Fluorescent color analysis of ascorbic acid by ratiometric fluorescent paper utilizing hybrid carbon dots-silica coated quantum dots

Highlights

• A ratiometric fluorescence probe with dual-emission of blue and red fluorescence was constructed.

• In the presence of ascorbic acid, the ratiometric fluorescence probe exhibits sensitive and wide color changes.

• Ratiometric fluorescent test paper is sensitively and selectively response to ascorbic acid.

• The test papers gave a clear judgment of ascorbic acid amount in fruit juice by visual discrimination.

Abstract

Monitoring the concentration of ascorbic acid (AA) in biological samples is essential to food quality and healthcare. Various fluorescence methods widely reported for the detection of AA are usually based on “turn-on” or “turn-off” principle. However, few ratiometric fluorescent sensors were reported for AA determination. Additionally, the application of ratiometric fluorescence probe is usually limited by the narrow range of color changes and insensitivity color change of the AA concentration. Herein, a novel ratiometric fluorescent test paper to achieve the continuous color changes from red to blue was developed for the fluorometric qualitative and quantitative determination of AA. The nanoprobe consists of blue-emitting carbon dots (C-dots) and red-emitting silica coated quantum dots (QDs@SiO₂) with certain ratiometric fluorescence intensity of blue to red. The fluorescence of blue-emitting CDs (peaking at 450 nm) is quenched by Fe³⁺, then the blue fluorescence is restored consecutively with the gradual increasing of AA concentration due to the reduction from Fe³⁺ to Fe²⁺ by using AA. While the fluorescence of red-emitting QD@SiO₂ (peaking at 630 nm) works as a stable internal standard. Therefore, a distinguish color transition from red to blue can be observed by naked eyes intuitively with the increasing of AA concentration. By calculating the ratiometric fluorescent intensity at 450 nm and 630 nm or observing the color of the mixture solution by naked eyes, the concentration of AA can be detected easily and accurately. Under optimized conditions, the fluorescent intensity ratio (I₄₅₀/I₆₃₀) shows linear relationship with the concentration of AA from 0 to 70 μM with the detection limit as low as 3.17 μM. Finally, a ratiometric fluorescent sensor paper was further developed for AA detection with satisfactory results in fruit juice. The method proposed here gives the wide applications of fluorescent test paper in food and biological assays.

Introduction

Ascorbic acid (AA), more well-known as vitamin C, has been widely used in food, chemical, nutraceutical, pharmaceutical and cosmetic fields.^[1] AA is a powerful antioxidant used to reduce the oxidative stress of peroxidase. AA functions as a key coenzyme in many metabolic pathways and plays an important role in human life. Many important biosynthesis processes also need the involving of AA. The intakes of AA are related to the states of people's health. For example, the deficiency of AA in body will result in scurvy, anemia, and decrease in immunity.^[[2], [3], [4]^] However, excessive intakes of AA also lead to urinary stone, diarrhea and stomach convulsion.^5,6 Therefore, the measurement of the content of AA in food and biological sample has great significance for food quality and healthcare.

In the last few years, many methods for the qualitative and quantitative determination of AA have been proposed, such as electrochemical detection,^[[7], [8], [9]^] capillary electrophoresis,^10,11 spectrophotometry ^[12] and high performance liquid chromatography.^13,14 Although these approaches have made important contributions to detecting the concentration of AA, there are still exist many deficiencies and limitations, such as the utilization of sophisticated and specialized equipments, the time-consuming processes and requirements for skilled technicians. Compared to these methods, fluorescence assay has attracted widespread attention due to its low cost, simple operation and high sensitivity. Recently, many fluorescent probes have been developed for AA detection, such as metal nanoparticle probe,^15,16 organic molecule probe,^[3] quantum dot,^[17] carbon dots (CDs) ^[[18], [19], [20]^] and conjugated polymer dots.^[21] For example, Liu et al. have developed a simple graphene quantum dots (GQDs)-based fluorescence sensing system for rapid detection of AA in the presence of copper ions.^[22] Fong's groups have successfully demonstrated unique fluorescence signalling strategy by the “turn-on” of CDs-Fe³⁺ sensing system that enabled highly specific detection of AA.^[23] Liu and co-workers have synthesized Gly-GQDs, the fluorescence of which can be quenched by Ce⁴⁺, by a simple and green pyrolysis methods to detect AA with “off-on” detection mode.^[24] Nevertheless, these fluorescent nanoprobes based on “turn-off” or “turn-on” principle proposed in the above work are called as “single fluorescence”, environmental fluctuation and high background interference resulting in false positive signals, which make the fluorescent nanoprobes less sensitive and less accurate. The emergence of ratiometric fluorescence nanoprobes seems to solve the problems of single fluorescence probes by the joint use of an internal standard segment and a responsive segment, providing a self-comparing of two emissions intensity ratio to detect the analyte concentration. Until now, there are still few reports on ratiometric fluorescent sensor for AA determination with broad color variation and more visual sensitivity.

Fluorescent test strips are easy to carry, and their detection results are directly visible, and do not need to rely on large and expensive analytical instruments, thus they are receiving more and more attention from research. Zhang's group uses 2-hydroxyethyldithiocarbamate (HDTC) modified CdSe@ZnS quantum dots to realize the detection of Hg²⁺ with high sensitivity and high selectivity.^[25] HDTC-QDs are added dropwise to cellulose paper to construct a paper sensor for detecting Hg²⁺ ions that can be directly observed by the naked eye. The detection limit of the sensor for Hg²⁺ ions can reach 0.2 ppm. However, in general, most fluorescent sensors are not suitable for the preparation of paper sensors. This is because the fluorescent materials are likely to lose optical or sensitive activity in the dry or aggregated state. In addition, how to fix the solution phase sensor on the substrate is also a problem. Zhang's group used the fluorescent graphene oxide test paper prepared by inkjet printing technology to successfully fix the fluorescent material evenly on the substrate.^[26] It is worth noting that the existing fluorescent test paper generally has the problems of low chromaticity resolution, narrow spectral evolution range, and inability to visually detect the target. Until now, fluorescent test papers that can visually and quantitatively detect the target are still relatively rare.

Herein, we developed a ratiometric fluorescent nanoprobe for detection of AA more intuitively by the distinguished color change. A ratiometric fluorescence probe has been firstly constructed by QD@SiO₂ with red fluorescence as the internal standard and CDs with blue fluorescence as signal unit. CD was specifically sensitive to Fe³⁺ ion due to the interaction of electron transfer from CD to Fe³⁺, resulting in the efficient quenching of CD fluorescence.^[27] In the presence of AA, the blue emission of CDs was restored through the reduction between Fe³⁺ and AA. While the red fluorescence of QD@SiO₂ keep constant during the whole experiment. Due to the changes in the intensity ratio of the two emission wavelength, a distinguish color transition from red to blue can be observed by naked eyes intuitively with the increasing of AA concentration. Printing the ratiometric fluorescent nanoprobe onto a piece of filter paper, a serial of color evolution from red to pink to purplish red to purple to blue was displayed with the addition of AA, and it was applied for the determination of AA in kiwi fruit with satisfactory result. The method we proposed possesses high sensitivity, excellent selectivity, simple operation and real-time detection in both aqueous solution and fruit juice sample by the naked eye.