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Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission
Analytica Chimica Acta xxx (xxxx) xxx
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Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregationinduced emission Man Zhang a, Yu Wang b, Xuan Sun b, Jialei Bai b, Yuan Peng b, Baoan Ning b, Zhixian Gao b, **, Baolin Liu a, * a
School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, People’s Republic of China Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environment and Operational Medicine, Tianjin, 300050, People’s Republic of China
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
This study firstly uses strand displacement amplification and DNA G-quadruplex with aggregationinduced emission (AIE) to detect small-molecule patulin toxin, achieving signal amplification to obtain a lower limit of detection. The AIE material provides a more convincing experimental validation. Compared with general fluorescent dyes, the combination effect is better and the sensitivity is higher. This work provided a new idea for the detection of mycotoxins, which is more convenient and faster than the traditional method. This approach provided a broader perspective on the establishment of rapid food safety testing methods.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2019 Received in revised form 25 January 2020 Accepted 29 January 2020 Available online xxx
A novel sensitive assay was established by using strand displacement amplification (SDA) and DNA Gquadruplex with aggregation-induced emission (AIE) for the detection of patulin (PAT) toxin. The complementary DNA (cDNA) of the aptamer and PAT competed for binding to aptamer-modified magnetic beads. The cDNA was obtained by magnetic separation and used as a primer in SDA to produce a large amount of G-base single-stranded DNA (ssDNA). They can form the G-quadruplex to be combined with the AIE of TTAPE dye, which features a special combination of G-quadruplex that amplify the fluorescent signals. This work can reach a lower detection limit of 0.042 pg mL1 with a wide linear range of 0.001e100 ng mL1 for PAT detection than other methods. The results also showed good recoveries of 97.8%e104% and 101.7%e105.3% in spiked apple and grape juices, respectively. The assay used for the
Keywords: Patulin SDA Aptamer G-quadruplex
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Z. Gao), [email protected] (B. Liu). https://doi.org/10.1016/j.aca.2020.01.064 0003-2670/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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TTAPE AIE
detection of PAT exhibits high sensitivity and good specificity. It also provides a stable and reliable platform for detecting other small-molecule toxins. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Patulin (PAT), 4-hydroxy-4H-furo[3,2c] pyran-2[6H]-one, is a kind of mycotoxin produced by fungi such as Aspergillus and Penicillium. It is widely found in various moldy fruits and silage, especially in mildewed apples and apple juice [1,2]. Due to PAT is teratogenic and can damage the respiratory and urinary systems, causing nerve palsy and pulmonary edema [3], the maximum residue limit of PAT has been set at 0.4 mg/kg body weight by the World Health Organization and the Food and Agriculture Organization [4]. There are many traditional methods have been established to detect PAT toxin, including high-performance liquid chromatography (HPLC) coupled with diode array [5], gas chromatographyemass spectrometry (GC-MS) [6], electrochemical biosensing [7], and quartz-crystal microbalance(QCM) [8]. Although these methods show good accuracy and reproducibility, some of them require expensive instruments and skilled operators. In particular, immunological methods show good sensitivity but require the preparation of specific antibodies to PAT, which is expensive and timeconsuming [9,10]. At present, the detection methods of PAT still need to be improved and optimized. Therefore, a fast and sensitive method for detecting PAT in cereal products is urgently needed. Aptamers are a piece of DNA or RNA sequence that can be obtained by systematic evolution of ligands by exponential enrichment (SELEX) [11]. Because of their high affinity and selectivity, the configuration changes accordingly when they bind to targets [12]. The aptamer of PAT was selected and identified by Wu et al. [14] Several researchers have used this aptamer to detect PAT and obtained good results [15,16]. In addition, DNA isothermal signal amplification and good materials can be used to improve the sensitivity of aptamer sensors [17,18]. Isothermal amplification is a simple system [19] that needs no temperature change [20], features quick operation, and can be combined with various systems [21]. A variety of isothermal amplification methods are widely used in the field of biosensing [22]. Strand displacement amplification (SDA) is simpler and more stable than other amplification methods [23]. The design of the template and primer is simple, and the reaction conditions are mild [24]. The SDA can produce large amounts of single-stranded DNA (ssDNA); hence, some researchers use DNA stain, such as SYBR Green I, to achieve a single output. However, this method generally shows poor specificity [25]. Eventually, researchers used ssDNA configuration to form a special secondary structure, such as the Gquadruplex, by hemin staining to achieve visual inspection [26], but this method features insufficient sensitivity. Subsequently, tetrahydrothiophene (ThT) [27] and N-methylmorpholine (NMM) [28] were combined with G-quadruplex to generate a fluorescent signal; however, real-time monitoring was impossible, and the control of reaction processes for forming the G-quadruplex lacked accuracy. AIE and DNA binding research received increasing attention [29]. AIE as a new type of fluorescent materials are non-emissive in solution but become highly emissive during aggregation so they can serve as good fluorescent bio-probes for biosensor detection [30]. For example, the designer can synthesize an AIE material that specifically binds to T-base-rich DNA [31] or ssDNA but cannot adsorb double-stranded DNA (dsDNA) [32]. Compared with other kinds of AIE materials that can bind to G-quadruplex [33,34], TTAPE is a new
dye that is highly affinitive to G-base-rich DNA and is completely soluble in water [35]. It can combine with G-base-rich DNA, whereas metal ions are then added to form the TTAPE/G-quadruplex, which features a strong fluorescence was measured at excitation and emission wavelengths of 350 and 490 nm, respectively. Therefore, TTAPE can be used as a good material for the combination with Gquadruplex, which bears importance in biological applications. Herein, we have established a method for SDAeDNA G-quadruplexeAIE to achieve the highly sensitive and rapid detection of PAT. In this work, we used aptamers to identify the target PAT and the cDNA. The cDNA competed with PAT through the aptamer and the system can rely on magnetic adsorption to separate the cDNA from the solution as a primer for SDA because the aptamers were modified on magnetic nanoparticles. Then, the primer can be used in SDA to produce considerable G-base-rich DNAs, which were combined with TTAPE to form TTAPE/G-DNA. After adding Kþ to form the G-quadruplex, the TTAPE/G-quadruplex complex emitted a strong fluorescence. This method exhibited high specificity and can determine PAT from actual samples. 2. Experimental 2.1. Reagents All HPLC-purified oligonucleotide sequences (Table S1) were synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Magnetic beads (streptavidin-modified) were purchased from Thermo Fisher Scientific Co., Ltd (Shanghai, China). Nb.BbvCI nicking enzyme, Klenow fragment (3ʹ/5ʹ exo) DNA polymerase (KF), CutSmart buffer, and deoxynucleotide triphosphates (dNTPs) were obtained from New England BioLabs (Ipswich, MA, U.S.A.). PAT, T-2, aflatoxins B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEN) were obtained from JLG Technology Co., Ltd (Beijing, China). TTAPE was purchased from AIEgen Biotech Co., Ltd (Hong Kong, China). All other chemicals (analytical reagent) used in this work were obtained from standard reagent companies and without additional purification. All buffers were prepared by using pure water (18.2 MU cm) obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, U.S.A). 2.2. Apparatus All fluorescence analysis data in the experiment were obtained by using a F97pro device fluorescence spectrophotometer (Shanghai Lengguang Technology Co., Ltd.). Aseptic operations in the tests were performed in an Esco Biotech Class II Biohazard Safety Cabinet (Jakarta, Indonesia). Centrifuges (Shanghai Anting Scientific Instrument Factory Co., Ltd., Shanghai, China), OSE-DB-02 refrigerated five-stage programmable metal bath (Tiangen Biochemical Technology Co., Ltd., Beijing, China), DYY-11 electrophoresis apparatus (Liuyi Instrument Factory, Beijing, China), and ImageQuant 350 gel imaging system (GE Healthcare, U.S.A.) were also used in the experiments. 2.3. Preparation of PAT aptamer-functionalized magnetic beads Streptavidin-modified magnetic beads (50 mL, 10 mg mL1) were
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
M. Zhang et al. / Analytica Chimica Acta xxx (xxxx) xxx
removed from the 4 C refrigerator and transferred into a lowadsorption centrifuge tube after concussion mixing. Then, an equivalent phosphate buffered saline solution (PBS) was added to form a 5 mg mL1 system. The frozen biotinylated PAT aptamer was retrieved at 20 C and centrifuged at 4000 rpm (537.6 rcf) for 60 s. The aptamer solution was mixed with the magnetic beads and incubated for 15 min in a vertical suspension meter at 37 C. Finally, the solution was placed in the magnetic frame for 3 min. The upper liquid was discarded, and the remaining solution was washed thrice with PBS. PAT aptamer-functionalized magnetic beads were obtained and stored at 4 C until use. 2.4. cDNA competes with PAT for aptamer PAT aptamer-functionalized magnetic beads (100 mL) were mixed with cDNA (100 mL) and various concentrations of PAT (200 mL) at 37 C for 60 min. The same steps as the previous magnetic separation were used in the operation, but the supernatant was retained. cDNA was obtained and used as the primer in SDA. 2.5. SDA reaction The SDA loading operation was performed in a biosafety station. The supernatant obtained above was selected as the primer for SDA reaction. Template DNA (20 mL) and 1 x CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 mg mL1 bovine serum albumin) were added and incubated at 95 C for 5 min and then slowly cooled to room temperature. Next, Nb.BbvCI nicking enzyme (10 U mL1), KF (5 U mL1), and dNTPs (10 mM) were added to the system. The 100 mL reaction volume was achieved by using pure water and then evenly mixing with the mixture. Finally, after incubation at 37 C for 60 min and 80 C for 20 min, the amplification was stopped. The amplification product was stored at 4 C for further use. 2.6. Native polyacrylamide gel electrophoresis (PAGE) Native PAGE (15%) was prepared with 1 TBE buffer (90 mM Tris-HCl, 2 mM EDTA, 90 mM boric acid, pH 7.8). Then, the gel was
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run at 120 V for 60 min and stained with gene green for 45 min. PAGE was performed to verify the SDA products and the feasibility of the experiment. 2.7. Synthesis of DNA G-quadruplex and AIE fluorometric detection After SDA reaction, the product was incubated with TTAPE (10 mM) in Tris-HCl (5 mM) buffer for 30 min at 4 C to form the TTAPE/G-DNA. The free TTAPE dye showed no emission but produced fluorescence at the excitation wavelength of 350 nm when combined with DNA rich in G-base. G-quadruplex can be formed by adding Kþ (1 M) for 30 min at room temperature. Then, fluorescence can be obtained because the TTAPE/G-quadruplex mixture exhibited a strong emission at 490 nm. 2.8. Detection of PAT in real sample The established method was verified to determine whether it can detect PAT in grape juice (water, sugar, 100% concentrated white grape juice) and apple juice (water, 100% concentrated apple juice) purchased from local supermarkets. The sample preparation steps were adopted from the work of Wu [14]. The samples were placed in a test tube, added with different concentrations of PAT toxin (0.05, 0.5, 5, and 50 ng mL1), mixed with an equal volume of ethyl acetate, and centrifuged at 5000 rpm (2348g) for 5 min at room temperature. The supernatant was retained, dried under nitrogen, and filtered through a 0.22 mm nylon syringe. The dried powder was dissolved in PBS buffer for subsequent experiments. Finally, a sample was obtained for the next detection. 3. Results and discussion 3.1. Principle of SDA and DNA G-quadruplex with AIE assay for PAT detection The principle of the assay is based on SDA with AIE strategy. As shown in Scheme 1, streptavidin magnetic beads were combined with a biotinylated aptamer to form an aptamer magnetic bead. Subsequently, this magnetic bead system was added to the cDNA
Scheme 1. Schematic of SDAeG-quadruplexeAIE assay is based on strand isothermal amplification and TTAPE/G-quadruplex strategy.
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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and PAT toxin for competitive reaction. Several aptamers bound to the complementary strand, whereas others bound to the target PAT. When aptamer, cDNA, and PAT had fully reacted, the cDNA of supernatant not bound to the aptamer was retained through magnetic separation and used as primer for SDA. SDA reaction can be carried out using the primer, template DNA, and enzymes to complete the efficient fluorescence signal amplification. The template DNA was divided into three parts: a part complementary to the supernatant primer, another part containing a specific cleavage site of the Nb.BbvCI nicking enzyme, and a part rich in C base. Primer and template DNA were complemented by KF and dNTPs, and Nb.BbvCI can cleave the ssDNA at specific sites of dsDNA to obtain a large number of G-base-enriched ssDNA repeatedly. TTAPE showed a good affinity for DNA rich in G-base. Therefore, no fluorescence effect can be observed when only TTAPE was present. However, TTAPE bound to G-DNA disrupted the internal mechanism of the molecules and emitted strong fluorescence. Moreover, the addition of Kþ caused the TTAPE/G-DNA mixture to form a large amount of TTAPE/G-quadruplex, and more TTAPE aggregated to fluoresce, resulting in a stronger fluorescent signal. However, the primer cannot be obtained when the target PAT was absent, preventing the SDA and the formation of TTAPE/G-DNA complex. Therefore, the fluorescent signal output is closely related to the concentration of PAT. Thus, the proposed method can quantitatively detect PAT. 3.2. Characterization and feasibility verification Circular dichroism (CD) spectroscopy was performed to verify the affinity of the selected PAT aptamer and characterize the DNA G-quadruplex. The aptamer, PAT, and PAT-aptamer conjugates were determined via MOS-450/AF-CD ultravioletevisible spectrometer; the corresponding spectra are shown in Fig. S1. When only PAT or aptamer was present, PAT exhibited no peak nor trough, whereas the aptamer presented a trough and a peak at 250 and 280 nm, respectively, which are characteristics of DNA. However, the mixture of PAT and aptamer displayed a weakly negative cotton peak at 250 nm and a positive cotton peak at 280 nm. The characteristic peak position was unchanged, but the intensity was weakened. This result indicates that the aptamer bound well to PAT because the base stacking and helical structure of the aptamer changed due to binding. The CD spectrum of the G-quadruplex showed a negative peak at 240 nm and a positive peak at 265 nm. By contrast, the CD spectrum of the DNA template strongly increased at 240 and 280 nm which proved that G-quadruplex was formed given the changes in the spatial conformation of the DNA template. In addition, the fluorescence spectrum of the incomplete SDA (Fig. 1A) verified the feasibility of the experiment. The fluorescence intensity was weak when no primer, template, nor any kind of enzyme was applied. Hence, SDA reaction could not be completed, the G-base-rich ssDNA could not be formed in large amounts, and the TTAPE/G-quadruplex complex could not be formed. Fluorescence intensity reached the highest value when the reaction system was integral. The SDA product was investigated via PAGE. The results were visualized through ImageQuant (Fig. 1B). The SDA reaction induced a large number of ssDNA and achieved amplification in the manner that we expected. The absence of primer, KF, Nb.BbvCI nicking enzyme, template DNA, or dNTPs could not produce the target ssDNA. Therefore, SDA can generate a large amount of G-base-rich ssDNA to form G-quadruplex, which should be combined with TTAPE to obtain a fluorescence signal. We also used PAGE to verify the SDA products of different concentrations (1 nM, 100 nM, 10 mM) were stained with SYBR Green I and TTAPE, respectively, and observed the bands under the gel imager (Fig. S7). It also illustrated that TTAPE was more sensitive to G-base-
Fig. 1. (A) Fluorescence spectra of a system without complete SDA. TTAPE/Gquadruplex cannot be formed to produce fluorescence. (B) M: 20 bp marker; lane 1: all in; lane 2: no KF; lane 3: no Nb. BbvCI; lane 4: no dNTPs; lane 5: no template DNA.
rich DNA staining and more conducive to obtaining highly sensitive signals.
3.3. Optimization of SDA reaction and the concentration of cDNA and aptamer We optimized the conditions in the reaction process accordingly to obtain more SDA products. As shown in Fig. S2, the following parameters were optimized: (a) concentration of Nb.BbvCI nicking enzyme, (b) concentration of KF polymerase, and (c) time of amplification. The single-variable principle was followed when optimizing one of the conditions while keeping the other conditions the same. We obtained the following results: (a) the best concentration of Nb. BbvCI nicking enzyme is 16 U, (b) the most suitable concentration of KF polymerase enzyme is 5 U, and (c) the optimal amplification time is 60 min. We also optimized the concentration of aptamer combined with streptavidin magnetic beads (Fig. S3). cDNA concentration was used to compete with the target as shown in Fig. S4. The results can be obtained from the fluorescence spectrum. The fluorescence intensity of the supernatant increased when the concentration of carboxyfluorescein (FAM)e aptamer was gradually increased. Combined with the measurement of the supernatant after coupling reaction by the nucleic acid meter, when the concentration of the aptamer was 10 nM, certain conditions were required for the reaction without wasting materials. In addition, the instructions for the purchased streptavidin
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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3.4. Analytical performance of the assay Fig. 2A shows the fluorescence intensity spectra obtained by adding different concentrations of PAT to the detection system. Fluorescence intensity gradually increased when the PAT concentration increased (0.001,0.005 0.01, 0.05,0.1, 0.5,1, 5,10,50 and 100 ng mL1) based on the above optimization conditions. In addition, the results in Fig. 2B show that fluorescence intensity featured a strong linear correlation in the range of
Fig. 2. (A) Fluorescence spectra of SDA-G-quadruplex with TTAPE for PAT detection at excitation and emission wavelengths of 350 and 490 nm, respectively, and the wide detection range of 0.001e100 ng mL1. (B) Linear relationship of the fluorescence (DF/ F0) and logarithm concentration of PAT. The linear equation is y ¼ 1.3481 þ 0.2086lgx (R2 ¼ 0.9946) in the range of 0.001e100 ng mL1.
magnetic beads indicated the optimum DNA concentration for coupling, that is, 1 mg magnetic beads can be coupled to 200 pM DNA. Thus, we selected the aptamer concentration of 10 nM. Fig. S4 shows that the remaining FAM-cDNA after binding to the aptamer increased in fluorescence intensity as the concentration of the cDNA increased. However, the amount of DNA remaining in the solution was small when the concentration of the cDNA was 1 or 10 nM. Almost all of the aptamers attached to the magnetic beads. The fluorescence value increased remarkably when the concentration reached 100 nM and considerably increased further when the concentration reached 1 and 10 mM, indicating the excessive amounts of the remaining primers. Therefore, the optimum connection concentration is 100 nM.
Fig. 3. (A) Fluorescence spectrum of specificity for the detection of different smallmolecule toxins with three different concentrations (10, 100, and 1000 ng mL1) by the proposed method; (B) changes in fluorescence intensity. PAT showed the highest fluorescence value, whereas other similar toxins (T-2, FB1, OTA, AFB1, and ZEN) produced poor fluorescence.
Table 1 Summarized methods and LODs for PAT toxin detection. Analytical methods
Linear range
LOD
Sample
Ref.
MIP-ESP AgNPs@ZnMOF-MIP NIR fluorescence assay LC-MS/MS GC-MS AuNP-BP NSs based aptasensing HPLC-Magnetic SPE SDA-G-quadruplex-AIE aptasensor
0.002e2 ng/mL 0.1e10 mM/L 0e2.4 pM 0.064e1.6 mM 40e2500 mg/L 0.1 nM-10 mM 0.006e2.6 mM 0.001e100 ng/mL
0.001 ng/mL 0.06 mmol/L 0.06 mg/L 0.035 mM 0.4 mg/L 0.03 nM 0.001 mM 0.042 pg/mL
Apple and grape juice Environmental water and apple juice Apple juice Apple juice Apple juice Apple juice Apple juice Apple and grape juice
[36] [37] [9] [38] [6] [39] [40] This work
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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Table 2 Recovery of PAT at different concentration levels in apple juice and grape juice (n ¼ 6). Sample
Apple juice
Grape juice
Spiked concentration (ng mL1)
0.05 0.5 5 50 0.05 0.5 5 50
SDA-G-quadruplex-AIE
HPLC
Recovery (%)
RSD(%)
Recovery (%)
RSD(%)
104 4.4 100 66 105.3 2.37 101.8 5.58
102 1.01 97.8 1.87 102.5 1.75 101.7 2.27
127.0 3.6 100.4 5.7 122.0 3.4 98 5.2
102.4 2.9 100.2 4.3 86.0 2.5 99.7 5.8
0.001e100 ng mL1, and the linear equation is y ¼ 1.3481 þ 0.2086lgx (R2 ¼ 0.9946). The limit of detection of the sensing system was as low as 0.042 pg mL1 (S/N ¼ 3). We also performed validation without SDA, and the fluorescence spectrum is shown in Fig. S5. When no amplification was carried out, adding different concentrations of target to obtain the supernatant primer was rarely sufficient to combine with TTAPE to produce a strong fluorescence, and thus, a good linear relationship was not obtained. Therefore, SDA substantially improved the sensitivity and effectively amplified the signal. We further explored the effect of Kþ on the reaction results in Fig. S6. Primers with different concentrations showed minimal effect on fluorescence intensity when no Kþ was present. This result indicated that the amplification products failed to form the G-quadruplex complex and cannot combine with TTAPE. The fluorescence value was enhanced after Kþ was added. Hence, Kþ considerably improved the binding of SDA products to TTAPE. To verify the reproducibility of the experiment, according to the established method, we selected (0.01 ng mL1, 1 ng mL1, 100 ng mL1) three different concentrations of the target PAT and performed six repetitive experiments (Fig.S8). The results were good, and the obtained fluorescence curves were also related to the same before. The proposed method was compared with other current methods (Table 1). The proposed assay is notably more sensitive and features a wider analytical range than other methods. 3.5. Evaluation of specificity We evaluated the specificity of the proposed sensing experiment for PAT detection by determining and comparing the fluorescent signal changes caused by PAT and its four kinds of structural analogues (FB1, OTA, ZEN, T-2, and AFB1). As illustrated in Fig. 3, toxins with three concentrations (10, 100, and 1000 ng mL1) were selected to compete with the cDNA of the PAT aptamer, and each measurement was repeated thrice. Based on the results, PAT exhibited an evident dramatic fluorescence intensity, whereas other similar toxins produced weak reactions. The changes in the fluorescence intensity may be due to the specific binding of the PAT aptamer to PAT molecules. Therefore, the established method features a good specificity for PAT. 3.6. Actual sample detection and methodological comparison Spiked recovery experiments and methodological comparisons were conducted to evaluate the stability and accuracy of the SDAeG-quadruplexeAIE assay. The concentrations of PAT in spiked apple and grape juices were detected using the developed method and HPLC. No significant difference was observed between the two methods, indicating their good compatibility. Table 2 summarizes the results of recoveries and relative standard deviations (RSDs). The apple and grape juices showed average recoveries of 97.8%e 104% and 101.7%e105.3%, The RSDs reached 1.01%e4.4% and 1.75%e
5.58%, respectively. These results revealed that our method possesses good accuracy and can sensitively determine PAT in real samples. HPLC analysis was conducted using an isocratic system at 0.8 mL/min. A constant volume (15 mL) of each sample was injected and eluted with 5% acetonitrile in water (retention time of 9 min). 4. Conclusions We believe that this work is the first to report the detection of PAT toxin based on SDA and G-quadruplex with AIE. Functionalized aptamer magnetic beads realized the recognition function of PAT, and cDNA competed with PAT for binding to the aptamer to provide the primer for SDA. High amounts of G-base-rich DNA were achieved by SDA. The combination of G-quadruplex and TTAPE achieved an extremely strong fluorescence intensity. TTAPE is a highly stable and simple probe that can considerably help in obtaining the signal output after forming the DNA secondary structure. It has been used for the detection of small-molecule toxins and has achieved high sensitivity and rapid detection. This study also showed that the combination of isothermal amplification of DNA with AIE can be useful in food safety testing, biomedical, and other fields. Funding This work was supported by the National Key Research and Development Program of China (No. 2018YFC1602903, 2017YFC1200903), and the Key Research and Development Program of Tianjin (No.18YFZCNC01260). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Man Zhang: Writing - original draft. Yu Wang: Software. Xuan Sun: Data curation. Jialei Bai: Project administration. Yuan Peng: Writing - review & editing. Baoan Ning: Writing - review & editing. Zhixian Gao: Funding acquisition. Baolin Liu: Funding acquisition. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2020.01.064. References [1] R. Steiman, et al., Production of patulin by micromycetes, Mycopathologia 105 (3) (1989) 129e133.
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Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregationinduced emission Man Zhang a, Yu Wang b, Xuan Sun b, Jialei Bai b, Yuan Peng b, Baoan Ning b, Zhixian Gao b, **, Baolin Liu a, * a
School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, People’s Republic of China Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environment and Operational Medicine, Tianjin, 300050, People’s Republic of China
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
This study firstly uses strand displacement amplification and DNA G-quadruplex with aggregationinduced emission (AIE) to detect small-molecule patulin toxin, achieving signal amplification to obtain a lower limit of detection. The AIE material provides a more convincing experimental validation. Compared with general fluorescent dyes, the combination effect is better and the sensitivity is higher. This work provided a new idea for the detection of mycotoxins, which is more convenient and faster than the traditional method. This approach provided a broader perspective on the establishment of rapid food safety testing methods.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2019 Received in revised form 25 January 2020 Accepted 29 January 2020 Available online xxx
A novel sensitive assay was established by using strand displacement amplification (SDA) and DNA Gquadruplex with aggregation-induced emission (AIE) for the detection of patulin (PAT) toxin. The complementary DNA (cDNA) of the aptamer and PAT competed for binding to aptamer-modified magnetic beads. The cDNA was obtained by magnetic separation and used as a primer in SDA to produce a large amount of G-base single-stranded DNA (ssDNA). They can form the G-quadruplex to be combined with the AIE of TTAPE dye, which features a special combination of G-quadruplex that amplify the fluorescent signals. This work can reach a lower detection limit of 0.042 pg mL1 with a wide linear range of 0.001e100 ng mL1 for PAT detection than other methods. The results also showed good recoveries of 97.8%e104% and 101.7%e105.3% in spiked apple and grape juices, respectively. The assay used for the
Keywords: Patulin SDA Aptamer G-quadruplex
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Z. Gao), [email protected] (B. Liu). https://doi.org/10.1016/j.aca.2020.01.064 0003-2670/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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TTAPE AIE
detection of PAT exhibits high sensitivity and good specificity. It also provides a stable and reliable platform for detecting other small-molecule toxins. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Patulin (PAT), 4-hydroxy-4H-furo[3,2c] pyran-2[6H]-one, is a kind of mycotoxin produced by fungi such as Aspergillus and Penicillium. It is widely found in various moldy fruits and silage, especially in mildewed apples and apple juice [1,2]. Due to PAT is teratogenic and can damage the respiratory and urinary systems, causing nerve palsy and pulmonary edema [3], the maximum residue limit of PAT has been set at 0.4 mg/kg body weight by the World Health Organization and the Food and Agriculture Organization [4]. There are many traditional methods have been established to detect PAT toxin, including high-performance liquid chromatography (HPLC) coupled with diode array [5], gas chromatographyemass spectrometry (GC-MS) [6], electrochemical biosensing [7], and quartz-crystal microbalance(QCM) [8]. Although these methods show good accuracy and reproducibility, some of them require expensive instruments and skilled operators. In particular, immunological methods show good sensitivity but require the preparation of specific antibodies to PAT, which is expensive and timeconsuming [9,10]. At present, the detection methods of PAT still need to be improved and optimized. Therefore, a fast and sensitive method for detecting PAT in cereal products is urgently needed. Aptamers are a piece of DNA or RNA sequence that can be obtained by systematic evolution of ligands by exponential enrichment (SELEX) [11]. Because of their high affinity and selectivity, the configuration changes accordingly when they bind to targets [12]. The aptamer of PAT was selected and identified by Wu et al. [14] Several researchers have used this aptamer to detect PAT and obtained good results [15,16]. In addition, DNA isothermal signal amplification and good materials can be used to improve the sensitivity of aptamer sensors [17,18]. Isothermal amplification is a simple system [19] that needs no temperature change [20], features quick operation, and can be combined with various systems [21]. A variety of isothermal amplification methods are widely used in the field of biosensing [22]. Strand displacement amplification (SDA) is simpler and more stable than other amplification methods [23]. The design of the template and primer is simple, and the reaction conditions are mild [24]. The SDA can produce large amounts of single-stranded DNA (ssDNA); hence, some researchers use DNA stain, such as SYBR Green I, to achieve a single output. However, this method generally shows poor specificity [25]. Eventually, researchers used ssDNA configuration to form a special secondary structure, such as the Gquadruplex, by hemin staining to achieve visual inspection [26], but this method features insufficient sensitivity. Subsequently, tetrahydrothiophene (ThT) [27] and N-methylmorpholine (NMM) [28] were combined with G-quadruplex to generate a fluorescent signal; however, real-time monitoring was impossible, and the control of reaction processes for forming the G-quadruplex lacked accuracy. AIE and DNA binding research received increasing attention [29]. AIE as a new type of fluorescent materials are non-emissive in solution but become highly emissive during aggregation so they can serve as good fluorescent bio-probes for biosensor detection [30]. For example, the designer can synthesize an AIE material that specifically binds to T-base-rich DNA [31] or ssDNA but cannot adsorb double-stranded DNA (dsDNA) [32]. Compared with other kinds of AIE materials that can bind to G-quadruplex [33,34], TTAPE is a new
dye that is highly affinitive to G-base-rich DNA and is completely soluble in water [35]. It can combine with G-base-rich DNA, whereas metal ions are then added to form the TTAPE/G-quadruplex, which features a strong fluorescence was measured at excitation and emission wavelengths of 350 and 490 nm, respectively. Therefore, TTAPE can be used as a good material for the combination with Gquadruplex, which bears importance in biological applications. Herein, we have established a method for SDAeDNA G-quadruplexeAIE to achieve the highly sensitive and rapid detection of PAT. In this work, we used aptamers to identify the target PAT and the cDNA. The cDNA competed with PAT through the aptamer and the system can rely on magnetic adsorption to separate the cDNA from the solution as a primer for SDA because the aptamers were modified on magnetic nanoparticles. Then, the primer can be used in SDA to produce considerable G-base-rich DNAs, which were combined with TTAPE to form TTAPE/G-DNA. After adding Kþ to form the G-quadruplex, the TTAPE/G-quadruplex complex emitted a strong fluorescence. This method exhibited high specificity and can determine PAT from actual samples. 2. Experimental 2.1. Reagents All HPLC-purified oligonucleotide sequences (Table S1) were synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Magnetic beads (streptavidin-modified) were purchased from Thermo Fisher Scientific Co., Ltd (Shanghai, China). Nb.BbvCI nicking enzyme, Klenow fragment (3ʹ/5ʹ exo) DNA polymerase (KF), CutSmart buffer, and deoxynucleotide triphosphates (dNTPs) were obtained from New England BioLabs (Ipswich, MA, U.S.A.). PAT, T-2, aflatoxins B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEN) were obtained from JLG Technology Co., Ltd (Beijing, China). TTAPE was purchased from AIEgen Biotech Co., Ltd (Hong Kong, China). All other chemicals (analytical reagent) used in this work were obtained from standard reagent companies and without additional purification. All buffers were prepared by using pure water (18.2 MU cm) obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, U.S.A). 2.2. Apparatus All fluorescence analysis data in the experiment were obtained by using a F97pro device fluorescence spectrophotometer (Shanghai Lengguang Technology Co., Ltd.). Aseptic operations in the tests were performed in an Esco Biotech Class II Biohazard Safety Cabinet (Jakarta, Indonesia). Centrifuges (Shanghai Anting Scientific Instrument Factory Co., Ltd., Shanghai, China), OSE-DB-02 refrigerated five-stage programmable metal bath (Tiangen Biochemical Technology Co., Ltd., Beijing, China), DYY-11 electrophoresis apparatus (Liuyi Instrument Factory, Beijing, China), and ImageQuant 350 gel imaging system (GE Healthcare, U.S.A.) were also used in the experiments. 2.3. Preparation of PAT aptamer-functionalized magnetic beads Streptavidin-modified magnetic beads (50 mL, 10 mg mL1) were
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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removed from the 4 C refrigerator and transferred into a lowadsorption centrifuge tube after concussion mixing. Then, an equivalent phosphate buffered saline solution (PBS) was added to form a 5 mg mL1 system. The frozen biotinylated PAT aptamer was retrieved at 20 C and centrifuged at 4000 rpm (537.6 rcf) for 60 s. The aptamer solution was mixed with the magnetic beads and incubated for 15 min in a vertical suspension meter at 37 C. Finally, the solution was placed in the magnetic frame for 3 min. The upper liquid was discarded, and the remaining solution was washed thrice with PBS. PAT aptamer-functionalized magnetic beads were obtained and stored at 4 C until use. 2.4. cDNA competes with PAT for aptamer PAT aptamer-functionalized magnetic beads (100 mL) were mixed with cDNA (100 mL) and various concentrations of PAT (200 mL) at 37 C for 60 min. The same steps as the previous magnetic separation were used in the operation, but the supernatant was retained. cDNA was obtained and used as the primer in SDA. 2.5. SDA reaction The SDA loading operation was performed in a biosafety station. The supernatant obtained above was selected as the primer for SDA reaction. Template DNA (20 mL) and 1 x CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 mg mL1 bovine serum albumin) were added and incubated at 95 C for 5 min and then slowly cooled to room temperature. Next, Nb.BbvCI nicking enzyme (10 U mL1), KF (5 U mL1), and dNTPs (10 mM) were added to the system. The 100 mL reaction volume was achieved by using pure water and then evenly mixing with the mixture. Finally, after incubation at 37 C for 60 min and 80 C for 20 min, the amplification was stopped. The amplification product was stored at 4 C for further use. 2.6. Native polyacrylamide gel electrophoresis (PAGE) Native PAGE (15%) was prepared with 1 TBE buffer (90 mM Tris-HCl, 2 mM EDTA, 90 mM boric acid, pH 7.8). Then, the gel was
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run at 120 V for 60 min and stained with gene green for 45 min. PAGE was performed to verify the SDA products and the feasibility of the experiment. 2.7. Synthesis of DNA G-quadruplex and AIE fluorometric detection After SDA reaction, the product was incubated with TTAPE (10 mM) in Tris-HCl (5 mM) buffer for 30 min at 4 C to form the TTAPE/G-DNA. The free TTAPE dye showed no emission but produced fluorescence at the excitation wavelength of 350 nm when combined with DNA rich in G-base. G-quadruplex can be formed by adding Kþ (1 M) for 30 min at room temperature. Then, fluorescence can be obtained because the TTAPE/G-quadruplex mixture exhibited a strong emission at 490 nm. 2.8. Detection of PAT in real sample The established method was verified to determine whether it can detect PAT in grape juice (water, sugar, 100% concentrated white grape juice) and apple juice (water, 100% concentrated apple juice) purchased from local supermarkets. The sample preparation steps were adopted from the work of Wu [14]. The samples were placed in a test tube, added with different concentrations of PAT toxin (0.05, 0.5, 5, and 50 ng mL1), mixed with an equal volume of ethyl acetate, and centrifuged at 5000 rpm (2348g) for 5 min at room temperature. The supernatant was retained, dried under nitrogen, and filtered through a 0.22 mm nylon syringe. The dried powder was dissolved in PBS buffer for subsequent experiments. Finally, a sample was obtained for the next detection. 3. Results and discussion 3.1. Principle of SDA and DNA G-quadruplex with AIE assay for PAT detection The principle of the assay is based on SDA with AIE strategy. As shown in Scheme 1, streptavidin magnetic beads were combined with a biotinylated aptamer to form an aptamer magnetic bead. Subsequently, this magnetic bead system was added to the cDNA
Scheme 1. Schematic of SDAeG-quadruplexeAIE assay is based on strand isothermal amplification and TTAPE/G-quadruplex strategy.
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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and PAT toxin for competitive reaction. Several aptamers bound to the complementary strand, whereas others bound to the target PAT. When aptamer, cDNA, and PAT had fully reacted, the cDNA of supernatant not bound to the aptamer was retained through magnetic separation and used as primer for SDA. SDA reaction can be carried out using the primer, template DNA, and enzymes to complete the efficient fluorescence signal amplification. The template DNA was divided into three parts: a part complementary to the supernatant primer, another part containing a specific cleavage site of the Nb.BbvCI nicking enzyme, and a part rich in C base. Primer and template DNA were complemented by KF and dNTPs, and Nb.BbvCI can cleave the ssDNA at specific sites of dsDNA to obtain a large number of G-base-enriched ssDNA repeatedly. TTAPE showed a good affinity for DNA rich in G-base. Therefore, no fluorescence effect can be observed when only TTAPE was present. However, TTAPE bound to G-DNA disrupted the internal mechanism of the molecules and emitted strong fluorescence. Moreover, the addition of Kþ caused the TTAPE/G-DNA mixture to form a large amount of TTAPE/G-quadruplex, and more TTAPE aggregated to fluoresce, resulting in a stronger fluorescent signal. However, the primer cannot be obtained when the target PAT was absent, preventing the SDA and the formation of TTAPE/G-DNA complex. Therefore, the fluorescent signal output is closely related to the concentration of PAT. Thus, the proposed method can quantitatively detect PAT. 3.2. Characterization and feasibility verification Circular dichroism (CD) spectroscopy was performed to verify the affinity of the selected PAT aptamer and characterize the DNA G-quadruplex. The aptamer, PAT, and PAT-aptamer conjugates were determined via MOS-450/AF-CD ultravioletevisible spectrometer; the corresponding spectra are shown in Fig. S1. When only PAT or aptamer was present, PAT exhibited no peak nor trough, whereas the aptamer presented a trough and a peak at 250 and 280 nm, respectively, which are characteristics of DNA. However, the mixture of PAT and aptamer displayed a weakly negative cotton peak at 250 nm and a positive cotton peak at 280 nm. The characteristic peak position was unchanged, but the intensity was weakened. This result indicates that the aptamer bound well to PAT because the base stacking and helical structure of the aptamer changed due to binding. The CD spectrum of the G-quadruplex showed a negative peak at 240 nm and a positive peak at 265 nm. By contrast, the CD spectrum of the DNA template strongly increased at 240 and 280 nm which proved that G-quadruplex was formed given the changes in the spatial conformation of the DNA template. In addition, the fluorescence spectrum of the incomplete SDA (Fig. 1A) verified the feasibility of the experiment. The fluorescence intensity was weak when no primer, template, nor any kind of enzyme was applied. Hence, SDA reaction could not be completed, the G-base-rich ssDNA could not be formed in large amounts, and the TTAPE/G-quadruplex complex could not be formed. Fluorescence intensity reached the highest value when the reaction system was integral. The SDA product was investigated via PAGE. The results were visualized through ImageQuant (Fig. 1B). The SDA reaction induced a large number of ssDNA and achieved amplification in the manner that we expected. The absence of primer, KF, Nb.BbvCI nicking enzyme, template DNA, or dNTPs could not produce the target ssDNA. Therefore, SDA can generate a large amount of G-base-rich ssDNA to form G-quadruplex, which should be combined with TTAPE to obtain a fluorescence signal. We also used PAGE to verify the SDA products of different concentrations (1 nM, 100 nM, 10 mM) were stained with SYBR Green I and TTAPE, respectively, and observed the bands under the gel imager (Fig. S7). It also illustrated that TTAPE was more sensitive to G-base-
Fig. 1. (A) Fluorescence spectra of a system without complete SDA. TTAPE/Gquadruplex cannot be formed to produce fluorescence. (B) M: 20 bp marker; lane 1: all in; lane 2: no KF; lane 3: no Nb. BbvCI; lane 4: no dNTPs; lane 5: no template DNA.
rich DNA staining and more conducive to obtaining highly sensitive signals.
3.3. Optimization of SDA reaction and the concentration of cDNA and aptamer We optimized the conditions in the reaction process accordingly to obtain more SDA products. As shown in Fig. S2, the following parameters were optimized: (a) concentration of Nb.BbvCI nicking enzyme, (b) concentration of KF polymerase, and (c) time of amplification. The single-variable principle was followed when optimizing one of the conditions while keeping the other conditions the same. We obtained the following results: (a) the best concentration of Nb. BbvCI nicking enzyme is 16 U, (b) the most suitable concentration of KF polymerase enzyme is 5 U, and (c) the optimal amplification time is 60 min. We also optimized the concentration of aptamer combined with streptavidin magnetic beads (Fig. S3). cDNA concentration was used to compete with the target as shown in Fig. S4. The results can be obtained from the fluorescence spectrum. The fluorescence intensity of the supernatant increased when the concentration of carboxyfluorescein (FAM)e aptamer was gradually increased. Combined with the measurement of the supernatant after coupling reaction by the nucleic acid meter, when the concentration of the aptamer was 10 nM, certain conditions were required for the reaction without wasting materials. In addition, the instructions for the purchased streptavidin
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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3.4. Analytical performance of the assay Fig. 2A shows the fluorescence intensity spectra obtained by adding different concentrations of PAT to the detection system. Fluorescence intensity gradually increased when the PAT concentration increased (0.001,0.005 0.01, 0.05,0.1, 0.5,1, 5,10,50 and 100 ng mL1) based on the above optimization conditions. In addition, the results in Fig. 2B show that fluorescence intensity featured a strong linear correlation in the range of
Fig. 2. (A) Fluorescence spectra of SDA-G-quadruplex with TTAPE for PAT detection at excitation and emission wavelengths of 350 and 490 nm, respectively, and the wide detection range of 0.001e100 ng mL1. (B) Linear relationship of the fluorescence (DF/ F0) and logarithm concentration of PAT. The linear equation is y ¼ 1.3481 þ 0.2086lgx (R2 ¼ 0.9946) in the range of 0.001e100 ng mL1.
magnetic beads indicated the optimum DNA concentration for coupling, that is, 1 mg magnetic beads can be coupled to 200 pM DNA. Thus, we selected the aptamer concentration of 10 nM. Fig. S4 shows that the remaining FAM-cDNA after binding to the aptamer increased in fluorescence intensity as the concentration of the cDNA increased. However, the amount of DNA remaining in the solution was small when the concentration of the cDNA was 1 or 10 nM. Almost all of the aptamers attached to the magnetic beads. The fluorescence value increased remarkably when the concentration reached 100 nM and considerably increased further when the concentration reached 1 and 10 mM, indicating the excessive amounts of the remaining primers. Therefore, the optimum connection concentration is 100 nM.
Fig. 3. (A) Fluorescence spectrum of specificity for the detection of different smallmolecule toxins with three different concentrations (10, 100, and 1000 ng mL1) by the proposed method; (B) changes in fluorescence intensity. PAT showed the highest fluorescence value, whereas other similar toxins (T-2, FB1, OTA, AFB1, and ZEN) produced poor fluorescence.
Table 1 Summarized methods and LODs for PAT toxin detection. Analytical methods
Linear range
LOD
Sample
Ref.
MIP-ESP AgNPs@ZnMOF-MIP NIR fluorescence assay LC-MS/MS GC-MS AuNP-BP NSs based aptasensing HPLC-Magnetic SPE SDA-G-quadruplex-AIE aptasensor
0.002e2 ng/mL 0.1e10 mM/L 0e2.4 pM 0.064e1.6 mM 40e2500 mg/L 0.1 nM-10 mM 0.006e2.6 mM 0.001e100 ng/mL
0.001 ng/mL 0.06 mmol/L 0.06 mg/L 0.035 mM 0.4 mg/L 0.03 nM 0.001 mM 0.042 pg/mL
Apple and grape juice Environmental water and apple juice Apple juice Apple juice Apple juice Apple juice Apple juice Apple and grape juice
[36] [37] [9] [38] [6] [39] [40] This work
Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064
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Table 2 Recovery of PAT at different concentration levels in apple juice and grape juice (n ¼ 6). Sample
Apple juice
Grape juice
Spiked concentration (ng mL1)
0.05 0.5 5 50 0.05 0.5 5 50
SDA-G-quadruplex-AIE
HPLC
Recovery (%)
RSD(%)
Recovery (%)
RSD(%)
104 4.4 100 66 105.3 2.37 101.8 5.58
102 1.01 97.8 1.87 102.5 1.75 101.7 2.27
127.0 3.6 100.4 5.7 122.0 3.4 98 5.2
102.4 2.9 100.2 4.3 86.0 2.5 99.7 5.8
0.001e100 ng mL1, and the linear equation is y ¼ 1.3481 þ 0.2086lgx (R2 ¼ 0.9946). The limit of detection of the sensing system was as low as 0.042 pg mL1 (S/N ¼ 3). We also performed validation without SDA, and the fluorescence spectrum is shown in Fig. S5. When no amplification was carried out, adding different concentrations of target to obtain the supernatant primer was rarely sufficient to combine with TTAPE to produce a strong fluorescence, and thus, a good linear relationship was not obtained. Therefore, SDA substantially improved the sensitivity and effectively amplified the signal. We further explored the effect of Kþ on the reaction results in Fig. S6. Primers with different concentrations showed minimal effect on fluorescence intensity when no Kþ was present. This result indicated that the amplification products failed to form the G-quadruplex complex and cannot combine with TTAPE. The fluorescence value was enhanced after Kþ was added. Hence, Kþ considerably improved the binding of SDA products to TTAPE. To verify the reproducibility of the experiment, according to the established method, we selected (0.01 ng mL1, 1 ng mL1, 100 ng mL1) three different concentrations of the target PAT and performed six repetitive experiments (Fig.S8). The results were good, and the obtained fluorescence curves were also related to the same before. The proposed method was compared with other current methods (Table 1). The proposed assay is notably more sensitive and features a wider analytical range than other methods. 3.5. Evaluation of specificity We evaluated the specificity of the proposed sensing experiment for PAT detection by determining and comparing the fluorescent signal changes caused by PAT and its four kinds of structural analogues (FB1, OTA, ZEN, T-2, and AFB1). As illustrated in Fig. 3, toxins with three concentrations (10, 100, and 1000 ng mL1) were selected to compete with the cDNA of the PAT aptamer, and each measurement was repeated thrice. Based on the results, PAT exhibited an evident dramatic fluorescence intensity, whereas other similar toxins produced weak reactions. The changes in the fluorescence intensity may be due to the specific binding of the PAT aptamer to PAT molecules. Therefore, the established method features a good specificity for PAT. 3.6. Actual sample detection and methodological comparison Spiked recovery experiments and methodological comparisons were conducted to evaluate the stability and accuracy of the SDAeG-quadruplexeAIE assay. The concentrations of PAT in spiked apple and grape juices were detected using the developed method and HPLC. No significant difference was observed between the two methods, indicating their good compatibility. Table 2 summarizes the results of recoveries and relative standard deviations (RSDs). The apple and grape juices showed average recoveries of 97.8%e 104% and 101.7%e105.3%, The RSDs reached 1.01%e4.4% and 1.75%e
5.58%, respectively. These results revealed that our method possesses good accuracy and can sensitively determine PAT in real samples. HPLC analysis was conducted using an isocratic system at 0.8 mL/min. A constant volume (15 mL) of each sample was injected and eluted with 5% acetonitrile in water (retention time of 9 min). 4. Conclusions We believe that this work is the first to report the detection of PAT toxin based on SDA and G-quadruplex with AIE. Functionalized aptamer magnetic beads realized the recognition function of PAT, and cDNA competed with PAT for binding to the aptamer to provide the primer for SDA. High amounts of G-base-rich DNA were achieved by SDA. The combination of G-quadruplex and TTAPE achieved an extremely strong fluorescence intensity. TTAPE is a highly stable and simple probe that can considerably help in obtaining the signal output after forming the DNA secondary structure. It has been used for the detection of small-molecule toxins and has achieved high sensitivity and rapid detection. This study also showed that the combination of isothermal amplification of DNA with AIE can be useful in food safety testing, biomedical, and other fields. Funding This work was supported by the National Key Research and Development Program of China (No. 2018YFC1602903, 2017YFC1200903), and the Key Research and Development Program of Tianjin (No.18YFZCNC01260). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Man Zhang: Writing - original draft. Yu Wang: Software. Xuan Sun: Data curation. Jialei Bai: Project administration. Yuan Peng: Writing - review & editing. Baoan Ning: Writing - review & editing. Zhixian Gao: Funding acquisition. Baolin Liu: Funding acquisition. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2020.01.064. References [1] R. Steiman, et al., Production of patulin by micromycetes, Mycopathologia 105 (3) (1989) 129e133.
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Please cite this article as: M. Zhang et al., Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.064