in Food Based on FRET Between Aptamers Functionalized

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Food Safety and Toxicology 2+

A Turn-on Fluoresence Sensor for Hg in Food Based on FRET Between Aptamers Functionalized Upconversion Nanoparticles and Gold Nanoparticles Yan Liu, Qin Ouyang, Huanhuan Li, Min Chen, Zheng-Zhu Zhang, and Quansheng Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00546 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Journal of Agricultural and Food Chemistry

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A Turn-on Fluoresence Sensor for Hg2+ in Food Based on

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FRET

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Nanoparticles and Gold Nanoparticles

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Yan Liu a, Qin Ouyang a, Huanhuan Li a, Min Chen a, Zhengzhu Zhang b, Quansheng

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Chen a,b,∗

Between

Aptamers

Functionalized

Upconversion

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a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

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b

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 210036, China

∗ Corresponding author. Tel.: +86-511-88790318. Fax: +86-511-88780201. E-mail: [email protected] (Q.S.Chen)

ACS Paragon Plus Environment


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ABSTRACT

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In this study, a turn-on nanosensor for detecting Hg2+ was developed based on the fluorescence

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resonance energy transfer (FRET) between long-strand aptamers functionalized upconversion

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nanoparticles (UCNPs) and short-strand aptamers functionalized gold nanoparticles (GNPs). In the

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absence of Hg2+, FRET between UCNPs and GNPs occurred owing to the specific matching between

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two aptamers, result in the fluorescence quenching of UCNPs. In the presence of Hg2+, long-stranded

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aptamers foldback into a hairpin structure due to the stable binding interactions between Hg2+ and

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thymine, leading to the release of GNPs from UCNPs, result in the quenched fluorescence restoration.

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Under the optimized conditions, the nanosensor achieved a linear detection range of 0.2 - 20 µM and

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a low detection limit (LOD) of 60 nM. Meanwhile, it showed good selectivity and has been applied

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to detecting Hg2+ in tap water and milk samples with good precision.

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KEYWORDS: Upconversion nanoparticles; Gold nanoparticle; Aptamers; Fluorescence resonance

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energy transfer; Turn-on fluoresence sensor; Hg2+


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INTRODUCTION

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Mercury ions (Hg2+), as common environmental pollutants, can enter human body through food

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chains and harm the health even at low concentration 1. Due to its toxic effects, many countries have

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developed strict restriction on the level of mercury residue in food, of which the lowest limitation

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standards limit of mercury is 0.02 mg/kg (ca. 10 µM). For satisfying the mercury limitation

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requirements, develop more sensitive detection method featured with simplicity and selectivity will

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become an urgent mission in food monitoring. To satisfied the low concentration detecting, several

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methods with intuitive results and simple operation for detecting Hg2+ have been proposed in recent

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years, such as colorimetric assays

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spectroscopy

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analytical method has become core keywords, with advantages of high sensitivity, low detection

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limit and high analytical precision. To date, various fluorescent chemosensors have been developed

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by generally using rhodamine

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downconversion fluorophores

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synthesis of these fluorophores and their poor water-solubility limited their practical application in

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food detection. Besides, conventional down-conversion emitting properties make them usually suffer

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from high background fluorescence of endogenous chromophores in samples. Therefore, developing

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a new simple sensor with low background and high sensitivity for Hg2+ detection is still a valuable

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challenge.

2, 3

14-18

, and surfance enhanced Raman scattering (SERS)

4-6

, benzothiazole derivatives

11-13

4-13

, fluorescent analytical method

, plasmon resonance

19-21

. In particular, fluorescent

7-10

, and other chemical synthesized

as fluorescent probes. However, the difficult and danger in the

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Comparing with the conventional fluorescent materials, such as organic molecules, C-dots and

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quantum dots (QDs), upconversion nanoparticles (UCNPs) possess many outstanding features such

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as displaying high Stokes shift, showing high resistance to photobleaching, providing continuous ACS Paragon Plus Environment


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luminescence, inducing only a very weak autofluorescence background, avoiding photo degradation

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in samples and enhancing signal-to-noise ratio22. More importantly, lanthanide-doped UCNPs can

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convert longer wavelength light to shorter wavelength light through a photon upconversion process

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by doping different ratios of rare earth ions23-25, further preventing background autofluorescence.

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Meanwhile, Easy tunability of emission wavelength and high sensitivity also extends the application

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field of the UCNPs. Among the application in Hg2+ detection, most are based and widespread

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applicability in FRET systems due to their high extinction coefficients, easy synthesis and surface

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chemistry modification. However, almost all of these biosensor are based on the mechanism of

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fluorescence quenching induced by FRET. Compared to these fluorescence quenching probes,

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fluoresence turn-on sensors are more suitable for practical applications, because they can effectively

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avoid the wrong response and have a higher signal-to-noise in dark background26, 27.

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Aptamers, are single-stranded DNA or RNA sequences, which can be isolated through systematic

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evolution of ligands by exponential enrichment (SELET) in vitro. They have functions similar to

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antibodies but have yet another merits of lower cost, easily synthesized and modified, and good

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stability. In addition, aptamers are able to bind with target molecules with high selectivity and

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specificity, so they have been widely used as recognition elements in biosensors. However, most

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biosensor based on aptamer were used to sensing bacteria, antibiotics, DNA and other molecules,

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few were sensing metal ion. Although the aptamer for Hg2+ have been designed based on the stable

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binding interactions between Hg2+ and thymine, there were no turn-on FRET systems that have been

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explored for Hg2+ detection.

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Thus, taking advantage of the unique properties of UCNPs, GNPs and aptamers, herein, we report

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a turn-on upconversion fluorescence biosensor for Hg2+ detection sensitively. Long-stranded aptamer ACS Paragon Plus Environment

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modified NaYF4:Tm3+, Yb3+ UCNPs serve as fluorescences donors, while the complementary

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short-stranded modified GNPs were employed as quenchers. Due to the complementary pairing of

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the short-stranded aptamer and long-stranded aptamer, the distance between UCNPs and GNPs was

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shortening, the FRET occurred because of spectral overlap between UCNPs fluorescence emission

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and GNPs absorption, leading to fluorecence quenching of UCNPs. Thus, the fluorescence signal of

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detection system was weak in the initial state. When Hg2+ was introduced, rich T-T mispairs in

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long-strand aptamer can selectively capture Hg2+ ions and form T-Hg2+-T base pairs, causing

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long-stranded aptamer folded back, a phenomenon which leads to the separate of GNPs from UCNPs,

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make the FRET diminish, thereby result in the recovery of fluorescence. On these bases, the propose

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sensor can realize sensitive and selective testing of Hg2+ with LOD of 60 nM. In addition, it is able to

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sense Hg2+ in food samples.

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2. EXPERIMENTAL SECTION

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2.1 Materials

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Rare-earth chloride hexahydrate: Ytterbium(III) chlorid hexahydrate (99.9%), yttrium (III) chlorid

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hexahydrate (99.9%), holmium (III) chlorid hexahydrate (99.9%), oleic acid (OA, 90 %),

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3-Aminopropyltriethoxysilane (APTES), 1-octadecene (ODE, 90 %), tetraethoxysilane (TEOS) were

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obtained from Sigma-Aldrich. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

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(EDC), N-Hydroxysuccinimide (NHS), succinic anhydride, hydrogen tetrachloroaurate hydrate

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(HAuCl4, 99.9%) were supplied by Macklin reagent Ltd. (Shanghai, China). All aptamers were

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supplied by Sangon Biotechnology Ltd. (Shanghai, China), the sequences of aptamers are as follows:

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(long-stranded aptamer) 5' NH2 C6-CTA CAG TTT CAC CTT TTC CCC CGT TTT GGT GTT T-3',



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(short-stranded aptamer) 5' SH C6-GAA ACT GTA G-3'. Methanol, ethanol, cyclohexane, sodium

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hydroxide, ammonium fluoride, ammonia (25%), methylbenzene and trisodium citrateand were

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standard analytical pure and provided by Sinopharm chemical reagent Co.,Ltd. (Shanghai, China).

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2.2 Instruments and characterizations

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The nanoparticles size and morphology were observed on a JEM-2100 (HR) transmission electron

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microscopy (Japan Electron Optics Laboratory, Japan) at 120 kV acceleration voltage by placing a

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drop of sample solution on a TEM copper grid. Powder X-ray diffraction (XRD) were determined on

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a D8 Advance X-ray Diffractometer (Bruker AXS, Ltd., Germany) to analyze the sample crystal

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structure. Fourier transform infrared (FT-IR) spectra was recorded on Nicolet Nexus 470 Fourier

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transform infrared spectrophotometer (Thermo Electron Co., U.S.A.) using KBr pellet pressing

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method to confirm the successful chemical modification of UCNPs. UV-2450 spectrophotometer

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(Shimadzu, Japan) was used to measure the UV-vis absorption spectra of GNPs and verify the

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aptamer modification. Upconversion fluorescence was tested on the assembled upconversion

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fluorescence spectrophotometer, it assembly diagram can be found in Supplementary Information,

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Fig. S1. The main components are 980 nm near infrared laser, SAC universal sample chamber,

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Omni-λ monochromator, PMTH-S1 side-on photomultiplier tube, DCS103 data collector and

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H1800V high voltage power supply. The zeta potential was measured using Malvern zetasizer Nano

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ZS90 (Malvern Instruments Ltd., U.K.).

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2.3 Development of biosensor

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The chemical route followed to obtain the DNA modified upconversion nanoparticles and complementary DNA modified GNPs is given in Supplementary Information, Fig. S2 and Fig. S3.


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2.3.1 Synthesis of aptamers-modified UCNPs

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The procedure for preparation of oleic acid-capped upconversion nanoparticles (OA-UCNPs) was

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performed based on previous research28 with some modifications. First, 0.2367 g of yttrium(III)

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chloride hexahydrate, 0.0775 g of ytterbium(III) chloride hexahydrate and 0.0066 g of holmium (III)

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chloride hexahydrate were weighed accurately and dispersed in 12 mL of methanol. Add this solution

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into the 250 mL three-neck round-bottom flask whic contains 18 mL of oleic acid and 42 mL of

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1-octadecene. A reflux condenser pipe whose lower end was fixedly arranged on the right above the

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flask, was fixedly connected with the T-connector. The other two outlets of T-connector was

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connected with inlet argon pipe and balloon. The other two necks of flask were attached to gas outlet

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pipe and temperature rod, respectively. Put the flask on the magnetic heated stirrer. In the argon

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protection environment, heat the above solution to 160 ºC under magnetic stirring and keep for 30

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min, cooling naturally to 50 ºC. Then, turn off the argon intake valve and remove the reflux

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condenser pipe from flask. Add 10 mL of methanol solution which involved 0.1482 g of NH4F and

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0.1 g of NaOH into the above flask drop by drop. Next, heat up to 70 ºC and maintained it long

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enough to promote full and evaporation of methanol. Then, re-attach the reflux condenser pipe on the

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flask, turn on the argon intake valve and raise the heating temperature to 300 ºC, stirring constantly.

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During this period, slow airflow velocity and inspect whether the bracket for the condensate pipe is

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reliable from time to time. After the reaction, the solution appeared as transparent straw yellow.

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Close the heating switch and allow the solution to cool down naturally to room temperature. The

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nanoparticles were separated from the final solution by centrifugation and washed with the mixture

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of cyclohexane and ethanol (v: v = 2: 1). Repeat three times. Dry the final product overnight in

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vacuum oven at 55 ºC.



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The Carboxyl modification of UCNPs was carried out using the sequential procedure, first coat the

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SiO2, second modify the amino groups and finally graft the carboxyl groups. The detail steps are

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described as following. First, suspend 0.2 g of OA-UCNPs in 60 mL of ethanol, treat with ultrasonic

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wave for 30 min to completely dissolve the power. Then, add 2.5 mL of 25% ammonia and 20 mL of

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distilled water into the solution, seal off the flask and heat it to 70 ºC with stirring vigorously. After

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keeping the temperature for 15 min, 200 µL of TEOS was added dropwise and the mixture was

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stirred for another 6 h. Next, add 200 µL of APTES dropwise, keep stirring for 3 h, and cool slowly

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to room temperature. The product was separated by centrifugation at 8000 rpm for 8 minutes, and

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washed with ethanol. Repeat this process for three times, obtain amino-modified upconversion

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nanoparticles (NH2-UCNPs). The resulting precipitate was dispersed in 15 mL methylbenzene, then

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5 mL 0.16 M succinic anhydride in methylbenzene was added into above solution. Under the

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protection of nitrogen, heating above solution to 80 ºC and continue to stir for a further 12 h. After

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cool to room temperature, the production was separated by centrifugation (11000 rpm, 15 min),

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washed with deionized water for three times. Finally, the precipitate (COOH-UCNPs) was added in a

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vacuum drying oven and then dry it at 60 ºC for 4 h. 29

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The procedure for the aptamer-functionalized UCNPs was adapted from a reported literature

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First, 5 mg of carboxyl-modified UCNPs and 4 mg EDC were dispersed in 5 mL ultrapure water. Stir

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for 15 min at room temperature to activating the catboxylic groups on the surface of UCNPs. Next, a

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NHS solution in PBS buffer (10 mM, pH=7.4) was added into above solution, stir for a further 5 min.

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Then, 700 µL 10 µM long-strand aptamer was added. As solution was stirred continuously,

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carbodiimide coupling reaction took place, making the long-stranded aptamers with -NH2 covalently

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attached to the COOH-UCNPs. After reaction for 12 h, the aptamers-modified UCNPs were purified


.

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by centrifuged at 12000 rpm for 10 min and washed with PBS buffer for three times. The final

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production was re-dispersed in 10 mL PBS buffer and stored at 4 ºC.

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2.3.2 Synthesis of aptamers-modified GNPs

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GNPs were synthesized with citrate reduction method. First, all glass vessels used were cleaned in

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aqua regia and washed under running ultrapure water. Then, the 250 mL round-bottom flask

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contained 50 mL 1 mmol/L HAuCl4·4H2O solution was equipped with a reflux condenser and

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incubated in the magnetic heated stirrer. Heat, stirring, until the solution boiled, add 5 mL 38.8

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mmol/L trisodium citrate solution quickly. At this point, the solution changed from pale yellow color

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to wine red. Allow it to boil for 30 min, cut down the power and leave the solution to cool naturally.

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Pour the solution into the amber laboratory bottle and stored at 4 ºC. Transfer 5 mL of the prepared

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GNPs solution to 10 mL centrifuge tube and add into 960 µL of 10 µM sulfhydryl modified

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short-stranded aptamers. After 16 h incubation at 50 ºC, adjust the pH and salt concentration of

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solution by introducing necessary salts, making the primary solution contain 0.1 M of NaCl and be at

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pH 7.4. Then, keep the mixed solution at 50 ºC for a further 40 h. To purify the aptamer modified

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GNPs, the above solution were centrifuged at 12000 rpm for 20 min, and the precipitate was washed

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with PBS buffer for three times. The final wine red pellet was re-suspended in 5 mL PBS buffer and

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stored at 4 ºC. Based on the Beer's law, the concentration of aptamers-modified GNPs was estimated

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to be 200 nM, which was calculated by using their extinction coefficient of 3.6×108 cm-1M-1 at 528

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nm and diameter of about 15 nm 30.

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2. 4 Detection of Hg2+ by UCNPs -aptamers-GNPs biosensors

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The upconversion fluorescence spectrophotometer was preheated for 30 min to obtain stable



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signals before testing. The process of detecting Hg2+ is as follows: first, 5 mg/L aptamers-modified

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UCNPs was mixed with aptamers-modified GNPs and hybridized for half an hour. Then, various

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amounts of Hg2+ or other test samples were added into the above mixtures. After 0.5 h incubation at

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room temperature, the mixture was measured using the assembled upconversion fluorescence

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spectrofluorometer.

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2. 5 Bio-detection of Hg2+ in spiked-in samples

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For demonstrating the viability of this method, Hg2+ in tap water and milk was analyzed by the

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constructed turn-on sensor. Typically, different concentrations of the standard solution of Hg2+ were

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spiked into the pretreated tap water and milk and quantitatively detected by the developed method.

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The pretreatment method of tap water and milk are as follow. Tap water was centrifuged and filtered

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through a 0.22 µm membrane. Milk samples were purchased from a local supermarket. 20 mL of

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milk was added to 4 mL of CHCl3 and 4 mL of 10% Cl3CCOOH solution. Then the mixture was

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vortexed for 10 min on lab-dancer and centrifuged to remove proteins in milk. Subsequently, the

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supernate was filtered through a 0.22 µM membrane to remove lipids. The gained filtrate was stored

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for further use.

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3. RESULTS AND DISCUSSION

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3.1. Synthesis and surface functionalization of UCNPs and GNPs

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TEM, XRD, FT-IR, UV-vis and zeta potential test were used to characterize the shape, size,

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crystal form and surface modification of the nanoparticles in this study. As seen in Fig. 1A, the

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morphology of OA-UCNPs dispersed in the hexane are highly monodisperse hexagonal polyhedra

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with a mean diameter of 50 ± 3 nm. By X-ray diffraction method, crystal structure and matter phase


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were obtain. It can be seen in Fig.1E, the XRD patterns of UCNPs shows pronounced peaks,

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indicating the high crystallinity of the synthesized material. In order to improve the water solubility

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of UCNPs to embroad their practical application, an efficient approach to modifying the surface of

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UCNPs was presented. As illustrated in Fig. S2, the whole procedure consists of three steps: first,

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coating silica shell on the UCNPs via the reverse microemulsion method; and then, modifying amine

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groups by adding APTES; finally, grafting carboxylic groups onto the amino groups through ring

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opening reaction of succinic anhydride. The inset in Fig. 1A depicts the TEM image of silica-coating

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UCNPs. The shell thickness is about 3 nm, and the whole shell is integrated and uniform. The silica

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shell could offer good hydrophilicity to UCNPs and serve as a platform for further modification. The

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surface structure of the UCNPs after each modification step were confirmed by FT-IR spectroscopy.

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As presented in Fig. 1F, the peak at 3400 cm−1 is corresponding to the stretching vibration of –OH

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groups, peaks at 2930 cm−1 and 2853 cm−1 are ascribed to the asymmetric and symmetric stretching

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vibrations of the methylene group (–CH2–), peaks at 1567 cm−1 and 1418 cm−1 are the characteristic

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bands of asymmetric and symmetric stretching vibrations of the –COOH group (curve a), confirming

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the existence of oleic acid on the surface of UCNPs. After modifying nanoparticles surface,

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characteristic peaks at 1625 and 1068 cm−1 (curve b) appeared, which are of the stretching and

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bending vibration of –NH2 groups and the stretching vibration of Si–O bond, validating successful

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coating of SiO2 and modification of –NH2 groups (named NH2-UCNPs). After grafting –COOH by

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reacting with succinic anhydride (named COOH-UCNPs), a new band of carbonyl groups at 1732

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cm−1 appeared in the FT-IR spectrum, and meanwhile, the band at 3400 cm−1 disappeared (curve c).

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This is because the successful graft of carboxylic groups onto amino groups and transform of

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hydroxyl groups into carboxylic groups. Additionally, the surface potentials (ζ) of nanoparticles are



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measured by zeta potential analyzer (Fig. 1I). Water-soluble NH2-UCNPs are positively-charged with

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zeta potential of 22.3 ± 2.48 mV. After carboxyl modification, the zeta potential of UCNPs changes

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into -26.7 ± 3.56 mV, reveals that the -COOH groups have been successfully grafted onto UCNPs.

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The zeta potential of UCNPs-aptamers positively shift to -14.6 ± 2.14 mV, which is because of the

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positive charges of amino-modified long-stranded aptamers. Besides, the synthesized GNPs show

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zeta potential of -27.6 ± 3.52 mV, attributed to the citrate stabilizing agent in the surface. Upon

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conjugation of the –SH-modified short-stranded aptamers, zeta potential of GNPs negatively shift to

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-32.2 ± 3.76 mV, suggesting that the short-stranded aptamers modified GNPs are more stable in

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water solution than the GNPs. Fig. 1B showes the TEM image of GNPs. GNPs have an average

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diameter of 15 nm, and show good water solubility and dispersibility. The aptamers modification of

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UCNPs and GNPs were verified from the results of UV-vis absorption spectroscopy (Fig. 1G). After

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aptamers modification, the absorbance spectra of UCNPs and GNPs solution exhibit new peak at 260

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nm, confirming the formation of long-stranded aptamers modified UCNPs and short-stranded

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aptamers modified GNPs.

[Here for Fig. 1]

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3.2. Sensing scheme

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A schematic of the turn-on fluorescent sensor for Hg2+ based on the FRET pair of UCNPs and

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GNPs was shown in Fig. 2. Two single-stranded aptamers (long-stranded aptamers and

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short-stranded aptamers) were modified in UCNPs and GNPs, respectively. In the absence of Hg2+,

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the total 10 complementary base pairs of short-stranded aptamers were hybridized with the

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corresponding bases of long-stranded aptamers, bringing GNPs to the proximity of UCNPs, leading


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to green fluorescence quenching of UCNPs. This is because the overlap between the absorption

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spectrum of GNPs and the fluorescence spectrum of UCNPs in 545 nm (inset a in Fig. 2) that cause

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FRET between them. When Hg2+ was introduced, long-strand aptamer on the surface of UCNPs

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folded into hairpin structure, which is attributed to the reason that T-T mispairs in long-strand

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aptamer can selectively capture Hg2+ ions and form T-Hg2+-T base pairs (inset b in Fig. 2). In this

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case, only six base pairs between long-stranded aptamer and short-stranded aptamer left. Besides,

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Since the van der Waals radius of mercury is ∼1.44 Å while the base pair spacing in DNA duplex is

243

∼3.4 Å, suggesting the T-Hg2+-T pair is more stable than the Watson-Crick A-T pair31. Therefore,

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GNPs modified with short-stranded aptamer release from UCNPs, resulting in the disappearance of

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FRET and the recover of upconversion fluorescence. The developed UCNPs-aptamers-GNPs

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detection system was characterized by TEM. In absence of Hg2+, many GNPs attaches to UCNPs,

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which can be seen in Fig. 1C. In presence of Hg2+, the number of GNPs attached in UCNPs

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significantly decreased (Fig. 1D), successfully indicated the sensing principle of the detection.

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[Here for Fig. 2]

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To investigate the process of fluorescence resonance energy transfer, as shown in Fig. 3A, the

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strong fluorescence of the UCNPs was gradually quenched with the increase of GNPs concentrations.

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This fluorescence quenching of UCNPs is attributed to the FRET from UCNPs to GNPs via the

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short-stranded aptamers modified on the GNPs surface binding the long-stranded aptamers on the

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UCNPs surface. The fluorescence quenching was defined by the following equation: E = (Fi－F0)/F0,

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in which Fi and F0 refer to the upconversion fluorescence intensities of the UCNPs in the presence

256

and absence of the GNPs. As seen in Fig. 3B, the quenching efficiency increased with the

257

concentration of GNPs. When the concentration of GNPs reached 170 nM, the quenching efficiency ACS Paragon Plus Environment


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tended to grow sluggishly. Therefore, 170 nM was selected as the final concentration of GNPs for the

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subsequent experiments. In this case, the fluorescence quenching efficiency reaches 85.4%, which

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offers favorable conditions for sensitive detection of target.

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[Here for Fig. 3]

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3.3. Optimization of detection conditions

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To obtain high performance of the developed sensor, the hybridization time of aptamers-modified

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UCNPs and aptamers-modified GNPs, and the reaction time after adding Hg2+ were optimized in this

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study. The fluorescence changes of FRET system in the presence of 5 mg/L aptamers-modified

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UCNPs and 170 nM aptamers-modified UCNPs for a fixed time interval of 3 min are shown in Fig.

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3C. The fluorescence intensity decreased gradually over time. When the hybridization time reached

268

18 min, the fluorescence intensity stopped decrease due to the almost complete hybridization of

269

aptamers-modified UCNPs and aptamers-modified GNPs. To ensure the fully hybridization, an

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optimum 21 min hybridization time was applied in the subsequent experiments. The latter part of Fig.

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3C shown the changing trend of fluorescence intensity with the reaction time after adding Hg2+. The

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fluorescence intensity was found to be increased gradually within the first 9 min, however, no

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prominent increase was observed after 9 min because almost all the Hg2+ had combined with

274

thymine in the long-strand aptamers to make the FRET disappear. Based on this study, 9 min reaction

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time was used in the further experiments. All the experiment were performed at room temperature

276

(25−28℃).

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3.4. Fluorescence detection of Hg2+ based on UCNPs-aptamers-GNPs FRET sensor

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To demonstrate the performance of the developed FRET sensor for Hg2+ detection, the detection


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range and LOD were investigated. Fig. 4A shows UC fluorescence spectra against the concentrations

280

of Hg2+ in the homogeneous assay. As the concentration of Hg2+ increased, the fluorescence intensity

281

at 545 nm increased gradually. The plot of fluorescence intensity against the concentrations of Hg2+

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is shown in Fig. 4B. A linear correlation between the fluorescence intensities and Hg2+ concentrations

283

is obtained in low concentration regions (0.2-20 µM), which can be fitted as y = 83.351x+414.86,

284

with R2 = 0.9947. The LOD can be calculated by 3S0/S, where S0 represents the relative standard

285

deviation of the measured value of nine blank samples, and S is the slope of the calibration curve (S0

286

= 1.69, S = 83.351). In this study, a value of 60 nM was obtained, which is lower than the safe level

287

for food (10 µM). Thus, this sensor can detect Hg2+ quantitatively at low concentration.

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289

[Here for Fig. 4]

3.5. Selectivity and anti-interfering of UCNPs-aptamers-GNPs FRET system

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To explore the selectivity of the developed system, several metal ions (K+, Na+, Mn2+, Ba2+, Cu2+,

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Ca2+, Cd2+, Mg2+, Zn2+, Pb2+ and Fe3+) were examined. As shown in Fig. 5, only Hg2+ resulted in a

292

high response, while none of these metal ions caused fluorescence “turn-on” even in 10 times

293

concentration, demonstrating the high selectivity of the FRET system. Besides, anti-interfering test

294

was investigated. The results in Fig. 5 shows that, fluorescence intensity restored even if other metal

295

ions existed. Besides, nearly no fluorescence intensity difference was observed when detecting 10

296

µM of Hg2+ by the FRET sensor whether other metal ions existed. Therefore, this detection system is

297

rather selective and other metal ions do not interfere with the Hg2+ detection, indicating that the

298

developed system can be used to detect Hg2+ in real samples.

299

[Here for Fig. 5]



300

3.6. Hg2+ determination in real samples

301

To test whether the developed sensor could be further applied in real samples, the spiked known

302

amount of Hg2+ into tap water and pre-treated milk samples were detected and investigated. The

303

prepared tap water and milk were spiked into Hg2+ on three different level of concentrations.

304

Through recording the corresponding fluorescence intensity values and substituted them in the linear

305

formula, we calculated the test values and further obtained the recoveries. Meanwhile, intra- and

306

inter-assay coefficients of variation (CV) were measured to analysis the reliability of the sensor. As

307

shown in Table 1, the quantitative detection of spiked samples with three different concentration of

308

Hg2+ was achieved with recoveries in the range of 95.18% to 108.22%, intra-assay CV in range of

309

2.03% to 7.07%. and inter-assay CV in the range of 5.31% to 8.57%. These results revealed the

310

feasibility of the developed method for Hg2+ detection in real samples. Furthermore, a comparison of

311

the developed method in this work with other published fluorescence methods for the detection of

312

Hg2+ ions is presented in Table S1. Compared with DAM and Eu3+ hybrid carbon dots32, 33, the

313

developed UCNPs-aptamers-GNPs exhibited higher sensitivity for Hg2+ ions detection. Even though

314

the sensitivity of the method for Hg2+ ion is not comparable with that of other methods in the

315

literature34-39, but it exhibits simplicity, accuracy and rapidity for detection of Hg2+ ions. Meanwhile,

316

its its excellent water-soluble performance can make it more extensive applications in practice.

[Here for Table 1]

317

318

ASSOCIATED CONTENT

319

Supporting Information

320

Assembly diagram of the instrument can be found in Fig. S1. The chemical route followed to


Page 16 of 29

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321

obtain the DNA modified upconversion nanoparticles and complementary DNA modified GNPs is

322

given in Fig. S2 and Fig. S3. A comparison of the developed method in this work with other

323

published fluorescence methods for the detection of Hg2+ ions is presented in Table S1.

324

AUTHOR INFORMATION

325

Corresponding Author

326

*Telephone.: +86-511-88790318.

327

Fax: +86-511-88780201.

328

E-mail: [email protected] (Q.S.Chen).

329

Funding This work has been financially supported by the National Natural Science Foundation of China

330 331

(31772063).

332

Notes

The authors declare no competing financial interest.

333

334

ACKNOWLEDGEMENT

The authors are thankful to the authorities of Jiangsu University for providing research facilities to

335 336

carry out this work.

337

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428

Figure Captions

429

In this work, five figures were constructed for further demonstration with brief descriptions as

430

follows:

431

Fig. 1. (A) TEM image of UCNPs (inset: TEM image of UCNPs@SiO2). (B) TEM image of GNPs.

432

(C) TEM image of UCNPs-aptamers-GNPs in absence of Hg2+. (D) TEM image of

433

UCNPs-aptamers-GNPs in presence of Hg2+. (E) XRD of UCNPs. (F) FT-IR spectra of (a) UCNPs,

434

(b) amino-modified UCNPs, (c) carboxyl-modified UCNPs. (G) a. UV-vis spectra of the UCNPs

435

before and after aptamers modification; b. UV-vis spectra of the GNPs before and after aptamers

436

modification. (H) Zeta potential of UCNPs-NH2, UCNPs-COOH, UCNPs-aptamers, GNPs and

437

GNPs-aptamers.

438

Fig. 2. Schematic Description of the UCNPs-aptamers-GNPs FRET sensor for Hg2+.

439

Fig. 3. (A) Upconversion fluorescence spectra of the 100 µL 5 mg/L UCNPs after addition of equal

440

volume of GNPs with different concentrations (0, 20, 30, 40, 50, 60, 70, 90, 100, 110, 120, 140, 170,

441

200 nM). (B) Plot of fluorescence quenching efficiency versus GNPs concentration. (C)

442

Optimization experiments of hybridization and reaction time (20 µM was selected as the Hg2+

443

concentration to determine the optimum hybridization and reaction time).

444

Fig. 4. (A) Upconversion fluorescence spectra of the UCNPs-aptamers-GNPs in the presence of

445

different concentrations of Hg2+. (B) Plot of upconversion fluorescence intensity at 545 nm versus

446

Hg2+ concentrations (0.2, 0.4, 0.8, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 µM).

447

Fig. 5. Selectivity and anti-interfering of the developed UCNPs-aptamers-GNPs FRET sensor. The



448

concentrations of Hg2+ and other metal ions are 10 µM and 100 µM. Every data point was the mean

449

of three measurements. The error bars are the standard deviation.

450


Page 22 of 29

Page 23 of 29

451


Table 1. Determination of Hg2+ in real samples using the developed method Intra-assay

Inter-assay

Sample Found±SD

Recovery

CVs

Found±SD

Recovery

CVs

(µM)

(%)

(%)

(µM)

(%)

(%)

0.2

0.216 ± 0.015

108.22%

7.07

0.196 ± 0.017

98.18

8.57

2

2.08 ± 0.11

104.08%

5.24

1.93 ± 0.14

96.61

7.19

20

20.90 ± 1.01

104.51%

4.81

20.62 ± 1.73

103.1

8.37

0.2

0.190 ± 0.009

95.18

4.76

0.199 ± 0.016

99.72

8.12

2

1.97 ± 0.11

98.45

5.96

2.07 ± 0.11

103.77

5.31

20

19.91 ± 0.40

99.55

2.03

19.92 ± 1.07

99.60

5.39

(µM)

Tap water

Milk

452

SD: standard deviation; CVs: coefficient of variations.

453



454 455

TOC Graphic. Schematic Description of the UCNPs-aptamers-GNPs FRET sensor for Hg2+

456 457


Page 24 of 29

Page 25 of 29


Fig. 1. (A) TEM image of UCNPs (inset: TEM image of UCNPs@SiO2). (B) TEM image of GNPs. (C) TEM image of UCNPs-aptamers-GNPs in absence of Hg2+. (D) TEM image of UCNPs-aptamers-GNPs in presence of Hg2+. (E) XRD of UCNPs. (F) FT-IR spectra of (a) UCNPs, (b) amino-modified UCNPs, (c) carboxyl-modified UCNPs. (G) a. UV-vis spectra of the UCNPs before and after aptamers modification; b. UV-vis spectra of the GNPs before and after aptamers modification. (H) Zeta potential of UCNPs-NH2, UCNPs-COOH, UCNPs-aptamers, GNPs and GNPs-aptamers. 115x202mm (300 x 300 DPI)



Fig. 2. Schematic Description of the UCNPs-aptamers-GNPs FRET sensor for Hg2+. 289x157mm (300 x 300 DPI)


Page 26 of 29

Page 27 of 29


Fig. 3. (A) Upconversion fluorescence spectra of the 100 µL 5 mg/L UCNPs after addition of equal volume of GNPs with different concentrations (0, 20, 30, 40, 50, 60, 70, 90, 100, 110, 120, 140, 170, 200 nM). (B) Plot of fluorescence quenching efficiency versus GNPs concentration. (C) Optimization experiments of hybridization and reaction time (20 µM was selected as the Hg2+ concentration to determine the optimum hybridization and reaction time). 227x59mm (300 x 300 DPI)



Fig. 4. (A) Upconversion fluorescence spectra of the UCNPs-aptamers-GNPs in the presence of different concentrations of Hg2+. (B) Plot of upconversion fluorescence intensity at 545 nm versus Hg2+ concentrations (0.2, 0.4, 0.8, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 µM). 166x65mm (300 x 300 DPI)


Page 28 of 29

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Fig. 5. Selectivity of the developed UCNPs-aptamers-GNPs FRET sensor. The concentrations of Hg2+ and other metal ions are 10 µM and 100 µM. Every data point was the mean of three measurements. The error bars are the standard deviation. 75x59mm (300 x 300 DPI)


in Food Based on FRET Between Aptamers Functionalized

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