A Ratiometric Fluorescent Hydrogel Test Kit for On-Spot Visual

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A Ratiometric Fluorescent Hydrogel Test Kit for On-Spot Visual Detection of Nitrite Yuanjin Zhan, Yanbo Zeng, Lei Li, Fang Luo, Bin Qiu, Zhenyu Lin, and Longhua Guo ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00125 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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A Ratiometric Fluorescent Hydrogel Test Kit for On-Spot

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Visual Detection of Nitrite

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Yuanjin Zhan,a1 Yanbo Zeng,b1 Lei Li,b* Fang Luo,c Bin Qiu,a Zhenyu Lin,a and

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Longhua Guoa, b*

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a

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Science of Food Safety and Biology; Fujian Provincial Key Laboratory of Analysis

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and Detection Technology for Food Safety; College of Chemistry, Fuzhou University,

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Fuzhou, 350116, China

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b

Institute of Nanomedicine and Nanobiosensing; MOE Key Laboratory for Analytical

College of Biological, Chemical Sciences and Engineering, Jiaxing University,

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Jiaxing 314001, China.

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c

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350116, China

College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian

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1

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Corresponding authors: E-mail: [email protected] (L Li); [email protected]

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(LH Guo).

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Tel: +86 15280077696; +86 591 22866141

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Fax: +86 591 22866135

Zhan and Zeng contributed equally to this work.

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ACS Paragon Plus Environment

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ABSTRACT

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In this work, we proposed a new method based on carbon dots (named m-CDs) for

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selective and efficient detection of nitrite (NO2-), which was based on the interaction

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between the amine group of m-CDs and NO2- via diazo reaction that produced

24

diazonium salts and induced the fluorescence quenching of m-CDs. The concentration

25

of NO2- shows a good linear relationship with the quenched fluorescent intensity from

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0.063 to 2.0 μM (R2 = 0.996) with a detection limit of 0.018 μM. In addition, a

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ratiometric

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electrostatic interaction by introducing the Ru(bpy)3Cl2·6H2O as an internal reference

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fluorescent reagent. Interestingly, a transition of the fluorescent color of the ratiometric

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probe from cyan to red could be visual observed upon increasing the concentration of

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NO2-. Based on these findings, a ratiometric fluorescent-based portable agarose

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hydrogel test kit was fabricated and applied for on-spot assessment of NO2- content

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within 10 minutes. As far as we known, this is the first ratiometric fluorescent sensors

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for visual detection of NO2-. It has broad application prospects in environmental

35

monitoring and food safety assessment.

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KEYWORDS: nitrite, carbon dots, ratiometric fluorescent probe, agarose hydrogel test

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kit, visualization

fluorescence

probe

(m-CDs@[Ru(bpy)3]2+)

was

constructed

via

38 39

Nitrate (NO3-) and nitrite (NO2-) are widely present in the environment and are the

40

most common nitrogenous compounds in nature. NO3- is widely used in agriculture as

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a nitrogen fertilizer for plant growth. NO2- has antiseptic properties and is often added 2


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to meat products as a color retention agent in the food processing industry to maintain

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a good appearance. Besides, it can prevent the production of clostridium botulinum and

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improve the safety of meat products.1 It should be noted that when NO3- reductase is

45

present, NO3- is reduced to NO2- in the digestive system and/or micro-organisms.2 NO2-

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can also be converted into N-nitrosamines in the acidic environment of the intestine to

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become a carcinogen that may cause esophagus cancer and stomach cancer.3-5 NO2- can

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oxidize normal oxygen-carrying hemoglobin in the blood into methemoglobin, thus

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losing oxygen carrying capacity and causing tissue hypoxia.6 It can be known that the

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presence of NO2- in water and food can cause various human diseases due to its high

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toxicity and pathogenicity. Therefore, the detection of NO2- content is of great

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significance for public health, environment protection, and food safety assessment.

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To date, many methods for aqueous NO2- detection have been developed, such as

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chemiluminescence,7 electrochemical methods,8, 9 colorimetric method,10, 11 capillary

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electrophoresis,12, 13 spectrophotometry,14 and chromatography.15, 16 Meanwhile, some

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sensitive and efficient methods are also reported. However, these reported methods

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either require expensive instruments, tedious detection procedure, poor visual semi-

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quantitative ability, or are time consuming. As a comparison, the fluorescence based

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methods are fast, sensitive, cheap, and capable of spatial and temporal resolution.

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Consequently, hitherto fluorescence based approaches have been widely used for

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biosensing, bioimaging, and target tracking.17, 18

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Traditional single emission fluorescence probe is subject to interference from

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biological backgrounds, instruments, environmental conditions, and fluorescent probes 3


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themselves.19, 20 The emergence of ratiometric fluorescence technology has overcome

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the above drawbacks. The ratiometric method is a novel analytical method for

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determining a target by measuring the ratio of the signal intensity as a basis for

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quantification with higher sensitivity and selectivity. At the same time, the observed

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color change of the ratiometric fluorescent probe can be used for semi-quantitative

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analysis based on the standard colorimetry chart, which lays a theoretical foundation

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for the development of portable fluorescent devices and standard detection.21-23

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Carbon quantum dots are a new type of carbon-based zero-dimensional

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nanomaterials with adjustable optical properties,17, 24, 25 good water solubility,26 low

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toxicity27, good anti-photobleaching,28,

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friendliness, wide source of raw materials, low cost,31 good biocompatibility32 and

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many other advantages. Due to the above merits, since its discovery, it has been widely

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used in many fields such as biosensing,33 biological imaging,34 LEDs,35 and energy.36

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high quantum yield,30 environmental

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In this work, we utilized m-phenylenediamine (mPD) as a raw material to synthesize

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carbon dots (named m-CDs) by a one-step hydrothermal carbonization method. m-CDs

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were found to selectively respond to NO2- via diazo reaction under strong acidic

80

conditions, which produced diazonium salts and induced the fluorescence quenching of

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m-CDs. As described in Scheme 1, a novel ratiometric fluorescence sensor (m-

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CDs@[Ru(bpy)3]2+) was fabricated by introducing Ru(bpy)3Cl2•6H2O as a reference

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fluorescent reagent for building in correction and affording multiple emissions. Under

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optimal conditions, upon adding the concentration of NO2- along with the decreasing

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of the ratio of the fluorescence intensity F494/F610 of the ratiometric probe, resulting in 4


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a transition of the fluorescent color of the ratiometric probe from cyan to red. Therefore,

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on the one hand, the ratio of fluorescence intensity can be used for quantitative analysis

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of NO2-; on the other hand, we can semi-quantitatively estimate NO2- based on the

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fluorescent color of the probe by naked eye under UV light. Besides, a portable test kit

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was prepared using agarose hydrogel as a matrix to mix and solidify m-

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CDs@[Ru(bpy)3]2+, enabling on-line, on-spot and visual detection of NO2- without the

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need for complex operations and instrumentation, indicating the proposed method has

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broad application prospects in environment protection and food safety assessment.

94 95

Scheme 1. The Principle for Visual Detection of NO2- Using m-CDs@[Ru(bpy)3]2+

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Ratiometric Probe.

97 98

MATERIALS AND METHODS

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Preparation of m-CDs. m-phenylenediamine (mPD, 0.60 g) was dissolved in

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absolute ethanol (60 mL) and sonicated for 5 min to make it homogeneous. The mixed 5


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solution was transferred to a 100 mL Teflon-lined autoclave and reacted for 12 h under

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180 °C, then naturally cooled to room temperature to obtain a brown clear solution,

103

which was further purified through a silica gel column and the fluorescent component

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was collected as a pure carbon dots.37

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Preparation of m-CDs@[Ru(bpy)3]2+ Ratiometric Probe and Portable Agarose

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Hydrogel Test Kit. m-CDs@[Ru(bpy)3]2+ ratiometric probe was prepared based on

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electrostatic interaction. Briefly, the negatively charged m-CDs (20 μg/mL) and the

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positively charged Ru(bpy)3Cl2·6H2O (40 μg/mL) were mixed at a volume ratio of 6:1

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and stirred at room temperature for 12 hours, followed by removal of unbound

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Ru(bpy)3Cl2·6H2O by a dialysis bag (1000 Da). Further, the agarose was dispersed in

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the above solution and heated in a microwave oven. After the agarose was completely

112

dissolved to form a gelatinous solution, a certain amount of the above solution was

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taken into the centrifuge tube cap. The agarose hydrogel was formed under room

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temperature for several minutes, and stored at 4 ℃ for later use.

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Determination of NO2-. m-CDs (100 μL, 20 μg/mL), sulfuric acid solution (50 μL,

116

0.1 M), and different concentrations of NO2- solution (50 μL) were added in sequence.

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After reacting for 40 minutes, the above reaction solution was removed to a fluorescent

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cuvette and measured by a F4600 fluorescence spectrometer.

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Analysis of Real Samples. NO2- was processed and extracted from milk powder and

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lunch meat quality control samples according to the procedure of China national

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standard (GB 5009.33-2010) and the solutions were stored at 4 ℃ before use. We used

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traditional Griess colorimetry and the proposed method to determine NO2- at the same 6


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RESULTS AND DISCUSSION

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Characterization of m-CDs. Figure 1A shows the high resolution transmission

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electron microscope (HRTEM) image of the as-prepared m-CDs. A typical dot-like

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spherical structure was observed with a particle size of about 5 nm (Figure 1B). The

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atomic force microscope (AFM) measurement shows that all m-CDs were

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homogeneous (Figure 1C), and the average height of nanodots was about 2.0 nm

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(Figure 1D). The functional groups in m-CDs were identified by Fourier transform

132

infrared spectroscopy (FT-IR) spectroscopy. As shown in Figure 1E, some new

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characteristic peaks in m-CDs appeared at about 2805–2958 (aliphatic C-H, υ), 1385.9

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(C-N=, υ), and 1245.8 cm–1 (C-O, υ) compared to the precursor mPD. The X-ray

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photoelectron spectroscopic (XPS) spectrum (Figure S1) also confirms that the

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elemental composition and chemical bonds of m-CDs are consistent with the FT-IR

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measurements. These results indicated that the as-synthesized m-CDs by hydrothermal

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carbonization was mainly formed through intermolecular polymerization and

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intramolecular cyclization of mPD.37 As shown in Figure 1F, m-CDs solution emitted

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cyan fluorescence under 365nm UV light. The UV absorption characteristic peak was

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located at 384 nm (red line), which belongs to n-π* transition. The optimal emission

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wavelength of m-CDs was located at 494 nm under 384 nm excitation, and the

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fluorescence emission centers of m-CDs were consistent with the UV absorption

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characteristic peaks. The result indicated that the m-CDs fluorescence was mainly due 7


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to the electron defects of the amine group. Fluorescence determination also found that

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m-CDs have a high quantum yield (31.58%, see Supporting Information) in water and

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the fluorescence lifetime was 8.80 ns (Figure S2).

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Figure 1. Characterization of m-CDs. (A) HRTEM. (B) Size distribution. (C) AFM.

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The scale bar on the right side of Figure 1C represents the height. (D) Height

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corresponds to the line in the Figure 1C. (E) FT-IR spectra. (F) Absorption (red),

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excitation (purple), and emission (cyan) spectra. The inset of Figure 1F is the

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corresponding color of the m-CDs solution under visible light (left) and 365 nm UV

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light (right).

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Determination of NO2- Based on m-CDs and Optimization of Experimental

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Parameters. Interestingly, we observed the fluorescence of m-CDs was quenched to

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varying degrees after reacting with different concentrations of NO2- under strong acidic

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condition (Figure 2A). Based on this phenomenon, we constructed a fluorescence

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sensor based on m-CDs for the detection of NO2-. To better understand the efficiency 8


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of m-CDs in response to NO2-, the fluorescence intensity versus time curve of m-CDs

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reacted with NO2- was examined. As shown in Figure 2B, m-CDs exhibited good

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photobleaching resistance after 60 min of photoexcitation under strongly acidic

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conditions. Under the same conditions, the fluorescence intensity of m-CDs rapidly

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quenched about 90% within 40 min after adding 10 μM NO2-, indicating that m-CDs

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reacted with NO2- quickly and efficiently. Therefore, we chose 40 min as the optimal

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reaction time.

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Moreover, the concentration of acid and temperature are the important factor for the

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sensing system. On the one hand, the acidity may affect the fluorescence properties of

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m-CDs. It can be seen in Figure 2C (red line), when the concentration of H2SO4 was

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greater than 0.1 M, the fluorescence intensity of m-CDs was significantly reduced due

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to the protonation of amine group in m-CDs, thereby changing the nature and rate of

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the non-radiative transition process that competes with the radiative transition process.

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As shown in Figure 2C (green line), the quenching efficiency F/F0 towards NO2–

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reached maximum while the concentration of H2SO4 was 0.1 M. Thereby, 0.1 M of

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H2SO4 was chosen for the subsequent study. In addition, the temperature can

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significantly affect the reaction rate and reaction process. Figure 2D (red line) shows

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that the temperature has no effect on the fluorescence intensity of m-CDs. And yet low

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temperature has great influence in the quenching rate and interaction between m-CDs

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and NO2-. The quenching efficiency F/F0 increases with increasing temperature and

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reaches its maximum at 30 ℃. So, 30 °C was selected as the optimized temperature in

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this study. 9


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Figure 2. Feasibility of the proposed method and optimization of experimental

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parameters. (A) is the fluorescent signals of m-CDs in response to different

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concentrations of NO2-; (B) is the time-dependant fluorescence intensity changes of m-

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CDs in the presence of 10 μM of NO2- in H2SO4 (0.1 M). Among them, m-CDs, H+ and

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NO2- in the green solid line are added in order, while in the blue dotted line, H+ and

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NO2- are premixed for 15 min, then m-CDs are added. The results show that the two

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kinetic curves are completely coincident, indicating that the m-CDs respond to NO2- as

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a result of the interaction of m-CDs, H+ and NO2-. (C) and (D) are acidity and

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temperature optimization, respectively.

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Analytical Performance of the NO2- Sensor. Under optimal conditions, the

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performance of the proposed method was examined. It can be seen in Figure 3A and

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3B that the fluorescence intensity of m-CDs at 494 nm decreased gradually with the 10


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addition of NO2-, and the inset image shows the evolution of corresponding colors from

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left to right under 365 nm UV light. It shows a good liner relationship between 0.0625

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to 2.0 μM (R2 = 0.996) with a detection limit of 0.018 μM (3σ/K, σ is the standard

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deviation of blank solution and K is the slope of the calibration curve). This outcome

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demonstrated that the performance of this sensor based on m-CDs is superior to the

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previously reported method for the detection of NO2- (Table S1).

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Figure 3. (A) Fluorescence spectra and the corresponding colors of m-CDs after adding

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different concentrations of NO2- (0–6.0 μM) in H2SO4 (0.1 M); (B) Plot of fluorescence

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intensity of m-CDs vs NO2- concentrations.

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Specificity and Selectivity of the Proposed Method towards NO2-. To verify the

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proposed method could be used for determining NO2- in real sample, the specificity and

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selectivity of the sensing platform need to be explored. On the one hand, control

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experiments were performed under the same conditions using NO2- and other

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nitrogenous compounds including NO2- under neutral conditions, NO3-, ONOO-, N3-,

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and NH4+. Figure 4A shows that the fluorescence intensity of m-CDs is significantly

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reduced after adding NO2- under 0.1 M H2SO4. In contrast, no obvious fluorescence

215

quenching was observed in the presence of other nitrogenous substances and their 11


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mixture. The result confirmed that the effect of nitrogenous compounds on m-CDs

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could be ignored, and the sensor for NO2- detection exhibited remarkable specificity.

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On the other hand, we further investigated the selectivity of the sensor. The

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fluorescence intensity of m-CDs was determined in the presence of some common ions

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(F-, Cl-, Br-, I-, SO42-, CO32-, NO3-, PO43-, Na+ , K+ , Ca2+, Mg2+, NH4+, Zn2+, Mn2+, Fe3+,

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Cu2+, Al3+, Ba2+, Co2+, and Ni2+) ) and common substances in biological fluids including

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glucose, AA, biothiols, and amino acids. Figure 4B shows that the effect of these

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interferents on the detection of NO2- based on m-CDs could be negligible. Apparently,

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these results strongly suggested that the sensor could be used for specific and selective

225

detection of NO2- in real sample.

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Figure 4. Specificity and selectivity of the proposed method towards NO2-. (A) is the

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sensing platform responses of different nitrogenous compounds and their mixture; (B)

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is the responses of other common interferents (from 1 to 29, concentrations of

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interferents were 0.25mM). (1) blank, (2) NO2-, (3) F-, (4) Cl-, (5) Br-, (6) I-, (7) SO42-,

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(8) CO32-, (9) NO3-, (10) PO43-, (11) Na+, (12) K+, (13) Ca2+, (14) Mg2+, (15) NH4+, (16)

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Zn2+, (17) Mn2+, (18) Fe3+, (19) Cu2+, (20) Al3+, (21) Ba2+, (22) Co2+, (23) Ni2+, (24)

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glucose, (25) ascorbic acid, (26) glutathione, (27) L-Cysteine, (28) H2O2, and (29)

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mixture. 12


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Determination of NO2- in Quality Control Samples and Meat Products. To

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further assess the performance and practicability of the sensor in the real sample

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detection, the proposed method based on m-CDs was used to evaluate NO2- contents in

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the quality control milk powder and lunch meat samples purchased from CFAPA

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Testing Technology Co., Ltd. (Dalian, China). NO2- extracted from quality control milk

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powder and lunch meat samples were detected to be 3.75 mg/kg and 4.35 mg/kg,

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respectively, which was close to the provided reference value. As it can be seen from

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Table 1, the recoveries and relative standard deviations (RSD) were in the range 94.3–

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107.0% and 4.3–5.9%, respectively. Therefore, the m-CDs based fluorescence platform

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could be an effective and reliable tool for the detection of NO2- in food samples.

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Table 1. Detection results of NO2- in milk powder and luncheon meat quality control

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samples using the proposed fluorescence sensor (n=5). Samples

Detected

Reference

Spiked

Total found

Recovery

RSD

(mg/kg)

(mg/kg)

(mg/kg)

(mg/kg)

(%)

(%)

Milk

3.77

3.50

3.52

7.14

95.7

4.3

powder

3.72

3.50

7.04

11.10

104.8

5.2

3.81

3.50

14.08

18.53

104.6

5.6

Luncheon

4.35

3.40

3.52

7.67

94.3

5.1

meat

4.39

3.40

7.04

11.92

107.0

5.9

4.31

3.40

14.08

18.32

99.5

4.7

248 13


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At the same time, to further investigate the practical application potential of

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fluorescence sensor in meat samples, four meat samples obtained from the local market

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were measured by using the proposed strategy and the traditional colorimetry method

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(Figure S3). The results were listed in Table 2. Based on these results, this method could

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be valuable for the detection of NO2- in food safety assessment as an optional scheme.

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Table 2. Detection results of NO2- in meat products obtained from the local market

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using the proposed fluorescence sensor (n=5). Samples

Colorimetry

Proposed

Spiked

Total

Recovery

RSD

detected

method

(mg/kg)

found

(%)

(%)

(mg/kg)

detected

5.3

(mg/kg)

(mg/kg) Cured

12.87

13.48

17.60

30.31

95.6

fish

12.79

13.67

35.20

48.06

97.7

4.4

Cured

1.58

1.63

17.06

19.79

106.5

5.3

beef

1.55

1.57

35.2

38.42

104.7

4.6

Sausage

23.36

24.87

17.60

41.63

95.2

6.1

23.47

24.64

35.20

60.82

102.8

5.2

43.77

45.91

17.60

64.41

105.1

5.4

43.92

45.68

35.20

82.07

103.4

4.8

Bacon

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Mechanism of the Sensing System. To further investigate the interaction between

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m-CDs and NO2- under 0.1 M H2SO4, transient fluorescence spectra at 494 nm before 14


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and after adding different concentrations of NO2- were scanned. Figure 5A shows that

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the fluorescence lifetime of m-CDs was remarkably shortened as the concentration of

261

NO2- increased, the result proclaimed that NO2- caused the fluorescence quenching of

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m-CDs mainly due to the dynamic quenching effect. Then, we studied the changes of

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the UV absorption peak at 384 nm of m-CDs after the addition of NO2-. The result is

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presented in Figure 5B and it clearly shows that the peak at 384 nm gradually weaken

265

and disappeared with the addition of NO2-, owing to the interaction destroyed the n-π*

266

transition process of m-CDs. Besides, the FT-IR spectra (Figure 5C) explicitly

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illuminates that the primary amine group bending vibration peak38, 39 at 1640 cm-1 in

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m-CDs disappeared and a new absorption peak was generated at 2260.4 cm-1 after

269

adding NO2- (5 μM), which was attributed to the triple bond or the double bond

270

accumulation stretching vibration peak.40, 41 These results strongly suggested that the

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fluorescence quenching of m-CDs is mainly due to amino groups participating in the

272

reaction and conversion to other functional groups that result in energy transfer.

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Interestingly, after adding N-(1-naphthyl) ethylenediamine dihydrochloride to the

274

reaction solution of m-CDs and NO2-, we observed that the mixed solution immediately

275

changed from colorless to magenta, and the absorbance of the magenta dye was positive

276

proportional to the concentration of NO2-, as shown in the inset of Figure 5D. These

277

data confirmed the diazonium reaction between m-CDs and NO2- under 0.1 M H2SO4

278

and produced diazonium salt.42-44 The diazonium salt was an electron-withdrawing

279

group and the electron cloud of n-electron lone was not coplanar with the π-electron

280

cloud on the benzene ring, and the transition from n to π* was a forbidden process. And 15


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the probability of intersystem crossing (ISC) transition between S1 and T1 was large,

282

inducing the fluorescence quenching of m-CDs.

283 284

Figure 5. Transient fluorescence spectra (A), Absorption spectra (B), and FT-IR

285

spectra (C) of m-CDs in H2SO4 (0.1 M) before and after the addition of NO2-. (D) shows

286

that the absorbance of the magenta dye is proportional to the concentration of NO2-.

287

The inset image in (D) shows the corresponding color variations of these solutions.

288 289

Response of Ratiometric Fluorescence Test kit (m-CDs@[Ru(bpy)3]2+) to NO2-.

290

As mentioned previously, single emission fluorescence is susceptible to biological

291

background, environment conditions and instrumentation. In order to eliminate these

292

shortcomings, and to achieve semi-quantitative visualization, herein a ratiometric

293

fluorescence probe (m-CDs@[Ru(bpy)3]2+) was constructed by introducing positively

294

charged Ru(bpy)3Cl2·6H2O as an internal reference fluorescent probe and combined 16


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with negatively charged m-CDs via electrostatic interaction. As shown in Figure 6A in

296

the supporting information (also shown below), the C-H stretching vibration is located

297

at 3070-3020 cm-1, and the stretching vibration (skeleton band) of the aromatic

298

heterocyclic ring at 1600- 1500 cm-1, and the out-of-plane bending vibration of aromatic

299

hydrogen at 900-700 cm-1. The above peak is attributed to the pyridine aromatic

300

heterocyclic ring in [Ru(bpy)3]2+. The C-H stretching vibration peak located at 2958-

301

2805 cm-1, the C-N= stretching vibration peak at 1386 cm-1 and a C-N stretching

302

vibration peak at 1051 cm-1. These three characteristic peaks belong to m-CDs. The

303

UV-vis absorption spectra (Figure 6B) of m-CDs@[Ru(bpy)3]2+ show that the

304

characteristic absorption peaks of λ=452 nm and λ=384 nm are derived from n-π*

305

transition of [Ru(bpy)3]2+ and m-CDs, respectively. DLS size distribution results show

306

that the m-CDs become larger after being combined with [Ru(bpy)3]2+ by electrostatic

307

interaction (Figure 6C). Figure 6D shows that the zeta potential of m-CDs is as high as

308

-24.7 mV, and the zeta potential of [Ru(bpy)3]2+ is +7.31 mV, and the zeta potential of

309

m-CDs@[Ru(bpy)3]2+ is -13.6 mV. These results confirmed the successful association

310

between m-CDs and [Ru(bpy)3]2+. As shown in Figure S4A and S4B, the [Ru(bpy)3]2+

311

has no response to NO2- and H2SO4 even at relatively high concentration. Besides, the

312

effect of common ions and other interferents on the detection of NO2- based on m-

313

CDs@[Ru(bpy)3]2+ could be negligible (Figure S4C). These results suggested that the

314

ratiometric probe based on m-CDs@[Ru(bpy)3]2+ could be used for specific and

315

selective detecting NO2-.

17


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316 317

Figure 6. Characterization of m-CDs@[Ru(bpy)3]2+. (A) FT-IR spectrum. (B) UV-vis

318

absorption spectrum. (C) Dynamic light scattering (DLS) size distribution. (D) Zeta

319

potential.

320 321

As presented in Figure 7A, the red fluorescence of [Ru(bpy)3]2+ at 610 nm remains

322

unchanged after gradually increasing the concentration of NO2- from 0 to 6 μM, while

323

the cyan fluorescence of m-CD at 494 nm gradually quenched, resulting in a transition

324

of the fluorescent colors of the ratiometric probe from cyan to red. And the fluorescence

325

intensity ratio F494/F610 shown a good linear relationship against NO2- concentration,

326

ranging from 0 to 2.0 μM (R2 = 0.997) (Figure 7B). The above data illustrated that the

327

ratiometric probe was superior to single emission m-CD for visual detection of NO2-.

328

Meanwhile, Commission Internationale de L’Eclairage (CIE) coordinates in Figure 7C 18


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and Table S2 have further confirmed these color variations. The sensing system had

330

cyan emission with CIE coordinates of (0.1508, 0.4302). As the NO2- concentrations

331

increased, a substantial red-shift tendency in the CIE coordinate was observed from

332

cyan (0.1807, 0.4253; 0.25 μM) to cyan-green (0.2092, 0.4054; 0.50 μM), green

333

(0.3015, 0.3793; 1.0 μM), green-yellow (0.3483, 0.3595; 2.0 μM), yellow (0.3832,

334

0.3450; 3.0 μM), orange (0.4496, 0.3217; 4.0 μM), orange-red (0.5208, 0.2988; 5.0

335

μM) and red (0.5992, 0.2806; 6.0 μM). These results indicated that the reliable of color

336

variation in the ratiometric fluorescence sensing system from another aspect.

337

Following the above strategy, we further fabricated a portable test kit to simplify the

338

detection process and shorten the detection time so that it can be applied for on-spot

339

detection. It is known that the agarose hydrogel has no fluorescent background, and has

340

controllable shape and large loading capacity. Therefore, agarose hydrogel was utilized

341

as a carrier to fabricate a uniform ratiometric fluorescence matrix for visual semi-

342

quantitative analysis of NO2-. Figure 7D shows that the fluorescent colors of the

343

hydrogel test kit change continuously with the concentration of NO2- in the range of

344

0−100 μM, which is convenient to distinguish the concentration of NO2- according to

345

the observed multicolor fluorescence under UV light. Figure S5 shows the

346

complementary colors of the corresponding colors in Figure 7D. As a consequent, it

347

can be used for semi-quantitative analysis of NO2- with the naked eye using a standard

348

colorimetry card. It is known that the detection efficiency is vital for on-spot detection.

349

Thus we investigated the color variations of the sensor with different reaction time. It

350

can be clearly seen that naked-eye distinguishable color change is observed when the 19


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351

concentrations of NO2- are equal or larger than 50 μM in terms of a reaction time of 10

352

minutes is selected (Figure 7Dc). The contrast ration can be further improved when the

353

reaction time is increased to 20 and 40 mins (Figure 7Dd and 7De). Due to the good

354

portability of the m-CDs@[Ru(bpy)3]2+-based agarose hydrogel and the vivid color

355

display, it could have great potential and prospects for on-spot visual detection of NO2-

356

in real samples.

357 358

Figure 7. (A) Fluorescence spectra and the corresponding colors of m-

359

CDs@[Ru(bpy)3]2+ ratiometric probe after adding different concentrations of NO2- (0–

360

6.0 μM) in H2SO4 (0.1 M); (B) Plot of the fluorescence intensity ratio F494/F610 against

361

NO2- concentration. (C) CIE 1931 (x,y) chromaticity diagram of the sensing system for

362

the detection of NO2- at different concentrations derived from fluorescence spectra

363

(black dots). (D) shows that the fluorescent colors of the hydrogel test kit change 20


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continuously with the concentration of NO2- in the range of 0−100 μM. (a) and (b) are

365

the pictures of m-CDs@[Ru(bpy)3]2+-based agarose hydrogel solid matrix under visible

366

light and under 365 nm UV light, respectively. (c), (d) and e) are the photos of the

367

agarose hydrogel test kit under 365 nm UV light after reacting for 10 minutes (c), 20

368

minutes (d), and 40 minutes (e), respectively.

369 370

CONCLUSIONS

371

In conclusion, we proposed a facile strategy for determining NO2- in food and

372

environmental matrix, which was based on the interaction between the amine group of

373

m-CDs and NO2- via diazo reaction that produced diazonium salts and induced the

374

fluorescence quenching of m-CDs. The diazo-reaction could well avoid the influence

375

of other common interferents. Moreover, we verified the accuracy of the proposed

376

method by measuring the content of NO2- in two kinds of quality control samples, and

377

further applied to the practical detection of NO2- in four kinds of meat products.

378

Following the above strategy, we chose the Ru(bpy)3Cl2·6H2O as an internal reference

379

fluorescent reagent to prepare a ratiometric fluorescent probe (m-CDs@[Ru(bpy)3]2+).

380

Upon the addition of NO2-, the intensity ratio of the two emission wavelengths is

381

changed, resulting in continuous changes in the fluorescence color of the ratiometric

382

probe. Compared with single emission m-CDs, the ratiometric probe can significantly

383

enhance the sensitivity and practicability of visual detection. On this basis, we further

384

fabricated a m-CDs@[Ru(bpy)3]2+ ratiometric fluorescence test kit using agarose

385

hydrogel as a carrier. The test kit is portable, low-cost and highly efficient, enabling 21


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386

rapid detection of NO2- on site and in situ. The vivid colors could be applied for visual

387

semi-quantitative detection of NO2- within 10 minutes with the naked eye. Thus the test

388

kit could have potential application for on-spot detection of NO2- in environmental and

389

food samples.

390 391

ASSOCIATED CONTENT

392

Supporting Information

393

Additional information about the chemical regents, instruments, experimental section,

394

XPS spectra, the fluorescence lifetime, traditional colorimetric method, specificity and

395

selectivity of m-CDs@[Ru(bpy)3]2+ towards NO2-, and the complementary colors of

396

agarose hydrogel test kit (Figures S1-S5). Comparison of the analytical performance of

397

different methods for the detection of NO2- (Table S1). The CIE coordinates value of

398

the ratiometric fluorescence sensing system (Table S2).

399 400

AUTHOR INFORMATION

401

Corresponding Author

402

*E-mail: [email protected] (L Li)

403

*E-mail: [email protected] (LH Guo)

404

Author Contributions

405

1

406

Notes

407

The authors declare no competing financial interest.

Zhan and Zeng contributed equally to this work.

22


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408 409

ACKNOWLEDGMENTS

410

This work was supported by the National Natural Science Foundation of China

411

(21675028, 21507041), Nature Sciences Funding of Fujian Province (2018J01682),

412

STS Key Project of Fujian Province (2017T3007), the Cooperative Project of

413

Production and Study in University of Fujian Province (2018Y4007), and the Program

414

for New Century Excellent Talents in Fujian Province University.

415 416

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For TOC only

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A Ratiometric Fluorescent Hydrogel Test Kit for On-Spot Visual

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