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High-throughput Low Background G-quadruplex Aptamer Chemiluminescence Assay for Ochratoxin A Using a Single Photonic Crystal Microsphere Peng Shen, Wei Li, Yan Liu, Zhi Ding, Yang Deng, Xuerui Zhu, Yanhao Jin, Yichen Li, Jianlin Li, and Tiesong Zheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03592 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017
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Analytical Chemistry
High-throughput
Low
Background
G-quadruplex
Aptamer
Chemiluminescence Assay for Ochratoxin A Using a Single Photonic Crystal Microsphere Peng Shen a,c, Wei Li
b, c
, Yan Liu
a, c
, Zhi Dinga, Yang Deng a, Xuerui Zhua, Yanhao Jina,
Yichen Lia, Jianlin Li a* and Tiesong Zheng a* a
Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China
b
Department of Electronic and Electrical Engineering, The University of Sheffield, Sheffield, S3 7HQ, United Kingdom
c
These authors contributed to this work equally and should be regarded as co-first authors
*Corresponding authors. Tel.: +86 25 83598286; fax: +86 25 83598901. E-mail addresses: [email protected], [email protected] [email protected]
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ABSTRACT: We reported a novel hemin-G-quadruplex aptamer chemiluminescence assay platform for ochratoxin A (OTA) using the single silica photonic crystal microsphere (SPCM). The oligonucleotide A sequence containing aptamer sequences of hemin and OTA is immobilized on the surface of SPCM. The other oligonucleotide B sequence containing partially complementary sequence with a part of OTA aptamer and a part of hemin aptamer is used blocking chain. The hybridization between chain A and chain B will be influenced by the presence or absence of OTA in the system, which will affect the bioactivity of DNAzyme. Thus, the chemiluminescence signal depends on the concentration of OTA in samples. In the single particle assay platform, the signal/noise is remarkably enhanced and the background signal can be ignored by separating hemin from the surface of SPCM. The limit of detection (LOD) of the new method reaches to pg/mL and linear detection range is in four orders of magnitude for OTA. The new assay platform can provide a sensitive, cost-efficient, simple and high throughput screening for OTA.
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Analytical Chemistry
The ochratoxin A (OTA), N-[(3R)-(5-chloro-8-hydroxy-3-methyl-1oxo-7-iso-chromanyl) carbonyl]-L-phenylalanine, is one of the most common mycotoxins produced by Aspergillus and penicillium fungi1. OTA is widely present in various agricultural products, food and feedstuffs when they are harvested, processed, stored and transported in improper conditions. OTA is a small molecule and very stable for heat. Therefore, it is difficult to be removed once it enters the food chain. Extensive studies have shown that OTA causes nephrotoxic, carcinogenic, heptotoxin, teratogenic, and immunotoxic effects to human being and animals2,3. The International Agency for Research on Cancer has classified OTA as possible human carcinogen (group 2B). The European Commission (ECNo.123/2005) and other countries have been set the maximum tolerated OTA levels4. To protect the safety of food chain of human being and animals from OTA contamination, the urgent task is to develop the rapid, simple and effective detection technique methods to accurate safety evaluation for OTA in the food and feeds. At present, chromatographic techniques, immunoassay and aptamer techniques have been employed to detect OTA. Chromatographic techniques mainly include gas chromatography (GC)5, high-performance liquid chromatography (HPLC)6, and HPLC-tandem mass spectrometry (HPLC-MS)7 or ultrahigh-pressure liquid chromatograph-MS/MS(UPLC-MS/MS)8.
The
chromatographic
apparatus
methods
are
time-consuming, expensive and required the trained personnel though they can provide high sensitivity, selection and good repeatability for OTA confirmed assays. Immunoassay methods for OTA are relatively simple, rapid and cost-effective in the practice. However, immunoassay methods require the specific antibody which are tedious to prepare, poor stability and cross reactivity2,9. Aptamers are a special group of nucleic acids that can bind various targets (e.g., small-molecule drugs, organic, inorganic or metal ions, peptides, proteins and cells) with high binding specificity and affinity10.Since aptamer of OTA was screened by Cruz-Aguado in 200811, various aptasensors have been reported to detect OTA, which included electrochemistry12,13, optical14, fluorescence9,15,16 and chemiluminesence17-21. Among aptasensors, DNAzymes, specifically, the hemin/G-quadruplex horseradish peroxidase (HRP) has attracted great interest in the recent years due to its simple and label-free protocol22-24. The hemin-aptamer complex with the peroxidase-like activity can catalyze the H2O2-mediated oxidation of 2,2' azino-bis ACS Paragon Plus Environment
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(3-ethylbenzthiazoline-6-sulfonic acid) ABTS2- to the colored product ABTS·- or the oxidation of luminol by H2O2 to produce chemiluminescence22,23,25,26. Although these DNAzyme assay systems for OTA displayed the good sensitivity and linear detection range, they have high background signal because hemin itself can catalyze ABTS2- or the oxidation of luminol by H2O2 to produce chemiluminescence. In addition, they often perform in the 96 well microplate which requires large volume reagents. Photonic crystal encoded microsphere suspension array is a powerful platform for bioassay27-29. The microspheres possess brilliant structural color and can be traced during the whole process of assay. In addition, they have three-dimensional porous structure and can accommodate much more probe molecules on their surfaces, which can improve the detection sensitivity. In our previous studies, the fluorescent immunoassay29, chemiluminescent immunoassay30,31 and fluorescence aptamer assay32,33 for multiplex mycotoxins have been established using the photonic crystal encoding microspheres. In this work, we designed a G-quadruplex aptamer chemiluminescence assay for OTA on the surface of the microsphere. The microsphere assay system based on G-quadruplex aptamer chemiluminescence displays a simple, low-background signal, wide linear detection range and high sensitivity for OTA.
EXPERIMENTAL SECTION Experimental Materials. OTA, ochratoxin B (OTB), aflatoxin B2 (AFB2) and fumonisin B1 (FB1) standard substances were purchased from Pribolab (Singapore). (3-aminopropyl) triethoxysilane (APTES), Tris(hydroxymethyl) aminomethane (Tris), succinic acid, and hemin were bought from Sigma-Aldrich (Shanghai, China). N-Hydroxysuccinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC)
were
from
TCI
Chemistry Co. (Shanghai, China). Luminol was bought from Sangon Biotech Co., Ltd (Shanghai, China). 4-Iodophenol (PIP) and dimethysulfoxide (DMSO) were bought from Sinopharm Chemical Reagent Co., Ltd., China. 30% H2O2, sodium bicarbonate, ammonia and absolute ethanol were from Nanjing Chemicals Ltd., China. Luminol stock solution (50 mM) was prepared in 0.05 M pH 10.3 carbonate buffer solution (CBS). PIP stock solution (4 mM) was prepared in 100 mL of DMSO. 0.1 M H2O2 stock solution was prepared in double distilled water with 30%H2O2. Prior to use, luminol, PIP and H2O2 stock solutions were mixed and diluted using
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Analytical Chemistry
0.05 M pH 8.2 Tris-HCl EDTA buffer solution to 0.5 mM, 0.4 mM and 4 mM for luminol, PIP and H2O2, respectively. OTA enzyme-linked immunosorbent assay (ELISA) kits were bought from Hongdu Biotech Co., Ltd (Shandong, China). The sequences of the oligomers are as follows: A: 5’-NH2-C6-TGG-GTA-GGG-CGG-GTT-GGG-AAA-GAT-CGG-GTG-TGG-GTG-GCG-TAA -AGG-GAG-CAT-CGG-ACA-3’ B: 5’-CAC ACC CGA AAA ATC CCA ACC C-3’ These sequences of the oligomers were synthesized, modified and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Cereal samples including rice, corn and wheat samples were bought from supermarket in local. The silica photonic crystal microspheres (SPCMs) were prepared in our lab according to our previous method29. The characteristics of SPCMs were seen in supporting information (Figure S1). The Modification of SPCM Surfaces. The hydroxyls on the surfaces of SPCMs were firstly activated with piranha solution (30% hydrogen peroxide and 70% sulfuric acid (v/v), 5 µL/each microsphere) for 6 h. After the SPCMs were washed with deionized water for three times, they were immersed in 2% APTES toluene solution (5 µL/each microsphere) and shaken at 60 °C at 8×g for 6 h. Then they were respectively washed with toluene, ethanol and double distilled water for 3 times and dried at an oven at 100 °C for 30 min. The aminated microspheres were immersed in succinic acid solution (100 mg succinic acid in 4.7 mL of DMSO and 300 µL of sodium bicarbonate solution, 5 µL/each microsphere) and shaken at 10×g at room temperature for 2 h. Then the microspheres were respectively washed with DMSO and double distilled water for 3 times. The above modified microspheres were immersed in 10 mg/mL EDC, 15 mg/mL NHS and 50 nM A chain in phosphate buffer solution (PBS) (5 µL/each microsphere) and shaken at 10×g at room temperature for 12 h. Then, the microsphere was washed with PBS and Tris-HCl for 3 times, respectively. Determination of OTA. A series of concentration of OTA and 100 nM hemin (5 µL/each microsphere) were respectively added in the centrifuge tubes containing the microspheres modified with A chain and shaken at 10×g at 25 °C for 1.5 h. Then the microspheres were washed with Tris-HCl buffer solution for 3 times. 50 nM B chain (5 µL/each microsphere) was
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added in the system and shaken at 10×g at 25 °C for 30 min. Then, the microspheres were washed with Tris-HCl buffer solution for 3 times. A single microsphere was added in a well of 384 black microplate and 20 µL of chemiluminescence substrate was added into each well. Then the emitted photons were measured with multifunctional microplate reader (expressed as relative light unit, RLU) (TECN, Infinite 200, Switzerland). The standard curves were obtained by plotting chemiluminescent intensities against the logarithm of analyte concentration. The Optimization of Experimental Conditions. The influences of the different concentrations of K+ and Mg2+ on the chemiluminescent signal intensity and reaction time between B chain and hemin-G-quadruplex on the chemiluminescent signal intensity have been investigated. Specificity Assay. The specificity assay was performed among OTA, OTB, AFB2 and FB1 according to the above method. Sample Preparation. The sample preparation was referenced to previous procedures29. Corn, rice and wheat samples were ground by a high-speed disintegrator and particles were less than 1mm. These have been confirmed without OTA by HPLC. A series of different concentration of OTA standard solutions were prepared with 100% methanol solution. The spiked samples were prepared by mixing the different concentration of OTA solution with 5 g samples and put in a fume hood overnight. 25 mL of 80% methanol (v/v0) and 1 g NaCl were added into the spiked samples and homogenized by homogenizer at 820×g for 1 min. The samples were shaken at 5×g for 30 min and filtrated with Whatman filter paper. The filtrate was further filtrated with 0.45 µm filter membrane and collected. In addition, the control samples were prepared according to above methods. ELISA Methods for Assays. The traditional ELISA for validation assays were carried out in according to ELISA kit instructions. Safety Precautions. The preparation of OTA solution and extraction should be carried out in a fume hood. The experimenters should avoid direct exposure to OTA sample and laboratory contamination. The experimenters should wear safety glasses, gloves, mouth-muffle, and laboratory coat when they operate above experiments. Data Extraction and Analysis. The test was repeated at least three times and all data was analyzed by Origin 8.5 software.
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The Principle of the Developed Method for OTA Detection. The principle of the designed assay for OTA on the surface of a single SPCM is showed in Scheme 1. DNA sequence of A chain containing aptamer sequences of hemin and OTA was immobilized on the surface of SPCM. In Scheme 1A, when the hemin and OTA are appeared in the sample, the G-quadruplex is formed by self-assembly of hemin and its aptamer. Meanwhile, OTA binds to its aptamer. The blocking DNA B chain is designed to contain partially complementary DNA aptamer sequences of OTA and hemin. When the blocking chain B is added in the above system, blocking B chain will not hybridize with its complementary DNA sequence because their weaker binding force (Scheme 1A). Then the luminol and H2O2 are added in the above system, the DNAzyme can be activated because hemin-G-quadruplex can catalyze luminol to produce chemiluminescence (Scheme 1A). On the contrary, without OTA in the sample, blocking B chain can easily hybridize with its complementary DNA and hemin-G-quadruplex is dismissed and hemin is released from the surface of microsphere (Scheme 1B). The chemiluminescence signal will not be produced because there is not hemin-G-quadruplex on the surface of microsphere (Scheme 1B).
Scheme 1. The G-quadruplex aptamer chemiluminescence assay for ochratoxin A on the surface of a single SPCM. A, Chemiluminescence signal will be activated when OTA is present in the sample; B, Chemiluminescence signal will not be activated when OTA is absent in the sample. Most of G-quadruplex aptamer chemiluminescence assay systems are performed in the 96 well-microplate without separation of hemin. In these systems, although hemin-G-quadruplex displays a highly enhanced catalytic activity compared with hemin itself, the chemiluminescence signal intensity resulting from hemin catalysis to ABTS2- or luminol cannot be ignored. Figure 1 shows that the influence of the background signal of hemin on the chemiluminescence intensity ACS Paragon Plus Environment
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on the surface of microsphere. When the different concentration of OTA and 100 nM hemin were added in the system without separation of hemin from the surface of single microsphere, the chemiluminescence signal intensities are more than 800000 even without OTA. This result indicates that hemin has a strong catalytic ability toward the luminol-H2O2 chemiluminescence and can induce a high background signal. Although the chemiluminescence signal intensities are increased as the increase of concentration of OTA, the values of signal/noise are low (Figure 1A). On the contrary, the background signal intensity is so low that it almost can be ignored when hemin is separated from the surface of single microsphere by washing microsphere (Figure 1B). The values of signal/noise are remarkably enhanced. The chemiluminescence signal results from that the hemin-G-quadruplex catalyzes luminol under H2O2.
Chemiluminescence intensity(a.u)
1200000
A
800000
400000
0
0 10 100 The concentration of OTA（ ng/mL）
Chemiluminescence intensity(a.u)
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40000
B
30000
20000 10000
0 0 10 100 The concentration of OTA（ ng/mL）
Figure 1. The influence of background signal on the chemiluminescence intensity for G-quadruplex aptamer chemiluminescence assay for OTA when the different concentration of OTA and 100 nM hemin were added in the system. A, Chemiluminescence intensity without separation of hemin from the surface of single microsphere; B, Chemiluminescence intensity separated hemin from the surface of single microsphere by washing microsphere. The Optimization of Experimental Conditions. Divalent and monovalent cations such as ACS Paragon Plus Environment
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Mg2+ or Ca2+and K+ were found to be essential for the specific recognition of OTA by aptamer4,11,20. Mg2+ or Ca2+can be as a bridging interaction mediation between the target and the oligonucleotide and enhances binding to the aptamer11. K+ can induce guanine-rich DNA folding into a stable G-quadruplex conformation because one G-quadruplex has a channel at its center with a diameter that correlates well with the K+ radius (1.3 Å)34. Here, the different concentrations of Mg2+and K+ were optimized and the results were showed in Figure 2A and 2B. The concentrations of Mg2+ significantly affect the chemiluminescence signal intensities. Under the presence of OTA in the system, signal intensities display a linear increase to a maximum value and then decrease in range of 0~45 mM Mg2+ (Figure 2A). However, the system shows a low chemiluminescence value under the absence of OTA. These results further indicate that Mg2+ can enhance the interaction between OTA and aptamer and improve the bioactivity of hemin-G-quadruplex in range of 0~30 mM Mg2+. As the increase of the concentration of K+, the chemiluminescence signal increases and reaches to a plateau (Figure 2B). The result can be ascribed to the benefit of K+ on the sable G-quadruplex. Therefore, 30 mM Mg2+ and 9 mM K+ are chosen as the optimum concentration, respectively. Another parameter, the reaction time between B chain and hemin-G-quadruplex also was optimized. The influence of different reaction times between B chain and hemin-G-quadruplex on the chemiluminescence signal intensities is showed in Figure 2C. During the short reaction times such as 0 and 10 min, the signal intensities are obviously higher than that of the long reaction times in the whole observed concentration of OTA. This is attributed to the higher enzyme activity in the samples of short reaction time. However, the background signals also are high and slopes of detection curves are low in these samples. 30 min is used as the optimal reaction time between B chain and hemin-G-quadruplex because the highest slope of detection curves is obtained in the condition.
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Chemiluminescence intensity(a.u)
45000
250 nM OTA 0 nM OTA
A 30000
15000
0
Chemiluminescence intensity(a.u)
0
10 20 30 40 The concentration of MgCl2 (mM)
50
B
40000
30000
20000
0
Chemiluminescence intensity(a.u)
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60000
2
C
4 6 8 The concentration of K+(mM)
10
12
0min 10min 30min 45min 60min
40000
20000
0 1E-5
1E-3 0.1 10 The concentration of OTA(ng/mL)
1000
Figure 2. The influences of the different concentration of Mg2+ (A) and K+ (B) on the chemiluminescence signal intensities; The influence of different reaction times between B chain and hemin-G-quadruplex on the chemiluminescence signal intensities (C). The Calibration of Curve. A series of concentrations of OTA standard solution were detected using the developed method under the optimal conditions. The calibration of curve for OTA is obtained and showed in Figure 3. Figure 3A shows that the chemiluminescence intensities display a dose-dependent S-shape increase relationship as the increase of the concentration of OTA. The Figure 3B displays that the wide linear detection range for OTA is from 0.01 to 100 ACS Paragon Plus Environment
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ng/mL with 0.996 coefficient of determination. The half maximum inhibitory concentration (IC50) reaches to be 0.38 ng/mL. The limit of detection (LOD) is estimated to be 4.28 pg/mL according to signal-to-noise ratio of 3. The LOD of the developed system is far lower than the values previously reported by aptamer hemin-G-quadruplex chemiluminescence system4,17,20. The comparison with other reported methods for OTA detection is listed in Table 1. The pg/mL LOD for OTA can be attributed to the reasons: one is the three-dimensional porous structural microsphere carrier which can accommodate much more probe molecules on its surface and enhance the kinetics of molecule reactions in the solution; the other is the high signal/noise chemiluminescence signal in the developed aptamer method. In addition, the LOD can satisfy the maximum tolerated OTA levels (2 ng/g for the European Commission4) of the most countries and
Chemiluminescence intensity(a.u)
regions.
A
45000
30000
15000
0 1E-4
45000
Chemiluminescence intensity(a.u)
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Analytical Chemistry
0.01 1 100 The concentration of OTA (ng/mL)
B
30000
y=7848.134x +24625.635 R2=0.996 15000
0.01
0.1 1 10 The concentration of OTA (ng/mL)
100
Figure 3. The calibration of curve for OTA assay. A, The relationship between the concentration of OTA and chemiluminescence intensities; B, The linear detection range for OTA.
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Table 1. The Comparison with Other Reported Methods for OTA Detection. Detection methods
Recognition
Matrix
component
Linear detection
LOD
range
Reagent
References
volume (µL)
Immunochemilumin
antibody
escence
Rice,
0.01-1ng/mL
-
100
35
0.05-10 pg/mL
0.05 pg/mL
100
36
coffee, beans
Immunofluorescence
antibody
Wheat, Corn rice
Fluorescence
aptamer
wine
0.05-20 ng/mL
13 pg/mL
500
9
Fluorescence
aptamer
wine
808-2019.1 ng/mL
808 ng/mL
50
37
Chemiluminescence
aptamer
coffee
0.01-100 ng/mL
0.27 ng/mL
200
17
Chemiluminescence
aptamer
coffee
0.1-100 ng/mL
0.22 ng/mL
180
16
Colorimetric
aptamer
wine
2.5-10 ng/mL
1.0 ng/mL
100
4
Luminescent
aptamer
-
0-24.23 ng/mL
2.0 ng/mL
500
2
Electrochemilumine
aptamer
wheat
0.02-3.0 ng/mL
7 pg/mL
500
38
scence Optical method
aptamer
corn
0.043-4.0 µg/mL
0.43 ng/mL
200
39
Chemiluminescence
aptamer
Rice,
0.01-100 ng/mL
4.28 pg/mL
5
This work
Wheat, corn
The Specificity Evaluation. The specificity of the developed method has been evaluated among the structural analogs of OTA, OTB and other mycotoxins such as AFB2 and FB1. The results are showed in Figure 4. In the linear detection range from 0.01 to 1 ng/mL, the signal values of OTA are higher that of OTB. But, the signal values of OTA are lower that of OTB in the linear detection range from 1 to 100 ng/mL (Figure 4A). The cross-reactivity between OTA and OTB was found around at 1 ng/mL. The result can be ascribed to the high similarity in molecule structure between OTA and OTB. Among OTA, AFB2 and FB1, there is not
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cross-reactivity and the new method shows a good specificity in the whole observed concentration range (Figure 4B). In addition, the signal values of OTA are far higher that of AFB2 and FB1. These results indicate that the developed method can specifically recognize OTA from the mycotoxins although there is cross-reactivity around at 1ng/mL between OTA and OTB.
Chemiluminescence intensity(a.u)
A
OTA OTB
60000
40000
20000
0 1E-3 0.1 10 1000 The concentration of OTA and OTB (ng/mL)
45000
Chemiluminescence intensity(a.u)
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Analytical Chemistry
B
OTA FB1 AFB2
30000
15000
0 0.01
0.1 1 10 100 1000 The concentration of mycotoxins (ng/mL)
Figure 4. The specificity evaluation of the developed method. A, The specificity between OTA and OTB; B, The specificity among the OTA, AFB2 and FB1. Application in the Real Samples. To confirm the validity of the developed method, the spiked cereal samples including rice, wheat and corn have been detected by the method. The recovery rates of OTA are showed in Figure 5A and original data are shown in Table S1, respectively. The most of them are more than 80% except one corn sample (72.19%) spiked at 0.01 ng/g OTA. The results show that the influence of the different substrate samples on the recovery rate is not serious.
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11 naturally contaminated cereal samples have been further detected in parallel using the traditional ELISA and the new method, respectively. Figure 5B shows that the results of the developed method are consistent with that of the ELISA method. Although the further work is needed to confirm the validity of the new method through the large of data from HPLC-MS/MS, these results indicate that the developed method has a great potential in high throughput screening for OTA in agricultural products. Compared with the traditional ELISA method, the new method has obvious advantages:1) label-free detection; 2) requirement of small-volume reagents; 3) cost-efficient; 4) simple operation process. In the previous G-quadruplex aptamer chemiluminescence assay systems for OTA, most of them have ignored the background chemiluminescence signal of hemin, which may reduce the sensitivity of the systems. On the contrary, the established method overcomes the problem and shows a high sensitivity and wide linear detection range for OTA. In addition, the platform can expand to the other G-quadruplex aptamer assay system.
Recovery rates(%)
120
Corn Wheat Rice)
A
80
40
0
0.01 0.1 1 The spiked concentration of OTA in samples (ng/g)
The concentration detected by the ELISA(ng/g)
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B 12
8 y=1.028x+0.279 R2=0.938
4
0 0
4
8
12
The concentration detected by the developed method(ng/g)
Figure 5. The application of the developed method in real samples. A, The recovery rates of ACS Paragon Plus Environment
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the spiked samples; B, The relationship between the developed method and traditional ELISA. CONCLUSION In this work, we established a new G-quadruplex aptamer chemiluminescence assay platform for OTA using a single SPCM. The activity of DNAzyme was depended on the concentrations of Mg2+ and K+. The reaction time between the blocking chain B and G-quadruplex aptamer can influence the chemiluminescence signal intensity. The LOD of the developed method reaches to pg/mL with a high signal/noise, low background signal and four orders of magnitude linear detection range. The recovery rates of the method in the real samples displayed agreement with that of the traditional ELISA method. The results demonstrated that the new platform has a great potential to be employed for high throughput screening for OTA in practice. ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Tel.: +86 25 83598286. Fax: +86 25 83598901. E-mail: [email protected]. E-mail:[email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS
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The authors are grateful to the support from National Natural Science Foundation of China no.31471642 and no.31071542, Natural Science Foundation of Jiangsu Province no. BK20131398 and 100 Talents Program of Nanjing Normal University. Supporting Information. Figure S1 for the characteristics of SPCM taken by the scanning electron microscope (SEM) and digital camera. Table S1 for the data of recovery rates of OTA in spiked samples. REFERENCES (1) Tang,
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(11) Jorge, A. C.A.; Gregory, P. J. Agric. Food Chem. 2008, 56, 10456-10461. (12) Yang, M.L.; Jiang, B.Y.; Xie, J.Q.; Xiang, Y.; Ruo Yuan, Chai, Y.Q. Talanta 2014,125, 45-50. (13) Yang, L.; Zhang, Y.; Li, R.B.; Lin, C.Y.; Guo, L.H.; Qiu, B.; Lin, Z.Y.; Chen, G.N. Biosens.
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Biosen. Bioelectron. 2014,58, 308-313. (18) Choa, S.; Park, L.; Chong, R.; Kim, Y. T.; Lee, J. H. Biosen. Bioelectron. 2014,52,310-316. (19) Yang, C.; Lates, V.; Prieto-Simón, B.; Marty, J. L.; Yang, X.R. Talanta 2013,116,520-526. (20) Freeman, R.; Liu, X.Q.; Willner, I. J. Am. Chem. Soc. 2011, 133, 11597-11604. (21) Park, L.; Kim, J.; Lee, J.H. Talanta 2013,116,736-742. (22) Liu, X.Q.; Freeman, R.; Golub, E.; Willner, I.; ACS Nano. 2011,5,7648-7655. (23) Leng, Y.K.; Sun, K.; Chen, X.Y.; Li, W.W. Chem. Soc. Rev.2015,44, 5552-5595. (24) Ma, D.L.; Wang, W.H.; Mao, Z.F.; Kang, T.S.; Han, Q.B.; Chan, P. W.H.; Leung, C.H. Chem.
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High-throughput Low Background G-quadruplex Aptamer Chemiluminescence Assay for Ochratoxin A Using a Single Photonic Crystal Microsphere Peng Shen, Wei Li, Yan Liu, Zhi Ding, Yang Deng, Xuerui Zhu, Yanhao Jin, Yichen Li, Jianlin Li, and Tiesong Zheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03592 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017
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Analytical Chemistry
High-throughput
Low
Background
G-quadruplex
Aptamer
Chemiluminescence Assay for Ochratoxin A Using a Single Photonic Crystal Microsphere Peng Shen a,c, Wei Li
b, c
, Yan Liu
a, c
, Zhi Dinga, Yang Deng a, Xuerui Zhua, Yanhao Jina,
Yichen Lia, Jianlin Li a* and Tiesong Zheng a* a
Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China
b
Department of Electronic and Electrical Engineering, The University of Sheffield, Sheffield, S3 7HQ, United Kingdom
c
These authors contributed to this work equally and should be regarded as co-first authors
*Corresponding authors. Tel.: +86 25 83598286; fax: +86 25 83598901. E-mail addresses: [email protected], [email protected] [email protected]
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ABSTRACT: We reported a novel hemin-G-quadruplex aptamer chemiluminescence assay platform for ochratoxin A (OTA) using the single silica photonic crystal microsphere (SPCM). The oligonucleotide A sequence containing aptamer sequences of hemin and OTA is immobilized on the surface of SPCM. The other oligonucleotide B sequence containing partially complementary sequence with a part of OTA aptamer and a part of hemin aptamer is used blocking chain. The hybridization between chain A and chain B will be influenced by the presence or absence of OTA in the system, which will affect the bioactivity of DNAzyme. Thus, the chemiluminescence signal depends on the concentration of OTA in samples. In the single particle assay platform, the signal/noise is remarkably enhanced and the background signal can be ignored by separating hemin from the surface of SPCM. The limit of detection (LOD) of the new method reaches to pg/mL and linear detection range is in four orders of magnitude for OTA. The new assay platform can provide a sensitive, cost-efficient, simple and high throughput screening for OTA.
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Analytical Chemistry
The ochratoxin A (OTA), N-[(3R)-(5-chloro-8-hydroxy-3-methyl-1oxo-7-iso-chromanyl) carbonyl]-L-phenylalanine, is one of the most common mycotoxins produced by Aspergillus and penicillium fungi1. OTA is widely present in various agricultural products, food and feedstuffs when they are harvested, processed, stored and transported in improper conditions. OTA is a small molecule and very stable for heat. Therefore, it is difficult to be removed once it enters the food chain. Extensive studies have shown that OTA causes nephrotoxic, carcinogenic, heptotoxin, teratogenic, and immunotoxic effects to human being and animals2,3. The International Agency for Research on Cancer has classified OTA as possible human carcinogen (group 2B). The European Commission (ECNo.123/2005) and other countries have been set the maximum tolerated OTA levels4. To protect the safety of food chain of human being and animals from OTA contamination, the urgent task is to develop the rapid, simple and effective detection technique methods to accurate safety evaluation for OTA in the food and feeds. At present, chromatographic techniques, immunoassay and aptamer techniques have been employed to detect OTA. Chromatographic techniques mainly include gas chromatography (GC)5, high-performance liquid chromatography (HPLC)6, and HPLC-tandem mass spectrometry (HPLC-MS)7 or ultrahigh-pressure liquid chromatograph-MS/MS(UPLC-MS/MS)8.
The
chromatographic
apparatus
methods
are
time-consuming, expensive and required the trained personnel though they can provide high sensitivity, selection and good repeatability for OTA confirmed assays. Immunoassay methods for OTA are relatively simple, rapid and cost-effective in the practice. However, immunoassay methods require the specific antibody which are tedious to prepare, poor stability and cross reactivity2,9. Aptamers are a special group of nucleic acids that can bind various targets (e.g., small-molecule drugs, organic, inorganic or metal ions, peptides, proteins and cells) with high binding specificity and affinity10.Since aptamer of OTA was screened by Cruz-Aguado in 200811, various aptasensors have been reported to detect OTA, which included electrochemistry12,13, optical14, fluorescence9,15,16 and chemiluminesence17-21. Among aptasensors, DNAzymes, specifically, the hemin/G-quadruplex horseradish peroxidase (HRP) has attracted great interest in the recent years due to its simple and label-free protocol22-24. The hemin-aptamer complex with the peroxidase-like activity can catalyze the H2O2-mediated oxidation of 2,2' azino-bis ACS Paragon Plus Environment
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(3-ethylbenzthiazoline-6-sulfonic acid) ABTS2- to the colored product ABTS·- or the oxidation of luminol by H2O2 to produce chemiluminescence22,23,25,26. Although these DNAzyme assay systems for OTA displayed the good sensitivity and linear detection range, they have high background signal because hemin itself can catalyze ABTS2- or the oxidation of luminol by H2O2 to produce chemiluminescence. In addition, they often perform in the 96 well microplate which requires large volume reagents. Photonic crystal encoded microsphere suspension array is a powerful platform for bioassay27-29. The microspheres possess brilliant structural color and can be traced during the whole process of assay. In addition, they have three-dimensional porous structure and can accommodate much more probe molecules on their surfaces, which can improve the detection sensitivity. In our previous studies, the fluorescent immunoassay29, chemiluminescent immunoassay30,31 and fluorescence aptamer assay32,33 for multiplex mycotoxins have been established using the photonic crystal encoding microspheres. In this work, we designed a G-quadruplex aptamer chemiluminescence assay for OTA on the surface of the microsphere. The microsphere assay system based on G-quadruplex aptamer chemiluminescence displays a simple, low-background signal, wide linear detection range and high sensitivity for OTA.
EXPERIMENTAL SECTION Experimental Materials. OTA, ochratoxin B (OTB), aflatoxin B2 (AFB2) and fumonisin B1 (FB1) standard substances were purchased from Pribolab (Singapore). (3-aminopropyl) triethoxysilane (APTES), Tris(hydroxymethyl) aminomethane (Tris), succinic acid, and hemin were bought from Sigma-Aldrich (Shanghai, China). N-Hydroxysuccinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC)
were
from
TCI
Chemistry Co. (Shanghai, China). Luminol was bought from Sangon Biotech Co., Ltd (Shanghai, China). 4-Iodophenol (PIP) and dimethysulfoxide (DMSO) were bought from Sinopharm Chemical Reagent Co., Ltd., China. 30% H2O2, sodium bicarbonate, ammonia and absolute ethanol were from Nanjing Chemicals Ltd., China. Luminol stock solution (50 mM) was prepared in 0.05 M pH 10.3 carbonate buffer solution (CBS). PIP stock solution (4 mM) was prepared in 100 mL of DMSO. 0.1 M H2O2 stock solution was prepared in double distilled water with 30%H2O2. Prior to use, luminol, PIP and H2O2 stock solutions were mixed and diluted using
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Analytical Chemistry
0.05 M pH 8.2 Tris-HCl EDTA buffer solution to 0.5 mM, 0.4 mM and 4 mM for luminol, PIP and H2O2, respectively. OTA enzyme-linked immunosorbent assay (ELISA) kits were bought from Hongdu Biotech Co., Ltd (Shandong, China). The sequences of the oligomers are as follows: A: 5’-NH2-C6-TGG-GTA-GGG-CGG-GTT-GGG-AAA-GAT-CGG-GTG-TGG-GTG-GCG-TAA -AGG-GAG-CAT-CGG-ACA-3’ B: 5’-CAC ACC CGA AAA ATC CCA ACC C-3’ These sequences of the oligomers were synthesized, modified and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Cereal samples including rice, corn and wheat samples were bought from supermarket in local. The silica photonic crystal microspheres (SPCMs) were prepared in our lab according to our previous method29. The characteristics of SPCMs were seen in supporting information (Figure S1). The Modification of SPCM Surfaces. The hydroxyls on the surfaces of SPCMs were firstly activated with piranha solution (30% hydrogen peroxide and 70% sulfuric acid (v/v), 5 µL/each microsphere) for 6 h. After the SPCMs were washed with deionized water for three times, they were immersed in 2% APTES toluene solution (5 µL/each microsphere) and shaken at 60 °C at 8×g for 6 h. Then they were respectively washed with toluene, ethanol and double distilled water for 3 times and dried at an oven at 100 °C for 30 min. The aminated microspheres were immersed in succinic acid solution (100 mg succinic acid in 4.7 mL of DMSO and 300 µL of sodium bicarbonate solution, 5 µL/each microsphere) and shaken at 10×g at room temperature for 2 h. Then the microspheres were respectively washed with DMSO and double distilled water for 3 times. The above modified microspheres were immersed in 10 mg/mL EDC, 15 mg/mL NHS and 50 nM A chain in phosphate buffer solution (PBS) (5 µL/each microsphere) and shaken at 10×g at room temperature for 12 h. Then, the microsphere was washed with PBS and Tris-HCl for 3 times, respectively. Determination of OTA. A series of concentration of OTA and 100 nM hemin (5 µL/each microsphere) were respectively added in the centrifuge tubes containing the microspheres modified with A chain and shaken at 10×g at 25 °C for 1.5 h. Then the microspheres were washed with Tris-HCl buffer solution for 3 times. 50 nM B chain (5 µL/each microsphere) was
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added in the system and shaken at 10×g at 25 °C for 30 min. Then, the microspheres were washed with Tris-HCl buffer solution for 3 times. A single microsphere was added in a well of 384 black microplate and 20 µL of chemiluminescence substrate was added into each well. Then the emitted photons were measured with multifunctional microplate reader (expressed as relative light unit, RLU) (TECN, Infinite 200, Switzerland). The standard curves were obtained by plotting chemiluminescent intensities against the logarithm of analyte concentration. The Optimization of Experimental Conditions. The influences of the different concentrations of K+ and Mg2+ on the chemiluminescent signal intensity and reaction time between B chain and hemin-G-quadruplex on the chemiluminescent signal intensity have been investigated. Specificity Assay. The specificity assay was performed among OTA, OTB, AFB2 and FB1 according to the above method. Sample Preparation. The sample preparation was referenced to previous procedures29. Corn, rice and wheat samples were ground by a high-speed disintegrator and particles were less than 1mm. These have been confirmed without OTA by HPLC. A series of different concentration of OTA standard solutions were prepared with 100% methanol solution. The spiked samples were prepared by mixing the different concentration of OTA solution with 5 g samples and put in a fume hood overnight. 25 mL of 80% methanol (v/v0) and 1 g NaCl were added into the spiked samples and homogenized by homogenizer at 820×g for 1 min. The samples were shaken at 5×g for 30 min and filtrated with Whatman filter paper. The filtrate was further filtrated with 0.45 µm filter membrane and collected. In addition, the control samples were prepared according to above methods. ELISA Methods for Assays. The traditional ELISA for validation assays were carried out in according to ELISA kit instructions. Safety Precautions. The preparation of OTA solution and extraction should be carried out in a fume hood. The experimenters should avoid direct exposure to OTA sample and laboratory contamination. The experimenters should wear safety glasses, gloves, mouth-muffle, and laboratory coat when they operate above experiments. Data Extraction and Analysis. The test was repeated at least three times and all data was analyzed by Origin 8.5 software.
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Analytical Chemistry
The Principle of the Developed Method for OTA Detection. The principle of the designed assay for OTA on the surface of a single SPCM is showed in Scheme 1. DNA sequence of A chain containing aptamer sequences of hemin and OTA was immobilized on the surface of SPCM. In Scheme 1A, when the hemin and OTA are appeared in the sample, the G-quadruplex is formed by self-assembly of hemin and its aptamer. Meanwhile, OTA binds to its aptamer. The blocking DNA B chain is designed to contain partially complementary DNA aptamer sequences of OTA and hemin. When the blocking chain B is added in the above system, blocking B chain will not hybridize with its complementary DNA sequence because their weaker binding force (Scheme 1A). Then the luminol and H2O2 are added in the above system, the DNAzyme can be activated because hemin-G-quadruplex can catalyze luminol to produce chemiluminescence (Scheme 1A). On the contrary, without OTA in the sample, blocking B chain can easily hybridize with its complementary DNA and hemin-G-quadruplex is dismissed and hemin is released from the surface of microsphere (Scheme 1B). The chemiluminescence signal will not be produced because there is not hemin-G-quadruplex on the surface of microsphere (Scheme 1B).
Scheme 1. The G-quadruplex aptamer chemiluminescence assay for ochratoxin A on the surface of a single SPCM. A, Chemiluminescence signal will be activated when OTA is present in the sample; B, Chemiluminescence signal will not be activated when OTA is absent in the sample. Most of G-quadruplex aptamer chemiluminescence assay systems are performed in the 96 well-microplate without separation of hemin. In these systems, although hemin-G-quadruplex displays a highly enhanced catalytic activity compared with hemin itself, the chemiluminescence signal intensity resulting from hemin catalysis to ABTS2- or luminol cannot be ignored. Figure 1 shows that the influence of the background signal of hemin on the chemiluminescence intensity ACS Paragon Plus Environment
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on the surface of microsphere. When the different concentration of OTA and 100 nM hemin were added in the system without separation of hemin from the surface of single microsphere, the chemiluminescence signal intensities are more than 800000 even without OTA. This result indicates that hemin has a strong catalytic ability toward the luminol-H2O2 chemiluminescence and can induce a high background signal. Although the chemiluminescence signal intensities are increased as the increase of concentration of OTA, the values of signal/noise are low (Figure 1A). On the contrary, the background signal intensity is so low that it almost can be ignored when hemin is separated from the surface of single microsphere by washing microsphere (Figure 1B). The values of signal/noise are remarkably enhanced. The chemiluminescence signal results from that the hemin-G-quadruplex catalyzes luminol under H2O2.
Chemiluminescence intensity(a.u)
1200000
A
800000
400000
0
0 10 100 The concentration of OTA（ ng/mL）
Chemiluminescence intensity(a.u)
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40000
B
30000
20000 10000
0 0 10 100 The concentration of OTA（ ng/mL）
Figure 1. The influence of background signal on the chemiluminescence intensity for G-quadruplex aptamer chemiluminescence assay for OTA when the different concentration of OTA and 100 nM hemin were added in the system. A, Chemiluminescence intensity without separation of hemin from the surface of single microsphere; B, Chemiluminescence intensity separated hemin from the surface of single microsphere by washing microsphere. The Optimization of Experimental Conditions. Divalent and monovalent cations such as ACS Paragon Plus Environment
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Analytical Chemistry
Mg2+ or Ca2+and K+ were found to be essential for the specific recognition of OTA by aptamer4,11,20. Mg2+ or Ca2+can be as a bridging interaction mediation between the target and the oligonucleotide and enhances binding to the aptamer11. K+ can induce guanine-rich DNA folding into a stable G-quadruplex conformation because one G-quadruplex has a channel at its center with a diameter that correlates well with the K+ radius (1.3 Å)34. Here, the different concentrations of Mg2+and K+ were optimized and the results were showed in Figure 2A and 2B. The concentrations of Mg2+ significantly affect the chemiluminescence signal intensities. Under the presence of OTA in the system, signal intensities display a linear increase to a maximum value and then decrease in range of 0~45 mM Mg2+ (Figure 2A). However, the system shows a low chemiluminescence value under the absence of OTA. These results further indicate that Mg2+ can enhance the interaction between OTA and aptamer and improve the bioactivity of hemin-G-quadruplex in range of 0~30 mM Mg2+. As the increase of the concentration of K+, the chemiluminescence signal increases and reaches to a plateau (Figure 2B). The result can be ascribed to the benefit of K+ on the sable G-quadruplex. Therefore, 30 mM Mg2+ and 9 mM K+ are chosen as the optimum concentration, respectively. Another parameter, the reaction time between B chain and hemin-G-quadruplex also was optimized. The influence of different reaction times between B chain and hemin-G-quadruplex on the chemiluminescence signal intensities is showed in Figure 2C. During the short reaction times such as 0 and 10 min, the signal intensities are obviously higher than that of the long reaction times in the whole observed concentration of OTA. This is attributed to the higher enzyme activity in the samples of short reaction time. However, the background signals also are high and slopes of detection curves are low in these samples. 30 min is used as the optimal reaction time between B chain and hemin-G-quadruplex because the highest slope of detection curves is obtained in the condition.
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Chemiluminescence intensity(a.u)
45000
250 nM OTA 0 nM OTA
A 30000
15000
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10 20 30 40 The concentration of MgCl2 (mM)
50
B
40000
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0
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C
4 6 8 The concentration of K+(mM)
10
12
0min 10min 30min 45min 60min
40000
20000
0 1E-5
1E-3 0.1 10 The concentration of OTA(ng/mL)
1000
Figure 2. The influences of the different concentration of Mg2+ (A) and K+ (B) on the chemiluminescence signal intensities; The influence of different reaction times between B chain and hemin-G-quadruplex on the chemiluminescence signal intensities (C). The Calibration of Curve. A series of concentrations of OTA standard solution were detected using the developed method under the optimal conditions. The calibration of curve for OTA is obtained and showed in Figure 3. Figure 3A shows that the chemiluminescence intensities display a dose-dependent S-shape increase relationship as the increase of the concentration of OTA. The Figure 3B displays that the wide linear detection range for OTA is from 0.01 to 100 ACS Paragon Plus Environment
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ng/mL with 0.996 coefficient of determination. The half maximum inhibitory concentration (IC50) reaches to be 0.38 ng/mL. The limit of detection (LOD) is estimated to be 4.28 pg/mL according to signal-to-noise ratio of 3. The LOD of the developed system is far lower than the values previously reported by aptamer hemin-G-quadruplex chemiluminescence system4,17,20. The comparison with other reported methods for OTA detection is listed in Table 1. The pg/mL LOD for OTA can be attributed to the reasons: one is the three-dimensional porous structural microsphere carrier which can accommodate much more probe molecules on its surface and enhance the kinetics of molecule reactions in the solution; the other is the high signal/noise chemiluminescence signal in the developed aptamer method. In addition, the LOD can satisfy the maximum tolerated OTA levels (2 ng/g for the European Commission4) of the most countries and
Chemiluminescence intensity(a.u)
regions.
A
45000
30000
15000
0 1E-4
45000
Chemiluminescence intensity(a.u)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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0.01 1 100 The concentration of OTA (ng/mL)
B
30000
y=7848.134x +24625.635 R2=0.996 15000
0.01
0.1 1 10 The concentration of OTA (ng/mL)
100
Figure 3. The calibration of curve for OTA assay. A, The relationship between the concentration of OTA and chemiluminescence intensities; B, The linear detection range for OTA.
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Table 1. The Comparison with Other Reported Methods for OTA Detection. Detection methods
Recognition
Matrix
component
Linear detection
LOD
range
Reagent
References
volume (µL)
Immunochemilumin
antibody
escence
Rice,
0.01-1ng/mL
-
100
35
0.05-10 pg/mL
0.05 pg/mL
100
36
coffee, beans
Immunofluorescence
antibody
Wheat, Corn rice
Fluorescence
aptamer
wine
0.05-20 ng/mL
13 pg/mL
500
9
Fluorescence
aptamer
wine
808-2019.1 ng/mL
808 ng/mL
50
37
Chemiluminescence
aptamer
coffee
0.01-100 ng/mL
0.27 ng/mL
200
17
Chemiluminescence
aptamer
coffee
0.1-100 ng/mL
0.22 ng/mL
180
16
Colorimetric
aptamer
wine
2.5-10 ng/mL
1.0 ng/mL
100
4
Luminescent
aptamer
-
0-24.23 ng/mL
2.0 ng/mL
500
2
Electrochemilumine
aptamer
wheat
0.02-3.0 ng/mL
7 pg/mL
500
38
scence Optical method
aptamer
corn
0.043-4.0 µg/mL
0.43 ng/mL
200
39
Chemiluminescence
aptamer
Rice,
0.01-100 ng/mL
4.28 pg/mL
5
This work
Wheat, corn
The Specificity Evaluation. The specificity of the developed method has been evaluated among the structural analogs of OTA, OTB and other mycotoxins such as AFB2 and FB1. The results are showed in Figure 4. In the linear detection range from 0.01 to 1 ng/mL, the signal values of OTA are higher that of OTB. But, the signal values of OTA are lower that of OTB in the linear detection range from 1 to 100 ng/mL (Figure 4A). The cross-reactivity between OTA and OTB was found around at 1 ng/mL. The result can be ascribed to the high similarity in molecule structure between OTA and OTB. Among OTA, AFB2 and FB1, there is not
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cross-reactivity and the new method shows a good specificity in the whole observed concentration range (Figure 4B). In addition, the signal values of OTA are far higher that of AFB2 and FB1. These results indicate that the developed method can specifically recognize OTA from the mycotoxins although there is cross-reactivity around at 1ng/mL between OTA and OTB.
Chemiluminescence intensity(a.u)
A
OTA OTB
60000
40000
20000
0 1E-3 0.1 10 1000 The concentration of OTA and OTB (ng/mL)
45000
Chemiluminescence intensity(a.u)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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B
OTA FB1 AFB2
30000
15000
0 0.01
0.1 1 10 100 1000 The concentration of mycotoxins (ng/mL)
Figure 4. The specificity evaluation of the developed method. A, The specificity between OTA and OTB; B, The specificity among the OTA, AFB2 and FB1. Application in the Real Samples. To confirm the validity of the developed method, the spiked cereal samples including rice, wheat and corn have been detected by the method. The recovery rates of OTA are showed in Figure 5A and original data are shown in Table S1, respectively. The most of them are more than 80% except one corn sample (72.19%) spiked at 0.01 ng/g OTA. The results show that the influence of the different substrate samples on the recovery rate is not serious.
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11 naturally contaminated cereal samples have been further detected in parallel using the traditional ELISA and the new method, respectively. Figure 5B shows that the results of the developed method are consistent with that of the ELISA method. Although the further work is needed to confirm the validity of the new method through the large of data from HPLC-MS/MS, these results indicate that the developed method has a great potential in high throughput screening for OTA in agricultural products. Compared with the traditional ELISA method, the new method has obvious advantages:1) label-free detection; 2) requirement of small-volume reagents; 3) cost-efficient; 4) simple operation process. In the previous G-quadruplex aptamer chemiluminescence assay systems for OTA, most of them have ignored the background chemiluminescence signal of hemin, which may reduce the sensitivity of the systems. On the contrary, the established method overcomes the problem and shows a high sensitivity and wide linear detection range for OTA. In addition, the platform can expand to the other G-quadruplex aptamer assay system.
Recovery rates(%)
120
Corn Wheat Rice)
A
80
40
0
0.01 0.1 1 The spiked concentration of OTA in samples (ng/g)
The concentration detected by the ELISA(ng/g)
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B 12
8 y=1.028x+0.279 R2=0.938
4
0 0
4
8
12
The concentration detected by the developed method(ng/g)
Figure 5. The application of the developed method in real samples. A, The recovery rates of ACS Paragon Plus Environment
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the spiked samples; B, The relationship between the developed method and traditional ELISA. CONCLUSION In this work, we established a new G-quadruplex aptamer chemiluminescence assay platform for OTA using a single SPCM. The activity of DNAzyme was depended on the concentrations of Mg2+ and K+. The reaction time between the blocking chain B and G-quadruplex aptamer can influence the chemiluminescence signal intensity. The LOD of the developed method reaches to pg/mL with a high signal/noise, low background signal and four orders of magnitude linear detection range. The recovery rates of the method in the real samples displayed agreement with that of the traditional ELISA method. The results demonstrated that the new platform has a great potential to be employed for high throughput screening for OTA in practice. ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Tel.: +86 25 83598286. Fax: +86 25 83598901. E-mail: [email protected]. E-mail:[email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS
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The authors are grateful to the support from National Natural Science Foundation of China no.31471642 and no.31071542, Natural Science Foundation of Jiangsu Province no. BK20131398 and 100 Talents Program of Nanjing Normal University. Supporting Information. Figure S1 for the characteristics of SPCM taken by the scanning electron microscope (SEM) and digital camera. Table S1 for the data of recovery rates of OTA in spiked samples. REFERENCES (1) Tang,
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