Binding-Induced DNA Dissociation Assay for Small Molecules

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Binding-Induced DNA Dissociation Assay for Small Molecules: Sensing Aflatoxin B1 Lin Xu, Hongquan Zhang, Xiaowen Yan, Hanyong Peng, Zhixin Wang, Qi Zhang, Peiwu Li, Zhaowei Zhang, and X. Chris Le ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00975 • Publication Date (Web): 15 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018

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Binding-Induced DNA Dissociation Assay for Small Molecules: Sensing Aflatoxin B1

Lin Xu1,2, Hongquan Zhang2, Xiaowen Yan2, Hanyong Peng2, Zhixin Wang2, Qi Zhang1, Peiwu Li,1*, Zhaowei Zhang1*, X. Chris Le2* 1

Key Laboratory of Biology and Genetic Improvement of Oil Crops, Key Laboratory of

Detection for Mycotoxins, Laboratory of Risk Assessment for Oilseeds Products (Wuhan), Ministry of Agriculture, and National Reference Laboratory for Biotoxin Test, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, P.R. China 2

Division of Analytical and Environmental Toxicology, Department of Laboratory

Medicine and Pathology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G3

* Corresponding author: [email protected] [email protected] [email protected]

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ABSTRACT: We describe a new fluorescence turn-on sensor for homogeneous detection of aflatoxin B1 (AFB1), a potent low molecular weight mycotoxin. A key innovation is the bindinginduced intramolecular interaction involving the following two sets of probes: (1) a gold nanoparticle (AuNP) immobilized with hundreds of assistant oligonucleotides (AO) and dozens of anti-AFB1 monoclonal antibodies (mAb), and (2) the AFB1-BSA antigen conjugated with fluorophore-labeled signal oligonucleotides (SO) that contained a short sequence complementary to AO. Specific binding of AFB1-BSA to the antibody brought the fluorophore very close to the surface of the AuNP through a stable intramolecular hybridization between AO and SO, resulting in efficient quenching of fluorescence. The improved fluorescence quenching substantially reduced the background, due to the binding-induced intramolecular hybridization,

and improved the signal-to-background

ratio by 390%. In the presence of AFB1 in a sample, competitive binding of AFB1 in the sample to the antibodies immobilized on the AuNP caused the release of the fluorophorelabeled AFB1-BSA from the AuNP, turning on fluorescence. A detection limit of 2.3 nM was achieved, which meets the requirement for AFB1 detection at regulatory levels. Analyses of rice samples using this assay showed recoveries of 86-102%. Incorporating appropriate antibody probes could extend the assay to the detection of other small molecules.

KEYWORDS: binding-induced DNA interaction; aflatoxin B1; homogeneous immunoassay; fluorescence quenching; DNA nanotechnology

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Aflatoxin B1 (AFB1), mainly produced by Aspergillus flavus and A. parasitic, is one of the most common and toxic food-contaminating mycotoxins.1-3 The International Agency for Research on Cancer (IARC) has classified AFB1 as a Group 1 human carcinogen.4 The food producers spend between $500 million and $1.5 billion per year for the management of mycotoxins.5 AFB1 is resistant to thermal and chemical treatment, and it is ubiquitous in agricultural production and food processing,6-7 albeit its concentrations vary. Many countries have set maximum tolerable levels of AFB1, or total aflatoxins (aflatoxin B1, B2, G1, and G2), in food and feed. These guideline values are very low, generally vary from 2 to 20 ng/g. The presence of trace concentrations of toxic AFB1 in various foods and animal feeds continues to challenge development of ultrasensitive assays. High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS)8-9 and immunoassays10-13 have been commonly used for the detection of AFB1. The HPLC-MS/MS methods require instrumentation that may not be available for on-site analysis. Most immunoassays for AFB1, such as enzyme-linked immunosorbent assay (ELISA), microchip, and lateral flow strip, require either extensive washing steps or complicated fabrication process.14-19 We aim to develop a simple homogeneous assay that could be potentially adapted for on-site analysis. While we chose AFB1 as the target molecule of interest, our overall objective of this research was to develop a generalizable strategy that will enable detection of small molecules. Our rationale for choosing a homogeneous assay strategy is to establish a mixand-read format that eliminates the need for separation or washing steps. The homogeneous mix-and-read assays will potentially be useful for on-site analysis. Due to the extensive use of DNA-based sensing in the toxin analysis20, we describe here the development of a binding-induced DNA dissociation technique, enabling homogenous detection of small molecules. This technique incorporates the principle of binding-induced DNA assembly (BINDA) with properties of fluorescence quenching by gold nanoparticles (AuNPs). BINDA is particularly useful for the detection of nucleic acids and proteins.21-24 Like proximity ligation assays (PLA),25-26 BINDA requires the binding of two affinity ligands (e.g. antibodies) to the same target molecule. Thus, proteins are particularly suitable for analysis by PLA and BINDA, because the large size of protein 3


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molecules allows for the binding of two antibodies. However, the size of small molecules is insufficient for simultaneous binding of two antibodies to a single small molecule. Therefore, a new strategy must be developed to assay for small molecules, such as AFB1. This paper illustrates the design principle of binding-induced DNA dissociation in response to competitive binding. It demonstrates an application of the assay to the detection of AFB1.

■ EXPERIMENTAL SECTION Materials and Reagents. All DNA oligonucleotides were synthesized, labeled, and purified by Integrated DNA Technologies (IDT, Coralville, IA). The DNA sequences and modifications are listed in Table S1 and Figure S1 of the Supporting Information (SI). AuNPs of 20 nm were purchased from TED PELLA (Redding, CA). Bovine serum albumin (BSA), chemical standard of AFB1, and AFB1-BSA antigen were obtained from SigmaAldrich (Oakville, ON).

Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), sodium

chloride (NaCl), magnesium chloride hexahydrate (MgCl2·6H2O), potassium carbonate (K2CO3), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHA-NHS), 5(6)carboxyfluorescein-N-hydroxysuccinimide ester (fluorophore-NHS), 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), and corning 96 well plates were bought from Sigma-Aldrich (Oakville, ON). Phosphate buffered saline (PBS), Tween-20, Coomassie plus (Bradford) assay reagent, and Millipore Amicon Ultra-0.5 Centrifugal Filter were acquired from Fisher Scientific (Nepean, ON). Micro Bio-Spin™ P-6 Gel Columns (BioRad, Hercules, CA) were used for the purification of conjugates. Water from an Ultrapure Milli-Q water system was autoclaved. All other reagents were of analytical grade. AntiAFB1 monoclonal antibodies (mAb) were produced as described previously.27 Rice powder was bought from a local market in Edmonton, Canada. Sorvall legend micro 21R micro-centrifuge and Multi-mode microplate reader Filter Max F5 were purchased from Thermo Fisher Scientific (Nepean, ON). Functionalization of AuNPs with monoclonal antibody (mAb) and assistant oligonucleotide (AO, Figure S1). Commercial AuNPs with an average diameter of 20 nm were functionalized with anti-AFB1 mAb. The generation of AuNPs-mAb conjugates was 4


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performed as follows. AuNPs solution (1mL, 1.16 nM) was adjusted to an optimal pH value (pH=8.8) with 20 mM K2CO3 (10 μL), and then mixed with 20 μL anti-AFB1 mAb (1 mg/mL). The mixed solution was mildly stirred for 30 min at room temperature, and the mAb-modified AuNPs were further functionalized using the following procedures. 5.8 μL of 100 μM AO was added to the above modified AuNPs solution. The mixture was incubated for 10 min, and it was then mixed with 100 μL of 20% (v/v) PEG 20000 and incubated for additional 30 min. Then, 100 μL of 5 M NaCl was added and the mixture was incubated at room temperature in the dark for 3 h. The mixture was centrifuged at 13,500 rpm for 15 min at 4ºC. The supernatant was removed and the AuNPs were collected. The AuNPs was washed with 0.05% Tween-20/PBS solution and centrifuged at 13,500 rpm for 15 min at 4 ºC, to remove the excess AO and antibodies. Finally, the functionalized AuNPs were dissolved in 500 μL of filtered PBS buffer containing 0.1% (w/v) BSA and stored at 4 ºC. Preparation of the AFB1-labeled DNA probe (Figure S2). AFB1-labeled DNA probe was prepared using a two-step process. The first step was to conjugate the AFB1-BSA antigen with the linker oligonucleotide (LO) to generate the AFB1-BSA-LO. Briefly, 20 μL of 30 μM AFB1-BSA solution was incubated with 6 μL of 10 mM MHA-NHS overnight at 4 ºC or for 2 h at room temperature. The excess unreacted MHA-NHS was removed using a 30-kDa ultra-0.5 Amicon filter. In another tube, 7.2 μL of 500 μM LO was incubated with 30 μL of 50 mM freshly prepared TCEP for 0.5 h at room temperature. After purification using a spin-6 column, the LO was added to the above active AFB1-BSA modified with MHA-NHS to generate the AFB1-BSA-LO conjugate. The second step of the process involved hybridization of the AFB1-BSA-LO probe with the signal oligonucleotide (SO). Briefly, 6 μL of 100 μM SO was added to the above AFB1-BSA-LO solution. Hybridization between the complementary domains of LO (C2) and SO (C2*) produced the fluorescent AFB1-labeled DNA probe (Figure S2). The excess SO was removed by filtrating using an ultra-0.5 Amicon filter. Fluorescence detection. Fluorescence measurements were performed on a multimode 5


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microplate reader (Thermo Fisher Scientific, Nepean, ON) using a corning 96-well plate. Excitation was 485 nm and fluorescence was measured at 535 nm. In a typical reaction assay, the mixture contained optimal volume of AFB1-labeled DNA probe and functionalized AuNPs, detection buffer and standard AFB1 solution or sample extraction. Optimization of the length of C1* in SO. Three SOs designs (SO6, SO7 and SO8) with varying length of complementary C1* (6, 7 and 8 n.t.) were prepared (Table S1) for the analysis of 20 nM AFB1. Each SO (1.2 nM) was added to 3 nM functionalized AuNPs to test whether there was any the DNA self-assembly. 1.2 nM of AFB1-labeled DNA probes formed with varying SO (SO6, SO7 or SO8) were mixed with 3 nM functionalized AuNPs in 0.05% Tween-20/PBS solution (pH=7.4). Either 20 nM AFB1 standard or buffer only (blank) was added to the mixture. After 1 h incubation at room temperature, 95 μL of the resulting solution was placed in 96-well plate, and fluorescence was measured using the microplate reader. Optimization of the density of antibody on AuNPs. AuNPs were functionalized with varying antibody loading amount to test the optimum antibody density. Briefly, 20 nm AuNPs (1.16 nM, 1 mL) was incubated with a fixed amount of AO (100 μM, 6 μL) and varying amounts of anti-AFB1 mAb (1 mg/mL, 20 μL and 2 μL). After conjugation, the antibody loading amount of varying modified AuNPs was detected using the Coomassie blue method. 3 nM functionalized AuNPs were mixed with 1.2 nM of AFB1-labeled DNA probe in 0.05% Tween-20/PBS solution. Either 20 nM AFB1 standard or buffer (blank) was added. After 1 h incubation at room temperature, 95 μL of the resulting solution was transferred to a 96-well plate, and fluorescence at 535 nm was measured using the microplate reader. Optimization of the ratio of antibody to fluorophore-labeled DNA probe. The antibody to fluorophore-labeled DNA probe ratio was optimized by analyzing 20 nM AFB1 and blank. AuNPs functionalized with different antibody loading amount (Table S2) were used in the analysis of 20 nM AFB1 or blank. 0.05% Tween-20/PBS solution was added to 1.2 6


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nM AFB1-labeled DNA probe and varying antibody concentrations (1.2, 2.4, 3, 3.6 and 4.8 nM) of the functionalized AuNPs. After 1 h incubation at room temperature, 95 μL of the resulting solution was placed in the microplate reader for the fluorescence measurements. Detection of AFB1 in buffer solution and in rice samples spiked with AFB1. A series of concentrations of AFB1 standard (0, 5, 10, 20, 30, 40, and 50 nM) were prepared in 0.05% tween-20/PBS buffer. Each standard solution was mixed with 1.2 nM of AFB1-labeled DNA probe and 3 nM of functionalized AuNPs. After 1 h incubation, 95 μL resulting solution was placed in the fluorescence microplate reader and fluorescence was detected. Rice powder samples were prepared as reported before with minor modification28. Briefly, rice powder samples (5 g each) were spiked with a series of concentrations of AFB1 standard (5, 10, 20, 30, 40, and 50 nM). Each of these samples was mixed with 10 mL of 70% (v/v) methanol/water and vigorously stirred for 5 min. An aliquot of 1 mL supernatant was diluted in 4 mL of 0.05% tween-20/PBS buffer. Each diluted sample was mixed with 1.2 nM AFB1-labeled DNA probe and 3 nM functionalized AuNPs. The resulting solution was placed in the microplate reader and fluorescence was measured. The concentration of AFB1 in the rice sample extract was determined against the calibration of the AFB1 standard. The recoveries of AFB1 from the three spiked samples were calculated by comparing the measured and the added amounts of AFB1.

■ RESULTS AND DISCUSSION Working principle of the assay. The assay involves binding of two components: a functionalized AuNP and a AFB1-labeled DNA probe (Figure 1). The functionalized AuNPs are conjugated with hundreds of assistant oligonucleotides (AO) and dozens of anti-AFB1 mAb to a single AuNP. Each AO contains a short (8 nucleotides) complementary domain C1 (Figure S1). The AFB1-labeled DNA probe is generated through a two-step process. A linker oligonucleotide (LO) is first conjugated to the AFB1BSA antigen where BSA provides reactive groups for LO conjugation. A longer 7


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complementary domain C2 (13 n.t.) of LO, is used for hybridization with a fluorophorelabeled signal oligonucleotide (SO). The SO comprises three functional domains: a C2* domain complementary to C2, a poly thymine (poly T) spacer, and a C1* domain complementary to C1. Because multiple LO strands can be conjugated to a single BSA molecule, mixing the AFB1-BSA-LO conjugate with SO at specific molar ratios allows a desirable number of the SO probe to be attached to each AFB1 molecule through hybridization between C2 and C2*. The use of LO offers three benefits, allowing examination of different SO constructs without the need of repeating the entire conjugation process, generating AFB1-BSA-LO-SO assembly with a precise molar ratio, which is challenging through direct conjugation of SO to the AFB1-BSA antigen, and extending to other small molecules by only altering the corresponding target-BSA. In the absence of target molecules (Figure 1, top right), specific binding of the AFB1 antigen in the DNA probe to the antibody on the AuNPs surface places C1* in close proximity to hundreds of AO on the AuNP surface, dramatically increasing the local effective concentrations of C1* and C1. As a consequence, the intramolecular interaction between C1* and C1 forms a stable C1*:C1 hybridization. The hybridization between C1* and C1 brings the fluorescent molecule very close to AuNPs, leading to efficient fluorescence quenching. Thus, the fluorescence is off in the absence of target molecules. In the presence of target molecules (Figure 1b), target AFB1 molecules compete with AFB1 of the DNA probe in binding to antibody on the AuNP, resulting in dissociation of AFB1 of the DNA probe from antibody. When AFB1 is dissociated from the antibody on the AuNP, the interaction between C1* and C1 becomes inter-molecular. The intermolecular hybridization between the short (≤8 n.t.) complementary sequences of C1* and C1 (Tm≤10 oC) is unstable and is dissociated at the temperature of the assay (25 oC). The dissociation of C1* from C1 further results in release of the DNA probe from the AuNP, generating a turn-on fluorescence signal which is then used for quantification of AFB1. The assay holds several appealing features that are beneficial to homogeneous and sensitive detection of small molecules. First, the traditional competitive assay format (Figure S3) cannot bring the fluorophore very close to the AuNP (Figure S4a), thus suffering from high background. Our new assay overcomes this problem (Figure S4b). 8


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Secondly, each AuNP is conjugated with dozens of antibody molecules, achieving the benefit of a multivalent antibody, and improving the affinity for binding to target molecules. Thirdly, the assay is conducted in a single tube without the need for separation or washing. In principle, alternations of antibody and antigen can expand the assay to detection of other small molecules, without the need for redesigning the probes (Figure S1 and S2). Background reduction and efficient fluorescence quenching. A key feature of our assay design is the reduced background as compared to the conventional competitive assay, because the use of binding-induced DNA hybridization allows the fluorescent molecule in the DNA probe to be placed very close to AuNP, leading to a high fluorescence quenching efficiency. Figure S4 schematically shows the distance between the fluorophore (F) and the AuNP quencher, comparing the conventional assays (Figure S4a) with the bindinginduced DNA hybridization assay (Figure S4b). In the conventional assays, the distance between the fluorophore and AuNP could be more than 18 nm because of the antibody and BSA present between the fluorophore and AuNP (Figure S4a). This relatively large distance between the fluorophore and the AuNP quencher hampered the fluorescence quenching efficiency, resulting in higher background. However, with the binding-induced DNA hybridization (Figure S4b), the distance between the fluorophore and the AuNP quencher was substantially reduced to less than 1.6 nm, corresponding to the length of 5 thymine nucleotides (approximately 0.33 nm per nucleotide). The shortened distance between the fluorophore and the AuNP quencher enhanced the fluorescence quenching efficiency, resulting in lower background. We have estimated apparent fluorescence quenching efficiency by determining the fluorescence intensities of the two fluorescence probes before and after they were bound with the AnNP quencher. When 1.2 nM of the conventional AFB1-BSA fluorescent probe (Figure S3) was mixed with 3 nM of the antibody-functionalized AuNP (Figure S4a), fluorescence intensity was approximately 51% of that from the initial 1.2 nM of the conventional AFB1-BSA fluorescent probe. These results corresponded to an overall apparent quenching efficiency of 49% (Figure 2). When the same concentration (1.2 nM) of our AFB1-BSA-DNA fluorescent probe (Figure 1) was mixed with 3 nM of the 9


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antibody-functionalized AuNP (Figure S4b), fluorescence intensity was approximately 13% of that from the initial 1.2 nM of the AFB1-BSA-DNA fluorescent probe. Thus the apparent overall quenching efficiency of our system was 87% (Figure 2). This measure took into account the overall process of affinity binding and fluorescence quenching. When we removed the unbound AFB1-BSA-DNA fluorescent probe from the mixture by ultracentrifugation and immediately measured the fluorescence of the complex, the fluorescence intensity was only 2% of the initial intensity of the unbound probe. These results suggest that the actual fluorescence quenching efficiency of our new system was 98% (Figure 2). A consequence of the improved fluorescence quenching efficiency was the substantially lower background. As compared to the conventional assay, our new assay improved the signal-to-background ratio by 390%, as indicated by the relative slopes of the two regression lines (Figure 3). Optimization of the key parameters involved in the assay. To demonstrate that the hybridization between C1 and C1* can be induced upon binding of the fluorophore-labeled DNA probe to antibody on the AuNPs and that such hybridization can be disassembled upon competitive binding of target molecules to the antibody, we measured fluorescence of three solutions, representing the un-assembled (free) probe, quenching after assembling of the probe, and recovery of fluorescence due to competitive binding (Figure 4). When there is no binding between the AuNP and the SO, the free SO fluoresces as expected (Figure 4, left bar). The substantial decrease in fluorescence (Figure 4, central bar) is due to binding of antibody to the AFB1 antigen and the binding-induced DNA assembly (BINDA) that brings the fluorophore to the functionalized AuNP. These results are consistent with the principle of our assay. These results also indicate that the hybridization between C1 and C1* is dependent on the binding of the fluorophore-labeled DNA probe to the antibody on the AuNP, and that competitive binding of target molecules to the antibody can disassemble hybridization between C1 and C1*. The length of C1* in SO plays a vital role in reducing background and improving the sensitivity because the length of C1* impacts its hybridization with C1 upon binding of the 10


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DNA probe to antibody on the AuNPs and its disassociation from C1 upon competitive binding of target molecules. We designed three SO probes, SO6, SO7 and SO8, containing 6, 7, or 8 n.t. for C1*, respectively. We then compared both background and signal of three analyses using the DNA probes prepared by three SOs (Figure 5). The background (B) denotes the fluorescence of blank; and the sample (S) is the fluorescence intensity upon the addition of 20 nM AFB1. The background (Figure 5a) decreases along with increase of the length of C1*, consistent with the expectation that the longer C1* enhances bindinginduced hybridization between C1* and C1. By comparison, the difference between signal and background is comparable from the use of SO6 and SO7, but it is about 2 times higher than that of SO8, suggesting that the 8-n.t. C1* domain is too long to efficiently dissociate from C1 upon the competitive target binding. The normalized fluorescence (Figure 5b), (SB)/B, shows that the use of SO7 resulted in the optimum signal-to-background ratio. These results indicate effective binding-induced intramolecular hybridization of the 7-n.t. complementary region between C1* and C1, as well as efficient dissociation of C1* from C1 upon competitive target binding. Multiple mAb molecules can be conjugated onto a single 20-nm AuNP. Such mAbcoated AuNP contains multiple target binding sites, similar to binding property of a multivalent antibody. The apparent affinity (avidity) of the multivalent antibody is equal to Kn, where n is the number of the combining sites, and K is the intrinsic association constant of the individual mAb.29 Therefore, it is anticipated that increase of the conjugation number of mAb to a single AuNP can improve avidity of individual AuNPs.30 We prepared two types of functionalized AuNPs conjugated respectively with 6 and 34 mAb molecules per AuNP (Table S2). We then compared background and signal of two analyses using these two types of AuNPs. The concentration of AuNPs was tailored to ensure that each analysis solution contained the same amount of mAb. The AuNPs conjugated with 34 mAb per AuNP resulted in a much lower background than AuNPs conjugated with 6 mAb per AuNP (Figure S5a), proving that the increase of the conjugation number of mAb per AuNP improves avidity of individual AuNPs. Because two analyses generated comparable signal difference (S-B), a larger normalized fluorescence was obtained for the analysis using AuNPs conjugated with the larger number of mAb (Figure 11


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S5b). The molar ratio of the functionalized AuNPs to the AFB1-labeled DNA probe has an impact on the background and sensitivity of the assay. Increasing this ratio represents more antibody molecules available for binding to antigen (AFB1) molecules in the DNA probe, thereby depleting the unbound DNA probe in the blank solution and reducing the background. On the other hand, a too high ratio results in the excess of antibody molecules that bind to target molecules without signal generation, decreasing detection sensitivity. We increased the ratio of the antibody to the DNA probe from 1 to 4 and tested background and signal of analyses using these ratios. As expected, increasing this ratio led to lower both background and signal (Figure S6a). The largest normalized fluorescence was obtained when the antibody-to-DNA probe ratio was 2.5 (Figure S6b). We also optimized other parameters, such as the order in which the three reagents were mixed: the functionalized AuNPs, the AFB1-labeled fluorescent DNA probe, and the target AFB1 molecules. We observed that direct mixing of the two probes with the sample was simple and effective (Figure S7). Determination of AFB1 in buffer and rice samples. To test the applicability of the assay, we determined the concentrations of AFB1 in buffer solutions and rice sample extracts. A linear dynamic range was observed from 5 to 50 nM AFB1 in buffer solutions (Figure S8), which focused on the determination of trace concentrations of AFB1. This dynamic range can be expanded to higher concentrations of AFB1, by proportionally increasing the concentrations of the functionalized AuNPs and the AFB1-labeled fluorescent DNA probe. A limit of detection of 2.3 nM was achieved, which is lower than most guideline values (maximum tolerable levels) of AFB1 in food and feed. We further applied the assay to the analysis of rice samples spiked with AFB1. Recoveries ranging from 86% to 102% were obtained (Table S3). These results suggest that the assay is applicable to the detection of AFB1 in real agriculture products. The assay is sensitive because of the low background and the use of a turn-on fluorescence signal for detection.

■ CONCLUSIONS 12


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We have developed a fluorescence turn-on assay, enabling sensitive and homogeneous detection of AFB1 without the need for separation or washing. Compared to other available assays for AFB1, this new assay has several appealing features. The binding-induced DNA assembly design brings the fluorophore closer to AuNPs, increasing fluorescence quenching efficiency and reducing background. The presence of dozens of antibody molecules on an AuNP exhibits a similar binding property of a multivalent antibody, improving the affinity for binding to the target molecules. The assay uses a mix-and-read format and fluorescence turn-on signal for simple detection. The assay can be readily adapted for the detection of other small molecules by altering appropriate antibodies.

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■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.??. Details of oligonucleotide sequences, gold nanoparticle functionalization, experimental optimization, and additional information shown in three tables and eight figures (PDF)

■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] ORCID X. Chris Le: 0000-0002-7690-6701 Notes The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS We thank the National Key R&D Program of China (2016YFE0119900), National Program for Support of Top-notch Young Professionals, the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Canada Research Chairs Program, Alberta Innovates, Alberta Health, and the China Scholarship Council for their support.

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■ REFERENCES (1) Marin, S.; Ramos, A.; Cano-Sancho, G.; Sanchis, V. Mycotoxins: occurrence, toxicology, and exposure assessment. Food Chem. Toxicol. 2013, 60, 218-237. (2) Stoev, S. D. Food safety and increasing hazard of mycotoxin occurrence in foods and feeds. Crit. Rev. Food Sci. 2013, 53 (9), 887-901. (3) Lee, H.J.; Ryu, D. Worldwide occurrence of mucotoxins in cereals and ceral-derived food products: public health perspectives of their co-occurrence. J. Agri. Food Chem. 2017, 65 (33), 7034-7051. (4) IARC (International Agency for Research on Cancer). Overall evaluations of carcinogenicity: an updating of IARC monographs, vol. 1 to 42. IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans: Suppl 7. IARC 1987, 7, 1-440. (5) Robens, J.; Cardwell, K. The Costs of Mycotoxin Management to the USA: Management of Aflatoxins in the United States. J. Toxicol. Toxin Rev. 2003, 22 (2-3), 139-152. (6) Kabak, B.; Dobson, A. D.; Var, I. Strategies to prevent mycotoxin contamination of food and animal feed: a review. Crit. Rev. Food Sci. 2006, 46 (8), 593-619. (7) Sharma, K. K.; Pothana, A.; Prasad, K.; Shah, D.; Kaur, J.; Bhatnagar, D.; Chen, Z.-Y.; Raruang, Y.; Cary, J. W.; Rajasekaran, K.; Sudini, H. K.; Bhatnagar-Mathur, P. Peanuts that keep aflatoxin at bay: a threshold that matters. Plant Biotechnol. J. 2018, 16 (5), 1024-1033. (8) Lai, X.-W.; Sun, D.-L.; Ruan, C.-Q.; Zhang, H.; Liu, C.-L. Rapid analysis of aflatoxins B-1, B-2, and ochratoxin A in rice samples using dispersive liquid-liquid microextraction combined with HPLC. J. Sep. Sci. 2014, 37 (1-2), 92-98. (9) Wei, R.; Qiu, F.; Kong, W.; Wei, J.; Yang, M.; Luo, Z.; Qin, J.; Ma, X. Co-occurrence of aflatoxin B-1, B-2, G(1), G(2) and ochrotoxin A in Glycyrrhiza uralensis analyzed by HPLC-MS/MS. Food Control 2013, 32 (1), 216-221. (10) Eiyazzadeh-Keihan, R.; Pashazadeh, P.; Hejazi, M.; de la Guardia, M.; Mokhtarzadeh, A. Recent advances in nanomaterial-mediated bio and immune sensors for detection of aflatoxin in food products. Trends Anal. Chem. 2017, 87, 112-128. (11) Lin, Y.X.; Zhou, Q.; Tang, D.P.; Niessner, R.; Knopp, D. Signal-on photoelectrochemical 15


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immunoassay for aflatoxin B-1 based on enzymatic product-ething MnO2 nanosheet for dissociation of carbon dots. Anal. Chem. 2017, 89 (10), 5637-5645. (12) Wang, X.; Niessner, R.; Tang, D.; Knopp, D. Nanoparticle-based immunosensors and immunoassays for aflatoxins. Anal. Chim. Acta 2016, 912, 10-23. (13) Sapsford, K. E.; Taitt, C. R.; Fertig, S.; Moore, M. H.; Lassman, M. E.; Maragos, C. A.; ShriverLake, L. C. Indirect competitive immunoassay for detection of aflatoxin B-1 in corn and nut products using the array biosensor. Biosens. Bioelectron. 2006, 21 (12), 2298-2305. (14) Li, D.; Ying, Y.; Wu, J.; Niessner, R.; Knopp, D. Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection. Microchim. Acta 2013, 180 (7-8), 711-717. (15) Li, X.; Li, P.; Zhang, Q.; Li, R.; Zhang, W.; Zhang, Z.; Ding, X.; Tang, X. Multi-component immunochromatographic assay for simultaneous detection of aflatoxin B 1, ochratoxin A and zearalenone in agro-food. Biosens. Bioelectron. 2013, 49 (22), 426-432. (16) Song, S.Q.; Liu, N.; Zhao, Z.Y.; Ediage, E.N.; Wu, S.L.; Sun, C.P.; De Saeger, S.; Wu, A.B. Multiplex lateral flow immunoassay for mycotoxin determination. Anal. Chem. 2014, 86 (10), 4995-5001. (17) Matabara, E.; Ishimwe, N.; Uwimbabazi, E.; Lee, H.H. Current immunoassay methods for the rapid detection of aflatoxin in milk and dairy products. Compreh. Rev. Food Sci. Food Safe. 2017, 16 (5), 808-820. (18) Xiong, Y.; Pei, K.; Wu, Y.; Xiong, Y. Colorimetric ELISA based on glucose oxidase-regulated the color of acid–base indicator for sensitive detection of aflatoxin B 1 in corn samples. Food Control 2017, 78, 317-323. (19) Guo, L.; Feng, J.; Fang, Z.; Xu, J.; Lu, X. Application of microfluidic "lab-on-a-chip" for the detection of mycotoxins in foods. Trends Food Sci. Technol. 2015, 46 (2), 252-263. (20) Esteban-Fernández de Ávila, B.; Lopez-Ramirez, M. A.; Báez, D. F.; Jodra, A.; Singh, V. V.; Kaufmann, K.; Wang, J. Aptamer-modified graphene-based catalytic micromotors: Off–on fluorescent detection of ricin. ACS Sens. 2016, 1 (3), 217-221. (21) Zhang, H.; Li, X. F.; Le, X. C. Binding-induced DNA assembly and its application to yoctomole detection of proteins. Anal. Chem. 2012, 84 (2), 877-884. 16


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(22) Li, J.; Zhong, X.; Zhang, H.; Le, X. C.; Zhu, J.-J. Binding-induced fluorescence turn-on assay using aptamer-functionalized silver nanocluster DNA probes. Anal. Chem. 2012, 84 (12), 51705174. (23) Zhang, H.; Li, F.; Dever, B.; Li, X. F.; Le, X. C. DNA-mediated homogeneous binding assays for nucleic acids and proteins. Chem. Rev. 2013, 113 (4), 2812-41. (24) Yan, X.; Le, X.C.; Zhang, H. Antibody-bridged beacon for homogeneous detection of small molecules. Anal. Chem. 2018, 90 (16), 9667–9672. (25) Cheng, S.; Shi, F.; Jiang, X.; Wang, L.; Chen, W.; Zhu, C. Sensitive detection of small molecules by competitive immunomagnetic-proximity ligation assay. Anal. Chem. 2012, 84 (5), 2129-32. (26) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gustafsdottir, S. M.; Ostman, A.; Landegren, U. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 2002, 20 (5), 473-477. (27) Zhang, D.; Li, P.; Yang, Y.; Zhang, Q.; Zhang, W.; Xiao, Z.; Ding, X. A high selective immunochromatographic assay for rapid detection of aflatoxin B₁. Talanta 2011, 85 (1), 736-742. (28) Zhang, D.; Li, P.; Zhang, Q.; Li, R.; Zhang, W.; Ding, X.; Li, C. M. A naked-eye based strategy for semiquantitative immunochromatographic assay. Anal. Chim. Acta 2012, 740, 74-9. (29) Chen, Y.; Chen, Q.; Han, M.; Zhou, J.; Gong, L.; Niu, Y.; Zhang, Y.; He, L.; Zhang, L. Development and optimization of a multiplex lateral flow immunoassay for the simultaneous determination of three mycotoxins in corn, rice and peanut. Food Chem. 2016, 213, 478-484. (30) Tobita, T.; Oda, M.; Azuma, T. Segmental flexibility and avidity of IgM in the interaction of polyvalent antigens. Mol. Immunol. 2004, 40 (11), 803-811.

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Figures

Figure 1 Schematic showing the principle of binding-induced fluorescence turn-on assay for a small molecule, aflatoxin B1. (a) The gold nanoparticle (AuNP) was functionalized with monoclonal antibody and assistant oligonucleotide (AO). The AFB1-labeled fluorescent DNA probe was formed by hybridizing the signal oligonucleotide (SO) with the linker oligonucleotide (LO). Specific binding of AFB1-BSA to the antibody brought the fluorophore very close to the surface of the AuNP through a stable intramolecular hybridization between AO and SO, resulting in efficient quenching of fluorescence. (b) Competitive binding of AFB1 in the sample to the antibody immobilized on the AuNP caused the release of the fluorophore-labeled AFB1-BSA from the AuNP, turning on fluorescence.

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Figure 2 Comparison of apparent overall fluorescence quenching efficiency obtained from the conventional assay and the new assay. The conventional assay used 1.2 nM of the AFB1-BSA fluorescent probe and 3 nM of the antibody-functionalized AuNPs (Figure S4a). The new assay used 1.2 nM of the AFB1-BSA-DNA fluorescent probe and 3 nM of antibody-functionalized AuNPs (Figure S4b). The apparent overall fluorescence quenching efficiency was estimated as (F0-B)/F0 X 100%, where Fo was the fluorescence intensity of the fluorescent probe alone and B was the fluorescence intensity from the reaction mixture of fluorescent probe and the functionalized AuNPs.

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Figure 3 Comparison of signal-to-background ratio (S/B) obtained from the conventional assay and the new assay for the detection of AFB1. The conventional assay used 1.2 nM of the AFB1-BSA fluorescent probe and 3 nM of the antibody-functionalized AuNPs (Figure S4a). The new assay used 1.2 nM of the AFB1-BSA-DNA fluorescent probe and 3 nM of antibody-functionalized AuNPs (Figure S4b).

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Figure 4 Results showing the relative fluorescence intensity from the unbound probe, formation of the binding-induced assembly (BINDA), and competitive binding to the target molecule AFB1. (1) No binding; the solution contained 3 nM functionalized AuNPs and 1.2 nM signal oligonucleotide (SO). (2) Formation of BINDA; resulting in fluorescence quenching. The solution contained 3 nM functionalized AuNPs and 1.2 nM AFB1-labeled fluorescent DNA probe. (3) Competitive binding of AFB1; resulting in release of the AFB1-labeled fluorescent DNA probe from the AuNP, restoring its fluorescence. The solution contained 3 nM functionalized AuNPs, 1.2 nM AFB1-labeled fluorescent DNA probe, and 20 nM AFB1. The signal in (3) did not resume to the full intensity as in (1) because the low concentration of AFB1 could not completely displace off all the bound fluorescent probes.

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Figure 5 Optimization of the length of C1* in signal oligonucleotide (SO). (a) Fluorescence intensity of the blank (B) and sample (S). (b) Normalized fluorescence represents the ratio of (S-B)/B. The blank solution contained 3 nM functionalized AuNPs and 1.2 nM AFB1-labeled fluorescent DNA probe (schematic in Figure S2). The sample solution contained 3 nM functionalized AuNPs, 1.2 nM AFB1-labeled fluorescent DNA probe, and 20 nM AFB1. 22


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For TOC only

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Binding-Induced DNA Dissociation Assay for Small Molecules

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