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High affinity truncated aptamers for ultra-sensitive colorimetric detection of bisphenol A with label-free aptasensor
Food Chemistry 317 (2020) 126459
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High affinity truncated aptamers for ultra-sensitive colorimetric detection of bisphenol A with label-free aptasensor
T
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Min Jia , Junyi Sha, Zhihua Li, Wenjing Wang, Hongyan Zhang Key Laboratory of Animal Resistance Biology of Shandong Province, Institute of Biomedical Sciences, Key Laboratory of Food Nutrition and Safety of Shandong Normal University, College of Life Science, Shandong Normal University, Jinan 250014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bisphenol A Truncated aptamers AuNPs Aptasensor
The widespread exposure of bisphenol A (BPA) presents a significant risk to human health. A rapid, ultrasensitive and label-free colorimetric aptasensor using high affinity truncated aptamers was developed for BPA detection. Truncated 38-mer and 12-mer aptamers specific for BPA were obtained through rationally truncation from 63-mer BPA aptamer. The dissociation constants (Kd) of 38-mer and 12-mer aptamers were determined to be 13.17 nM and 27.05 nM. Then, truncated aptamers were used in label-free colorimetric detection assays based on gold nanoparticles (AuNPs). The limit of detections of aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamer, respectively. The recovery rates in milk, orange juice and mineralized water samples were 93.88% to 107.30%. Therefore, the developed BPA colorimetric aptasensor using truncated aptamers has great application prospects in food safety control and environmental monitoring.
1. Introduction Bisphenol A (BPA) is an important monomer for the manufacturing of epoxy resins and polycarbonate plastic, which are used as food packaging materials and containers (Sun et al., 2016). Due to the widespread uses, BPA can permeate into the food chain and environment leading to significant human exposure dose. BPA is one of the most active endocrine-disrupting compounds which can mimic the functions of estradiol, disrupt normal endocrine function and cause adverse human health effects (Authority, 2015). To protect public health, BPA is restricted to use in plastic infant feeding bottles and the maximum limits of BPA in plastic materials were also established in many countries (European Union, 2011; U. S. Food and Drug Administration, 2013). Therefore, development of detection methods for BPA in food samples and food containers is an urgent undertaking for assuring human health. Traditional detection techniques for the quantification of BPA are mainly focused on instrument-based methods with high sensitivity (Cao & Popovic, 2018; Dreolin, Aznar, Moret, & Nerin, 2019). However, expensive analytical equipments, complicated and time-consuming pretreated procedures and proficient operators were required for instrument-based methods. On the other hand, rapid methods were also used for BPA detection due to the characteristics of convenient and inexpensive. Immunoassay-based methods (Lei et al., 2013; Peng et al., ⁎
2018), one kind of the most universal rapid detection methods, were strongly dependent on the quality of antibody. Hence, the nonspecific binding to analogues and stability of the antibody would seriously affect the detection performance. Therefore, novel sensors with efficient recognition elements for ultrasensitive, selective and rapid detection of BPA were highly desired. Aptamers are recognized as an ideal antibody alternative for their high specific and affinity, excellent stability, easy to synthesis and modification. Thus, aptamers have been widely used as “chemical antibody” in food safety analysis and detection field (Zhang et al., 2018). Aptamers are specific single-stranded oligonucleotides with high affinity for target including small organic molecules (Ma et al., 2020), metal ions (Zhou, Lin, & Gan, 2017), proteins (Chinnappan et al., 2020), or even bacteria (Zou, Duan, Wu, Shen, & Wang, 2018) and cancer cells (Yang et al., 2014). The sequences of ligands were obtained by in vitro screening called systematic evolution of ligands by exponential enrichment (SELEX) strategy (Wang, Chen, Larcher, Barrero, & Veedu, 2019). A DNA aptamer for BPA was screened through SELEX strategy by Jo et al. (2011) with the length of 63 nucleotides, which showed high affinity and specificity. Subsequently, several detection methods, such as surface-enhanced Raman scattering assay (Marks, Pishko, Jackson, & Coté, 2014), electrochemical detection method (Yu et al., 2019), fluorescence analysis (Lee et al., 2018), and circular dichroism (CD)
Corresponding author. E-mail address: [email protected] (M. Jia).
https://doi.org/10.1016/j.foodchem.2020.126459 Received 13 August 2019; Received in revised form 4 February 2020; Accepted 19 February 2020 Available online 20 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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server (http://www.bioinfo.rpi.edu/applications/mfold/) (Heiat, Najafi, Ranjbar, Latifi, & Rasaee, 2016). A molecular docking study was performed to predict the binding mechanism between BPA and 38-mer aptamer using the AutoDock 4.2 program (Trott & Olson, 2010). To determine the dissociation constants (Kd) for the original and truncated aptamers to BPA, the MST experiments were performed (Svobodová et al., 2017). All the aptamers were labeled by Cy5-C6 at 5′end with a constant concentration of 100 nM throughout the experiment. A serial dilution of BPA in binding buffer (25 mM Tris-HCl buffer, pH 8.0, containing 100 mM NaCl, 25 mM KCl, 10 mM MgCl2 and 5% ethanol) was prepared and mixed with the labeled aptamers in the volume ratio of 1:1. Due to the different expected Kd of the aptamers, the starting concentrations of BPA were varied (Schax, Lönne, Scheper, Belkin, & Walter, 2015). After incubated in room temperature for 20 min, the experiments were performed on a Monolith NT.115 instrument (NanoTemper Technologies, Germany) at 25 °C with 20% LED power and 40% MST power. The recorded fluorescence was normalized to fraction of aptamer bound. The Kd of the BPA and aptamers were calculated from the resulting sigmoidal curves using the built-in Hill1 sigmoidal fit in Origin 8.0. The CD spectra were performed on a MOS-500 CD chiroptical spectrometer (Bio-Logic, France). The scanning spectrum was recorded in the wavelength range of 200–350 nm at 1 nm interval with a 1 cm path length quartz cuvette. The CD data were averaged from four successive scans at a scanning rate of 100 nm/min with an appropriately corrected baseline. The molar concentrations of the 63-mer, 38mer, and 23-mer aptamers used in CD measurements was 2 μM. For the 12-mer aptamer, the molar concentrations of 6 μM was used to obtain appropriate ellipticity signal. The data were collected in units of millidegrees versus wavelength.
spectrum analysis (Kuang et al., 2014) were developed for the detection of BPA by using this BPA aptamer. Due to the original BPA aptamer possessed too long sequence in the perspective of application, further optimization was necessary to obtain effective aptamers. Unnecessary nucleotide sequence would introduce uncertainty in tertiary conformation and make unstable binding with targets in various buffer solutions. Therefore, truncation of aptamers was one of the most efficient strategies to improve the affinity and reduce cost in detection (Nie et al., 2018; Tian, Wang, Sheng, Li, & Li, 2016). In several instances, detection methods performed lower limit of detection (LOD) and excellent sensitivity by using truncated aptamers (Alsager et al., 2015; Chinnappan et al., 2017). However, systematic revelation of the changing of binding characteristic, secondary and tertiary structures transformation and target-binding domain identification before and after the truncation of aptamers were few. Meanwhile, the direct comparisons of the performance of truncated versus original aptamers used in the same detection method were rarely reported. Colorimetric methods have been proved to be a simple and convenient sensing pattern for the development of aptasensors, enabling color visualization without special instrument (He & Yang, 2018). With no need of complicated and time consuming steps, colorimetric aptasensor based on target molecules induced the aggregation of gold nanoparticals (AuNPs) has been widely used in numbers of rapid detections (Feng, Shen, Wu, Dai, & Wang, 2019; Zhang, McKelvie, Cattrall, & Kolev, 2016). Therefore, AuNPs-based colorimetric aptasensor has been utilized for ultra-sensitive and rapid detection of BPA. Meanwhile, the applicability and performance of truncated versus original BPA aptamers in detection method were also investigated. In the present study, we reported novel aptamers with 12-mer and 38-mer, which were generated from the reported BPA aptamer by rationally truncation. The changing of binding and structure characteristics before and after the truncation of aptamers was investigated. The binding affinities of BPA and aptamers were confirmed by MicroScale Thermophoresis (MST). Besides, the structural properties of truncated aptamers were characterized by secondary structure analysis and 3-D molecular docking. Then these aptamers were used to improve the performance of AuNPs-based colorimetric aptasensor of BPA. The detection conditions were also optimized to increase the sensitivity of the assay. The sensitivity, specificity and precision of the aptasensors using original and truncated aptamers were compared. Finally, the proposed AuNPs-based colorimetric aptasensor was further applied to determinate the concentrations of BPA in real food samples. This study utilized the truncated aptamers to develop a rapid and sensitive colorimetric method for detecting BPA in food simples, which has a great potential application in food safety detection field.
2.3. Synthesis of AuNPs AuNPs (~15 nm) were prepared by the reduction of HAuCl4 with sodium citrate according to previously methods with slight modification (Jia, Liu, Zhang, & Zhang, 2019). Briefly, an aqueous solution of sodium citrate (3 mL, 1% (w/w)) was added immediately into a boiling solution of HAuCl4 (100 mL, 0.01% (w/w)) with vigorous stirring. After the color of the mixture changed to wine red, the solution was heated under reflux for another 20 min, and kept stirring until cooled down to room temperature. The size of the AuNPs was characterized by transmission electron microscopy (TEM, Hitachi HT-7800, Japan) operating at 80 kV. The concentration of AuNPs was calculated to be approximately 2.0 nM according to the Beer-Lambert law. 2.4. ζ-Potential measurements
2. Materials and methods The surface ζ-potentials of AuNPs-aptamer without and with BPA were measured a Zetasizer Nano ZS90 equipment (Malvern Instruments, Worcestershire, UK). 120 μL samples of bare AuNPs, AuNP–aptamer, and AuNP–aptamer with 500 nM BPA were centrifuged at 10,000×g for 20 min and resuspended in 1 mL ultrapure water to remove excess aptamer. A minimum of triplicate measurements per sample were made.
2.1. Materials BPA and its analogues were purchased from Aladdin Industrial Corporation (Shanghai, China). Chloroauric acid tetrahydrate (HAuCl4·4H2O), trisodium citrate dihydrate (C6H5Na3O7·2H2O), sodium chloride (NaCl) and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The oligonucleotides were synthesized and HPLC-purified by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the oligonucleotides used in the experiment were list in Table S1. All other reagents were of analytical grade. Ultrapure water (18.2 MΩ·cm, Milli-Q plus, Millipore, MA, USA) was used for preparing solutions.
2.5. Detection of BPA with AuNPs-based colorimetric aptasensor To minimize effect of the citrate stabilization, AuNPs solution was centrifuged at 10,000×g for 30 min (Eppendorf centrifuge 5810R, Germany) and resuspended with ultrapure water. The purified AuNPs were mixed with 63-mer, 38-mer, and 12-mer aptamers to get the aptamer/particle ratio of 3:1 and incubated at room temperature for 1 h, respectively. Twenty microliter target solutions with different concentrations or sample solutions were added to 100 μL AuNPs-aptamer mixture, and the final AuNPs concentration was 9.26 nM. After incubated for 15 min, 0.5 M NaCl solution was added and mixed
2.2. Truncation and characterization of the aptamer All the truncated aptamers were shortened from the original 63-mer BPA-specific aptamer based on the structural analysis. The predicted secondary structures of all the aptamers were obtained by Mfold web 2
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thoroughly using the pipet to obtain final salt concentration of 33.3 mM. After incubated for another 5 min, the UV–vis absorption spectra of the solutions were measured by multi-mode microplate reader (Molecular Devices SpectraMax M5, USA) and the color changes were recorded by a digital camera. Standard curves were made according to the ΔA650/A520 value and the concentrations of BPA. ΔA650/ A520 = the absorption ratio A650/A520 in sample solutions containing BPA – the absorption ratio A650/A520 in blank solutions without BPA.
redundant sequences. Even the 12-mer aptamer with only 12 nucleotides still have high affinity with BPA, which indicated the 12-mer stem-loop region was the essential domains for BPA binding. These results partly be ascribed that eliminating redundant sequences can increase the stabilization of the 3D conformation of aptamer and improve the affinity for the target molecules. CD spectra technique was involved as a powerful tool to illuminate the changes of the secondary structure of aptamers (Paul & Guchhait, 2011). To investigate the effects of truncation on aptamer conformation, the intrinsic CD profiles of aptamers before and after flanking nucleotides elimination were compared (Fig. S1A). There were a positive peak at around 270 nm and a negative peak at around 244 nm emanated from the base stacking interaction and the helical suprastructure respectively (Cheng, Liu, Shi, & Zhao, 2018), which indicated the typical right-hand B-form structures primarily existed in the original 63-mer aptamer (Huo et al., 2016). After truncation, the 38-mer, 23-mer and 12-mer aptamer showed distinctively different CD spectra. A new shoulder peak of aptamer was observed in the positive peak of 38-mer aptamer. The negative peak of 23-mer aptamer was blue-shifted to 239 nm, and the 270 nm positive peak was split into two peak at 263 and 291 nm. For 12-mer aptamer, the negative peak was slightly blueshifted to 246 nm, and a significant red-shifted to 279 nm was observed in positive peak. Compared to the original one, the second structures of truncated aptamers had changed. The secondary structural changes of aptamers upon binding to BPA were also investigated (Fig. S1B–D). In the addition of BPA, the intensity of the positive band was increased without the peak position shifted obviously in 63-mer, 38-mer and 12-mer aptamers. This revealed that the presence of BPA can induce the conformations of aptamers shifted from B-form to A-form structures with the changes of base stacking and the right-handedness (Lin et al., 2011). The small molecular targets tended to bury in the hydrophobic pocket of the aptamers. To further investigate interactions of BPA and aptamer, the possible binding mode of 12-mer aptamer and BPA was investigated by the molecular docking method. The BPA was docked in the binding site of 38-mer aptamer with the result shown in Fig. 1C. The BPA-aptamer interactions rely on the structural compatibility and the combination of binding forces, such as hydrogen bonding, T-shaped interaction and hydrophobic effect (Cai et al., 2018). BPA was located in the hydrophobic active pocket of 38mer aptamer surrounding by the side chains of the nucleotides G-14, G15, G-16, C-26, G-27, C-28 to form stable hydrophobic bonds. Between BPA and 38-mer aptamer, five hydrogen bonds were formed between the nucleotide G-15, G-16, G-27, C-28. The hydroxyl “O” of BPA formed T-shaped interaction with the amino group of nucleotide G-27, with a bond length of 2.3 Å. The combination of these binding forces can mediate the BPA-aptamer interaction and help the formation of the stable binding complex. The binding free energy of BPA and aptamer evaluated with AutoDock Tools 1.5.6 was −4.90 kcal/mol. The truncated 38-mer and 12-mer aptamers were obtained with improved affinity through removing the redundant sequence. The changes of structural and binding properties were characterized to investigate the effect of truncation. To illustrate the performances of the truncated aptamers in detection method, the 63-mer, 38-mer and 12mer aptamers were then used in the establishment of AuNPs-based colorimetric aptasensors. The sensitivity and specificity of the aptasensors for BPA detection were investigated and compared.
2.6. Samples preparation For validating the practicability in real samples, milk sample, orange juice and mineralized water purchased from RT-mart supermarket (Jinan, China) were assayed by AuNPs-based colorimetric method using the truncated 38-mer aptamer. All samples were spiked with desired concentrations of BPA and allowed to stand for 5 min. The milk sample was centrifuged at 10,000×g for 5 min to remove the upper suction fat. Then the milk sample was mixed with the same volume of ethanol for protein precipitation and centrifuged at 10,000×g for 5 min. The supernatant was transferred to a Ultra-0.5 Centrifugal Filters (MWCO 3 kDa, Millipore, MA, USA). The filtrate solution was diluted 5-fold with ethanol. For orange juice and mineralized water, the samples were diluted for 10-fold with ethanol and filtered through 0.22-µm ultrafiltration membrane to remove the pulps and suspended particles before use. 3. Results and discussion 3.1. Truncation and characterization of the aptamers The full length sequence of aptamer usually contained the nonessential nucleotides that do not interact with target molecular nor act as the contact supporting role (Soheili et al., 2016). These nonessential nucleotides might introduces uncertainty in aptamer tertiary structure which can lead to unstable affinity for targets in different solutions. Meanwhile, too long sequence would also increase the cost of synthesis. Therefore, although the affinity and specificity of original BPA-specific aptamer were basically satisfactory, the length of the 63-mer sequence limited its potential application. To reduce cost of detection and improve the affinity and performance, the truncation of the original 63-mer aptamer was carried out based on the structural analysis. The predicated secondary structures of the aptamers obtained from the Mfold web server were shown in Fig. 1A. Some studies have shown that the region with specific secondary structure such as stem-loop structure, G-quartet loops, bulges and/or pseudoknots are often contributing to the direct binding with the target molecules. There are two stem-loop structures and some flanking sequences existing in the original 63-mer aptamer. Therefore, the aptamer was truncated in three different ways. In the first truncation, two stem-loop structures were kept and the flanking sequences were eliminated in 38-mer aptamer to retain the putative binding domains to the target. In the second truncation, one of the stem-loop structures was kept to get 12-mer aptamer. In the third truncation, the other stem-loop structure was kept to get 23-mer aptamer. The sequences of the original and truncated aptamers were presented in Table S1. Then, systematic revelation of the changing of binding characteristic, secondary and tertiary structures transformation and targetbinding domain identification before and after the truncation of 63-mer aptamer were carried out. The Kd values of the 63-mer, 38-mer, 12-mer and 23-mer aptamers were determined by using MST experiments, which were 491.69 nM, 13.17 nM, 27.05 nM and 1190.61 nM (Table S1). The binding affinities of 38-mer and 12-mer aptamers were enhanced by 37.3-fold and 18.2fold, respectively. The non-linear regression curves of the four aptamers were shown in Fig. 1B. Compared with 63-mer aptamer, the affinity of 38-mer and 12-mer aptamers was obviously improved by removing the
3.2. Construction and characterization of the BPA colorimetric aptasensors The principle of the proposed strategy for BPA colorimetric aptasensor is shown in Fig. 2A. The single stranded DNA aptamers can be absorbed on the surface of AuNPs through van der Waals force and DNA base-gold interaction (Mirau et al., 2018), which protected AuNPs from salt-induced aggregation. When BPA exists in the system, BPA aptamers would specifically bind to BPA to form BPA-aptamer complex due to the 3
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Fig. 1. Characterization of aptamers. (A) The predicted secondary structure models of 63-mer, 38mer, 23-mer and 12-mer BPA-specific aptamers by Mfold. (B) Binding curves of 63-mer, 38-mer, 23mer and 12-mer aptamers with BPA. Error bars: standard deviation (SD), n = 3. (C) The prediction of the binding sites of 12-mer aptamers for BPA by molecular docking.
experimental conditions were monitored by UV–vis spectrometer. As shown in Fig. 2B, the characteristic surface plasmon resonance absorption of AuNPs at 520 nm was decreased remarkably when NaCl was added to the unmodified AuNPs, which indicated that AuNPs aggregated due to salt-induction. Correspondingly, the color of AuNPs solution changed from wine-red to blue. However, if the aptamer exists in AuNPs solution, the stability of AuNPs will be improved and the absorption peak of AuNPs at 520 nm maintained under high salt condition. These phenomena indicated that the aptamer could prevent AuNPs to from aggregation. When the BPA was added, the absorbance at 520 nm decreased dramatically accompanied by a new absorption peak at around 650 nm. The color of the solution changed from winered to blue. It indicated that the added BPA may specifically bind with BPA aptamer, making the AuNPs released and aggregate under high salt condition. To characterize the morphology change of AuNPs at different experimental conditions, TEM technology was utilized (Fig. 2C). The bare AuNPs had a spherical morphology and dispersed evenly. After appropriate NaCl was added, AuNPs were aggregated heavily and formed an irregular network structure. In addition, AuNPs were still well-dispersed in the presence of aptamer under above NaCl concentration existence. However, AuNPs started to aggregate into clusters and the shape was changed obviously with the addition of BPA. Hence, these results were consistent with the proposed principle and the feasibility of this detection method was proved.
high affinity compared with the nonspecific absorption. AuNPs that separated from aptamers will aggregate under the high-salt condition. The color of the solution would gradually change from the original wine-red into purple or blue with the increasing concentrations of BPA. For small target molecules such as BPA, the interaction between the aptamer and the target molecule occurs in a specific binding domain that might be a part of aptamer sequences according to the molecular docking result. The excess flanking sequences outside of the binding region still nonspecifically interacted with the surface of AuNPs when BPA-aptamer complex formed, suppressing the complete dissociation of aptamer from AuNPs. Due to the adherence of flanking sequences, the aggregation of AuNPs was prevented resulting in undesired sensitivity. Therefore, we hypothesized that after the truncation of the aptamers, the sensitivity of colorimetric aptasensor would be enhanced with the improvement of the affinity and the decrease of nonspecific interaction. To investigate the interacting of AuNPs, aptamer and BPA, the surface ζ-potentials of bare AuNPs, AuNPs-aptamer without and with BPA were determined (Table S2). The surface ζ-potentials of AuNPs was decreased after the 63-mer aptamers association from −27.4 mV to −38.8 mV, which was indicate that aptamers were successful adsorbed on AuNPs. After incubation with 500 nM BPA, the ζ-potentials of AuNPs was increased to −33.2 mV, confirming that the aptamers was partly dissociate from the AuNPs when binding to BPA. For random 63-mer sequence, the decreased ζ-potentials confirmed that the sequence can adsorb on the AuNPs after random sequence added. However, there was no significant change in ζ-potentials when incubate with BPA. For the truncated aptamers, more negative ζ-potentials were obtained when AuNPs were incubated with aptamers, from −27.4 mV to −35.4 mV and −33.8 mV for 38-mer and 12-mer aptamers, respectively. When the AuNPs-aptamer mixtures were incubated with 500 nM BPA, the ζpotentials increased to −28.3 mV and −28.0 mV for 38-mer and 12mer aptamers, respectively. The ζ-potentials are close to the original ζpotentials of bare AuNPs (−27.4 mV), which were different from that of 63-mer aptamers. These results indicated that less residual interactions between BPA-bound aptamers and AuNPs were achieved after the truncation of 63-mer aptamer. Therefore, the truncated aptamers dissociated from AuNPs more completely in contrast with the original aptamer, which might be enhance the detection sensitivity. To investigate the feasibility and mechanism of the aptasensors, the characteristic absorption peaks of AuNPs solutions under different
3.3. Optimization of experimental parameters According to the principle of BPA colorimetric aptasensor, experimental conditions such as NaCl concentration, aptamer/AuNPs stoichiometric ratio, and ethanol concentration had a great effect on performance of the colorimetric method. Therefore, these key parameters were optimized by a series of systematic experiments. Due to the variation of the length of the aptamers, the parameters of NaCl concentrations and aptamer-AuNPs stoichiometric ratios were tested with the usage of the 63-mer, 38-mer and 12-mer aptamers, respectively. Controlling the aggregation of AuNPs by NaCl-induced approach is critical for the establishment of AuNPs-based colorimetric aptasensor. Many studies have verified the AuNPs protective effect of aptamer under certain NaCl concentration range (Feng et al., 2019; Mao et al., 4
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Fig. 2. (A) Schematic illustration for AuNP-based colorimetric aptasensor and the mechanism for ultra-sensitive detection of BPA by using the truncated aptamer. (B) UV–vis absorption spectra of AuNPs under different experimental conditions. (C) TEM image of AuNPs under different experimental conditions. Scale bar: 50 nm.
Fig. 3. Optimization of experimental parameters for 63-mer aptamer. (A) NaCl concentration. (B) Ratio of aptamer/AuNPs, n = 3. (C) Ethanol concentration. Error bars: SD, n = 3.
higher aptamer coverage resulted better protection efficiency of aptamer against salt-induced aggregation. Therefore, the concentrations of NaCl optimized for 63-mer aptamer/AuNPs with the ratio of 6:1 and 9:1 were 37.5 and 41.7 mM, respectively. Meanwhile, the NaCl concentrations for 38-mer and 12-mer were also optimized (Fig. S2, Supporting Information). To determine the optimal stoichiometric ratio of aptamer to AuNPs that can yield the highly sensitive detection of BPA, 20 μL BPA of different concentrations was added to complexes with the aptamer/AuNPs ratio of 3:1, 6:1 and 9:1. As shown in Fig. 3B, lower 63-mer aptamer/ AuNPs ratio result in higher sensitivity to salt and sharp transition to aggregation, which can avoid the excess protection of AuNPs toward
2017). Thus, NaCl concentration was optimized for reliable detection. For this purpose, different volumes of 0.5 M NaCl were added to 100 μL samples of bare AuNPs, AuNP-aptamer blended with different aptamer/ AuNPs ratio of 3:1, 6:1 and 9:1, respectively. The relative absorption at 520 nm (A520 (rel.)) was measured to evaluate the degree of AuNPs aggregation. As results shown in Fig. 3A, for the 63-mer aptamer/ AuNPs with the ratio of 3:1, relative absorption at 520 nm was decreased obviously after the concentration of NaCl reached to 33.3 mM. Therefore, the optimal NaCl concentration was 33.3 mM for BPA detection, whereby the AuNPs-aptamer dispersion was on the edge of stability and could be easily disrupted by the introduction of BPA. The 5
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Fig. 4. Absorption spectra of BPA detection using (A) 63-mer aptamers, (C) 38-mer aptamers, (E) 12-mer aptamers. The relationship between the ΔA650/A520 value and the concentration of BPA using (B) 63-mer, (D) 38-mer and (F) 12mer aptamers; Inset shows the linear relationship. Error bars: SD, n = 3. 95% confidence bands are delimited by blue dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the ethanol concentration. These results demonstrated that the addition of ethanol can enhance the recognition efficiency of aptamers. After the concentration of ethanol reached 15% (v/v), the ΔA650/A520 value increased slowly. Therefore, 15% (v/v) was chosen as optimal ethanol concentration.
target-induced aggregation. Therefore, aptamer/AuNPs stoichiometric ratio of 3:1 was defined as the optimal ratio for 63-mer aptamer. Meanwhile, the optimal ratios for 38-mer and 12-mer were also 3:1 as shown in Fig. S2. Organic solvent containing solutions were usually used as binding buffer in aptamer SELEX or aptamer-target binding property evaluation, especially for poorly soluble targets. We assumed that the use of organic solvent would enhance the solubility of target and could participate to the stabilization of the aptamer-target complex. Therefore, common organic solvent ethanol was utilized to improve the sensitivity of BPA detection. For this purpose, different concentrations of ethanol were used in 20 μL BPA sample solution to obtain final ethanol concentration from 0 to 16.7% (v/v) in detection system. ΔA650/A520 value of 20 nM and 60 nM BPA samples were calculated. As shown in Fig. 3C, an increasing trend for ΔA650/A520 value was observed by increasing
3.4. Quantification performance of the BPA colorimetric aptasensors After the optimization of the experimental parameters, the quantification performances of BPA colorimetric aptasensor using the 63-mer, 38-mer and 12-mer aptamers were validated using a series of BPA solutions with different concentrations, respectively. The UV–vis absorption spectrum of BPA colorimetric aptasensors against different BPA concentrations were recorded in Fig. 4. With the increase of the BPA concentration, the absorption peak of 520 nm decreased gradually, 6
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higher LOD (14.41 pM) than 38-mer. The correlation coefficient was 0.9934 in the range of 20 pM to 100 pM. Consistent with our hypothesis, the AuNPs-based colorimetric aptasensor achieved better performance using truncated aptamers. Combined with the results of ζ-potential measurements, the improvement of truncation in quantification performance might be attributed to the enhancement of affinity interaction for BPA when the excess sequences were eliminated and suppressed nonspecific absorption of aptamers on the surface of AuNPs. As shown in Table S3, the performance of this method and other reported methods were compared and summarized. After truncation, the LOD of aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamer, respectively. From the comparisons with other methods, the sensitivity of aptasensor using 38mer aptamer has the merit of a much lower LOD for BPA detection except the GC–MS method (Cao & Popovic, 2018) and electrochemical method (Yu et al., 2019). Although the sensitivity is not as good as these methods, our aptasensor possessed two major advantages. First, the detection was low-cost, because the use of special equipments or modified aptamers was avoided. Second, the detection process was simple and rapid by using this one-step, label-free aptasensor method, without considering expert personnel and time-consuming procedure. Therefore, the method here is capable of offering high detection sensitivity with less cost and simple detection process. 3.5. Specificity of the BPA colorimetric aptasensors The specificity of the aptasensor for BPA detection was also investigated. A variety of BPA analogues, such as bisphenol B (BPB), bisphenol F (BPF), bisphenol S (BPS), estrone (E1), 17β-estradiol (E2), estriol (E3), hexestrol (HES), diethylstilbestrol (DES), dienestrol (DI), progesterone (P) and tetrabromobisphenol A (TBBA) were individually added to the BPA colorimetric aptasensors using the 63-mer, 38-mer and 12-mer aptamers, and the values of ΔA620/A520 were calculated (Fig. 5A). The molecular structures of these analogues were shown in Fig. 5B and revealed the similar chemical structural characteristics as BPA. The truncated aptamers of 38-mer and 12-mer showed similar specificity characteristic as the original 63-mer. As expect, the ΔA650/ A520 values of BPA samples and the mixtures containing BPA had significant increases; however, other analogues did not result in an obvious ΔA650/A520 value increase. The results indicated that all of the established aptasensors using three aptamers were highly specific to BPA and has insignificant cross-reactivity with its analogues.
Fig. 5. (A) Specificity of BPA detection using AuNPs-based colorimetric aptasensor, n = 3. (B) Structures of components used in (A).
and the absorption of 650 nm increased correspondingly. For 63-mer aptamer, the results exhibited in Fig. 4B that ΔA650/A520 value had a good linear correlation with the increase of BPA concentration, which can be fitted as y = 0.01652x + 0.03785. The correlation coefficient was 0.9804 in low-concentration range of 5 nM to 60 nM. The LOD of this method was calculated as 2.02 nM by the concentration corresponding to the ΔA650/A520 value at three times standard deviation of 10 blank samples without BPA. Incubation of BPA with bare AuNPs and with AuNPs modified with a random 63-mer ssDNA did not show any interaction. The result of titration of BPA colorimetric aptasensor using 38-mer aptamer against BPA revealed a 265-fold enhancement of sensitivity compared with the aptasensor using 63-mer aptamer (Fig. 4D). BPA was detected down to 7.60 pM, and a linear response (R2 = 0.9911) was observed between 10 pM and 100 pM. The minimalist 12-mer also being effective, Fig. 4F showed that 12-mer system delivered a slightly
3.6. Repeatability and reproducibility of the BPA colorimetric aptasensors Precision of three BPA colorimetric aptasensors was assessed in terms of repeatability (intra-day precision) and reproducibility (interday precision). Repeatability (r) was calculated as the repeatability relative standard deviation (RSDr) of six consecutive measurements of the same sample of BPA solution (25 nM and 50 pM for the aptasensors
Table 1 Detection of BPA in real samples using BPA colorimetric aptasensor and HPLC-MS (n = 6). Samples
Milk
Orange juice
Mineralized water
Spiked (nM)
2 5 20 2 5 20 2 5 20
Proposed method
HPLC-MS
Found (nM)
Recovery (%) ± RSD (%)
Bias (%)
Found (nM)
Recovery (%) ± RSD (%)
Bias (%)
1.94 4.78 18.78 2.06 4.87 19.65 1.97 5.37 20.56
97.08 ± 6.82 95.60 ± 4.07 93.88 ± 4.56 102.83 ± 7.42 97.33 ± 3.89 98.26 ± 2.99 98.33 ± 8.12 107.30 ± 3.93 102.78 ± 4.15
−2.92 −4.40 −6.12 2.83 −2.67 −4.53 −1.67 7.30 2.78
1.93 5.09 19.56 1.96 4.92 20.85 2.07 5.28 19.79
96.42 ± 5.85 101.83 ± 5.68 97.81 ± 4.76 97.92 ± 3.81 98.43 ± 5.55 101.68 ± 3.06 103.58 ± 3.76 105.50 ± 4.57 98.93 ± 2.04
−3.58 1.83 −2.19 −2.08 −1.57 1.67 3.58 5.50 −1.07
7
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influence the work reported in this paper.
of 63-mer aptamer and truncated aptamers, respectively) within the same day. Similarly, reproducibility (R) was calculated as the reproducibility relative standard deviation (RSDR) of eighteen measurements obtained across three days of analyses. Values of RSDr and RSDR showed good precision for three aptasensors (Table S4). RSDr ranged from 3.37% to 6.07%, and RSDR ranged from 4.45% to 5.22%, which demonstrated good precision of the proposed method.
This study was financially supported by National Key R&D Program of China (2016YFD0401101) and National Natural Science Foundation of China (31871874).
3.7. Detection of BPA in real samples
Appendix A. Supplementary data
To eliminate interference of food matrix, a brief pretreatment was developed for sample preparation. According to the result of the experimental optimization, ethanol was utilized as diluent, protein precipitate and BPA extractant instead of water. BPA colorimetric aptasensor using 38-mer aptamer was employed to investigate the feasibility in real samples analysis. The results of food sample experiments were summarized in Table 1 and compared with those obtained using HPLC-MS. The recovery rate were 93.88%–107.30% vs. 96.42%–105.50% by HPLC-MS, whereas the relative standard deviation (RSD) by this method were 2.99%– 8.12% vs. 2.04%–5.85% by HPLCMS. Trueness is explained by computing bias%. Bias% were −6.12%–7.30% vs. −3.58% to 5.50% by HPLC-MS. A t-test was also performed and the calculated concentrations of BPA colorimetric aptasensor method were not statistically different from that of HPLC-MS method, with a 95% of confidence interval. Therefore, the developed BPA colorimetric aptasensor might be initially applied for the determination of BPA in real samples.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2020.126459.
Acknowledgements
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4. Conclusion In this study, aptamers with the length of 38-mer and 12-mer for BPA were obtained through rationally designed truncation. A systematic comparison of original and truncated aptamers on binding and structure characteristic were investigated and the result indicated that both 38-mer and 12-mer aptamers showed high affinity toward BPA. The molecular docking assay offered rational explanations of the interactions between BPA and aptamer. Then the 63-mer, 38-mer and 12mer aptamers were used as recognition elements to detect BPA through AuNPs-based colorimetric method. The AuNPs-based colorimetric aptasensors achieved high performance using truncated aptamers. The LOD of the aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamers, respectively. Meanwhile, the aptasensors using 38-mer and 12-mer aptamers showed high selectivity against other 11 kinds of BPA analogues. More importantly, the practicability of the colorimetric aptasensor was verified by detection of BPA in milk, orange juice and water samples. With excellent binding characteristics, optimized length and interference resistance in the detection, the truncated 38-mer and 12-mer aptamers exhibit great application potential in food safety control and environmental monitoring. Meanwhile, appropriate design of sensing configuration and the application of other unique nanomaterials, the performance of detection may be further improved. CRediT authorship contribution statement Min Jia: Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision. Junyi Sha: Validation, Formal analysis, Investigation, Writing - original draft. Zhihua Li: Validation, Investigation. Wenjing Wang: Validation, Investigation. Hongyan Zhang: Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to 8
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(2018). Structured DNA Aptamer Interactions with Gold Nanoparticles. Langmuir, 34(5), 2139–2146. Nie, J., Yuan, L., Jin, K., Han, X., Tian, Y., & Zhou, N. (2018). Electrochemical detection of tobramycin based on enzymes-assisted dual signal amplification by using a novel truncated aptamer with high affinity. Biosensors and Bioelectronics, 122, 254–262. Paul, B. K., & Guchhait, N. (2011). Exploring the strength, mode, dynamics, and kinetics of binding interaction of a cationic biological photosensitizer with DNA: Implication on dissociation of the drug–DNA complex via detergent sequestration. The Journal of Physical Chemistry B, 115(41), 11938–11949. Peng, X., Kang, L., Pang, F., Li, H., Luo, R., Luo, X., & Sun, F. (2018). A signal-enhanced lateral flow strip biosensor for ultrasensitive and on-site detection of bisphenol A. Food and Agricultural Immunology, 29(1), 216–227. Schax, E., Lönne, M., Scheper, T., Belkin, S., & Walter, J.-G. (2015). Aptamer-based depletion of small molecular contaminants: A case study using ochratoxin A. Biotechnology and Bioprocess Engineering, 20(6), 1016–1025. Soheili, V., Taghdisi, S. M., Hassanzadeh Khayyat, M., Fazly Bazzaz, B. S., Ramezani, M., & Abnous, K. (2016). Colorimetric and ratiometric aggregation assay for streptomycin using gold nanoparticles and a new and highly specific aptamer. Microchimica Acta, 183(5), 1687–1697. Sun, F., Kang, L., Xiang, X., Li, H., Luo, X., Luo, R., ... Peng, X. (2016). Recent advances and progress in the detection of bisphenol A. Analytical and Bioanalytical Chemistry, 408(25), 6913–6927. Svobodová, M., Skouridou, V., Botero, M. L., Jauset-Rubio, M., Schubert, T., Bashammakh, A. S., ... O’Sullivan, C. K. (2017). The characterization and validation of 17β-estradiol binding aptamers. The Journal of Steroid Biochemistry and Molecular Biology, 167, 14–22. Tian, Y., Wang, Y., Sheng, Z., Li, T., & Li, X. (2016). A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length. Analytical Biochemistry, 513, 87–92.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
High affinity truncated aptamers for ultra-sensitive colorimetric detection of bisphenol A with label-free aptasensor
T
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Min Jia , Junyi Sha, Zhihua Li, Wenjing Wang, Hongyan Zhang Key Laboratory of Animal Resistance Biology of Shandong Province, Institute of Biomedical Sciences, Key Laboratory of Food Nutrition and Safety of Shandong Normal University, College of Life Science, Shandong Normal University, Jinan 250014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bisphenol A Truncated aptamers AuNPs Aptasensor
The widespread exposure of bisphenol A (BPA) presents a significant risk to human health. A rapid, ultrasensitive and label-free colorimetric aptasensor using high affinity truncated aptamers was developed for BPA detection. Truncated 38-mer and 12-mer aptamers specific for BPA were obtained through rationally truncation from 63-mer BPA aptamer. The dissociation constants (Kd) of 38-mer and 12-mer aptamers were determined to be 13.17 nM and 27.05 nM. Then, truncated aptamers were used in label-free colorimetric detection assays based on gold nanoparticles (AuNPs). The limit of detections of aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamer, respectively. The recovery rates in milk, orange juice and mineralized water samples were 93.88% to 107.30%. Therefore, the developed BPA colorimetric aptasensor using truncated aptamers has great application prospects in food safety control and environmental monitoring.
1. Introduction Bisphenol A (BPA) is an important monomer for the manufacturing of epoxy resins and polycarbonate plastic, which are used as food packaging materials and containers (Sun et al., 2016). Due to the widespread uses, BPA can permeate into the food chain and environment leading to significant human exposure dose. BPA is one of the most active endocrine-disrupting compounds which can mimic the functions of estradiol, disrupt normal endocrine function and cause adverse human health effects (Authority, 2015). To protect public health, BPA is restricted to use in plastic infant feeding bottles and the maximum limits of BPA in plastic materials were also established in many countries (European Union, 2011; U. S. Food and Drug Administration, 2013). Therefore, development of detection methods for BPA in food samples and food containers is an urgent undertaking for assuring human health. Traditional detection techniques for the quantification of BPA are mainly focused on instrument-based methods with high sensitivity (Cao & Popovic, 2018; Dreolin, Aznar, Moret, & Nerin, 2019). However, expensive analytical equipments, complicated and time-consuming pretreated procedures and proficient operators were required for instrument-based methods. On the other hand, rapid methods were also used for BPA detection due to the characteristics of convenient and inexpensive. Immunoassay-based methods (Lei et al., 2013; Peng et al., ⁎
2018), one kind of the most universal rapid detection methods, were strongly dependent on the quality of antibody. Hence, the nonspecific binding to analogues and stability of the antibody would seriously affect the detection performance. Therefore, novel sensors with efficient recognition elements for ultrasensitive, selective and rapid detection of BPA were highly desired. Aptamers are recognized as an ideal antibody alternative for their high specific and affinity, excellent stability, easy to synthesis and modification. Thus, aptamers have been widely used as “chemical antibody” in food safety analysis and detection field (Zhang et al., 2018). Aptamers are specific single-stranded oligonucleotides with high affinity for target including small organic molecules (Ma et al., 2020), metal ions (Zhou, Lin, & Gan, 2017), proteins (Chinnappan et al., 2020), or even bacteria (Zou, Duan, Wu, Shen, & Wang, 2018) and cancer cells (Yang et al., 2014). The sequences of ligands were obtained by in vitro screening called systematic evolution of ligands by exponential enrichment (SELEX) strategy (Wang, Chen, Larcher, Barrero, & Veedu, 2019). A DNA aptamer for BPA was screened through SELEX strategy by Jo et al. (2011) with the length of 63 nucleotides, which showed high affinity and specificity. Subsequently, several detection methods, such as surface-enhanced Raman scattering assay (Marks, Pishko, Jackson, & Coté, 2014), electrochemical detection method (Yu et al., 2019), fluorescence analysis (Lee et al., 2018), and circular dichroism (CD)
Corresponding author. E-mail address: [email protected] (M. Jia).
https://doi.org/10.1016/j.foodchem.2020.126459 Received 13 August 2019; Received in revised form 4 February 2020; Accepted 19 February 2020 Available online 20 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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server (http://www.bioinfo.rpi.edu/applications/mfold/) (Heiat, Najafi, Ranjbar, Latifi, & Rasaee, 2016). A molecular docking study was performed to predict the binding mechanism between BPA and 38-mer aptamer using the AutoDock 4.2 program (Trott & Olson, 2010). To determine the dissociation constants (Kd) for the original and truncated aptamers to BPA, the MST experiments were performed (Svobodová et al., 2017). All the aptamers were labeled by Cy5-C6 at 5′end with a constant concentration of 100 nM throughout the experiment. A serial dilution of BPA in binding buffer (25 mM Tris-HCl buffer, pH 8.0, containing 100 mM NaCl, 25 mM KCl, 10 mM MgCl2 and 5% ethanol) was prepared and mixed with the labeled aptamers in the volume ratio of 1:1. Due to the different expected Kd of the aptamers, the starting concentrations of BPA were varied (Schax, Lönne, Scheper, Belkin, & Walter, 2015). After incubated in room temperature for 20 min, the experiments were performed on a Monolith NT.115 instrument (NanoTemper Technologies, Germany) at 25 °C with 20% LED power and 40% MST power. The recorded fluorescence was normalized to fraction of aptamer bound. The Kd of the BPA and aptamers were calculated from the resulting sigmoidal curves using the built-in Hill1 sigmoidal fit in Origin 8.0. The CD spectra were performed on a MOS-500 CD chiroptical spectrometer (Bio-Logic, France). The scanning spectrum was recorded in the wavelength range of 200–350 nm at 1 nm interval with a 1 cm path length quartz cuvette. The CD data were averaged from four successive scans at a scanning rate of 100 nm/min with an appropriately corrected baseline. The molar concentrations of the 63-mer, 38mer, and 23-mer aptamers used in CD measurements was 2 μM. For the 12-mer aptamer, the molar concentrations of 6 μM was used to obtain appropriate ellipticity signal. The data were collected in units of millidegrees versus wavelength.
spectrum analysis (Kuang et al., 2014) were developed for the detection of BPA by using this BPA aptamer. Due to the original BPA aptamer possessed too long sequence in the perspective of application, further optimization was necessary to obtain effective aptamers. Unnecessary nucleotide sequence would introduce uncertainty in tertiary conformation and make unstable binding with targets in various buffer solutions. Therefore, truncation of aptamers was one of the most efficient strategies to improve the affinity and reduce cost in detection (Nie et al., 2018; Tian, Wang, Sheng, Li, & Li, 2016). In several instances, detection methods performed lower limit of detection (LOD) and excellent sensitivity by using truncated aptamers (Alsager et al., 2015; Chinnappan et al., 2017). However, systematic revelation of the changing of binding characteristic, secondary and tertiary structures transformation and target-binding domain identification before and after the truncation of aptamers were few. Meanwhile, the direct comparisons of the performance of truncated versus original aptamers used in the same detection method were rarely reported. Colorimetric methods have been proved to be a simple and convenient sensing pattern for the development of aptasensors, enabling color visualization without special instrument (He & Yang, 2018). With no need of complicated and time consuming steps, colorimetric aptasensor based on target molecules induced the aggregation of gold nanoparticals (AuNPs) has been widely used in numbers of rapid detections (Feng, Shen, Wu, Dai, & Wang, 2019; Zhang, McKelvie, Cattrall, & Kolev, 2016). Therefore, AuNPs-based colorimetric aptasensor has been utilized for ultra-sensitive and rapid detection of BPA. Meanwhile, the applicability and performance of truncated versus original BPA aptamers in detection method were also investigated. In the present study, we reported novel aptamers with 12-mer and 38-mer, which were generated from the reported BPA aptamer by rationally truncation. The changing of binding and structure characteristics before and after the truncation of aptamers was investigated. The binding affinities of BPA and aptamers were confirmed by MicroScale Thermophoresis (MST). Besides, the structural properties of truncated aptamers were characterized by secondary structure analysis and 3-D molecular docking. Then these aptamers were used to improve the performance of AuNPs-based colorimetric aptasensor of BPA. The detection conditions were also optimized to increase the sensitivity of the assay. The sensitivity, specificity and precision of the aptasensors using original and truncated aptamers were compared. Finally, the proposed AuNPs-based colorimetric aptasensor was further applied to determinate the concentrations of BPA in real food samples. This study utilized the truncated aptamers to develop a rapid and sensitive colorimetric method for detecting BPA in food simples, which has a great potential application in food safety detection field.
2.3. Synthesis of AuNPs AuNPs (~15 nm) were prepared by the reduction of HAuCl4 with sodium citrate according to previously methods with slight modification (Jia, Liu, Zhang, & Zhang, 2019). Briefly, an aqueous solution of sodium citrate (3 mL, 1% (w/w)) was added immediately into a boiling solution of HAuCl4 (100 mL, 0.01% (w/w)) with vigorous stirring. After the color of the mixture changed to wine red, the solution was heated under reflux for another 20 min, and kept stirring until cooled down to room temperature. The size of the AuNPs was characterized by transmission electron microscopy (TEM, Hitachi HT-7800, Japan) operating at 80 kV. The concentration of AuNPs was calculated to be approximately 2.0 nM according to the Beer-Lambert law. 2.4. ζ-Potential measurements
2. Materials and methods The surface ζ-potentials of AuNPs-aptamer without and with BPA were measured a Zetasizer Nano ZS90 equipment (Malvern Instruments, Worcestershire, UK). 120 μL samples of bare AuNPs, AuNP–aptamer, and AuNP–aptamer with 500 nM BPA were centrifuged at 10,000×g for 20 min and resuspended in 1 mL ultrapure water to remove excess aptamer. A minimum of triplicate measurements per sample were made.
2.1. Materials BPA and its analogues were purchased from Aladdin Industrial Corporation (Shanghai, China). Chloroauric acid tetrahydrate (HAuCl4·4H2O), trisodium citrate dihydrate (C6H5Na3O7·2H2O), sodium chloride (NaCl) and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The oligonucleotides were synthesized and HPLC-purified by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the oligonucleotides used in the experiment were list in Table S1. All other reagents were of analytical grade. Ultrapure water (18.2 MΩ·cm, Milli-Q plus, Millipore, MA, USA) was used for preparing solutions.
2.5. Detection of BPA with AuNPs-based colorimetric aptasensor To minimize effect of the citrate stabilization, AuNPs solution was centrifuged at 10,000×g for 30 min (Eppendorf centrifuge 5810R, Germany) and resuspended with ultrapure water. The purified AuNPs were mixed with 63-mer, 38-mer, and 12-mer aptamers to get the aptamer/particle ratio of 3:1 and incubated at room temperature for 1 h, respectively. Twenty microliter target solutions with different concentrations or sample solutions were added to 100 μL AuNPs-aptamer mixture, and the final AuNPs concentration was 9.26 nM. After incubated for 15 min, 0.5 M NaCl solution was added and mixed
2.2. Truncation and characterization of the aptamer All the truncated aptamers were shortened from the original 63-mer BPA-specific aptamer based on the structural analysis. The predicted secondary structures of all the aptamers were obtained by Mfold web 2
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thoroughly using the pipet to obtain final salt concentration of 33.3 mM. After incubated for another 5 min, the UV–vis absorption spectra of the solutions were measured by multi-mode microplate reader (Molecular Devices SpectraMax M5, USA) and the color changes were recorded by a digital camera. Standard curves were made according to the ΔA650/A520 value and the concentrations of BPA. ΔA650/ A520 = the absorption ratio A650/A520 in sample solutions containing BPA – the absorption ratio A650/A520 in blank solutions without BPA.
redundant sequences. Even the 12-mer aptamer with only 12 nucleotides still have high affinity with BPA, which indicated the 12-mer stem-loop region was the essential domains for BPA binding. These results partly be ascribed that eliminating redundant sequences can increase the stabilization of the 3D conformation of aptamer and improve the affinity for the target molecules. CD spectra technique was involved as a powerful tool to illuminate the changes of the secondary structure of aptamers (Paul & Guchhait, 2011). To investigate the effects of truncation on aptamer conformation, the intrinsic CD profiles of aptamers before and after flanking nucleotides elimination were compared (Fig. S1A). There were a positive peak at around 270 nm and a negative peak at around 244 nm emanated from the base stacking interaction and the helical suprastructure respectively (Cheng, Liu, Shi, & Zhao, 2018), which indicated the typical right-hand B-form structures primarily existed in the original 63-mer aptamer (Huo et al., 2016). After truncation, the 38-mer, 23-mer and 12-mer aptamer showed distinctively different CD spectra. A new shoulder peak of aptamer was observed in the positive peak of 38-mer aptamer. The negative peak of 23-mer aptamer was blue-shifted to 239 nm, and the 270 nm positive peak was split into two peak at 263 and 291 nm. For 12-mer aptamer, the negative peak was slightly blueshifted to 246 nm, and a significant red-shifted to 279 nm was observed in positive peak. Compared to the original one, the second structures of truncated aptamers had changed. The secondary structural changes of aptamers upon binding to BPA were also investigated (Fig. S1B–D). In the addition of BPA, the intensity of the positive band was increased without the peak position shifted obviously in 63-mer, 38-mer and 12-mer aptamers. This revealed that the presence of BPA can induce the conformations of aptamers shifted from B-form to A-form structures with the changes of base stacking and the right-handedness (Lin et al., 2011). The small molecular targets tended to bury in the hydrophobic pocket of the aptamers. To further investigate interactions of BPA and aptamer, the possible binding mode of 12-mer aptamer and BPA was investigated by the molecular docking method. The BPA was docked in the binding site of 38-mer aptamer with the result shown in Fig. 1C. The BPA-aptamer interactions rely on the structural compatibility and the combination of binding forces, such as hydrogen bonding, T-shaped interaction and hydrophobic effect (Cai et al., 2018). BPA was located in the hydrophobic active pocket of 38mer aptamer surrounding by the side chains of the nucleotides G-14, G15, G-16, C-26, G-27, C-28 to form stable hydrophobic bonds. Between BPA and 38-mer aptamer, five hydrogen bonds were formed between the nucleotide G-15, G-16, G-27, C-28. The hydroxyl “O” of BPA formed T-shaped interaction with the amino group of nucleotide G-27, with a bond length of 2.3 Å. The combination of these binding forces can mediate the BPA-aptamer interaction and help the formation of the stable binding complex. The binding free energy of BPA and aptamer evaluated with AutoDock Tools 1.5.6 was −4.90 kcal/mol. The truncated 38-mer and 12-mer aptamers were obtained with improved affinity through removing the redundant sequence. The changes of structural and binding properties were characterized to investigate the effect of truncation. To illustrate the performances of the truncated aptamers in detection method, the 63-mer, 38-mer and 12mer aptamers were then used in the establishment of AuNPs-based colorimetric aptasensors. The sensitivity and specificity of the aptasensors for BPA detection were investigated and compared.
2.6. Samples preparation For validating the practicability in real samples, milk sample, orange juice and mineralized water purchased from RT-mart supermarket (Jinan, China) were assayed by AuNPs-based colorimetric method using the truncated 38-mer aptamer. All samples were spiked with desired concentrations of BPA and allowed to stand for 5 min. The milk sample was centrifuged at 10,000×g for 5 min to remove the upper suction fat. Then the milk sample was mixed with the same volume of ethanol for protein precipitation and centrifuged at 10,000×g for 5 min. The supernatant was transferred to a Ultra-0.5 Centrifugal Filters (MWCO 3 kDa, Millipore, MA, USA). The filtrate solution was diluted 5-fold with ethanol. For orange juice and mineralized water, the samples were diluted for 10-fold with ethanol and filtered through 0.22-µm ultrafiltration membrane to remove the pulps and suspended particles before use. 3. Results and discussion 3.1. Truncation and characterization of the aptamers The full length sequence of aptamer usually contained the nonessential nucleotides that do not interact with target molecular nor act as the contact supporting role (Soheili et al., 2016). These nonessential nucleotides might introduces uncertainty in aptamer tertiary structure which can lead to unstable affinity for targets in different solutions. Meanwhile, too long sequence would also increase the cost of synthesis. Therefore, although the affinity and specificity of original BPA-specific aptamer were basically satisfactory, the length of the 63-mer sequence limited its potential application. To reduce cost of detection and improve the affinity and performance, the truncation of the original 63-mer aptamer was carried out based on the structural analysis. The predicated secondary structures of the aptamers obtained from the Mfold web server were shown in Fig. 1A. Some studies have shown that the region with specific secondary structure such as stem-loop structure, G-quartet loops, bulges and/or pseudoknots are often contributing to the direct binding with the target molecules. There are two stem-loop structures and some flanking sequences existing in the original 63-mer aptamer. Therefore, the aptamer was truncated in three different ways. In the first truncation, two stem-loop structures were kept and the flanking sequences were eliminated in 38-mer aptamer to retain the putative binding domains to the target. In the second truncation, one of the stem-loop structures was kept to get 12-mer aptamer. In the third truncation, the other stem-loop structure was kept to get 23-mer aptamer. The sequences of the original and truncated aptamers were presented in Table S1. Then, systematic revelation of the changing of binding characteristic, secondary and tertiary structures transformation and targetbinding domain identification before and after the truncation of 63-mer aptamer were carried out. The Kd values of the 63-mer, 38-mer, 12-mer and 23-mer aptamers were determined by using MST experiments, which were 491.69 nM, 13.17 nM, 27.05 nM and 1190.61 nM (Table S1). The binding affinities of 38-mer and 12-mer aptamers were enhanced by 37.3-fold and 18.2fold, respectively. The non-linear regression curves of the four aptamers were shown in Fig. 1B. Compared with 63-mer aptamer, the affinity of 38-mer and 12-mer aptamers was obviously improved by removing the
3.2. Construction and characterization of the BPA colorimetric aptasensors The principle of the proposed strategy for BPA colorimetric aptasensor is shown in Fig. 2A. The single stranded DNA aptamers can be absorbed on the surface of AuNPs through van der Waals force and DNA base-gold interaction (Mirau et al., 2018), which protected AuNPs from salt-induced aggregation. When BPA exists in the system, BPA aptamers would specifically bind to BPA to form BPA-aptamer complex due to the 3
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Fig. 1. Characterization of aptamers. (A) The predicted secondary structure models of 63-mer, 38mer, 23-mer and 12-mer BPA-specific aptamers by Mfold. (B) Binding curves of 63-mer, 38-mer, 23mer and 12-mer aptamers with BPA. Error bars: standard deviation (SD), n = 3. (C) The prediction of the binding sites of 12-mer aptamers for BPA by molecular docking.
experimental conditions were monitored by UV–vis spectrometer. As shown in Fig. 2B, the characteristic surface plasmon resonance absorption of AuNPs at 520 nm was decreased remarkably when NaCl was added to the unmodified AuNPs, which indicated that AuNPs aggregated due to salt-induction. Correspondingly, the color of AuNPs solution changed from wine-red to blue. However, if the aptamer exists in AuNPs solution, the stability of AuNPs will be improved and the absorption peak of AuNPs at 520 nm maintained under high salt condition. These phenomena indicated that the aptamer could prevent AuNPs to from aggregation. When the BPA was added, the absorbance at 520 nm decreased dramatically accompanied by a new absorption peak at around 650 nm. The color of the solution changed from winered to blue. It indicated that the added BPA may specifically bind with BPA aptamer, making the AuNPs released and aggregate under high salt condition. To characterize the morphology change of AuNPs at different experimental conditions, TEM technology was utilized (Fig. 2C). The bare AuNPs had a spherical morphology and dispersed evenly. After appropriate NaCl was added, AuNPs were aggregated heavily and formed an irregular network structure. In addition, AuNPs were still well-dispersed in the presence of aptamer under above NaCl concentration existence. However, AuNPs started to aggregate into clusters and the shape was changed obviously with the addition of BPA. Hence, these results were consistent with the proposed principle and the feasibility of this detection method was proved.
high affinity compared with the nonspecific absorption. AuNPs that separated from aptamers will aggregate under the high-salt condition. The color of the solution would gradually change from the original wine-red into purple or blue with the increasing concentrations of BPA. For small target molecules such as BPA, the interaction between the aptamer and the target molecule occurs in a specific binding domain that might be a part of aptamer sequences according to the molecular docking result. The excess flanking sequences outside of the binding region still nonspecifically interacted with the surface of AuNPs when BPA-aptamer complex formed, suppressing the complete dissociation of aptamer from AuNPs. Due to the adherence of flanking sequences, the aggregation of AuNPs was prevented resulting in undesired sensitivity. Therefore, we hypothesized that after the truncation of the aptamers, the sensitivity of colorimetric aptasensor would be enhanced with the improvement of the affinity and the decrease of nonspecific interaction. To investigate the interacting of AuNPs, aptamer and BPA, the surface ζ-potentials of bare AuNPs, AuNPs-aptamer without and with BPA were determined (Table S2). The surface ζ-potentials of AuNPs was decreased after the 63-mer aptamers association from −27.4 mV to −38.8 mV, which was indicate that aptamers were successful adsorbed on AuNPs. After incubation with 500 nM BPA, the ζ-potentials of AuNPs was increased to −33.2 mV, confirming that the aptamers was partly dissociate from the AuNPs when binding to BPA. For random 63-mer sequence, the decreased ζ-potentials confirmed that the sequence can adsorb on the AuNPs after random sequence added. However, there was no significant change in ζ-potentials when incubate with BPA. For the truncated aptamers, more negative ζ-potentials were obtained when AuNPs were incubated with aptamers, from −27.4 mV to −35.4 mV and −33.8 mV for 38-mer and 12-mer aptamers, respectively. When the AuNPs-aptamer mixtures were incubated with 500 nM BPA, the ζpotentials increased to −28.3 mV and −28.0 mV for 38-mer and 12mer aptamers, respectively. The ζ-potentials are close to the original ζpotentials of bare AuNPs (−27.4 mV), which were different from that of 63-mer aptamers. These results indicated that less residual interactions between BPA-bound aptamers and AuNPs were achieved after the truncation of 63-mer aptamer. Therefore, the truncated aptamers dissociated from AuNPs more completely in contrast with the original aptamer, which might be enhance the detection sensitivity. To investigate the feasibility and mechanism of the aptasensors, the characteristic absorption peaks of AuNPs solutions under different
3.3. Optimization of experimental parameters According to the principle of BPA colorimetric aptasensor, experimental conditions such as NaCl concentration, aptamer/AuNPs stoichiometric ratio, and ethanol concentration had a great effect on performance of the colorimetric method. Therefore, these key parameters were optimized by a series of systematic experiments. Due to the variation of the length of the aptamers, the parameters of NaCl concentrations and aptamer-AuNPs stoichiometric ratios were tested with the usage of the 63-mer, 38-mer and 12-mer aptamers, respectively. Controlling the aggregation of AuNPs by NaCl-induced approach is critical for the establishment of AuNPs-based colorimetric aptasensor. Many studies have verified the AuNPs protective effect of aptamer under certain NaCl concentration range (Feng et al., 2019; Mao et al., 4
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Fig. 2. (A) Schematic illustration for AuNP-based colorimetric aptasensor and the mechanism for ultra-sensitive detection of BPA by using the truncated aptamer. (B) UV–vis absorption spectra of AuNPs under different experimental conditions. (C) TEM image of AuNPs under different experimental conditions. Scale bar: 50 nm.
Fig. 3. Optimization of experimental parameters for 63-mer aptamer. (A) NaCl concentration. (B) Ratio of aptamer/AuNPs, n = 3. (C) Ethanol concentration. Error bars: SD, n = 3.
higher aptamer coverage resulted better protection efficiency of aptamer against salt-induced aggregation. Therefore, the concentrations of NaCl optimized for 63-mer aptamer/AuNPs with the ratio of 6:1 and 9:1 were 37.5 and 41.7 mM, respectively. Meanwhile, the NaCl concentrations for 38-mer and 12-mer were also optimized (Fig. S2, Supporting Information). To determine the optimal stoichiometric ratio of aptamer to AuNPs that can yield the highly sensitive detection of BPA, 20 μL BPA of different concentrations was added to complexes with the aptamer/AuNPs ratio of 3:1, 6:1 and 9:1. As shown in Fig. 3B, lower 63-mer aptamer/ AuNPs ratio result in higher sensitivity to salt and sharp transition to aggregation, which can avoid the excess protection of AuNPs toward
2017). Thus, NaCl concentration was optimized for reliable detection. For this purpose, different volumes of 0.5 M NaCl were added to 100 μL samples of bare AuNPs, AuNP-aptamer blended with different aptamer/ AuNPs ratio of 3:1, 6:1 and 9:1, respectively. The relative absorption at 520 nm (A520 (rel.)) was measured to evaluate the degree of AuNPs aggregation. As results shown in Fig. 3A, for the 63-mer aptamer/ AuNPs with the ratio of 3:1, relative absorption at 520 nm was decreased obviously after the concentration of NaCl reached to 33.3 mM. Therefore, the optimal NaCl concentration was 33.3 mM for BPA detection, whereby the AuNPs-aptamer dispersion was on the edge of stability and could be easily disrupted by the introduction of BPA. The 5
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Fig. 4. Absorption spectra of BPA detection using (A) 63-mer aptamers, (C) 38-mer aptamers, (E) 12-mer aptamers. The relationship between the ΔA650/A520 value and the concentration of BPA using (B) 63-mer, (D) 38-mer and (F) 12mer aptamers; Inset shows the linear relationship. Error bars: SD, n = 3. 95% confidence bands are delimited by blue dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the ethanol concentration. These results demonstrated that the addition of ethanol can enhance the recognition efficiency of aptamers. After the concentration of ethanol reached 15% (v/v), the ΔA650/A520 value increased slowly. Therefore, 15% (v/v) was chosen as optimal ethanol concentration.
target-induced aggregation. Therefore, aptamer/AuNPs stoichiometric ratio of 3:1 was defined as the optimal ratio for 63-mer aptamer. Meanwhile, the optimal ratios for 38-mer and 12-mer were also 3:1 as shown in Fig. S2. Organic solvent containing solutions were usually used as binding buffer in aptamer SELEX or aptamer-target binding property evaluation, especially for poorly soluble targets. We assumed that the use of organic solvent would enhance the solubility of target and could participate to the stabilization of the aptamer-target complex. Therefore, common organic solvent ethanol was utilized to improve the sensitivity of BPA detection. For this purpose, different concentrations of ethanol were used in 20 μL BPA sample solution to obtain final ethanol concentration from 0 to 16.7% (v/v) in detection system. ΔA650/A520 value of 20 nM and 60 nM BPA samples were calculated. As shown in Fig. 3C, an increasing trend for ΔA650/A520 value was observed by increasing
3.4. Quantification performance of the BPA colorimetric aptasensors After the optimization of the experimental parameters, the quantification performances of BPA colorimetric aptasensor using the 63-mer, 38-mer and 12-mer aptamers were validated using a series of BPA solutions with different concentrations, respectively. The UV–vis absorption spectrum of BPA colorimetric aptasensors against different BPA concentrations were recorded in Fig. 4. With the increase of the BPA concentration, the absorption peak of 520 nm decreased gradually, 6
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higher LOD (14.41 pM) than 38-mer. The correlation coefficient was 0.9934 in the range of 20 pM to 100 pM. Consistent with our hypothesis, the AuNPs-based colorimetric aptasensor achieved better performance using truncated aptamers. Combined with the results of ζ-potential measurements, the improvement of truncation in quantification performance might be attributed to the enhancement of affinity interaction for BPA when the excess sequences were eliminated and suppressed nonspecific absorption of aptamers on the surface of AuNPs. As shown in Table S3, the performance of this method and other reported methods were compared and summarized. After truncation, the LOD of aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamer, respectively. From the comparisons with other methods, the sensitivity of aptasensor using 38mer aptamer has the merit of a much lower LOD for BPA detection except the GC–MS method (Cao & Popovic, 2018) and electrochemical method (Yu et al., 2019). Although the sensitivity is not as good as these methods, our aptasensor possessed two major advantages. First, the detection was low-cost, because the use of special equipments or modified aptamers was avoided. Second, the detection process was simple and rapid by using this one-step, label-free aptasensor method, without considering expert personnel and time-consuming procedure. Therefore, the method here is capable of offering high detection sensitivity with less cost and simple detection process. 3.5. Specificity of the BPA colorimetric aptasensors The specificity of the aptasensor for BPA detection was also investigated. A variety of BPA analogues, such as bisphenol B (BPB), bisphenol F (BPF), bisphenol S (BPS), estrone (E1), 17β-estradiol (E2), estriol (E3), hexestrol (HES), diethylstilbestrol (DES), dienestrol (DI), progesterone (P) and tetrabromobisphenol A (TBBA) were individually added to the BPA colorimetric aptasensors using the 63-mer, 38-mer and 12-mer aptamers, and the values of ΔA620/A520 were calculated (Fig. 5A). The molecular structures of these analogues were shown in Fig. 5B and revealed the similar chemical structural characteristics as BPA. The truncated aptamers of 38-mer and 12-mer showed similar specificity characteristic as the original 63-mer. As expect, the ΔA650/ A520 values of BPA samples and the mixtures containing BPA had significant increases; however, other analogues did not result in an obvious ΔA650/A520 value increase. The results indicated that all of the established aptasensors using three aptamers were highly specific to BPA and has insignificant cross-reactivity with its analogues.
Fig. 5. (A) Specificity of BPA detection using AuNPs-based colorimetric aptasensor, n = 3. (B) Structures of components used in (A).
and the absorption of 650 nm increased correspondingly. For 63-mer aptamer, the results exhibited in Fig. 4B that ΔA650/A520 value had a good linear correlation with the increase of BPA concentration, which can be fitted as y = 0.01652x + 0.03785. The correlation coefficient was 0.9804 in low-concentration range of 5 nM to 60 nM. The LOD of this method was calculated as 2.02 nM by the concentration corresponding to the ΔA650/A520 value at three times standard deviation of 10 blank samples without BPA. Incubation of BPA with bare AuNPs and with AuNPs modified with a random 63-mer ssDNA did not show any interaction. The result of titration of BPA colorimetric aptasensor using 38-mer aptamer against BPA revealed a 265-fold enhancement of sensitivity compared with the aptasensor using 63-mer aptamer (Fig. 4D). BPA was detected down to 7.60 pM, and a linear response (R2 = 0.9911) was observed between 10 pM and 100 pM. The minimalist 12-mer also being effective, Fig. 4F showed that 12-mer system delivered a slightly
3.6. Repeatability and reproducibility of the BPA colorimetric aptasensors Precision of three BPA colorimetric aptasensors was assessed in terms of repeatability (intra-day precision) and reproducibility (interday precision). Repeatability (r) was calculated as the repeatability relative standard deviation (RSDr) of six consecutive measurements of the same sample of BPA solution (25 nM and 50 pM for the aptasensors
Table 1 Detection of BPA in real samples using BPA colorimetric aptasensor and HPLC-MS (n = 6). Samples
Milk
Orange juice
Mineralized water
Spiked (nM)
2 5 20 2 5 20 2 5 20
Proposed method
HPLC-MS
Found (nM)
Recovery (%) ± RSD (%)
Bias (%)
Found (nM)
Recovery (%) ± RSD (%)
Bias (%)
1.94 4.78 18.78 2.06 4.87 19.65 1.97 5.37 20.56
97.08 ± 6.82 95.60 ± 4.07 93.88 ± 4.56 102.83 ± 7.42 97.33 ± 3.89 98.26 ± 2.99 98.33 ± 8.12 107.30 ± 3.93 102.78 ± 4.15
−2.92 −4.40 −6.12 2.83 −2.67 −4.53 −1.67 7.30 2.78
1.93 5.09 19.56 1.96 4.92 20.85 2.07 5.28 19.79
96.42 ± 5.85 101.83 ± 5.68 97.81 ± 4.76 97.92 ± 3.81 98.43 ± 5.55 101.68 ± 3.06 103.58 ± 3.76 105.50 ± 4.57 98.93 ± 2.04
−3.58 1.83 −2.19 −2.08 −1.57 1.67 3.58 5.50 −1.07
7
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influence the work reported in this paper.
of 63-mer aptamer and truncated aptamers, respectively) within the same day. Similarly, reproducibility (R) was calculated as the reproducibility relative standard deviation (RSDR) of eighteen measurements obtained across three days of analyses. Values of RSDr and RSDR showed good precision for three aptasensors (Table S4). RSDr ranged from 3.37% to 6.07%, and RSDR ranged from 4.45% to 5.22%, which demonstrated good precision of the proposed method.
This study was financially supported by National Key R&D Program of China (2016YFD0401101) and National Natural Science Foundation of China (31871874).
3.7. Detection of BPA in real samples
Appendix A. Supplementary data
To eliminate interference of food matrix, a brief pretreatment was developed for sample preparation. According to the result of the experimental optimization, ethanol was utilized as diluent, protein precipitate and BPA extractant instead of water. BPA colorimetric aptasensor using 38-mer aptamer was employed to investigate the feasibility in real samples analysis. The results of food sample experiments were summarized in Table 1 and compared with those obtained using HPLC-MS. The recovery rate were 93.88%–107.30% vs. 96.42%–105.50% by HPLC-MS, whereas the relative standard deviation (RSD) by this method were 2.99%– 8.12% vs. 2.04%–5.85% by HPLCMS. Trueness is explained by computing bias%. Bias% were −6.12%–7.30% vs. −3.58% to 5.50% by HPLC-MS. A t-test was also performed and the calculated concentrations of BPA colorimetric aptasensor method were not statistically different from that of HPLC-MS method, with a 95% of confidence interval. Therefore, the developed BPA colorimetric aptasensor might be initially applied for the determination of BPA in real samples.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2020.126459.
Acknowledgements
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4. Conclusion In this study, aptamers with the length of 38-mer and 12-mer for BPA were obtained through rationally designed truncation. A systematic comparison of original and truncated aptamers on binding and structure characteristic were investigated and the result indicated that both 38-mer and 12-mer aptamers showed high affinity toward BPA. The molecular docking assay offered rational explanations of the interactions between BPA and aptamer. Then the 63-mer, 38-mer and 12mer aptamers were used as recognition elements to detect BPA through AuNPs-based colorimetric method. The AuNPs-based colorimetric aptasensors achieved high performance using truncated aptamers. The LOD of the aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamers, respectively. Meanwhile, the aptasensors using 38-mer and 12-mer aptamers showed high selectivity against other 11 kinds of BPA analogues. More importantly, the practicability of the colorimetric aptasensor was verified by detection of BPA in milk, orange juice and water samples. With excellent binding characteristics, optimized length and interference resistance in the detection, the truncated 38-mer and 12-mer aptamers exhibit great application potential in food safety control and environmental monitoring. Meanwhile, appropriate design of sensing configuration and the application of other unique nanomaterials, the performance of detection may be further improved. CRediT authorship contribution statement Min Jia: Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision. Junyi Sha: Validation, Formal analysis, Investigation, Writing - original draft. Zhihua Li: Validation, Investigation. Wenjing Wang: Validation, Investigation. Hongyan Zhang: Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to 8
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