Single-Particle LRET Aptasensor for the Sensitive Detection of

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Single-Particle LRET Aptasensor for the Sensitive Detection of Aflatoxin B1 with Upconversion Nanoparticles Fuyan Wang, Yameng Han, Shumin Wang, Zhongju Ye, Lin Wei, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02599 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Analytical Chemistry

Single-Particle LRET Aptasensor for the Sensitive Detection of Aflatoxin B1 with Upconversion Nanoparticles Fuyan Wang,† Yameng Han,‡ Shumin Wang,‡ Zhongju Ye,‡ Lin Wei,*,† and Lehui Xiao*,‡

†Key

Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of

Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China. ‡State

Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and

Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071, China. *Corresponding Author: [email protected] and [email protected]

Abstract Contamination of foods and feeds by aflatoxins is a universal yet serious problem all over the world. Particularly, aflatoxin B1 (AFB1) is the most primary form and readily leads to terrible damages to human health. In this work, we construct a sensitive aptasensor based on single-particle detection (SPD) to analyze AFB1 in peanut samples with luminescence resonance energy

transfer

(LRET)

between

the

aptamer

modified

upconversion

nanoparticles

(UCNPs-aptamer) and gold nanoparticles (GNPs). The UCNP-aptamer plays as the luminescence donor, while GNP acts as the energy acceptor. In the absence of AFB1, GNPs would adsorb onto the surface of UCNPs-aptamer because of the association between aptamers and GNPs, leading to luminescence quenching. However, the luminescence of UCNPs-aptamer is recovered gradually in the presence of AFB1 because the aptamers possess stronger affinity toward AFB1 than GNPs.

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Through statistically counting the number of luminescent particles on the glass slide surface, the concentration of AFB1 in solution is accurately determined. The linear dynamic range for AFB1 detection is from 3.13 to 125.00 ng/mL. The limit-of-detection (LOD) is 0.17 ng/mL, which is much lower than the allowable concentration in foods. As a result, this method would provide promising application for the sensitive detection of AFB1 in foods and feeds, which might make a meaningful contribution to food safety and public health in the future.


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Introduction Aflatoxins are important and toxic mycotoxins, which are produced by Aspergillus flavus and Aspergillus parasiticus during the process of grain growth and storage.1 Generally, the four common forms of aflatoxins are known as aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2).2 Among all of the aflatoxins, AFB1 is identified as one of the most potent toxic form due to its immunosuppressive, teratogenic, carcinogenic, and mutagenic effects, which can increase the incidence of liver cancer for human.3 Thus, AFB1 is classified as group I carcinogen by the International Agency for Research on Cancer (IARC, 2002).4 In addition, the proliferation of AFB1 in grains and grain derived products may lead to terrible diseases such as acute cirrhosis, cell necrosis, and carcinoma.5 Especially, the AFB1 can be delivered to the human body through the food chain. Therefore, the maximum tolerant limit of the AFB1 in crop products is controlled between 0.05 and 20 ng/mL in many countries.6 The U.S. Food and Drug Administration (FDA) established that the standard of AFB1 in poultry feeds must be less than 300 ng/mL.7 Based on these health issues, it is a critical requirement for the development of sensitive detection method for AFB1 assay in human foods and animal feeds. Until now, a series of interesting methods based on aptamer-target interaction for the assay of AFB1 have been reported, for example, fluorescence,8 electrochemistry,9 liquid-liquid microextraction,10 surface-enhanced Raman scattering (SERS)1 and colorimetry.11 Although satisfactory results have been obtained from these methods for AFB1 detection, some challenges still exist, including tedious experimental procedure, poor signal-to-noise ratios, lower sensitivity, and abundant sample consumption, which greatly limit their potential practical applications. Therefore, considerable efforts have been devoted to design sensitive and facile approaches to



monitor AFB1 in foods and feeds. In recent years, single-particle detection (SPD), as a robust analytical strategy, has attracted a wide range of attention due to the capability of detecting small molecules and biomarkers at single-particle (or -molecule) level.12-19 Unlike the bulk sample measurement, SPD method collects optical signals generated from individual particles (or molecules), which can avoid the ensemble averaging effect from the tested samples and greatly improve the sensitivity and accuracy of the determined results. So far, many fluorescent nanoparticles have been exploited as efficient nanoprobes to detect target objects with SPD, including quantum dots and fluorescent polymer dots.20-22 However, the unstable optical properties (photo-bleaching and photo-blinking) of these fluorescent nanoparticles would influence the accuracy and specificity of the experimental results. Besides, the auto-fluorescence of biological samples might lead to the lower sensitivity and poor signal-to-noise ratio. Because of these shortages, it is of great important to develop new probes for SPD assay. Recently, lanthanide-doped UCNPs have attracted considerable attention due to their unique chemophysical merits, such as low toxicity, minimal photobleaching, high chemical stability and sharp emission band.23-25 Especially, the UCNPs display exclusive anti-Stokes emission characteristic, which can effectively avoid background interferences from biological samples and get rid of possible false positive signal output.26-28 Therefore, UCNPs are desirable luminescent nanomaterials for single-particle imaging in complex biological samples. In this study, we designed a UCNPs-based LRET aptasensor for the quantitative detection of AFB1 in peanut samples with SPD. The principle of the detection is illustrated in Scheme 1. This aptasensor consists of aptamers modified UCNPs and GNPs, where UCNP-aptamer serves as the luminescence donor and GNP acts as the acceptor. In brief, the mechanism of LRET is an energy


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transfer phenomenon between donors and acceptors. Only when the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor and the distance between each other is close enough, the luminescence of the donors could be effectively quenched.23 The aptamer is a short-sequence single-stranded oligonucleotide obtained by in vitro screening, which has specific recognition ability and high affinity with the corresponding target.29 Owing to its cost-effectiveness, facile structure-controlled synthesis, and high stability in non-physiological environment storage, aptamer has attracted widespread attentions.30-31 It can be labeled with enzymes, fluorescent dyes, and nanostructures to detect different targets. Normally, the aptamer always maintains a random coil structure in solution and the nucleic acids are exposed to the outside. In the absence of AFB1, UCNPs-aptamer would interact with GNPs because of the formation of coordination bonds between nitrogen and gold atoms,32 leading to the quenched luminescence from UCNPs-aptamer. On the contrary, in the presence of AFB1, UCNPs-aptamer could specifically bind with AFB1, resulting in the dissociation of GNPs from UCNPs-aptamer. A dosage-dependent fluorescence recovery process can be established. Therefore, by quantifying the number of fluorescent particles on the glass slide surface with SPD method, a linear range from 3.13 to 125.00 ng/mL for AFB1 assay is achieved with the LOD of 0.17 ng/mL in solution. Because of the high specificity of the aptasensor, the interference signals from other aflatoxins such as AFB2, AFG1, and AFG2 are negligible. As a consequence, we believe that the designed aptasensor based on SPD will create a unique platform for the precise and ultrasensitive assay of biomolecules in diverse areas in the future.

Experimental Section



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Reagents and Instruments Sodium citrate, ammonium fluoride (NH4F), Tris-HCl, ethanol (CH3CH2OH), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), sodium hydroxide (NaOH), sodium dihydrogen phosphate (NaH2PO4), and cyclohexane were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Yttrium(III) chloride hexahydrate (YCl3·6H2O), ytterbium(III)

chloride

hexahydrate

(YbCl3·6H2O),

erbium(III)

chloride

hexahydrate

(ErCl3·6H2O), methanol (CH3OH), chloroauric acid (HAuCl4·3H2O), 1-octadecene (1-ODE), poly(acrylic acid) (PAA, MW: 6000), bovine serum albumin (BSA), streptavidin (SA), oleic acid (OA),

N-hydroxysuccinimide

(NHS),

3-aminopropyltriethoxysilane

(APTES),

and

1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride (EDC) were purchased from Aladdin (Shanghai, China). Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were obtained from Guangzhou Analysis Center Keli Technology (Guangzhou, China). Biotin-modified AFB1 aptamer oligonucleotide (5′-biotin-AAAAAAAAAA GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3′) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Peanut samples were obtained from local grain market (Tianjin, China). All other reagents were commercially available as analytical purity. The ultrapure water was used throughout the experiments. The luminescence spectra of UCNPs were measured by a fluorescence spectrometer (F-4500, Japan) that was equipped with a 980 nm cw laser. The UV-vis absorption spectrum of GNPs was studied using a spectrophotometer (UV-2450, Japan). Dynamic light scattering (DLS) experiments of UCNPs and GNPs were determined by a Zetasizer Nano ZS system (Malvern, U.K.). The size and morphology characterizations were recorded on a transmission electron microscope (TEM,


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JEM2100, JEOL, Japan). Fourier transform infrared (FT-IR) spectra of the OA modified UCNPs (UCNPs-OA) and PAA modified UCNPs (UCNPs-PAA) were collected with a Spectrum One (B) spectrometer (AVATAR-370, U.S.A.). In this experiment, we utilized a home-built upright optical microscope (Olympus, Japan) for the single-particle imaging study. The 980 nm cw laser beam from a fiber coupled laser was expanded with a collimating lens and projected to the back port of the filter cube. Then the light was reflected by a short-pass dichroic mirror and focused at the back focal plane of the objective. The emission signal of the UCNPs was filtered by a band-pass filter and collected by the 40× objective (NA = 0.65). The fluorescent images of the samples were captured with sCMOS camera (Orcaflash 4.0, Hamamastu, Japan) for the SPD measurements. The pixel size of the sCMOS camera is 6.5 µm × 6.5 µm. The dark-field imaging experiments were carried out on an upright dark-field optical microscope (Ni-U, Nikon, Japan). All experiments were repeated three times. All of the images were analyzed with ImageJ (http://rsbweb.nih.gov/ij/). Preparation of UCNPs-PAA Oil phase UCNPs-OA were prepared according to the previously reported methods.33 Briefly, YCl3·6H2O (0.78 mM), YbCl3·6H2O (0.20 mM) and ErCl3·6H2O (0.02 mM) were dissolved in a three-neck flask containing 8 mL of OA and 15 mL of 1-ODE with continuously stirring. The mixture was heated to 160 °C to turn into a transparent solution under atmosphere of N2 and stirred for 30 min, followed by cooling to room temperature. Then, 10 mL of methanol solution containing NaOH (0.25 M) and NH4F (0.40 M) was injected to the flask and the mixture was stirred for 30 min. Subsequently, the reaction system was heated to 100 °C to remove excessive methanol. After that, the mixture was continuously heated to 300 °C and maintained for 1 h. After



cooling to room temperature, the obtained UCNPs-OA were precipitated and washed several times with ethanol and cyclohexane, and dried at 50 °C for 12 h. Next, water phase UCNPs-PAA were prepared by the ligand exchange method.34 Typically, 10 mg of UCNPs-OA was incubated in a mixed solution containing 10 mL of cyclohexane and 5 mL of ethanol. In addition, 20 mg of PAA was dissolved in 10 mL of ultrapure water and heated at 50 °C with vigorous stirring for 30 min. Subsequently, the UCNPs-OA mixture was added dropwise into the above PAA solution with continuously stirring for 4 h. The UCNPs-PAA were purified and washed with ethanol twice by centrifugation at 9000 rpm for 15 min. After that, the product was re-suspended in 10 mL ultrapure water and stored at 4 °C before use. Synthesis of GNPs The preparation of the uniform GNPs was carried out by the seed-mediated growth method.35 In brief, the synthesis procedure was divided into two steps including the seed preparation and particle growth. For seed (with diameter ~18 nm) preparation, 1.03 mL of HAuCl4·3H2O (24.28 mM) was incubated with 98.97 mL of ultrapure water at 110 °C for 10 min under vigorous stirring. And then 10 mL of sodium citrate (14.55 mM) was injected into the above solution and the mixture reacted at 110 °C for 20 min until cooling to room temperature. To obtain larger GNPs, 30 mL of sodium citrate solution (13 mM) was added into the prepared seed solution and the mixture was heated to 120 °C for 10 min. Subsequently, 20 mL of HAuCl4·3H2O (2.35 mM) was spiked into the above mixture gradually and the solution was heated for another 10 min, followed by cooling to room temperature. Finally, the synthesized GNPs (75 pM) were collected in a glass bottle and stored at 4 °C before use. Detection of AFB1 at the Single-Particle Level


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In order to detect AFB1, the aptasensor was constructed firstly. Briefly, 14 µL of SA (1.73 µM) and 10 µL of aptamers (10 µM) were incubated in 26 µL of filtered Tris-buffered saline (20 mM, pH 7.5) under gently shaking for 3 h. For the conjugation, excessive EDC/NHS (1 mg/mL) in a ratio of 1:3 was used to activate the UCNPs-PAA under room temperature for 30 min. Then, the above modification solution was added to 20 µL of UCNPs-PAA solution (2 mg/mL) and incubated for 3 h at room temperature with slowly shaking. Subsequently, 2 µL of ethanolamine solution (2 mM) was added and further incubated for 1 h to block nonspecific site. Eventually, the UCNPs-aptamer were collected and purified by centrifugation at 7000 rpm for 10 min and re-suspended in phosphate buffer (PB, 10 mM, pH 7.4). The final concentration of UCNPs-aptamer was 0.5 mg/mL. For AFB1 assay, 10 µL of concentrated GNPs (20×, 1.5 nM) was mixed with 5 µL of UCNPs-aptamer (0.5 mg/mL) with mildly shaking for 30 min. After that, different concentrations of AFB1 (e.g., with a final concentration of 0, 3.13, 9.38, 46.88, 78.13, 93.80, 125.00, and 156.25 ng/mL, respectively) were spiked into the mixture solution and incubated for 30 min at room temperature with shaking slowly. After that, the samples were measured by SPD method with the home-built UCNPs imaging system. For the in situ dark-field imaging experiments, 20 µL of GNPs (7.5 pM) were added into the flow channel (modified with amino groups). After 5 min, the channel was rinsed 3 times with purified water to remove unadsorbed GNPs in the solution. GNPs exhibit bright green color in the dark-field image. Subsequently, 15 µL of BSA solution (5 wt. %) was added into the flow channel and incubated for 3 h to block nonspecific binding sites. Next, 20 µL of UCNPs-aptamer (0.025 mg/mL) were added into the prepared channel and incubated with GNPs for 40 minutes. To



monitor the dissociation process, 15 µL of AFB1 (125.00 ng/mL) solution were injected into the channel and incubated for 2 h. Detection of AFB1 in Peanut Samples To demonstrate the potential application of the method, recovery assay was applied to detect AFB1 in peanut samples. The peanut samples were pretreated based on the reported method.5 Firstly, 10 g of fresh peanut samples were crushed by a laboratory mill. Peanut powder was dissolved in 30 mL of extraction solvent (methanol: water = 6:4 (v/v)) and stirred vigorously for 20 min. After that, the supernatant was obtained by centrifugation (3500 rpm, 10 min) and further purified with a 0.22 μm syringe filter. The extract was diluted 5 times with ultrapure water. Then, different concentrations of AFB1 (with a final concentration at 0, 12.52, 31.30, and 62.60 ng/mL) were added into the tested samples, respectively. The detection processes are the same as that in PB except that the AFB1 is diluted by the peanut samples.

Results and Discussion Characterization and Fabrication of Aptasensor for AFB1 Assay In this work, ~50 nm GNPs are chosen as the luminescence acceptors due to their surface stability, biosafety and particularly the large absorption cross-section.36 The GNPs were prepared according to the seed-mediated growth method.35 The size and morphology of GNPs are characterized by TEM in Figure 1a. Evidently, the particles show uniform spherical morphology and good dispersibility with a narrow size distribution (50.35 ± 1.26 nm), which is further confirmed by DLS characterization (with peak value at 51.25 nm) as shown in Figure 1b. The UCNPs-PAA were synthesized through ligand exchange method. Firstly, UCNPs-OA was


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obtained via the thermolysis strategy. Because of the presence of OA ligands on the surface of UCNPs-OA, the UCNPs-OA are hydrophobic which is unfavourable for surface modification. Therefore, ligand exchange procedure is required for the phase conversion. PAA is a polymer with good biocompatibility and stability in water, which contains many carboxyl groups (-COOH) in the polymer chain. Therefore, the OA ligand on the UCNPs surface can be readily replaced by PAA. After the ligand exchange process, UCNPs-PAA show good hydrophilicity and can be further modified with biomolecules. The UCNPs-PAA in the TEM image exhibit good monodispersity with diameter of 43.56 ± 1.14 nm (Figure 1c). Additionally, the DLS measurement (50.19 nm) in Figure 1d also demonstrates that the particles are uniform in size distribution in water. The UCNPs-PAA in the fluorescent image displays good single-particle dispersibility (Figure 1e), confirming the potential capability for SPD with optical microscopy. Furthermore, the coating of PAA polymer on the surface of UCNPs is confirmed by the FT-IR spectroscopic measurements as shown in Figure 1f. Before the ligand exchange procedure, two distinct peaks are found at 2879 and 2960 cm-1, which are ascribed to the symmetric and asymmetric stretching vibrations of -CH2 from OA ligand, respectively. It is worth noting that an obvious peak appears at 1735 cm-1 in the FT-IR spectrogram after the PAA ligand exchange process. This characteristic peak (1735 cm-1) is attributed to the symmetrical stretching vibration of the -COOH from PAA ligand. The successful modification of PAA polymer on the UCNPs surface not only ensures the monodispersity of UCNPs in biological medium but also contributes a large number of -COOH for the subsequent conjugation process with aptamers. To modify aptamers onto the surface, streptavidin (SA) was cross-linked on the surface of UCNPs based on the EDC/NHS chemistry. The biotin-modified AFB1 aptamers can then assemble onto the UCNPs



surface through specific biotin-streptavidin (SA) interaction to form the sandwiched UCNP-SA-AFB1 aptamer nanostructure. As depicted in Figure 1g, the emission spectrum of UCNPs-PAA is largely overlapped with the absorption spectrum of GNPs. According to the LRET concept, the degree of LRET is closely related to the spatial distance (7-10 nm) of the donors and acceptors. The length of a 10 spacer adenine linker is around 3 nm.26 Therefore, the LRET between UCNPs-aptamer and GNPs can be performed efficiently, and the luminescent signal may be well quenched once the UCNPs-aptamer and GNPs associate together successfully. Feasibility of AFB1 Detection with SPD Method To demonstrate the feasibility of the concept for AFB1 detection at the single-particle level, 15 µL of UCNPs-aptamer (0.025 mg/mL) were firstly injected into the flow channel (modified with amino groups) and incubated for 30 minutes. The monodispersed UCNPs-aptamer are readily observed in the flow channel (Figure 2a) after being washed 3 times with pure water and twice with PB (10 mM, pH 7.4). To avoid nonspecific adsorption, 10 µL of BSA solution (5 wt. %) was injected into the flow channel and incubated for 3 h to block those nonspecific binding sites. Subsequently, this channel was cleaned 3 times with pure water. Then 15 µL of GNPs (7.5 pM) was added into the channel and incubated for 40 min. Next, unbound GNPs were washed 3 times with pure water. Some of the fluorescent spots disappear as shown in Figure 2b. Given 15 µL of AFB1 (125.00 ng/mL) solution was slowly added into the flow channel, many fluorescent spots recovered again (Figure 2c). These observations indicate that AFB1 can successfully bind with the aptamers on the UCNPs surface. The much stronger affinity guarantees the efficient dissociation of GNPs from the UCNPs surface, resulting in the recovery of fluorescent signal at the single-particle level.


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As an alternative evidence to directly clarify the recognition process, dark-field imaging experiments at the single-particle level were also performed. Since the localized surface plasmon resonance of single GNPs is sensitive to the dielectric change of the local environment, the color or scattering intensity of GNPs will be changed (red-shift) once the UCNPs-aptamer binding onto the surface of GNPs. Initially, the GNPs adsorbed on the glass slide surface show characteristic green color in the dark-field image (Figure 2d). After passing through UCNPs-aptamer in the flow channel, the scattering color from some of the GNPs was changed from green to yellow, as illustrated in Figure 2e. More interestingly, given AFB1 was added into the flow channel, the color of those GNPs was recovered again as shown in Figure 2f. These observations are in good consistent with the fluorescence results as noted above. In addition to the measurement at the single-particle level, the fluorescence spectroscopic measurements in bulk solution from the above three fluorescent measurement samples were also performed to confirm this scenario (Figure 2g). Meanwhile, the resulted morphologies of the nanostructures from the mixture solution of UCNPs-aptamer and GNPs without and in the presence of AFB1 were characterized by TEM images. As shown in Figure 2h, without AFB1, heterodimers were noted. In the presence of AFB1, the hybridized nanostructures were disassembled (Figure 2i). The optical stability of nanoparticles was an important parameter for developing SPD strategies. Interestingly, the UCNPs-PAA display more attractive optical features on the aspect of excellent photostability in comparing with other fluorescent probes (such as quantum dots and fluorescent polymer dots). The single-particle fluorescent images of the UCNPs-PAA and quantum dots are shown in Figure 3a and b. The fluorescence intensity of the UCNPs-PAA is



constant and no photobleaching occurs under a long observation time Figure 3c. However, the fluorescence intensity of quantum dots is unstable under the same imaging condition as shown in Figure 3d. Therefore, the UCNPs-PAA are particularly suitable for single-particle counting-based ultra-sensitive assay. Quantitative Detection of AFB1 Based on the SPD Method Because of the robust recognition capability of aptamer toward AFB1, an AFB1 concentration dependent fluorescent particle number change in the microscopic image can be readily obtained. On this basis, we studied the detection performance of the aptasensor for the quantification of AFB1 with SPD method. Representative AFB1 concentration dependent fluorescent images are shown in Figure 4a. With the increase of AFB1 concentration in the reaction samples from 0 to 156.25 ng/mL, the number of fluorescent particles on the coverslip surface gradually increased. To avoid the artificial errors during the digital counting process, a ratio method is established for the AFB1 concentration assay instead of directly counting the absolute number in the fluorescent image. The ratio equation is expressed as 𝑅 = (𝑁𝑖 ― 𝑁0)/𝑁0, where 𝑁𝑖 and 𝑁0 present the counted number of UCNPs-aptamer with and without AFB1 in the samples, respectively. Therefore, the potential errors from different batches of samples and nonhomogeneous particles adsorption on the cover glass surface can be effectively avoided. As shown in Figure 4b, the dynamic range for AFB1 assay is obtained from 0 to 156.25 ng/mL. A linear calibration curve is realized over a concentration range from 3.13 to 125.00 ng/mL with a regression equation of 𝑦 = 0.045𝑥 + 0.76 (𝑅2 = 0.99) in Figure 4c. The LOD (0.17 ng/mL, 𝑆/𝑁 = 3) is lower and the linear range for AFB1 detection is wider than most of the reported methods as shown in Table 1.37-41 It is worth noting that the LOD is much lower than the


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permissible concentration in health foods and safe feeds (< 20 ng/mL), which demonstrates that this LRET-based aptasensor would be a promising option for AFB1 assay in food samples. The specificity is an essential criterion to evaluate the capability of the aptasensor for actual sample assay. To evaluate the selectivity of the SPD aptasensor for AFB1 detection, AFB2, AFG1 and AFG2 (with the same concentration of 625.00 ng/mL) were selected as interfering agents in the control experiments under the same reaction conditions. As shown in Figure 5a-b, these interfering agents don't cause obvious fluorescent signal recovery except in the presence of target molecule AFB1 (125.00 ng/mL), which is attributed to the specific recognition ability of aptamer with AFB1. The quantitative analysis results also confirm that interfering agents can not affect the measurement results (Figure 5c). Taken together, this SPD-based aptasensor possesses excellent selectivity and anti-interference ability for AFB1 detection. Detection of AFB1 in Peanut Samples Until now, AFB1 is still a huge potential threat to food safety in many developing countries. It is important to develop sensitive and accurate methods for the detection of AFB1. Encouraged by the excellent sensitivity and specificity, we further explored the practical application of the aptasensor for AFB1 detection in peanut samples. The peanuts were obtained from local grain market. Different concentrations of AFB1 (with a final concentration of 0, 12.52, 31.30, and 62.60 ng/mL) were added to the diluted (5×) peanut samples. As shown in Figure 6a, the number of fluorescent spots increases gradually as the concentration of the AFB1 increases. The spiked analysis results of the AFB1 in the peanut sample are obtained in Figure 6b. It is worth noting that the fluorescent signal does not increase in the blank peanut sample. The excellent recoveries from 97.60 to 101.53% are obtained via the above calibration curve in Figure 6c, indicating that the



designed method possesses reliable accuracy for AFB1 detection in agriculture products. These results show that the aptasensor developed in this work can be adopted as the qualitative and quantitative method for AFB1 assay in complex agricultural products. It is worth emphasizing that this analytical method can also be readily extended to detect other mycotoxins in crop products (e.g., ochratoxin, zearalenone, deoxynivalenol, and so on).

Conclusions In summary, we demonstrate a novel and sensitive aptasensor for AFB1 assay by counting the number of UCNPs-aptamer in the fluorescent image with SPD method. In the presence of AFB1, the specific recognition between the aptamers and AFB1 makes the donor escape away from the acceptor, leading to the luminescence recovery from UCNPs-aptamer. This signal-off-on aptasensor holds excellent accuracy, sensitivity and anti-interference ability for AFB1 detection. Under optimal reaction conditions, a linear range from 3.13 to 125.00 ng/mL and a LOD of 0.17 ng/mL for AFB1 assay are readily obtained by quantitatively counting the luminescent particles on the glass slide surface. Furthermore, no interference signal is observed from other aflatoxins, including AFB2, AFG1 and AFG2. Based on the outstanding selectivity of this probe, the developed aptasensor shows great specificity toward the AFB1 in complex crop samples. In addition, this method exhibits several advantages in contrast to traditional fluorescence assay, for example, good accuracy, high sensitivity, excellent signal-to-noise ratio, and ultra-low sample consumption. Owing to the merits as noted above, this sensitive and robust digital enumeration strategy can be potentially applied for the quantitative detection and screening of aflatoxins in food samples in the future.


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Conflicts of Interest The authors declare no competing financial interest. Acknowledgements This work was supported by National Natural Science Foundation of China (NSFC, Project no. 21522502), the Fundamental Research Funds for the Central Universities, and the Excellent Youth Scholars of Hunan Provincial Education Department (17B155).

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Scheme 1. The schematic diagram of the aptasensor for AFB1 detection with the SPD method.



Figure 1. (a) TEM image and (b) DLS size distribution of GNPs. (c) The corresponding TEM image and (d) DLS size distribution of UCNPs-PAA. (e) The fluorescent image of UCNPs-PAA. (f) The FT-IR spectra of UCNPs-OA (red line) and UCNPs-PAA (green line). (g) The luminescence spectrum of UCNPs-PAA (red line) and UV-vis absorption spectrum of GNPs (green line).


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Figure 2. (a) The fluorescent image of UCNPs-aptamer on the coverslip surface. (b) The fluorescent image of UCNPs-aptamer from the same region after the addition of GNPs. (c) The fluorescent image of UCNPs-aptamer from the same area in Figure b after the addition of AFB1. (d) The dark-field image of GNPs. (e) and (f) The dark-field images of GNPs from the same area in the absence and presence of AFB1 (125.00 ng/mL) after adding UCNPs-aptamer, respectively. (g) The luminescence spectroscopic characterizations of the UCNPs-aptamer in different samples (a, b, and c correspond to the samples as noted in Figure 2a-c, respectively). (h) and (i) TEM images of the aptasensors in the absence and presence of AFB1 (125.00 ng/mL), respectively.



Figure 3. (a) and (b) Representative single-particle fluorescent images of UCNPs-PAA and quantum dots with long observation time. (c) and (d) The time-dependent fluorescence tracks from individual particles as marked in figure (a) and (b), respectively.


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Figure 4. (a) Representative fluorescent images of UCNPs-aptamer in the presence of different AFB1 concentrations. (b) The dynamic range (from 0 to 156.25 ng/mL) and (c) the linear range (from 3.13 to 125.00 ng/mL) for AFB1 assay, respectively.



Figure 5. (a) and (b) The fluorescence images of the probes for AFB1 detection without and with AFB1 in the presence of interfering substances, respectively. (c) The specificity assay of AFB1 detection.


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Table 1. Comparison of the sensitivity of different methods for the detection of AFB1



Figure 6. (a) Representative fluorescent images of the recovery estimation in peanut samples with different AFB1 concentrations. (b) The determined ratios in in peanut samples after adding different concentrations of AFB1. (c) Recovery assay for AFB1 in peanut samples.


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