Simultaneous sensing of carbidopa and levodopa by a novel strategy based on dual-emission ratiometric assay of modified carbon dots.

Rapid control of the content of Parkinson's drugs in biological fluids and pharmaceutical formulations is of great importance because changes in the concentration of these drugs affect their bioavailability and biopharmaceutical properties. Therefore, we presented a simple and convenient method for the ratiometric detection of carbidopa and levodopa for carbon dots (CDs) dual-fluorescent emission. Dual-emission CDs were prepared from chitosan using a microwave method, following which the surface was chemically modified with terephthalaldehyde. CDs had two strong well-separated peaks at 445 and 510 nm. The relative measurement of carbidopa and levodopa was based on the static extinction of CDs at 445 nm and increase at 510 nm, respectively. The linear range for carbidopa measurement was 2.5-300 nM, with a limit of detection (LOD) of 2.1 nM, and a relative standard deviation (RSD) of 1.68%. Further, the linear range for levodopa measurement was equal to 3.0-400 nM, with LOD and RSD% of 2.8 nM and 3.5%, respectively. Also, selectivity of ratiometric sensor in the presence of interferences was investigated, which showed that the recovery of carbidopa and levodopa in serum and urine samples has changed between 96.80% and 116.24% with RSD% 0.11-0.77. CDs also provided good results for the determination of carbidopa and levodopa in real samples, and had high selectivity in the presence of possible interferences.

Activatable Probes for Ratiometric Imaging of Endogenous Biomarkers In Vivo.

Dynamic variations in the concentration and abnormal distribution of endogenous biomarkers are strongly associated with multiple physiological and pathological states. Therefore, it is crucial to design imaging systems capable of real-time detection of dynamic changes in biomarkers for the accurate diagnosis and effective treatment of diseases. Recently, ratiometric imaging has emerged as a widely used technique for sensing and imaging of biomarkers due to its advantage of circumventing the limitations inherent to conventional intensity-dependent signal readout methods while also providing built-in self-calibration for signal correction. Here, the recent progress of ratiometric probes and their applications in sensing and imaging of biomarkers are outlined. Ratiometric probes are classified according to their imaging mechanisms, and ratiometric photoacoustic imaging, ratiometric optical imaging including photoluminescence imaging and self-luminescence imaging, ratiometric magnetic resonance imaging, and dual-modal ratiometric imaging are discussed. The applications of ratiometric probes in the sensing and imaging of biomarkers such as pH, reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione (GSH), gas molecules, enzymes, metal ions, and hypoxia are discussed in detail. Additionally, this Review presents an overview of challenges faced in this field along with future research directions.

Ratiometric optical probes for biosensing.

Biosensing by optical probes is bringing about a revolution in our understanding of physiological and pathological states. Conventional optical probes for biosensing are prone to inaccurate detection results due to various analyte-independent factors that can lead to fluctuations in the absolute signal intensity. Ratiometric optical probes provide built-in self-calibration signal correction for more sensitive and reliable detection. Probes specifically developed for ratiometric optical detection have been shown to significantly improve the sensitivity and accuracy of biosensing. In this review, we focus on the advancements and sensing mechanism of ratiometric optical probes including photoacoustic (PA) probes, fluorescence (FL) probes, bioluminescence (BL) probes, chemiluminescence (CL) probes and afterglow probes. The versatile design strategies of these ratiometric optical probes are discussed along with a broad range of applications for biosensing such as sensing of pH, enzymes, reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione (GSH), metal ions, gas molecules and hypoxia factors, as well as the fluorescence resonance energy transfer (FRET)-based ratiometric probes for immunoassay biosensing. Finally, challenges and perspectives are discussed.

Smartphone-based on-site detection of hydrogen peroxide in milk by using a portable ratiometric fluorescent probe.

The on-site detection of hydrogen peroxide (H2O2) is important for maintaining food safety as the ingestion of H2O2 can lead to serious pathological conditions. However, most reported fluorescent probes require a fluorometer to ensure readable signal output and reliable detection result. Consequently, the fluorescent detection of H2O2 can be realized only within a standard laboratory setting. Herein, we report a novel supramolecular strategy that can successfully convert the typical off-on response to H2O2 into a ratiometric response, which allows the on-site detection of H2O2 when used in conjunction with a smartphone-based 3D-printed miniaturized testing system. This method has acceptable sensitivity, good anti-interference ability, and desirable accuracy compared to a standard detection method. More importantly, this portable ratiometric method can be used to detect H2O2 residue in commercial milk samples with the simple testing apparatuses.

Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells.

The cross talk between mitochondrial dynamic structure, determined primarily by mitochondrial fission and fusion events, and mitochondrial function of energetics, primarily ATP and ROS production, is widely appreciated. Understanding the mechanistic details of such cross talk between mitochondrial structure and function needs integrated quantitative analyses between mitochondrial dynamics and energetics. Here we describe our recently designed approach of mito-SinCe2 that involves high resolution confocal microscopy of genetically expressed ratiometric fluorescent probes targeted to mitochondria, and its quantitative analyses. Mito-SinCe2 analyses allows for quantitative analyses of mitochondrial structure-function relationship in single cells toward understanding the role of mitochondria and their heterogeneity in various physiological and pathological conditions.

Determination of Ag[I] and NADH Using Single-Molecule Conductance Ratiometric Probes.

The sensing platform based on single-molecule measurements provides a new perspective for constructing ultrasensitive systems. However, most of these sensing platforms are unavailable for the accurate determination of target analytes. Herein, we demonstrate a conductance ratiometric strategy combing with the single-molecule conductance techniques for ultrasensitive and precise determination. A single-molecule sensing platform was constructed with the 3,3',5,5'-tetramethylbenzidine (TMB) and oxidized TMB (oxTMB) as the conductance ratiometric probes, which was applied in the detection of Ag[I] and nicotinamide adenine dinucleotide (NADH). It was found that the charge transport properties of TMB and oxTMB were distinct with more than an order of magnitude change of the conductance, thus enabling conductance ratiometric analysis of the Ag[I] and NADH in the real samples. The proposed method is ultrasensitive and has an anti-interference ability in the complicated matrix. The limit of detection can be as low as attomolar concentrations (∼34 aM). We believe that the proposed conductance ratiometric approach is generally enough to have a promising potential for broad and complicated analysis.

Environmentally Sensitive Color-Shifting Fluorophores for Bioimaging.

We introduce color-shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, thereby enabling the generation of ratiometric fluorescent probes and biosensors. Specifically, we developed a ratiometric biosensor for imaging calcium ions (Ca2+ ) in living cells, ratiometric probes for different proteins, and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate.

Using Native Chemical Ligation for Site-Specific Synthesis of Hetero-bis-lanthanide Peptide Conjugates: Application to Ratiometric Visible or Near-Infrared Detection of Zn2.

The interest in ratiometric luminescent probes that detect and quantify a specific analyte is growing. Owing to their special luminescence properties, lanthanide(III) cations offer attractive opportunities for the design of dual-color ratiometric probes. Here, the design principle of hetero-bis-lanthanide peptide conjugates by using native chemical ligation is described for perfect control of the localization of each lanthanide cation within the molecule. Two zinc-responsive probes, r-LZF1Tb|Cs124|Eu and r-LZF1Eu|Cs124|Tb are described on the basis of a zinc finger peptide and two DOTA (DOTA=1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) complexes of terbium and europium. Both display dual-color ratiometric emission in response to the presence of Zn2+ . By using a screening approach, anthracene was identified for the sensitization of the luminescence of two near-infrared-emitting lanthanides, Yb3+ and Nd3+ . Thus, two novel zinc-responsive hetero-bis-lanthanide probes, r-LZF3Yb|Anthra|Nd and r-LZF3Nd|Anthra|Yb were assembled, the former offering a neat ratiometric response to Zn2+ with emission in the near-infrared around 1000 nm, which is unprecedented.

Analyzing Intracellular Gradients in Pollen Tubes.

Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.

Fluorescent, colourimetric, and ratiometric probes based on diverse fluorophore motifs for mercuric(II) ion (Hg2+) sensing: highlights from 2011 to 2019.

Though it has not been shown to deliver any biological importance, mercuric(II) ion (Hg2+) is a deleterious cation which poses grievous effects to the human body and/or the ecosystem, hence, the need for its sensitive and selective monitoring in both environmental and biological systems. Over the years, there has been a great deal of work in the use of fluorescent, colourimetric, and/or ratiometric probes for Hg2+ recognition. Essentially, the purpose of this review article is to give an overview of the advances made in the constructions of such probes based on the works reported in the period from 2011 to 2019. Discussion in this review work has been tailored to the kinds of fluorophore scaffolds used for the constructions of the probes reported. Selected examples of probes under each fluorophore subcategory were discussed with mentions of the typically determined parameters in an analytical sensing operation, including modulation in fluorescence intensity, optimal pH, detection limit, and association constant. The environmental and biological application ends of the probes were also touched where necessary. Important generalisations and conclusions were given at the end of the review. This review article highlights 196 references.

A Strategy of NIR Dual-Excitation Upconversion for Ratiometric Intracellular Detection.

Intracellular detection is highly desirable for biological research and clinical diagnosis, yet its quantitative analysis with noninvasivity, sensitivity, and accuracy remains challenging. Herein, a near-infrared (NIR) dual-excitation strategy is reported for ratiometric intracellular detection through the design of dye-sensitized upconversion probes and employment of a purpose-built NIR dual-laser confocal microscope. NIR dye IR808, a recognizer of intracellular analyte hypochlorite, is introduced as energy donor and Yb,Er-doped NaGdF4 upconversion nanoparticles are adopted as energy acceptor in the as-designed nanoprobes. The efficient analyte-dependent energy transfer and low background luminescence endow the nanoprobes with ultrahigh sensitivity. In addition, with the nonanalyte-dependent upconversion luminescence (UCL) excited by 980 nm as a self-calibrated signal, the interference from environmental fluctuation can be alleviated. Furthermore, the dual 808/980 nm excited ratiometric UCL is demonstrated for the quantification of the level of intracellular hypochlorite. Particularly, the intrinsic hypochlorite with only nanomolar concentration in live MCF-7 cells in the absence of exogenous stimuli is determined. Such an NIR dual-excitation ratiometric strategy based on dye-sensitized UCL probes can be easily extended to detect various intracellular analytes through tailoring the reactive NIR dyes, which provides a promising tool for probing biochemical processes in live cells and diagnosing diseases.

A ratiometric fluorescent probe for sensitive determination of the important glycopeptide antibiotic vancomycin.

A novel sensitive and selective probe for the important antibiotic vancomycin (Van) has been synthesized by integrating a coumarin and a fluorescein as dual fluorescence reporters and a Van binding peptide D-Ala-D-Ala. Only weak green fluorescence was initially observed, which was mostly attributed to fluorescence self-quenching induced by fluorophore stacking. Upon the binding of Van with the D-Ala-D-Ala peptide, the fluorescence turned on, probably due the disaggregation of fluorophores. The intensity ratio of the dual emission bands I519/I446 exhibited an excellent linear relationship with the concentration of Van increasing from 0-20 µM in synthetic urine. The lowest detection limit was calculated to be 92.8 nM in urine, which made the probe applicable in clinically relevant concentration ranges. The synthetic probe has also shown the potential for Van detection in human serum. More interestingly, this probe has been successfully applied for in vivo imaging of Van in zebrafish. Graphical Abstract.

An ESIPT-Based Ratiometric Fluorescent Probe for Highly Sensitive and Rapid Detection of Sulfite in Living Cells.

The novel ratiometric fluorescent probe HPQRB with an ESIPT effect based on Michael addition for highly sensitive and fast detection of sulfite in living HepG2 cells is reported. HPQRB can be easily synthesized by a two-step condensation reaction. HPQRB has a large emission shift (Δλ=116 nm), which is beneficial for fluorescence imaging research, and its sulfite-responsive site is based on a rhodamine-like structure with the emission peak at 566 nm, which decreases with increasing sulfite concentration. and its HPQ structure always has an ESIPT effect throughout the reaction process, keeping the emission peak at 450 nm as a self-reference. In particular, HPQRB has high selectivity for sulfite and responds quickly (within 30 s) with a low detection limit (44 nM). Furthermore, HPQRB has been successfully used for fluorescence imaging of sulfite in HepG2 cells, demonstrating the superior ability to detect sulfite under physiological conditions.

Visual Assay of Glutathione in Vegetables and Fruits Using Quantum Dot Ratiometric Hybrid Probes.

Future food safety monitoring with simple, fast, and visual methods has become increasingly important. Accordingly, this work was designed to construct a new-style dual-emission ratiometric fluorescent probe (CdSe@SiO2@CdTe) for visual assay of glutathione (GSH) with a "turn on" strategy. After adding Hg2+, the red fluorescence of the outer CdTe quantum dots (QDs) was quenched through both electron transfer and ion-binding processes. Upon the addition of GSH, the red fluorescence occurred again owing to the strong affinity between GSH and Hg2+, whereas the inner green fluorescence of CdSe QDs was unchanged, leading to a clearly recognizable fluorescence color change (from green to orange-red). In the concentration range from 0.1 to 10 µM, the relative fluorescence intensity ratios ( I619/ I535) showed an excellent linear correlation with the concentration of GSH, and the detection limit was as low as 42 nM under optimal conditions. Meanwhile, the ratiometric hybrid probes were successfully applied for direct visual sensing GSH in real vegetable and fruit samples.

A Reversible Fluorescent Probe for Real-Time Quantitative Monitoring of Cellular Glutathione.

The ability to monitor and quantify glutathione (GSH) in live cells is essential in order to gain a detailed understanding of GSH-related pathological events. However, owing to their irreversible response mechanisms, most existing fluorescent GSH probes are not suitable for this purpose. We have developed a ratiometric fluorescent probe (QG-1) for quantitatively monitoring cellular GSH. The probe responds specifically and reversibility to GSH with an ideal dissociation constant (Kd ) of 2.59 mm and a fast response time (t1/2 =5.82 s). We also demonstrate that QG-1 detection of GSH is feasible in a model protein system. QG-1 was found to have extremely low cytotoxicity and was applied to determine the GSH concentration in live HeLa cells (5.40±0.87 mm).

Ratiometric NanoCluster Beacon: A Label-Free and Sensitive Fluorescent DNA Detection Platform.

Although researches until now have emphasized the influence of an oligonucleotide sequence on the fluorescence of oligonucleotide-stabilized silver nanoclusters (AgNCs), this influence has been explored as a novel ratiometric fluorescent signal transduction in this work. This study builds on our original discovery of a template-transformation phenomenon, which demonstrated that the connection of a special DNA fragment (5'-CACCGCTTT-3') with a green-emitting AgNC nucleation sequence (GNuS, 5'-TGCCTTTTGGGGACGGATA-3') creates a red-emitting AgNC nucleation sequence (RNuS, 5'-CACCGCTTTTGCCTTTTGGGGACGGATA-3'). Attempts to expand this idea and construct elegant ratiometric NanoCluster Beacons (NCBs) for DNA sequence detection are not straightforward, and, thus, we carried out a series of investigations with the goal of understanding the mechanism of this template-transformation phenomenon. Experimental results showed that the six-nucleotide fragment (5'-CACCGC-3') at the 5'-end of RNuS acts as a template convertor and takes full responsibility for the template transformation from GNuS to RNuS. Moreover, we found that the appropriate proximity of the convertor to GNuS also plays a significant role in the template transformation. We then show that the insights gained here for the template-transformation mechanism allow us to construct ratiometric NCBs by simply appending the convertor and the GNuS onto a rationally designed stem-loop probe. This new type of NCB emits intense red fluorescence without the addition of a target DNA and emerges as a new, bright green emission only when hybridized to its target DNA. By measuring the distinct variation in the fluorescence intensity ratios of green and red emission, this ratiometric NCB was demonstrated to sensitively detect Hepatitis-A virus gene sequences, a proof-of-concept target in this work, with good selectivity.

Phenazine-Based Ratiometric Hg2+ Probes with Well-Resolved Dual Emissions: A New Sensing Mechanism by Vibration-Induced Emission (VIE).

Phenazines exhibit intriguing vibration-induced emission (VIE) owing to the fast intrinsic vibration of benzo[a,c]phenazine moiety. For the first time, a phenazine-based ratiometric fluorescent probe DBPST is developed for recognizing Hg2+ via restriction of VIE. Upon binding with Hg2+ , DBPST demonstrates two well-resolved emission peaks (over 130 nm) with a wide tuning color and affords a large signal-to-background ratio.

Quantitative generalized ratiometric fluorescence spectroscopy for turbid media based on probe encapsulated by biologically localized embedding.

PEBBLE (probe encapsulated by biologically localized embedding) nanosensor encapsulating an intensity-based fluorescence indicator and an inert reference fluorescence dye inside the pores of stable matrix can be used as a generalized wavelength-ratiometric probe. However, the lack of an efficient quantitative model render the choices of inert reference dyes and intensity-based fluorescence indicators used in PEBBLEs based generalized wavelength-ratiometric probes rather limited. In this contribution, an extended quantitative fluorescence model was derived specifically for generalized wavelength-ratiometric probes based on PEBBLE technique (QFMGRP) with a view to simplify the design of PEBBLEs and hence further extend their application potentials. The effectiveness of QFMGRP has been tested on the quantitative determination of free Ca(2+) in both simulated and real turbid media using a Ca(2+) sensitive PEBBLE nanosensor encapsulating Rhod-2 and eosin B inside the micropores of stable polyacrylamide matrix. Experimental results demonstrated that QFMGRP could realize precise and accurate quantification of free Ca(2+) in turbid samples, even though there is serious overlapping between the fluorescence excitation peaks of eosin B and Ca(2+) bound Rhod-2. The average relative predictive error value of QFMGRP for the test simulated turbid samples was 5.9%, about 2-4 times lower than the corresponding values of partial least squares calibration model and the empirical ratiometric model based on the ratio of fluorescence intensities at the excitation peaks of Ca(2+) bound Rhod-2 and eosin B. The recovery rates of QFMGRP for the real and spiked turbid samples varied from 93.1% to 101%, comparable to the corresponding results of atomic absorption spectrometry.

A New Turn on Fluorescent Probe for Selective Detection of Cysteine/Homocysteine.

A new fluorescent probe (probe 1) was developed for recognition of cysteine (Cys) and homocysteine (Hcy). Probe 1 exhibited a large absorption peak blue-shift (107 nm) as well as enhanced fluorescence responses to Cys/Hcy based on cyclization of thiol containing amino acids with aldehydes, inhibiting the C = N isomerization-induced quenching process by an intramolecular hydrogen bond. The detection mechanism was proved by (1)H NMR and mass spectrometry analysis.

Wavelength-ratiometric probes for saccharides based on donor-acceptor diphenylpolyenes.

The spectroscopic and photophysical properties of two donor-acceptor derivatives of 1,4-diphenylbuta-1,3-diene and 1,6-diphenylhexa-1,3,5-diene are described. Both compound posses a dimethylamino and a boronic acid groups as electron-donor and electron-withdrawing groups, respectively. Solvent polarity effects on the steady-state and fluorescence intensity decay are presented and show the formation of an excited-state charge-transfer (CT) state for both compounds. The formation of the anionic form of the boronic acid group at high pH induces a blue shift and an increase of the intensity in the emission spectra for both compounds. These spectral changes are interpreted as the lost of the electron-withdrawing property of the anionic form of the boronic acid group. The observed pK a of both compounds is around 8.8 and decrease to ~6 and ~7 in presence of fructose and glucose, respectively. Both compounds display a decrease of the mean lifetime at higher pH. Effects of the sugars on the fluorescence spectra and fluorescence lifetimes are also presented. For both compounds, a blue shift and an increase of the intensity are observed. These spectral changes lead to a wavelength-ratiometric method for the sugar recognition and analysis. Titration curves against fructose, galactose and glucose and dissociation constants are presented. Both compounds show a higher affinity for fructose. The affinity decreases for galactose and for glucose, respectively. Sugar effects on the fluorescence intensity decay are also presented. Both compounds display a decrease of the mean lifetime after addition of sugar.