWIR, 850–2500 nm) window has become an indispensable tool in biomedical research because it has weaker absorption, less light scattering, and less background fluorescence in this window. As we know, the design and synthesis of fluorescent dyes and fluorescent probes are the cores of fluorescence imaging and biosensing. Thus, their photophysical mechanisms exploring and bioanalytical applications are multidisciplinary and cutting-edge research topics. In this regard, we have been working on high-performance fluorescent dyes and fluorescent probes for years. In short, we have developed a series of bright, stable, aggregation-resistant, SWIR fluorescent dyes ECXs, based on a carbon-bridged spiro ring strategy. We also developed a series of high-performance SWIR fluorescent dyes CXs and Chrodols, which combine the structural advantages of cyanine and xanthenoid dyes. Based on these new SWIR scaffolds, we further constructed some activatable SWIR fluorescent probes with OFF-ON or ratiometric properties for biosensing in vivo. Therefore, the main line of our work is to gain an in-depth understanding of the photophysical mechanisms of fluorescent dyes, to create high-performance luminescent dyes, and to further develop fluorescent probes for bioimaging and biosensing.1 Introduction2 ECX Dyes Based on a Carbon-Bridged Spiro Ring Strategy3 Fluorescent Dyes and Fluorescent probes Combining the Structures of Cyanine and Xanthenoid Dyes3.1 CX Series Dyes3.2 A Functionalized Modified CX probe NRh3.3 CX-like Dyes with a Secondary Amino Py-23.4 Chrodol Series Dyes4 Conclusion and Future Prospects

Neuraminidase-Activatable NIR Fluorescent Probe for Influenza Virus Ratiometric Imaging in Living Cells and Colorimetric Detection on Cotton Swabs

Influenza, as one of the most serious communicable respiratory infectious diseases, is caused by influenza viruses. Neuraminidase (NA), also referred to as sialidase, is an important protein on the influenza virus surface. Because of its enzymatic function, NA has been selected as a promising target to develop influenza virus diagnosis reagents or drugs. Herein, a neuraminidase-responsive, light-up, near-infrared (NIR) fluorescence probe (SA-DCM) was reported that possesses both ratiometric and colormetric properties for different applications. Benefitting from the ratiometric properties, SA-DCM showed an increased anti-interference capacity for environmental fluctuation and enhanced detection accuracy with a large Stokes shift (>150 nm) and outstanding sensitivity and selectivity. Due to its colorimetric property, a color shift of the product HO-DCM from yellow to purple occurs when the pH value is adjusted. Therefore, a colorimetric approach through direct color conversion on a cotton swab to detect NA was established, and the result could be directly read by the naked eye with simplicity and practicality.

An ESIPT-based fluorescent turn-on probe with isothiocyanate for detecting hydrogen sulfide in environmental and biological systems

A probe with an isothiocyanate group was synthesized and evaluated for its H2S sensing ability. Upon addition of H2S, the probe exhibited ratiometric properties during absorption with a red-shift. The probe exhibited fluorescent off–on responses towards H2S via the ESIPT process, due to the conversion of isocyanate into amine. UV–vis, fluorescence, and 1H NMR spectroscopic analyses were performed to investigate the sensing mechanism. The probe has a large Stokes shift, short response time, and low detection limit. It can be used to estimate H2S levels within the range of 0–36 nM. The practical applicability of the probe was demonstrated using water samples and living cells.

Dual-Stimuli-Responsive Probes for Detection of Ovarian Cancer Cells and Quantification of Both pH and Enzyme Activity

Abstract Many physiological processes involve multiple coordinated chemical and/or biological events. Therefore, it is considered urgent to develop dual-responsive probes for a more comprehensive understanding of the synergistic effects between multiple analytes in complex cellular environments. In this study, we developed a dual-responsive probe βgal-BP-PMB (β-galactosyl–3,3′-dihydroxy-2,2′-bipyridyl–p-methoxybenzyl), the photoluminescence of which can be activated by β-galactosidase (β-gal) and acidic conditions. The overexpression of β-gal is an important feature of senescent and ovarian cancer cells. Single-input activatable probes for detecting β-gal activity in ovarian cancer cells can induce a false positive response from senescent cells. Because the lysosomal pH in senescent cells is increased, probe βgal-BP-PMB can be specifically activated in ovarian cancer cells, but silenced in senescent cells. Probe βgal-BP-PMB has a small molecular size, high sensitivity towards targeted stimuli and unique ratiometric properties, thereby enabling the quantification of both pH and enzyme activity. Such dual-responsive probes could earn a unique place in the field of bioimaging, where multiple analytes should be accurately and simultaneously monitored.

Dual-Stimulus Responsive Near-Infrared Reversible Ratiometric Fluorescent and Photoacoustic Probe for In Vivo Tumor Imaging.

Tumor-specific imaging is a major challenge in clinical tumor resection. To overcome this problem, several activatable probes have been developed for use in tumor imaging. However, most of these probes are activated based on a single-factor stimulation and are irreversible. Therefore, false signals that make tumor-specific imaging difficult are easily generated. We have developed a new dual-stimulus responsive near-infrared (NIR) reversible adenosine 5'-triphosphate (ATP)-pH probe for fluorescence and photoacoustic ratiometric imaging of tumors. Since the H+ and ATP content is significantly higher in the tumor microenvironment than that in normal tissues, the Förster resonance energy transfer-based probe ATP-pH was constructed with silicon rhodamine as the donor, CS dye as the acceptor, and ATP/H+ recognition units that could only be activated when both H+ and ATP were connected to the acceptor. The ATP-pH probe is reversibly activated by both the H+ and ATP, which effectively reduces the cumulative response of the probe in circulation after intravenous injection. Further, the NIR ratiometric property of the probe makes it suitable for in vivo imaging. Finally, our probe was successfully utilized in ratiometric photoacoustic and fluorescence tumor imaging and ratiometric fluorescence imaging-guided tumor resection.

A ratiometric fluorescent probe for mitochondrial esterase specific detection in living cells

Monitoring esterase activities in living cells are beneficial to predict the metabolism of various esters and evaluate the efficacy of therapeutic prodrugs. Up to now, subcellular esterase in cytoplasm/lysosome has been evaluated by fluorescence technology. However, accurate and reliable mitochondrial esterase detection remains a challenge, due to the lack of organelle specificity. Here, we demonstrate a mitochondrial directed esterase probe (Naph-Ester) which allows for the quantitative detection of mitochondrial esterase, as well as monitoring esterase content change in real time. Naph-Ester manifests remarkably ratiometric and red-shifted (about 127 nm) fluorescence in the presence of esterase. Benefiting from the ratiometric property, high specificity and low cytotoxicity, Naph-Ester provides reliable detection of mitochondrial esterase in living cells. Notably, its internalization path was identified to be energy dependent endocytosis, avoiding from hydrolysis by cytostromatic esterase. Overall, the present work demonstrates an effective esterase probe which can be applied as a reliable tool to monitor mitochondrial esterase.

A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems

The specific detection of cysteine (Cys) over homocysteine (Hcy), glutathione (GSH) and other amino acids is of great significance for studying its biological functions as well as for the diagnosis of related diseases. Chloroacetyl group was often used as a reaction site for cysteine fluorescent probes for its sensitivity and selectivity. However, high background fluorescence and low stability are common problems encountered by such probes. Here, four chloroacetyl group based fluorescent probes (C1, C2, C3, and H4) was synthesized for a comparative study. We found that the inefficient quenching ability of chloroacetyl group turned into an advantage when connected with a ratiometric fluorophore. With the modification of chloroacetyl group, probe H4 displayed excellent ratiometric property and great selectivity for Cys, the stability was also improved. Additionally, the probe was successfully applied for quantitative detection of Cys in fetal bovine serum and real-time imaging in living HeLa cells with low toxicity.

Two-component ratiometric sensor for Cu2+ detection on paper-based device.

The copper(II) ion (Cu2+) has played an indispensable role in diverse kinds of functional physiological processes of organisms, which has become of growing interest. Despite the fact that numerous Cu2+ test papers using fluorescent probes have been fabricated, sensors featuring the ratiometric property that integrates quenched probes and an inner standard dye are rarely reported. Herein, a two-component ratiometric sensor in a paper-based device is proposed to realize highly selective Cu2+ detection. To overcome shortcomings such as low signal-to-noise ratio and incorrect response of the quenching probe, a novel BODIPY-based turn-off probe (P2017) is designed and introduced into the paper-based device with better water solubility and selectivity for Cu2+ detection. Furthermore, a reference dye (B001), exhibiting an emission at 690nm when the excitation wavelength is 480nm, is also introduced into the paper-based device. These two components can enhance the quality of the signal as P2017 is sensitively quenched by Cu2+, while B001 with a photostable property, serving as an internal benchmark, is unable to react with Cu2+. The results indicated that the two components provided a new concept for optimizing paper-based device fabrication and developing accurate, simple, and inexpensive Cu2+ detection methods, which could be potentially applied to monitor human health and the environment in remote areas. Graphical abstract.

Fluorescence Enhancement of Terminal Amine Assembled on Gold Nanoclusters and Its Application to Ratiometric Lysine Detection.

Ratiometric fluorescent sensors have emerged as an attractive tool for analytical sensing and optical imaging due to their providing a built-in self-calibration for environmental effects. However, cumbersome processes of nanoparticles modified with fluorophores for constructing traditional ratiometric sensors limit their further application. Herein, we report a facile and label-free strategy for constructing a ratiometric sensor based on an aggregation-induced-emission (AIE)-active amine-terminated small molecule on the surface of gold nanoclusters (AuNCs). Intrinsic fluorescence of the terminal primary amine of the small molecule lysine resulting from AIE was first observed in the presence of glutathione-stabilized gold nanoclusters (GSH-AuNCs). Using lysine as both the fluorophore and the analyte, the synthesized GSH-AuNCs showed a good lysine-responsive ratiometric property. The AIE-active dual-emitting fluorescence property of the GSH-AuNCs/lysine complex made it feasible to achieve ratiometrically detection of the analyte without conjugated fluorogen. This AIE-active GSH-AuNC-based biosensor possesses high selectivity, rapid response, and excellent photostability. Moreover, the strategy opens a new pathway for the construction of a label-free ratiometric fluorescent sensor with various applications.

Endoplasmic Reticulum-Directed Ratiometric Fluorescent Probe for Quantitive Detection of Basal H2O2.

The endoplasmic reticulum (ER) has a central role in the fine-tuning of environmental and internal stimuli. We herein report a ratiometric fluorescent probe, α-Naph, capable of determining basal H2O2 in the ER. The probe specifically responds to H2O2. The limit of detection of the probe is as low as 38 nM, making it a feasible sensor to image intracellular basal H2O2. In addition, utilizing its ratiometric property, we are able to measure the concentration of H2O2 in the ER quantitatively, eliminating the error caused by the probe concentration and environment. The intracellular concentration of H2O2 in the ER is calculated to be 0.692 μM under normal conditions and 1.26 μM under the stimulation of phorbol myristate acetate.

High-Efficiency Cell-Penetrating Helical Poly(phenyl isocyanide) Chains Modified Cellular Tracer and Nanovectors with Thiol Ratiometric Fluorescence Imaging Performance

In order to precisely tail the endocytosis process and determine the internal location of drug carriers in cells, fluorescent tracers with high sensitivity and versatility are one of the most powerful tools. Nevertheless, conventional single fluorescent probes always suffered from the interference of background fluorescence or the lack of long-time monitoring capability, resulting in the low resolution and efficiency. To overcome this drawback, nanocarriers capable of multicolor fluorogenic and ratiometric properties became an urgently needed solution. In this contribution, starting from pentafluorophenyl ester (PFP)- and tetraphenylethene (TPE)-functionalized phenyl isocyanide (PI) monomers as well as l-hydrophilic (HP) PI monomers, a type of well-defined amphiphilic block copolymer, P(PFPPI-co-TPEPI-co-HPPI)-b-HPPPI, with controlled molecular weights and tunable compositions was prepared through sequential living copolymerization with phenylethynyl Pd(II) complex as a single catalyst in one pot. Disulfi...

Identification of lesion subtypes in biopsies of ductal carcinoma in situ of the breast using biomarker ratio imaging microscopy.

Although epidemiological studies propose aggressive and non-aggressive forms of ductal carcinoma in situ (DCIS), they cannot be identified with conventional histopathology. We now report a retrospective study of human biopsy samples using biomarker ratio imaging microscopy (BRIM). Using BRIM, micrographs of biomarkers whose expression correlates with breast cancer aggressiveness are divided by micrographs of biomarkers whose expression negatively correlates with aggressiveness to create computed micrographs reflecting aggressiveness. The biomarker pairs CD44/CD24, N-cadherin/E-cadherin, and CD74/CD59 stratified DCIS samples. BRIM identified subpopulations of DCIS lesions with ratiometric properties resembling either benign fibroadenoma or invasive carcinoma samples. Our work confirms the existence of distinct subpopulations of DCIS lesions, which will likely have utility in breast cancer research and clinical practice.

Assembly of Tetraphenylethene Containing Amine and Crown Ether Units and Aggregation-Induced Emission Properties

Dibenzo-24-crown-8 (DB24C8) and dibenzylammonium (DBA) are always utilized for the construction of supramolecular assemblies. We reported a new kind supramolecular assembly polymer constructed by M1 which was synthesized from tetraphenylethene (TPE) linked with DB24C8 and DBA through click reaction. The chemical structures of all the compounds were fully characterized by H NMR, C NMR, and MS. The H NMR titration showed that the supramolecular assembly was successfully constructed in acidic condition. The fluorescent results showed that the assemblies displayed ratio-metric properties with the changes of aggregation-induced emission and monomer-emission, and fluorescence quenching with addition of Pd.

Interferences of resveratrol with fura-2-derived fluorescence in intracellular free-Ca(2+) concentration determinations.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is an antioxidant widely employed in cell physiology studies. It has been reported that it interferes with fura-2-derived fluorescence, making the employment of this dye nonviable. In this work, the interference of resveratrol with fura-2 determinations of intracellular free-Ca(2+) concentration ([Ca(2+)]c) was examined. Solutions containing different concentrations of resveratrol, with or without fura-2, in the presence or in the absence of Ca(2+), were analyzed by spectrofluorimetry. AR42J tumor cells were employed to study the influence of resveratrol on fura-2 fluorescence in living cells, by single cell fluorimetry. Resveratrol impaired the detection of fura-2-fluorescence emission (510nm) at the 340, 360 and 380nm excitation wavelengths. Resveratrol emitted fluorescence at 510nm when lighted at all three excitation wavelengths. In addition, resveratrol emitted fluorescence at 380nm when excited at 340nm. Our observations suggest that the employment of the ratiometric properties of fura-2 to follow changes in [Ca(2+)]c in the presence of resveratrol is not viable. However, we think that the 380nm excitation light could be employed. Results could be expressed as F0/F380, where F0 is the resting fluorescence and F380 is the value of fluoresce at a certain time point. We could follow changes in [Ca(2+)]c evoked by CCK-8, and we also detected Ca(2+) mobilization by 100µM resveratrol in AR42J cells. This investigation presents evidence demonstrating that resveratrol interferes with fura-2 fluorescence spectra. Nevertheless, a chance still exists if the 380nm excitation wavelength is employed in the middle or low micromolar concentrations of resveratrol.

Two‐photon microscopy enables non‐invasive, in vivo, longitudinal study of arterial Ca2+ and diameter in optical biosensor mice during the development of hypertension (856.7)

Here, we report that use of two‐photon microscopy of ear arteries in exMLCK biosensor mice enables non‐invasive, in vivo, serial examination of vascular smooth muscle Ca2+ during the development of hypertension. ExMLCK mice express a FRET‐type biosensor of Ca2+/Calmodulin based on smooth muscle myosin light chain kinase (MLCK) and its ratiometric properties permit direct quantification of intracellular Ca2+ concentration. Anesthetized biosensor mice were positioned on a custom platform, facilitating imaging of selected arterial branches within the intact depilated ear, and studied before and during chronic infusion of angiotensin‐II (ANGII; 800 µg/g/day) via mini‐osmotic pump. Acute responsiveness to i.p. phenylephrine (PE; 30µg/g) was determined in a separate group of mice equipped with arterial blood pressure radio telemetry. Eight days of chronic ANGII increased intracellular Ca2+ from 294±47nM to 501±92nM (P<0.05) and decreased artery diameter by ‐14±2% (P<0.05). Acute PE induced robust vasomotion and 2‐3 fold oscillations in spatially averaged Ca2+. In summary, this new methodology permits for the first time the simultaneous study of Ca2+ signaling, structural remodeling and vascular reactivity in the intact living mouse during hypertension development.Grant Funding Source: Supported by NHLBI RO1091969, RO1HL107654, RO1DK077140, and T32HL072751.

Fluorescence Ratiometric Properties Induced by Nanoparticle Plasmonics and Nanoscale Dye Dynamics

Nanoscale transport of merocyanine 540 within/near the plasmon field of gold nanoparticles was recognized as an effective inducer of single-excitation dual-emission ratiometric properties. With a high concentration of the signal transducer (ammonium), a 700% increase in fluorescence was observed at the new red-shifted emission maximum, compared to a nanoparticle free sensor membrane. A previously nonrecognized isosbestic point is demonstrated at 581.4 ± 0.1 nm. The mechanism can be utilized for enhanced and simplified ratiometric optical chemical sensors and potentially for thin film engineering to make solar cells more effective and stable by a broader and more regulated absorption.

ChemInform Abstract: Supramolecular Chemical Sensors Based on Pyrene Monomer—Excimer Dual Luminescence.

AbstractReview: 131 refs.