Dual-Terminal Stemmed Aptamer Beacon for Label-Free Detection of

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Dual-terminal stemmed aptamer beacon for label-free detection of aflatoxin B1 in broad bean paste and peanut oil via aggregation-induced emission Xuhan Xia, Haibo Wang, Hao yang, Sha Deng, Ruijie Deng, Yi Dong, and Qiang He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05217 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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Journal of Agricultural and Food Chemistry

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Dual-terminal stemmed aptamer beacon for label-free detection of

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aflatoxin

3

aggregation-induced emission

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Xuhan Xia, Haibo Wang, Hao Yang, Sha Deng, Ruijie Deng*, Yi Dong and Qiang He

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College of Light Industry, Textile and Food Engineering, Healthy Food Evaluation Research

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Center and Key Laboratory of Food Science and Technology of Ministry of Education of

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Sichuan Province, Sichuan University, Chengdu 610065, China

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* Corresponding author:

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e-mail: [email protected]

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B1

in

broad

bean

paste

and

peanut

oil

via

Tel: +86 028 85467382

1 ACS Paragon Plus Environment


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ABSTRACT

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Aflatoxin B1 (AFB1) contamination ranks as one of the most critical food safety issues, and

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assays for its on-site monitoring is highly demanded. Herein, we propose a label-free,

14

one-tube, homogeneous and cheap AFB1 assay based on a finely tunable dual-terminal

15

stemmed aptamer beacon (DS aptamer beacon) and aggregation-induced emission (AIE)

16

effects. DS aptamer beacon structure could provide terminal protection of aptamer probe

17

against exonuclease I, and confer specific and quick response to target AFB1. Compared to

18

conventional molecule beacon structure, the stability of DS aptamer beacon could be finely

19

tuned by adjusting its two terminal stems, allowing elaborately optimizing probe affinity and

20

selectivity. By the utilization of AIE-active fluorophore which would be lighted-up by

21

aggregating to negatively charged DNA, AFB1 could be determined in label-free manner. The

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proposed method could quantify AFB1 in one test-tube using two unlabelled DNA strands.

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And it has been successfully applied for analyzing AFB1 in peanut oil and broad bean sauce,

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with total recoveries ranging from 92.75% to 118.70%. Thus, the DS aptamer beacon-based

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assay could potentially facilitate real-time monitoring and controlling of AFB1 pollution.

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KEYWORDS

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aflatoxin B1; aptamer; aggregation-induced emission; label-free; homogeneous analysis


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INTRODUCTION

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Aflatoxin B1 (AFB1) pollution is one of the most critical issues of food safety. AFB1

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is recognized as the most toxic mycotoxin due to its mutagenic, teratogenic,

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immunosuppressive, and carcinogenic effects.1,2 Thus, it has been categorized as group

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I carcinogens by the International Agency for Research on Cancer (IARC, 2002).1

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Especially, AFB1 is ubiquitous in plenty of crops, and can be produced and

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contaminate food products in all processes including growth, harvest, storage, or

35

processing. Therefore, it is highly demanded to develop cheap, fast and large

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instrument-independent on-site detection technology for AFB1 determination to

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achieve real-time monitoring and controlling of AFB1 contaminations.3 Conventional

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chromatography technologies, such as high-performance liquid chromatography

39

(HPLC)4 and liquid chromatography-mass spectrometry (LC-MS)5 are used as the

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golden methods for AFB1 analysis owing to their high reproducibility, precision and

41

accuracy. However, they longstanding suffer from the requirements of time-consuming

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sample pretreatment steps, well-trained personnel and sophisticated equipment,6 thus

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leading to low timeliness and disability for on-spot and rapid detection. Immunoassay,

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based on specific recognition between antibodies and AFB1, can surmount these

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obstacles to some extent.3,7,8 It could confer highly sensitive detection independent of

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large instruments.9,10 Nevertheless, the performance of immunoassay is highly reliant

47

on antibodies, while the preparation of antibody is fairly laborious and expensive,

48

especially for low immunogenic small molecules,11,12 such as AFB1. Besides,



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antibodies are hard to preserve because of its susceptibility to temperature or chemical

50

modifications.

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Fortunately, aptamer, a species of man-made antibody has been emerged, and is

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recognized as competitive affinity reagents in lieu of antibodies in some application

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occasions.13-15 In particular, aptamers can be acquired by in vitro selection which

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would be independent of target antigenicity,16-18 thus are well fit for acting as

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recognition module for low immunogenic small molecules such as AFB1.11,12,19

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Compared with antibodies, aptamers present some novel features such as high stability,

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low cost and easy to synthesize.20,21 In particular, As nucleic acids possess superior

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controllability, programmability, designability and responsiveness.22-26 Aptamers with

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their

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structure-switching probes, such as aptamer beacon, antisense displacement probe.13

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These probes employ binding-induced structural change to output the signal of target

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presence, avoiding the need for washing and separation processes.27,28 However, to the

63

best of our knowledge, these homogenous assays for AFB1 are built based on the

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labelled aptamer probe.29 The labelling process could complicate the detection process

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and sharply increase the cost. In particular, the labelling may potentially exert a

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negative effect on the binding ability of aptamer, thus hampering the recognizing

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process.30

inherently

nucleic

acid

nature,

can

be

engineered

into

numerous

68

Recently, a novel series of fluorescent dyes with aggregation‐induced emission

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(AIE) properties, have been developed and widely used in label‐free and fluorescent

70

analysis.31-33 These fluorescent dyes are initially non-emissive, and will be lighted-up 4 ACS Paragon Plus Environment

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when aggregation triggered by target molecule.31 AIE probe with positive charges was

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recently demonstrated to be able to interact strongly with DNA strands,34 and the

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fluorescence turn-on response accommodates it for label-free detection of DNA. In

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particular, we found that AIE dye was distinctly outperformed DNA intercalation dye

75

on detecting mixture containing single-strand (ss) and double-strand (ds) DNAs

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(Figure 2C and D).

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In order to construct label-free, cheap, convenient, and homogeneous AFB1 assays,

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herein, coupling with a finely tunable dual-terminal stemmed aptamer beacon (DS

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aptamer beacon), AIE dye was introduced to detect AFB1 contaminations. DS aptamer

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beacon, a terminal protected aptamer probe against exonuclease I (Exo. I), was

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designed to be able to specifically and efficiently identify target AFB1. Besides, instead

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of labelling the aptamer probes, the detection of AFB1 was indicated by the digestion

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of aptamer probe using AIE effects. Benefited from the specificity of aptamer, this

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assay could distinguish AFB1 with other mycotoxins and analogues. And only two

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unlabeled DNA sequences were required in this assay due to the utilization of AIE

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probe. All this detection process was conducted in one-test tube at constant

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temperature, eliminating complex separation process. This assay has been applied to

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detect AFB1 in complex peanut oil and bean sauce samples, and holds great promise

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for constructing a universal platform for on-site detection food contaminations.

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MATERIAL AND METHODS

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AFB1 detection procedures. Terminal-protected aptamer probe (DS aptamer

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beacon) was prepared in a volume of 16μL containing 2 μL aptamer (10 μM), 2 μL 5 ACS Paragon Plus Environment


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anti-aptamer (10 μM), 2 μL 10 × Exo. Ⅰ reaction buffer and 10 μL H2O. Then, the

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mixture was heated to 90 ℃ for 5 min and incubated at room temperature for 35 min. 2

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μL AFB1 solution was introduced into the above solution and incubated at 25 ℃ for 1

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h. Next, 1 μL Exo. Ⅰ (20 U/μL) was added to digest the DNA probe. The digest

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process was keeping at 37 ℃ for 1 h.

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Fluorescence and real-time fluorescence analysis. The above reaction mixture

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was mixed with 1 μL 9,10-distyrylanthracene (DSA) derivative with short alkyl chains

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(DSAI) solution (100 μM), 7 μL 10×PBS and 43 μL H2O. The fluorescent spectra were

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measured using F-7000 fluorophotometer (Hitachi, Japan). The excitation wavelength

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was 405 nm, with emission spectra recorded ranging from 425 nm to 650 nm. The

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real-time fluorescence analysis was carried on fluorescence microplate reader Synergy

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H1 (BioTek, USA). The excitation wavelength was 405 nm, and the emission

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wavelength was 405 nm.

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Detecting AFB1 in broad bean paste and peanut oil samples. 5 g of samples

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(broad bean paste or peanut oil) in 20 mL of methanol-water (70:30 v/v) was added to

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a 50-mL centrifuge tube. Extraction of the samples carried out by shaking for 60 min

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followed by 10 min centrifugation at 6000 rpm at room temperature. The supernatant

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was transferred to a 1.5-mL tube which subsequently used as analytical samples.

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RESULTS AND DISCUSSION

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Working principle of dual-terminal stemmed aptamer beacon. The key

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innovation is the design of dual-terminal stemmed aptamer beacon (DS aptamer

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beacon) (Scheme 1), which would specifically respond to target AFB1 due to the 6 ACS Paragon Plus Environment

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intrinsic advantages of aptamers on specificity and programmability. Besides, we

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found that AIE dyes would outperform the traditional DNA intercalation dyes on the

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displaying of the recognition process (Figure 2C and D). Specifically, DS aptamer

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beacon

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(aptamer:anti-ptamer hybridization), possessing two stems at 3’ and 5’ terminal, and

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two symmetrical loops in the middle region. Compared to traditional molecule beacon,

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the stability of DS aptamer beacon could be finely tuned by adjusting both two of stem

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sequences, allowing elaborately optimizing probe affinity and selectivity. As

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exonucleases, like Exo. I could only removal of nucleotides from 3’ terminal, DS

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aptamer beacon structure would initially confer the protection of aptamer probe to be

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digested by Exo. I. Target AFB1 would competitively bind to aptamer, leading to the

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structure-switching of double-strands aptamer beacons to be two single strands. Upon

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the disassembly of DS aptamer beacon, Exo. I would efficiently digest the

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single-strand aptamer and anti-aptamer. The recognition process could be monitored

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by nucleic acids dyes in a label-free manner. Herein, a kind of positively charged AIE

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dyes is adopted to display the presence of digestion products. The DSAI dye would

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only light up upon the binding to DNA strands by AIE effects, thus the presence of

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target AFB1 would lower down the fluorescence intensity. In this strategy, DS aptamer

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beacon would confer strikingly specificity for identifying target AFB1. With the

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adoption of AIE probe, a label-free, homogenous assay has been constructed, which

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could be applied to determinate AFB1 in complex samples like broad bean paste and

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peanut oils with simple pretreatment process.

is

designed

as

a

double-stranded

molecule

beacon

structure



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DNA presence independent AIE feature of DSAI. Firstly, the AIE dye was

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synthesized and its fluorescence features responding to DNA were investigated.

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Typical

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tetraphenylethene (TPE), are nonemissive in the dissolved states, while exert highly

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fluorescent emission when they are in the aggregate states.31 Herein, we synthesized a

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cationic DSA derivative (DSAI) as the AIE-active probe according to the published

143

work.35 The positively charged DSAI would confer high binding ability to negatively

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charged DNA, thus possessing the ability to the display both ssDNA and dsDNA

145

sequences. 1H NMR spectra and mass spectra were used to confirm the structure of

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obtained DSAI (Figure 1A and B, the synthesis procedures are detailed in the

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supporting information). Then, the AIE effects of DSAI were tested. The fluorescence

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intensity of DSAI would sharply increase in the presence of ssDNA sequences (Figure

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1C). And DSAI would display a large stokes shift of 130 nm (maximum excitation

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wavelength is 405 nm with a maximum emission wavelength 535 nm). The

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fluorescence imaging further confirmed the efficient light-up effects of DSAI upon the

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addition of DNA sequences (Figure 1D). The DNA presence independent AIE effects

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of DSAI provide the possibility of its utilization for constructing label-free aptasensor.

AIE-active

fluorophores,

such

as

9,10-distyrylanthracene

(DSA),

154

The recognition process using DS aptamer beacon. The successive validation of

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recognition process of DS aptamer beacon was carried by electrophoresis analysis and

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fluorescence analysis. The binding of target molecules would result in the formation of

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G-quadruplex of aptamer,13,36 thus leading to the increase of fluorescence intensity of

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aptamers using DNA intercalation dyes (Gelred dye). Thus, the enhancement of 8 ACS Paragon Plus Environment

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fluorescence intensity of aptamer with the addition of AFB1 indicates the success of

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binding of AFB1 to aptamers (line 1 and line 2 Figure 2A). Besides, the electrophoresis

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image clearly presents the disassembly of DS aptamer beacon to be single-strand upon

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addition of target AFB1 (line 4 and line 5 Figure 2A). In turn, Exo. I would digest most

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of ssDNA strands (line 6 and line 7 Figure 2A). Thus, the presence of AFB1 would

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lead to less digested DNA remained. The formation of partial secondary structure of

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aptamer or anti-aptamer may result in the remaining of slight DNA after digestion.

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To further confirm recognition mechanism, we constructed DS aptamer beacons

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with different lengths of stem (10 nt-12 nt, 10 nt-13 nt, 11 nt-13 nt, 11 nt-14 nt, 12

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nt-14 nt). As shown Figure 2B, higher contents of DS aptamer beacon were remained

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upon adding AFB1 with the increase of the stem length (line 6-10), which resulted

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from the enhanced stability of DS aptamer beacons. DS aptamer beacon with a longer

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stem would be more stable, thus conferring increased resistance to the binding process

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of AFB1 to aptamers. Further, the different binding efficiency of AFB1 towards

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aptamer would significantly influence the digestion process (line 16-20). Therefore,

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reducing of the stability of DS aptamer beacon would increase the possibility of AFB1

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binding. However, less stable DS aptamer beacons (especially DS aptamer beacons

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with a stem length of 10 nt-12 nt) could not completely form double-strand structure

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(line 1-5), which may result in higher background.

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The AIE dye DSAI was further adopted to detect the digested DNA products. As

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shown Figure 2C, the formation of double-strand structure of DS aptamer beacon

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would resist to the digestion of Exo. I, as only a slight reduction of fluorescence upon 9 ACS Paragon Plus Environment


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addition of Exo. I. While the addition of target AFB1 would significantly lower down

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the fluorescence of DNA products, indicating the success of binding of AFB1. The

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remaining fluorescence may be from aptamer with regions of secondary structure

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which could not be digested by Exo. I. Besides, the involvement of digestion process

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using Exo. I was demonstrated to be able to greatly improve the fluorescence response

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of DS aptamer beacon towards AFB1 (Figure S1). In particular, we found that AIE dye

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DSAI was outperformed DNA intercalation dye on detecting the digestion process

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(Figure 2D). This may be resulted from the different light-up mechanism of these dyes.

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DNA intercalation dye can only bind to dsDNA sequence, while DSAI would bind

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both ssDNA and dsDNA via electrostatic adsorption. As DS aptamer beacon was with

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the structure of loop (single strand) and stem (double-strand), DSAI would present

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higher efficient response to the concentration of DS aptamer beacon. Therefore, AIE

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dyes may be more competitive candidate for constructing label-free aptasensor.

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Fine tunability of DS aptamer beacon. The stability of DS aptamer beacon was

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indicated to significantly affect the recognition process of AFB1. The increased

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stability of DS aptamer would cut down the background, while compromising the

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recognition efficiency of DS aptamer beacon. Compared to conventional molecule

198

beacon, the stability of DS aptamer beacon could be finely tuned by adjusting both two

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of stems. The ‘granularity’ of melt temperature Tm of DS aptamer beacons ranges in

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0.7-3.4 ℃ when tuning the length of stem length (Figure 3A, Table S2, seven DS

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aptamer beacon with different stem length, 10 nt-12 nt, 10 nt-13 nt, 11 nt-13 nt, 11

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nt-14 nt, 12 nt-14 nt were constructed, Table S1, Figure S2), while the ‘granularity’ for 10 ACS Paragon Plus Environment

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conventional molecule beacon is over 10 ℃ (17.8 and 11.5 ℃) (Table S3).37 Even

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with relatively low tuned stability, the increase of stem length would obviously

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increase both background (without the addition of AFB1) to signal (with the addition of

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AFB1) (Figure 3A). Especially, when the DS aptamer beacons were with Tm over

207

60 ℃, the binding of AFB1 was significantly hurdled, as there was a sharp increase of

208

signals upon adding AFB1. The optimized DS aptamer beacon structure would be with

209

a stem length of 11 nt-13 nt based on the ratio of background to signal (Figure S3).

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Further, the effects of ratio of anti-aptamer to aptamer strands were investigated. The

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highest fluorescence ratio of DS aptamer beacon was obtained with the ratio of

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anti-aptamer to aptamer 1:1 (Figure 3B, Figure S4). Higher concentration of

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anti-aptamer would reinforce block effect from the anti-aptamer, thus resulted in low

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background (high fluorescence intensity without the addition of AFB1), whereas

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lowering down recognition efficiency.

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Quantification performance. After optimizing the sequence of DS aptamer beacon

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and digestion time (Figure S5), the quantification performance using DS aptamer

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beacon was validated by using a series of AFB1 solutions with different concentrations.

219

As shown in Figure 4A, the fluorescence intensity of digested DNA products by

220

adding DSAI dye steadily increased with the concentration of AFB1 from 10 ng/mL to

221

1700 ng/mL, suggesting that DS aptamer beacons design for AIE dye readout is highly

222

dependent on the concentration of target AFB1. The remaining fluorescence upon

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adding high concentration of AFB1 may result from the formation of partial secondary

224

structure of aptamer which could not be digested by Exo. I. Figure 4B displayed a 11 ACS Paragon Plus Environment


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calibration curve by plotting the signal increase versus the AFB1 concentration. The

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linear regression equation was calculated as A= -2.97B+3354.56 with a correlation

227

coefficient of 0.990, where A and B represented the fluorescence intensity and AFB1

228

concentration, respectively. LOD was defined as the concentration corresponding to

229

the fluorescence signal at three times standard deviation of blank without AFB1, and

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the LOD of the assay is 27.3 ng/mL. The assay using DNA-intercalation dye EvaGreen

231

conferred a LOD of 90.9 ng/mL with a correlation coefficient of 0.875 (Figure S6).

232

The adoption of AIE dye would greatly facilitate the improvement of sensitivity of DS

233

aptamer beacon for AFB1 detection. All the assay was carried in homogeneous solution

234

in one-test tube. Specifically, only two strand DNA without modification was used for

235

AFB1 detection, the cost for probe was estimated to be less than 1 cent (calculation is

236

based on the price provided by Integrated DNA Technologies, Inc.).

237

Specificity test. The specificity was examined by detecting fluorescence signal

238

changes of AFB1 and its analogues or concomitant components in different

239

concentrations (Figure 5A). As AFM1 is metabolite of AFB1, there is only an OH

240

group difference between these two molecules. And AFB2 is different from AFB1 by

241

only one bond (Figure 5B). Nevertheless, no component would lead to a nonnegligible

242

signal change besides AFB1, and all these interference components outputted signal

243

number the same as that of blank control in all these three concentrations (1000, 1500

244

and 2000 nM). The remarkable selectivity was contributed from the specificity of

245

aptamer. Besides, to investigate the effects of positive charged components in the

246

substrate on the homogeneous assay, the fluorescence response of DS aptamer beacon 12 ACS Paragon Plus Environment

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towards arginine and histidine was further tested. The addition of these positively

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charged components with concentrations ranging from 1000 to 2000 nM conferred no

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obvious effect on the signals of DS aptamer beacon using AIE dyes. Therefore, DS

250

aptamer beacons could strictly identify AFB1, potentially accommodating it for the

251

application in detecting AFB1 in real sample which may contain complex composition.

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Detecting AFB1 in broad bean paste and peanut oil samples. Finally, to assess

253

the feasibility of detecting AFB1 in complex matrixes such as fluid sample or liquid

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sample, we applied DS aptamer beacons to detect spiked AFB1 in broad bean paste and

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peanut oils. Broad bean paste, mainly made from broad bean and soybean, is a

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traditional condiment which is popular in Sichuan province (China). And peanut oils

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are very susceptible to be contaminated by AFB1. For targets with a concentration

258

falling in the detection dynamic range of the assay, the recovery ratios were fall in

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92.75% to 118.70% (Figure 6, Table S4) for detection in both broad bean paste and

260

peanut oils. All these samples were pretreated by simple extraction and centrifugation.

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Compared to other methods, DS aptamer beacon could achieve homogeneous detection

262

of AFB1 with label-free DNA probes in one-test tube (Table 1). Thus, this DS aptamer

263

beacon-based assay holds great potential for on-site detection of AFB1 both benefiting

264

from its low cost and simple pretreatment process.

265

In summary, a finely tunable aptamer beacon coupling with AIE dye has been

266

engineered to construct a label-free, one-tube and homogeneous AFB1 assay, and its

267

application for AFB1 detection in complex samples, peanut oil and bean sauce has been

268

demonstrated. This assay presents novel features: 1) Quick and specific recognition. 13 ACS Paragon Plus Environment


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the aptamer beacon could confer quick response to target AFB1 with high specificity; 2)

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Label-free. the introducing of AIE eliminates complex and costly labelling process of

271

DNA probe, thus only cheap DNA strands are needed; 3) One-tube and isothermal test.

272

All detection process is conducted in one test-tube at a constant temperature,

273

eliminating the separation process, thus well fit for on-site AFB1 detection; 4) Potential

274

universal platform. the simplicity of design for other food contaminations such as

275

ochratoxin A, oxytetracycline and staphylococcus aureus, using different aptamers.

276

The successful applications of this assays for determining AFB1 peanut oil and bean

277

sauce indicate its robustness in quantification performance in real samples. Therefore,

278

the assay offers broad prospects for on-site assessment of various food contaminations

279

such as mycotoxin, antibiotic residues and pathogenic bacteria, thus facilitating the

280

insurance of food safety in the production chain.

281

Funding

282

This work was supported by National Natural Science Foundation of China (No.

283

21804095,

No.

51773129),

China

Postdoctoral

Science

Foundation

(No.

284

2018M631079) and the Fundamental Research Funds for the Central Universities (No.

285

2018SCU12048, No. 1083304121001).

286

Supporting Information

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The Supporting Information is available free of charge on the ACS

288

Publications website. Supplementary methods, the effects of digestion process on

289

AFB1 detection, oligonucleotide sequences, the secondary structures of DS aptamer 14 ACS Paragon Plus Environment

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beacon, the tuning of stability of DS aptamer beacon, the tuning of stability of

291

conventional aptamer beacon, optimization of the length of anti-aptamer, optimization

292

of ratios of anti-aptamer to aptamer, optimization of digestion times, Quantification of

293

AFB1 using EvaGreen, determination of AFB1 spiked in the broad bean paste and

294

peanut oils.

295

Notes

296

The authors declare no conflict of interest.

297

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receptors. Nat. Chem. 2014, 6, 1003-1008.

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photoelectrochemical determination of tetracycline using a spindle-like ZnO-CdS@Au

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Separation-Based Multiple SELEX for Effectively Selecting Aptamers against

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Saxitoxin, Domoic Acid, and Tetrodotoxin. J. Agric. Food Chem. 2018, 66,

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Page 22 of 40

431

FIGURE CAPTIONS

432

Figure 1. Characterization of AIE dye DSAI. (A) 1H NMR spectra of DSAI; (B) Mass

433

spectra of DSAI; (C) Fluorescence spectra of DSAI with or without additions of DNA

434

sequence; (D) Fluorescence images of samples in (C).

435

Figure 2. Demonstration of the recognition process using DS aptamer beacon. (A)

436

Electrophoresis analysis of the recognition of AFB1 by DS aptamer beacon; (B)

437

Electrophoresis analysis of DS aptamer beacons with increased stem length (10 nt-12

438

nt, 10 nt-13 nt, 11 nt-13 nt, 11 nt-14 nt, 12 nt-14 nt) (line 1-5), in the presence of target

439

AFB1 (lanes 6-10), with digestion process (lanes 11-15), with both target AFB1

440

presence and digestion process (lanes 16-20); (C) Fluorescence analysis of the

441

response DS aptamer beacon to target AFB1 using AIE dye DASI; (D) Fluorescence

442

analysis of the response DS aptamer beacon to target AFB1 using DNA-intercalation

443

dye EvaGreen.

444

Figure 3. Investigation of the effects of stability of DS aptamer beacon on AFB1

445

detection. (A) The fluorescence intensity of DS aptamer beacon with stem lengths (9

446

nt-11 nt, 9 nt-12 nt, 10 nt-12 nt, 10 nt-13 nt, 11 nt-13 nt, 11 nt-14 nt, 12 nt-14 nt) in the

447

presence of DSAI with or without the addition of AFB1; (B) The fluorescence intensity

448

of DS aptamer beacon with different ratios of anti-aptamer to aptamer in the presence

449

of DSAI with or without the addition of AFB1.

450

Figure 4. Quantification of AFB1 using DS aptamer beacon. (A) Typical fluorescence

451

spectra of the sensing system upon addition of different concentrations of AFB1 (0, 10

452

,40 ,100, 200, 300, 500, 800, 1200, 1700 ng/mL); (B) The relationship between target

453

concentration and fluorescence response. Inner: The linear relationship between AFB1 22 ACS Paragon Plus Environment

Page 23 of 40


454

concentration and fluorescence response. The error bars indicate the standard deviation

455

of three parallel measurements for each concentration of target AFB1.

456

Figure 5. Specificity of AFB1 detection using DS aptamer beacon. (A) The

457

fluorescence intensity changes with additions of AFB1 and analogues or concomitant

458

components (AFB2, AFM1, OTA, ZEA, Tyr, Leu, Arg and His); (B) Structures of

459

components used in (A).

460

Figure 6. Recovery of AFB1 in the broad bean paste and peanut oils.



461

Page 24 of 40

Table 1. Comparison of analytical performance of aptamer-based AFB1 assay Methods

Label -free

Homogeneous detection

One-tube detection

Assay temperature

LOD (ng/mL)

Dynamic range (ng/mL)

Samples

Ref

DS aptamer beacon; AIE

Yes

Yes

Yes

Room temperature and 37 ℃

27.3

40-300

Broad bean paste; peanut oil

This work

FRET-based aptasensor

No

Yes

Yes

Room temperature

1.6

5-100

Infant rice cereal

38

No

Yes

Yes

Room temperature

1.06

3.12-1247

Rice; peanut

39

Room temperature

31.2

62.4-6240

Corn; peanut

40

0.11

0.1-10

Corn

41

15

5-50

Corn

42

2.5×10-5

5×10-5-5

Rye hay; rice cereal

43

3×10-4

0.001-0.05

Wheat

44

FRET-based aptasensor using aptamer-conjugated QDs Aptamer-cross-linked hydrogel-based aptasensor Chemiluminescence competitive aptasensor Nuclease cleavage amplified aptasensor

No

No

Yes

Yes

No

No

No

Yes

Yes

PCR-based aptasensor

Yes

Yes

No

DNA/silver nanoculsters-based aptasensor

Yes

No

No

4 ℃, 30 ℃ and 37 ℃ Room temperature and 4 ℃ 45 ℃, 60 ℃ and 95 ℃ Room temperature and 45 ℃


Page 25 of 40

463


SCHEME

464 465

Scheme 1. Schematic illustration of the design of dual-terminal stemmed aptamer beacon (DS

466

aptamer beacon) and its application for label-free detection of AFB1 via aggregation-induced

467

emission.



Page 26 of 40

468 469

Figure 1. Characterization of AIE dye DSAI. (A) 1H NMR spectra of DSAI; (B) Mass spectra of

470

DSAI; (C) Fluorescence spectra of DSAI with or without additions of DNA sequence; (D)

471

Fluorescence images of samples in (C).


Page 27 of 40


472 473

Figure 2. Demonstration of the recognition process using DS aptamer beacon. (A) Electrophoresis

474

analysis of the recognition of AFB1 by DS aptamer beacon; (B) Electrophoresis analysis of DS

475

aptamer beacons with increased stem length (10 nt-12 nt, 10 nt-13 nt, 11 nt-13 nt, 11 nt-14 nt, 12

476

nt-14 nt) (line 1-5), in the presence of target AFB1 (lanes 6-10), with digestion process (lanes

477

11-15), with both target AFB1 presence and digestion process (lanes 16-20); (C) Fluorescence

478

analysis of the response DS aptamer beacon to target AFB1 using AIE dye DASI; (D) Fluorescence

479

analysis of the response DS aptamer beacon to target AFB1 using DNA-intercalation dye

480

EvaGreen.



Page 28 of 40

481 482

Figure 3. Investigation of the effects of stability of DS aptamer beacon on AFB1 detection. (A) The

483

fluorescence intensity of DS aptamer beacon with stem lengths (9 nt-11 nt, 9 nt-12 nt, 10 nt-12 nt,

484

10 nt-13 nt, 11 nt-13 nt, 11 nt-14 nt, 12 nt-14 nt) in the presence of DSAI with or without the

485

addition of AFB1; (B) The fluorescence intensity of DS aptamer beacon with different ratios of

486

anti-aptamer to aptamer in the presence of DSAI with or without the addition of AFB1.


Page 29 of 40


487 488

Figure 4. Quantification of AFB1 using DS aptamer beacon. (A) Typical fluorescence spectra of

489

the sensing system upon addition of different concentrations of AFB1 (0, 10 ,40 ,100, 200, 300,

490

500, 800, 1200, 1700 ng/mL); (B) The relationship between target concentration and fluorescence

491

response. Inner: The linear relationship between AFB1 concentration and fluorescence response.

492

The error bars indicate the standard deviation of three parallel measurements for each concentration

493

of target AFB1.



Page 30 of 40

494 495

Figure 5. Specificity of AFB1 detection using DS aptamer beacon. (A) The fluorescence intensity

496

changes with additions of AFB1 and analogues or concomitant components (AFB2, AFM1, OTA,

497

ZEA, Tyr, Leu, Arg and His); (B) Structures of components used in (A).


Page 31 of 40


498 499

Figure 6. Recovery of AFB1 in the broad bean paste and peanut oils.



500

Page 32 of 40

TABLE OF CONTENTS

501


Page 33 of 40


Scheme 1. Schematic illustration of the design of dual-terminal stemmed aptamer beacon (DS aptamer beacon) and its application for label-free detection of AFB1 via aggregation-induced emission. 119x53mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 1. Characterization of AIE dye DSAI. (A) 1H NMR spectra of DSAI; (B) Mass spectra of DSAI; (C) Fluorescence spectra of DSAI with or without additions of DNA sequence; (D) Fluorescence images of samples in (C). 119x93mm (300 x 300 DPI)


Page 34 of 40

Page 35 of 40


Figure 2. Demonstration of the recognition process using DS aptamer beacon. (A) Electrophoresis analysis of the recognition of AFB1 by DS aptamer beacon; (B) Electrophoresis analysis of DS aptamer beacons with increased stem length (10 nt-12 nt, 10 nt-13 nt, 11 nt-13 nt, 11 nt-14 nt, 12 nt-14 nt) (line 1-5), in the presence of target AFB1 (lanes 6-10), with digestion process (lanes 11-15), with both target AFB1 presence and digestion process (lanes 16-20); (C) Fluorescence analysis of the response DS aptamer beacon to target AFB1 using AIE dye DASI; (D) Fluorescence analysis of the response DS aptamer beacon to target AFB1 using DNA-intercalation dye EvaGreen. 160x88mm (300 x 300 DPI)



Figure 3. Investigation of the effects of stability of DS aptamer beacon on AFB1 detection. (A) The fluorescence intensity of DS aptamer beacon with stem lengths (9 nt-11 nt, 9 nt-12 nt, 10 nt-12 nt, 10 nt13 nt, 11 nt-13 nt, 11 nt-14 nt, 12 nt-14 nt) in the presence of DSAI with or without the addition of AFB1; (B) The fluorescence intensity of DS aptamer beacon with different ratios of anti-aptamer to aptamer in the presence of DSAI with or without the addition of AFB1. 70x108mm (300 x 300 DPI)


Page 36 of 40

Page 37 of 40


Figure 4. Quantification of AFB1 using DS aptamer beacon. (A) Typical fluorescence spectra of the sensing system upon addition of different concentrations of AFB1 (0, 10 ,40 ,100, 200, 300, 500, 800, 1200, 1700 ng/mL); (B) The relationship between target concentration and fluorescence response. Inner: The linear relationship between AFB1 concentration and fluorescence response. The error bars indicate the standard deviation of three parallel measurements for each concentration of target AFB1. 70x114mm (300 x 300 DPI)



Figure 5. Specificity of AFB1 detection using DS aptamer beacon. (A) The fluorescence intensity changes with additions of AFB1 and analogues or concomitant components (AFB2, AFM1, OTA, ZEA, Tyr, Leu, Arg and His); (B) Structures of components used in (A). 75x92mm (300 x 300 DPI)


Page 38 of 40

Page 39 of 40


Figure 6. Recovery of AFB1 in the broad bean paste and peanut oils. 70x57mm (300 x 300 DPI)



TABLE OF CONTENTS 84x47mm (300 x 300 DPI)


Page 40 of 40

Dual-Terminal Stemmed Aptamer Beacon for Label-Free Detection of

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