Ratiometric Fluorescent Probe For Detection Research Articles

Synthetic ratiometric fluorescent probes for detection of ions.

Metal cations and anions are essential for versatile physiological processes. Dysregulation of specific ion levels in living organisms is known to have an adverse effect on normal biological events. Owing to the pathophysiological significance of ions, sensitive and selective methods to detect these species in biological systems are in high demand. Because they can be used in methods for precise and quantitative analysis of ions, organic dye-based ratiometric fluorescent probes have been extensively explored in recent years. In this review, recent advances (2015-2019) made in the development and biological applications of synthetic ratiometric fluorescent probes are described. Particular emphasis is given to organic dye-based ratiometric fluorescent probes that are designed to detect biologically important and relevant ions in cells and living organisms. Also, the fundamental principles associated with the design of ratiometric fluorescent probes and perspectives about how to expand their biological applications are discussed.

A benzothiazole-based ratiometric fluorescent probe for detection of formaldehyde and its applications for bioimaging.

We presented a new benzothiazole-based fluorescent probe for ratiometric sensing of formaldehyde. Upon treatment with formaldehyde, the alkylamine-functionalized probe can be converted to its aldehyde analogue via the target-mediated 2-aza-Cope rearrangement, which led to significant shifts in both absorption (from 392 to 452nm) and emission (from 492 to 552nm) bands. The sensing mechanism was confirmed by HPLC, UV/Vis and fluorescence spectroscopy. The probe is capable of sensing formaldehyde under physiological conditions with high selectivity over potentially competing biological analytes. The probe also displayed sensitive ratiometric fluorescence response (up to 35.7 fold) for formaldehyde with a low limit detection of 0.58μM. Furthermore, the probe was successfully employed for ratiometric imaging of formaldehyde in living cells as well as in zebrafish.

A Benzothiazole-Based Fluorescent Probe for Ratiometric Detection of Al3+ and Its Application in Water Samples and Cell Imaging.

An easily prepared benzothiazole-based probe (BHM) was prepared and characterized by general spectra, including 1H NMR, 13C NMR, HRMS, and single-crystal X-ray diffraction. Based on the synergistic mechanism of the inhabitation of intramolecular charge transfer (ICT), the BHM displayed high selectivity and sensitivity for Al3+ in DMF/H2O (v/v, 1/1) through an obvious blue-shift in the fluorescent spectrum and significant color change detected by the naked eye, respectively. The binding ratio of BHM with Al3+ was 1:1, as determined by the Job plot, and the binding details were investigated using FT-IR, 1H NMR titration, and ESI-MS analysis. Furthermore, the BHM was successfully applied in the detection of Al3+ in the Songhua River and on a test stripe. Fluorescence imaging experiments confirmed that the BHM could be used to monitor Al3+ in human stromal cells (HSC).

A Förster Resonance Energy Transfer Switchable Fluorescent Probe With H2S-Activated Second Near-Infrared Emission for Bioimaging.

Real-time and accurate detection of endogenous hydrogen sulfide is of great biomedical significance. Here, a FRET-based fluorescent probe for ratiometric detection of H2S was designed to comprise an AIE luminophore TPE as an energy donor and a monochlorinated BODIPY dye as an energy acceptor and H2S-responsive site. Such a designed probe showed H2S-dependent ratiometric and light-up NIR-II emission, enabling accurate imaging of H2S-rich cancer cells and identification of H2S-rich tumors with high resolution.

A new mitochondria-targeting fluorescent probe for ratiometric detection of H2O2 in live cells

With this research we presented a ratiometric and mitochondria-target fluorescent probe (Mito-HT) for detection of H2O2 both in vitro and in live cells. Mito-HT was constructed by direct conjugation of aryl boronate to fluorophore with three synthetic steps. The borate group is cleaved from Mito-HT in the presence of H2O2, resulting in the exposure of the hydroxyl group of the electron donating group. Then the ICT mechanism was turned on, and the fluorescence emission of Mito-HT at 493 nm was red-shifted to 562 nm, thereby achieving radiometric detection of H2O2. Mito-HT exhibited a highly selectivity towards H2O2, and this interaction can be completed within 40 min. Mito-HT could be used for quantitative detection of H2O2 (0–200 μM) through ratiometric fluorescence signal readout. And limit of detection (LOD) is approximately 0.33 μM. The relatively high stability and medium fluorescence quantum yield of Mito-HT (0.39) and Mito-HT-OH (0.43) enable clear mitochondria localization and dual-channel fluorescence imaging of H2O2 in live cells with confocal microscopy.

A Ratiometric Fluorescent Probe for Detection and Quantification of Endogenous Hydrogen Peroxide in Living Cells

We propose a promising capillary immunosensor by combining porous-layer surface modification of an open-tubular capillary and streptavidin-biotin-peroxidase nano-complex signal amplification of chemiluminescence read-out. The immunosensor, namely Porous Layer Open Tubular-Signal Amplification (PLOT-SA) sensor, is successfully applied for ultra-sensitive sensing of procalcitonin (PCT) in human serum samples. The results show the PLOT-SA sensor exhibits essential features for PCT determination, including a wide linear detection range of 0.1 pg/mL-100 ng/mL, an extremely low limit-of-quantitation of 0.01 pg/mL, excellent intra-day and inter-day stability and reproducibility with RSDs less than 4.5%. Our study provides a reliable and robust approach for developing capillary sensors that can be used in analysis of complex biologic samples.

A novel pyridinium-based fluorescent probe for ratiometric detection of peroxynitrite in mitochondria.

Peroxynitrite (ONOO-) is a primary kind of reactive oxygen species. Excessive ONOO- can induce oxidative damage to biomolecules and further results in various diseases. So, quantitative monitoring ONOO- with excellent selectivity and sensitivity is imperative for elucidating its role in biological processes. In this study, a novel pyridinium fluorescent ONOO- probe (CPC) has been constructed base on ICT-modulated by combining coumarin fluorophore and diphenylphosphinate recognition group. The fluorescence response of CPC for ONOO- is realized via the removal of diphenylphosphinate group. The probe CPC shows prominent features for detection of ONOO- including fast response rate (within 3 min), excellent selectivity and sensitivity, distinct colorimetric (red to green), and a large emission wavelength shift (105 nm). The emission intensity ration (I538/I643) exhibits 153-fold enhancement along with the increasing ONOO- and the detection limit is as low as 1.60 × 10-8 M. These good response properties make CPC possible to quantitative detection of ONOO- concentration. By using the strategy, the ratiometric CPC has been employed to detection of mitochondrial ONOO- in live cell successfully.

A novel nopinone-based colorimetric and ratiometric fluorescent probe for detection of bisulfite and its application in food and living cells

A colorimetric and ratiometric fluorescent probe DTF for HSO3− was synthesized from natural and renewable nopinone. The fluorescent probe DTF can quantitatively detect HSO3− with high selectivity, sensitivity and fast response time (60 s). The detection process was accompanied with the visible color change from yellow to colorless and the fluorescent color change from green to blue. The fluorescence intensity ratio (F430/F520) was linear with the concentration of HSO3− in the range of 0–80 μM, with a limit detection of 79.2 nM. The sensing mechanism was confirmed by 1H NMR and HRMS spectra. Above all, the novel fluorescent probe DTF was successfully applied to determine the concentration of HSO3− in food samples and HeLa cells.

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria.

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.

A ratiometric fluorescent probe for detection of uric acid based on the gold nanoclusters-quantum dots nanohybrid.

Herein, we developed a simple strategy for the preparation of dual-emission fluorescent nanohybrid constructed of gold nanoclusters (Au NCs) and quantum dots (QDs). The bovine serum albumin-capped Au NCs can be directly used as the stabilizers to prepare CdS QDs. The synthesized bovine serum albumin-capped Au NCs and CdS QDs nanohybrid (BSA-Au NCs/QDs) displayed dual emission bands respectively at 490 nm and 685 nm. An obvious fluorescence quenching around 685 nm was detected with the addition of hydrogen peroxide (H2O2) in the presence of Fe2+ ions, and the fluorescence emission peak at 490 nm was not affected. Uricase can break down uric acid to produce H2O2, and we further used the BSA-Au NCs/QDs as a ratiometric fluorescent probe for determination of uric acid with the linear range from 0.67 to 60 μmol·L-1 and the detection limit of 0.21 μmol·L-1.

A FRET ratiometric fluorescent probe for detection of Hg2+ based on an imidazo[1,2-a]pyridine-rhodamine system

A novel imidazo[1,2-a]pyridine-rhodamine ratiometric fluorescent probe IP-Hg for Hg2+ based on a fluorescence resonance energy transfer mechanism has been developed. The probe has been proved to show high sensitivity and high selectivity toward Hg2+. Furthermore, it could be used for imaging Hg2+ in cells and in polluted water.

A Mn-doped ZnS quantum dots-based ratiometric fluorescence probe for lead ion detection and “off-on” strategy for methyl parathion detection

In the present study, a novel ratiometric fluorescence probe based on dual-emitting Mn-doped ZnS quantum dots (Mn:ZnS QDs) was established for detection of Pb2+ and methyl parathion (MP). The dual-emitting Mn:ZnS QDs was aqueous synthesised in one pot and played as a ratiometric fluorescence probe for the detection of Pb2+ with high sensitivity. The fluorescence intensity ratio (F450nm/F585nm) of the probe against the concentration of Pb2+ showed a good linear relationship from 10 nM to 60 nM with a detection limit of 0.5 nM. Furthermore, the Mn:ZnS QDs/Pb2+ system was established as a turn-on fluorescence sensing system to quantitative measure the MP concentration. The fluorescence intensity at 450 nm exhibited a good linear relationship in the MP concentration range from 0.19 to 0.95 μM with a detection limit of about 0.02 μM. It also had a satisfying performance in fruit and environmental water samples. Therefore, the prepared Mn:ZnS QDs-based sensor has been established as a ratiometric fluorescence probe and turn-on method for quantitative detection of lead ion and MP, respectively.

A ratiometric fluorescent probe for detection of endogenous and exogenous hydrogen sulfide in living cells

A ratiometric visualized fluorescent probe of H2S of intramolecular charge transfer (ICT) and excited intramolecular proton transfer (ESIPT) mechanisms due to solvation effects has been designed and synthesized. This chemosensor shows the distinct signal changes with dual-emission in blue and green fluorescence spectral channel (from 495 nm to 525 nm) upon the addition of H2S in a single wavelength excitation. This chemosensor exhibits the low detection limit (91 nM) and high sensitivity and selectivity. Based on this, this chemosensor was successfully used not only to monitor H2S exogenously but also used to detect and image the endogenously generated H2S in HeLa cells with excellent performance.

A far-red FRET fluorescent probe for ratiometric detection of l-cysteine based on carbon dots and N-acetyl-l-cysteine-capped gold nanoparticles.

A novel far-red fluorescence resonance energy transfer (FRET) fluorescent probe for ratiometric detection of l-cysteine (l-Cys) has been designed. The system was established a FRET assembly by positively charged carbon dots (CDs) and negatively charged N-acetyl-l-cysteine capped gold nanoparticles (NAC-AuNPs). The fluorescence of CDs at 539 nm could be effectively quenched in the presence of NAC-AuNPs owing to FRET process, while the emission of NAC-AuNPs at 630 nm was appeared. Subsequently, the interactions between l-Cys and NAC-AuNPs resulted in the decreased emission intensity of NAC-AuNPs, but the emission intensity of CDs kept almost constant due to the continuous FRET efficiency. The ratio of emission intensities at 539 and 630 nm (I539/I630) exhibited a linear correlation to the l-Cys concentration in the range of 1.0-110 μM with the detection limit of 0.16 μM. Moreover, this far-red ratiometric sensor also revealed excellent selectivity toward l-Cys over other amino acids, which showed very high potential in the practical application for diagnosing of cysteine-related disease.

A FRET based ratiometric fluorescent probe for detection of sulfite in food.

A new fluorophore pyrido[1,2-a]benzimidazole based ratiometric fluorescent probe for the selective detection of sulfite ions in water was investigated. It shows large (pseudo) Stokes shifts (260 nm), high FRET efficiency, high selectivity and sensitivity. A distinct color change from red to colorless was observed and importantly, it proves to be a convenient and efficient tool to detect the sulfite levels in sugar samples.

A ratiometric fluorescent probe for detection of exogenous mitochondrial SO2 based on a FRET mechanism.

A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO2 was designed and synthesized. The probe is based on a fluorescence resonance energy transfer (FRET) mechanism. It exhibits high selectivity and sensitivity towards SO32− with a fast response time (3 min) and detection limit of 0.13 μM. Further, it showed low cytotoxicity and was successfully applied to image exogenous mitochondrial SO2 in cells.

Near-infrared mito-specific fluorescent probe for ratiometric detection and imaging of alkaline phosphatase activity with high sensitivity.

Fluorescent probe-based analytical methods for biological species have gained increasing attention for their powerful detection and imaging capabilities. However, it is still occasionally problematic for accurate analyses due to the influence of the probe concentration, intrinsic fluorescence, instrumental factors or environmental conditions, and intense laser irradiation is usually harmful to biological systems. An NIR ratiometric fluorescent probe can eliminate the influence of these factors and lead to a higher sensing performance. We propose the introduction of a reasonable masking group onto the meso oxygen atom of a cyanine framework (Cy-O) to achieve a NIR ratiometric probe by modulating the conjugated polymethine π-electron system of the cyanine dye. Herein, we report a proof-of-the-concept attempt for a highly sensitive and selective mito-specific NIR ratiometric fluorescent sensing strategy for ALP, and the phosphate group was chosen as the functional group for ALP catalyzed hydrolysis. Upon treatment with ALP, the probe Cy-OP exhibits excellent recognition properties such as high selectivity, high sensitivity (with a low detection limit of 0.16 mU mL-1), short response time (within 30 min) and a large spectral shift (from 736 to 516 nm in the absorption spectra and 766 to 616 nm in the emission spectra) with a low fluorescence background. Moreover, the ratio of emission (I616nm/I766nm) of the enzymatic residue was favorable for its application in a complex bio-system. Consequently, the well-designed probe was successfully applied for the detection of ALP in the living cells with satisfactory results.

Fast and sensitive fluorescent probe for ratiometric detection of hydrogen sulfide in mitochondria

A hemicyanine-based fluorescent dye, Mito-BT, was developed to ratiometrically detect H2S. This dye was of high selectivity, photostability and sensitivity with a rapid response time of 10 seconds. In addition, Mito-BT was localized in mitochondria, making it a promising probe for detecting H2S in cells.

A novel ratiometric fluorescent probe for detection of iron ions and zinc ions based on dual-emission carbon dots

Herein, we developed a facile route for the preparation of dual-emission carbon dots (DCDs) by carbonizing glutathione in the water/formamide mixture. The synthesized DCDs displayed dual emission band respectively at 470 nm and 655 nm under the single-wavelength excitation at 400 nm, which can be used for the detection iron ions (Fe3+) and zinc ions (Zn2+) in different pH environments. An obvious fluorescence quenching around 655 nm was detected with the addition of Fe3+ in acidic environments, and a linear range of 2.5–30 μmol·L−1 and a detection limit of 0.8 μmol L−1 for Fe3+ were achieved. Moreover, another fluorescence quenching at 470 nm was also observed with the addition of Zn2+ in alkaline environments, and a linear range of 2.5–50 μmol L−1 and a detection limit of 1.2 μmol L−1 for Zn2+ were achieved. The prepared DCDs was used as a ratiometric fluorescent probe for determination of Fe3+ and Zn2+ ions in serum samples.

A mitochondria-targeted and FRET-based ratiometric fluorescent probe for detection of SO2 derivatives in water

A mitochondria-targeted ratiometric fluorescent probe based on the fluorophore pyrido[1,2-a]benzimidazole was developed. It showed high sensitivity and selectivity toward SO2 derivatives in water. The probe was also successfully applied to detect intrinsically generated SO2 derivatives in living glioma cells, which may be helpful in ascertaining the biological roles of SO2 in mitochondria.