A ratiometric approach for pH optosensing with a single fluorophore indicator

A new fiber-optic prototype of luminometer has been designed in order to perform ratiometric-based measurements for optical sensing purposes. The coupling of a pH-selective sensing phase to the fiber-optic prototype has been evaluated for robust pH optosensing in drinking water. The pH-sensitive material has been synthesized by entrapping a pH-sensitive luminescent indicator (mercurochrome) in a sol-gel inorganic matrix. The pH optosensing is based on the detection of pH-induced reversible changes in the mercurochrome fluorescent emission and in the light reflected by the sensing phase.The instrument has been constructed using low-cost and simple optoelectronic components. The active phase was excited by means of a visible 470 nm high intensity light emitting diode (LED). The radiant power of the LED was modulated using a sinusoidal function so that scattered light due to light sources of different frequency than the modulating signal (e.g. sunlight) can be easily removed by adequate electronic filtering of the emission signal. Both the fluorescence emission from the dye and the sensing phase reflected light were collected in a bifurcated fiber-optic to allow the ratiometric measurement.Two different ratiometric approaches have been evaluated. The analytical performance of the pH optrode using both measurement methods have been compared, between them and with simple fluorescence intensity measurements, in terms of sensitivity, measurement range, response time, repeatability and insensitivity to changes in excitation light intensity.The applicability of the developed pH optrode and methods has been tested for pH analysis in tap and bottled still mineral water samples. The results obtained showed good agreement with the corresponding pH values provided by a commercial glass electrode.In this work, pH was selected as a model analyte to evaluate the performance of the proposed methodology, although other optical sensors for different applications/analytes could benefit of this approach.     

SciFinder-guided rational design of fluorescent carbon dots for ratiometric monitoring intracellular pH fluctuations under heat shock

A novel SciFinder-guided strategy for the preparation of pH responsive carbon dots (CDs-pH) for ratiometric intracellular imaging was developed.     

Wavelength encoded analytical imaging and fiber optic sensing with pH sensitive CdTe quantum dots

CdTe quantum dots (QDs), capped with mercaptopropionic acid (MPA), were synthesized and the variation of their fluorescence properties (steady state and lifetime) with pH was assessed in solution and when immobilized in a sol-gel host. Three different sizes of CdTe QDs with excited state lifetimes ranging from 42 to 48 ns and with emission maximum at 540 nm (QD₅₄₀), 580 nm (QD₅₈₀) and 625 nm (QD₆₂₅) were selected. The solution pH affects the maximum emission wavelength (shifts to higher wavelengths of 23, 24 and 27 nm for QD₅₄₀, QD₅₈₀ and QD₆₂₅, respectively), the excited state lifetime and the fluorescence intensity in a reversible way. Linearization of the maximum emission wavelength variation with the pH allows the estimation of an apparent ionization constant (pK_a) for each QD: 6.5 ± 0.1 (QD₅₄₀), 6.1 ± 0.5 (QD₅₈₀) and 5.4 ± 0.3 (QD₆₂₅). The variation of the QDs fluorescence properties was further explored using confocal laser scanning microscopy allowing the implementation of a new calibration method for pH imaging in solution. QDs were successfully immobilized on the tip of an optical fiber by dip-coating using sol-gel procedure. The immobilized QDs showed a similar pH behaviour to the one observed in solution and an apparent lifetime of 80, 68 and 99 ns, respectively. The proposed QDs based methodology can be successfully used to monitor pH using wavelength encoded data in imaging and fiber optic sensing applications.     

SciFinder-guided rational design of fluorescent carbon dots for ratiometric monitoring intracellular pH fluctuations under heat shock

Intracellular pH plays a significant role in various biological processes, including cell proliferation, apoptosis, metabolism, enzyme activity and homeostasis. In this work, a novel design strategy for the preparation of pH responsive carbon dots (CDs-pH) for ratiometric intracellular imaging was reported. By using SciFinder database, fluorescent CDs-pH with the required pK_a value of 6.84 were rationally designed, which is vital important for precise sensing of intracellular pH. As a result, the synthesized CDs-pH demonstrated robust ability to test pH fluctuations within the physiological range of 5.4-7.4. The CDs-pH was further utilized for fluorescent ratiometric imaging of pH in living HeLa cells, effectively avoided the influence of autofluorescence from native cellular species. Moreover, real-time monitoring of intracellular pH fluctuation under heat shock was successfully realized. This SciFinder-guided design strategy is simple and flexible, which has a great potential to be used for the development of other types of CDs for various applications.     

Field-portable ratiometric fluorescence imaging of dual-color label-free carbon dots for uranyl ions detection with cellphone-based optical platform

Under the public spotlight, uranyl (UO₂²⁺) ions has attracted considerable attention for the extreme radioactive and chemical toxicity to ourselves and our environment. Herein, we present a simple and effective ratiometric fluorescence imaging method for the visualizing and quantitative detection UO₂²⁺ ions by cellphone-based optical platform. The sensing solution was prepared by mixing label-free red carbon dots (r-CDs) and blue carbon dots (b-CDs) together with a fixed photoluminescence intensity ratio of 4:1. When UO₂²⁺ ions were added, the fluorescence of r-CDs can be selectively quenched, while the fluorescence of b-CDs remains stable without spectral changes. With the gradually increase the amounts of UO₂²⁺ ions, the different response of dual-color CDs resulted in a signification color evolution from deep red to dark purple under the ultraviolet (UV) light illumination. Then, a cellphone-based optical platform was constructed for directly imaging the color change of the samples, and the built-in Colorpicker APP quickly output the red, green and blue (RGB) channel values of these images within one second. Interesting, there was a linear relationship between the ratio of red and blue (R/B) channel values and UO₂²⁺ ions concentration from 0 μmol/L to 30.0 μmol/L (R² = 0.92804) with the detection limit of ∼8.15 μmol/L (signal-to-noise ratio of 3). In addition, the optical platform has also been applied to the quantification of UO₂²⁺ ions in tap water and river water sample. With the advantage of low-cost, portable, easy to operation, we anticipate that this method would greatly improve the accessibility of UO₂²⁺ ions detection even in resource-limited areas.     

Field-portable ratiometric fluorescence imaging of dual-color label-free carbon dots for uranyl ions detection with cellphone-based optical platform

Under the public spotlight, uranyl (UO₂²⁺) ions has attracted considerable attention for the extreme radioactive and chemical toxicity to ourselves and our environment. Herein, we present a simple and effective ratiometric fluorescence imaging method for the visualizing and quantitative detection UO₂²⁺ ions by cellphone-based optical platform. The sensing solution was prepared by mixing label-free red carbon dots (r-CDs) and blue carbon dots (b-CDs) together with a fixed photoluminescence intensity ratio of 4:1. When UO₂²⁺ ions were added, the fluorescence of r-CDs can be selectively quenched, while the fluorescence of b-CDs remains stable without spectral changes. With the gradually increase the amounts of UO₂²⁺ ions, the different response of dual-color CDs resulted in a signification color evolution from deep red to dark purple under the ultraviolet (UV) light illumination. Then, a cellphone-based optical platform was constructed for directly imaging the color change of the samples, and the built-in Colorpicker APP quickly output the red, green and blue (RGB) channel values of these images within one second. Interesting, there was a linear relationship between the ratio of red and blue (R/B) channel values and UO₂²⁺ ions concentration from 0 μmol/L to 30.0 μmol/L (R² = 0.92804) with the detection limit of ~8.15 μmol/L (signal-to-noise ratio of 3). In addition, the optical platform has also been applied to the quantification of UO₂²⁺ ions in tap water and river water sample. With the advantage of low-cost, portable, easy to operation, we anticipate that this method would greatly improve the accessibility of UO₂²⁺ ions detection even in resource-limited areas.     

基于聚乙烯亚胺碳纳米点的桑色素比率型荧光探针

利用水热合成法制备了一种基于聚乙烯亚胺的荧光碳纳米点,用光谱方法研究了其对桑色素的识别作用.结果表明,该碳纳米点对桑色素具有比率型荧光响应,且响应速度快、选择性和灵敏度高、抗干扰能力强.将桑色素加入到碳纳米点溶液中后,碳纳米点自身的荧光(460 nm)立即被猝灭,同时在555 nm处出现新的荧光发射峰并逐渐增强;其新发射峰(555 nm)与原发射峰(460 nm)的强度比值在此过程中呈线性增加.基于此,在1.0×10^-6～4.0×10^-5mol/L浓度范围内,可实现对桑色素的检测,该比率荧光响应非常灵敏,检出限可达3.0×10^-8mol/L.另外,该碳纳米点与常见金属离子、氨基酸及其它具有类似桑色素结构的黄酮醇均不反应,显示出对桑色素的高选择性.该检测方法的荧光强度随桑色素浓度的增加而逐渐增强,并伴随着由蓝色到黄色的荧光颜色变化,可实现对桑色素的可视化检测;同时该碳纳米点还可用于稀释胎牛血清中桑色素的定量检测.     

Boron Doped Carbon Dots with Unusually High Photoluminescence Quantum Yield for Ratiometric Intracellular pH Sensing

Herein we report that boron doping in carbon dots results in increased photoluminescence (PL) quantum yield, which could be used for ratiometric intracellular pH sensing in cancer cell lines. Using a mixture of citric acid monohydrate, thiourea, and boric acid, microwave-assisted synthesis of boron doped blue emitting carbon dots (B-Cdots) with an average size of 3.5±1.0 nm was achieved. For B-Cdots, the maximum quantum yield (QY) was observed to be 25.8 % (11.1 % (w/w) H₃BO₃ input concentration), whereas, the same was calculated to be 16.9 % and 11.4 % for Cdots (synthesized from citric acid monohydrate and thiourea only) and P-Cdots (phosphorus doped carbon dots; synthesized using citric acid monohydrate, thiourea and phosphoric acid) (11.1 % (w/w) H₃PO₄ input concentration), respectively. The observed luminescence efficiencies as obtained from steady state and time-resolved photoluminescence measurements suggest an alternative emission mechanism due to boron/phosphorus doping in carbon dots. We furthermore demonstrated facile composite formation using B-Cdots and another carbon dots with orange emission in presence of polyvinyl alcohol (PVA), resulting in white light emission (0.31, 0.32; λ_ex 380 nm). The white light emitting composite enabled ratiometric pH sensing in the aqueous medium and showed favorable uptake properties by cancerous cells for intracellular pH sensing as well.     

CdSe/CdS量子点荧光猝灭法测定芹黄素的研究

以巯基乙酸为稳定剂,在水溶液中合成了具有特殊光学性质的水溶性CdSe/CdS量子点。以该量子点为荧光探针,基于荧光猝灭法对芹黄素进行了定量检测。考察了缓冲体系、反应时间、量子点浓度等多种因素的影响。实验结果表明,在0.001 mol/L、pH为6.80的KH2PO4-Na2HPO4缓冲液中,当量子点浓度为1.2×10^-4mol/L、反应时间为20 min时,该方法的线性范围为0.16-27.02μg/mL,其线性回归方程为F0/F=0.99665＋0.11067ρ（μg/mL）,相关系数r=0.998,检出限为0.13μg/mL,并用于合成样品的分析。     

Multiway chemometric decomposition of EEM of fluorescence of CdTe quantum dots obtained as function of pH

The effect of the pH (from 3 to 10) on the excitation emission matrices (EEMs) of fluorescence of CdTe quantum dots (QDs), capped with mercaptopropionic acid (MPA), were analyzed by multiway decomposition methods of parallel factor analysis (PARAFAC), a variant of the parallel factor analysis method (PARAFAC2) and multivariate curve resolution alternating least squares (MCR-ALS). Three different sized CdTe QDs with emission maximum at 555 nm (QDa), 594 nm (QDb) and 628 nm (QDc) were selected for analysis. The three-way data structures composed of sets of EEMs obtained as function of the pH (EEMs, pH) do not have a trilinear structure. A marked deviation to the trilinearity is observed in the emission wavelength order—the emission spectra suffers wavelength shift as the pH is varied. The pH-induced variation of the fluorescence properties of QDs is described with only one-component PARAFAC2 or MCR-ALS models—other components are necessary to model scattering and/or other background signals in (EEMs, pH) data structures. Bigger sized QDs are more suitable tools for analytical methodologies because they show higher Stokes shifts (resulting in simpler models) and higher pH range sensitivity. The pH dependence of the maximum wavelength of the emission spectra is particularly suitable for the development of QDs/EEMs wavelength-encoded pH sensor bioimaging or biological label methodologies when coupled to multiway chemometric decomposition.     

水溶性CdSe量子点的合成及其作为荧光探针对大肠杆菌的快速检测

采用水相法以谷胱甘肽为稳定剂合成高稳定性的CdSe量子点,利用化学偶联剂的作用使得量子点表面基团与菌体之间的成功结合,对偶联的条件进行了优化.基于荧光分析法建立了一种快速简便的大肠杆菌检测定量分析方法.研究结果表明:合成的量子点具有稳定、荧光性能良好等突出优点.通过偶联剂量子点能与大肠杆菌结合,其荧光强度与大肠杆菌浓度...     

An ultrasensitive method for the determination of melamine using cadmium telluride quantum dots as fluorescence probes

An ultrasensitive and simple method for the determination of melamine was developed based on the fluorescence quenching of thioglycolic acid (TGA) capped CdTe quantum dots (QDs) at pH 11.0. In strong alkaline aqueous solution, the selectivity of the method has been greatly improved due to most heavy metal ions show no interference as they are in the precipitation form or in their anion form. Furthermore, CdTe quantum dots have higher quantum yields at higher pH. The method has a wider concentration range and lower detection limit. The influence factors on the determination of melamine were investigated and the optimum conditions were determined. Under optimum conditions, the fluorescence intensity change of TGA coated CdTe quantum dots was linearly proportional to melamine over a concentration range from 1.0 × 10⁻¹¹ to 1.0 × 10⁻⁵ mol L⁻¹ with a correlation coefficient of 0.9943 and a detection limit of 5 × 10⁻¹² mol L⁻¹. The mechanism of fluorescence quenching of the QDs has been proposed based on the infrared spectroscopy information and electrophoresis experiments in presence of melamine under alkaline condition. The proposed method was employed to detect trace melamine in milk powder and pet feeds with satisfactory results.     

Facile synthesis of N-acetyl-L-cysteine capped ZnS quantum dots as an eco-friendly fluorescence sensor for Hg2+

This paper described an investigation of a novel eco-friendly fluorescence sensor for Hg²⁺ ions based on N-acetyl-l-cysteine (NAC)-capped ZnS quantum dots (QDs) in aqueous solution. By using safe and low-cost materials, ZnS QDs modified by NAC were easily synthesized in aqueous medium via a one-step method. The quantitative detection of Hg²⁺ ions was developed based on fluorescence quenching of ZnS QDs with high sensitivity and selectivity. Under optimal conditions, its response was linearly proportional to the concentration of Hg²⁺ ions in a range from 0 to 2.4 × 10⁻⁶ mol L⁻¹ with a detection limit of 5.0 × 10⁻⁹ mol L⁻¹. Most of common physiologically relevant cations and anions did not interfere with the detection of Hg²⁺. The proposed method was applied to the trace determination of Hg²⁺ ions in water samples. The possible quenching mechanism was also examined by fluorescence and UV-vis absorption spectra.     

Developing a novel ratiometric fluorescent probe based on ESIPT for the detection of pH changes in living cells

As an important parameter of intracellular metabolism, pH plays important roles in maintaining normal physiological processes. The abnormal pH could cause disorder of cell function which may cause neurological diseases. Herein, we present two novel ratiometric fluorescent probes to detect pH changes. The probes employed 2-(2′-hydroxyphenyl)benzothiazole as fluorescent platform, and displayed desirable fluorescence response to pH on the basis of excited state intramolecular proton transfer (ESIPT) process. The probe BtyC-1 showed green fluorescence at 546 nm under acidic conditions, while it displayed strong blue fluorescence at 473 nm and weak green fluorescence at 546 nm under alkaline conditions. Biological experiments demonstrated that the probe BtyC-1 could be successfully applied for the ratiometric imaging of cellular pH and the NH₄Cl-induced pH changes in living cells.     

氮硫共掺杂碳量子点对胃液pH值的精确检测

以水杨酸和硫脲为原料,通过水热法一步合成水溶性好、稳定性强、量子产率高(36.23%)、呈蓝光的氮硫共掺杂碳量子点(N,S-CQDs).在302 nm波长激发下,N,S-CQDs在发射波长409 nm处的荧光强度与pH值(在2.01～5.11范围内)线性关系良好,相关系数为0.9931,可原位、实时检测胃液内pH值在Δ0.01范围内的变化.可用于胃癌早期诊断和新生儿健康防护.400μg/m L N,S-CQDs孵育铜绿微囊藻细胞48 h,存活率82.8%,其可作为人体胃细胞荧光探针,经人体排泄进入水环境,对淡水浮游生物毒性低,环境友好.     

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.     

Carbon dots as a fluorescent probe for label-free detection of physiological potassium level in human serum and red blood cells

A unique photoluminescence carbon dots (CDs) with larger size were prepared by microwave-assisted method. Complex functional groups on the surface of the CDs facilitate the nanoparticles to form affinity with some metal ions. Taking advantage of the effective fluorescence quenching effect of K⁺, a highly sensitive CD-based fluorescence analytical system for label-free detection of K⁺ with limit of detection (LOD) 1.0 × 10⁻¹² M was established. The concentrations of potassium ion in biological samples such as human serum are usually found at millimolar levels or even higher. The proposed method begins with a substantial dilution of the sample to place the K⁺ concentration in the dynamic range for quantification, which covers 3 orders of magnitude. This offers some advantages: the detection of K⁺ only needs very small quantities of biological samples, and the dilution of samples such as serum may effectively eliminate the potential interferences that often originate from the background matrix. The determined potassium levels were satisfactory and closely comparable with the results given by the hospital, indicating that this fluorescent probe is applicable to detection of physiological potassium level with high accuracy. Compared with other relative biosensors requiring modified design, bio-molecular modification or/and sophisticated instruments, this CD-based sensor is very simple, cost-effective and easy detection, suggesting great potential applications for successively monitoring physiological potassium level and the change in biological system.     

Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg²⁺. The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg²⁺, the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg²⁺ in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg²⁺ contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg²⁺ quantification related biological systems.     

两类荧光可调碳点的制备及在防伪油墨上的应用

以不同的氯代烃和胺类为反应原料,在相同反应条件下制备了2类荧光发射波长随激发波长变化而变化的碳点.其中一类碳点的荧光强度随着激发波长的红移呈现类似于高斯曲线的变化趋势(类型Ⅰ),另一类碳点的荧光强度随激发波长变化不规律(类型Ⅱ).通过荧光光谱、透射电子显微镜、X射线光电子能谱和红外光谱对2类碳点的结构和性质进行了表征和比较.考察了类型Ⅱ碳点作为一种新型的荧光油墨在多重防伪领域的应用性能.     

Quantum dots as donors in fluorescence resonance energy transfer for the bioanalysis of nucleic acids,proteins, and other biological molecules

Quantum dots (QDs) have a number of unique optical properties that are advantageous in the development of bioanalyses based on fluorescence resonance energy transfer (FRET). Researchers have used QDs as energy donors in FRET schemes for the analysis of nucleic acids, proteins, proteases, haptens, and other small molecules. This paper reviews these applications of QDs. Existing FRET technologies can potentially be improved by using QDs as energy donors instead of conventional fluorophores. Superior brightness, resistance to photobleaching, greater optimization of FRET efficiency, and/or simplified multiplexing are possible with QD donors. The applicability of the Förster formalism to QDs and the feasibility of using QDs as energy acceptors are also reviewed.

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Figure A ligand capped core/shell quantum dot acting as energy donor in a FRET process with aconjugated Cy3 labeled oligonucleotide