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Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe
Accepted Manuscript Title: Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe Authors: Lei Lv, Donghao Li, Renjie Liu, Chengbi Cui, Zhijun Guo PII: DOI: Reference:
S0925-4005(17)30370-2 http://dx.doi.org/doi:10.1016/j.snb.2017.02.143 SNB 21874
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
12-10-2016 22-2-2017 23-2-2017
Please cite this article as: Lei Lv, Donghao Li, Renjie Liu, Chengbi Cui, Zhijun Guo, Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.02.143 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe Lei Lva,b, Donghao Lia, Renjie Liuc, Chengbi Cui a,b*, Zhijun Guoa,b* a
Key Laboratory of Natural Resources of Changbai Mountain and Functional
Molecules, Yanbian University, Ministry of Education, YanJi, 133002, China. b
Department of Food Science and Engineering, Yanbian University, YanJi, 133002,
China. c
Institute of food science and engineering, Jilin agricultural university, Changchun,
130118, China. E-mail: [email protected]; [email protected] Fax: (+86) 433-2435549; Tel: (+86) 433-2435549
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Highlights 1. A label-free fluorescent aptasensor for OTA detection was designed. 2. The aptasensor shows high selectivity toward OTA with a low limit of detection 3. The proposed sensing system can be applied to the analysis of Ochratoxin A in real samples. 4. The principle can be extended to the detection of other targets.
Abstract: A label-free sensing strategy employing aptamers, SYBR Gold, and exonuclease I (Exo I) for ochratoxin A (OTA) detection was designed. In the presence of target molecules (OTA), the conformation of the aptamer specific for OTA is switched from a random coil to an antiparallel G-quadruplex. Subsequently, Exo I is added into the mixture to digest the unfolded aptamers selectively, which are the preferred substrates of Exo I. Following the addition of SYBR Gold as probe, a strong fluorescence intensity is obtained. This aptasensor shows high selectivity toward OTA with a low limit of detection (16.5 nM). The validity of the procedure and applicability of the aptasensor are successfully assessed through the detection of OTA in spiked red wine and beer without interference from the sample matrix. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples indicates its usefulness as a tool for verifying the effectiveness of mycotoxin control strategies.
Keywords: Ochratoxin A, Label-free, SYBR Gold, Aptamer, G-quadruplex
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1. Introduction Mycotoxins are toxic metabolites produced by fungi, mostly by saprophytic molds growing on various foodstuffs, including that of animal feeds, and by many plant pathogens [1]. Ochratoxin A (OTA) is one of the mycotoxins (a natural, toxic secondary metabolite) produced by several species of the fungi Penicillium and Aspergillus. OTA has been widely detected in cereal-derived products, dried fruits, spices, beer, and wine. OTA is potentially carcinogenic to human beings. This mycotoxin is also weakly mutagenic and can cause immunosuppression and immunotoxicity [2]. To avoid the risk of OTA consumption, the detection and quantification of OTA level in contaminated raw materials are considerably useful. Various analytical methods have been established for the determination of OTA. OTA analysis is typically performed via conventional chromatographic methods, such as thin layer chromatography, high-performance liquid chromatography (HPLC), or gas chromatography coupled to ultraviolet visible, fluorescence, or mass spectrometry [3-8]. However, these methods generally require multiple steps, including extraction, extensive sample cleaning, preconcentration, and analyte derivatization, prior to detection. Such sample treatment not only makes the analysis time consuming and costly but also requires trained personnel. Immunoassays provide a simple and economical alternative to instrumental methods for OTA analysis. A common alternative for OTA detection is the immunoassay based on antigen–antibody interactions, such as enzyme-linked immunosorbent assay (ELISA). Several groups have previously established immunoassays for OTA [9-13]. Nevertheless, ELISA is a
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heterogeneous method and involves multiple washing steps. In addition, the storage and application conditions of the antibody, such as temperature, pH, and ionic strength, are strictly defined. All these conditions have limited the ability of the existing antibody-based methods to satisfy the current detection requirements. Aptamers are single-stranded DNA or RNA molecules that can bind specifically to their targets and selected via an in vitro process called systematic evolution of ligands by exponential enrichment [14]. Aptamers have been considered a good candidate alternatives to replace antibodies. Furthermore, aptamers can be selected for a broad range of targets, including small molecules, proteins, nucleic acids, cells, tissues, and organisms. The binding ability of aptamers is as good as that of antibodies, but their synthesis, maintenance, and delivery are easier. Thus, aptamers are promising molecular receptors for bioanalytical applications [15, 16]. Recently, a series of assays using aptamers against OTA have been developed. These assays mainly involved fluorescence [17, 18], electrochemical [19, 20], and colorimetric transducers [21, 22], etc. Among these assays, electrode processing is troublesome for the electrochemical method, and the sample color may interfere with the test results for the colorimetric method. Fluorescence methods have received increasing attention because of their distinct advantages, such as high sensitivity, rapid analysis, and little damage to sample [23-27]. Biosensing via fluorescence is one of the well known methods for the design of aptamer-based assays, largely because of the ease of detection, good sensitivity, and potential for high-throughput analysis. However, frequently in these assays, the aptamer needs to be labeled with
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fluorophores. The process of DNA modification is laborious and expensive. [28-30]. Moreover, these fluorescent and/or quenching molecules may even alter the binding properties of aptamers [31-33]. In addition, the majority of current labeled dyes, particularly near-infrared dyes, suffer from poor photostability and low emission intensities, ultimately limiting the further improvement of fluorescence methods [34, 35]. Therefore, the development of label-free methods for fluorescent detection is highly desirable. A few label-free fluorescent aptasensors for OTA detection have been reported. For example, Zhang et al. have reported a fluorescent aptasensor based on Tb3+ and structure-switching aptamer for label-free detection of ochratoxin A [36]. However, the Tb3+ is poisonous, and the performance is tedious. Lv et al. have reported a simple and sensitive label-free fluorescent aptasensor for ochratoxin A detection [37]. However, the sensing systems are in turn-off mode, and such turn-off assays might compromise specificity because other quenchers or environmental stimuli might lead to fluorescence quenching, thereby leading to “false positive” results . Considering these problems, we designed a label-free turn-on fluorescent aptasensor for OTA detection based on exonuclease I (Exo I) enzyme and SYBR Gold dye. SYBR Gold was applied as fluorescent probe because of its high sensitivity and specificity for nucleic acids, as well as its low toxicity and good stability [38]. The aptamer specific for OTA can fold to form antiparallel G-quadruplex structure upon exposure to OTA [39]. The formation of antiparallel G-quadruplex structure is resistant to Exo I digestion. The amount of aptamer left after nuclease reaction is proportional to OTA
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concentrations, thereby making it proportional to the fluorescence intensities from SYBR Gold that can only stain nucleic acids but not their digestion products, nucleoside monophosphates. The results of our experiment also confirmed that our design strategy was very successful. The major advantage of combining Exo I enzyme and SYBR Gold with aptamer is the elimination of the need for DNA modification, thereby significantly reducing the cost. This fluorometric method is low cost, highly sensitivity, and involves a simple performance. The elements used in the assays are cheap and commercially available. Furthermore, the method is a nearly universal one because of the exceptional structure selectivity of Exo I. The principle can be extended to the detection of other targets, such as ions, small molecules, and proteins.
2. Materials and methods 2.1 Materials The OTA aptamers (5’-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3’) [40] were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The fluorescent dye SYBR Gold (10 000× concentrated) was purchased from Invitrogen (CA, USA). Exo I was bought from TaKaRa Biotechnology Inc. (Dalian, China). OTA, ochratoxin B (OTB), ochratoxin C (OTC), aflatoxin B1 (AFB1), and zearalenone (ZEN) were purchased from Fermentek (Jerusalem,
Israel).
Poly
(allylamine)
hydrochloride
(PAH),
poly
(diallyldimethylammonium chloride) (PDDA) were purchased from Sigma Aldrich (Shanghai, China). Binding buffer (10 mM Tris, pH 8.5, 120 mM NaCl, 5 mM KCl,
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10 mM MgCl2, and 20 mM CaCl2) was used for the binding reaction between the aptamer and OTA. The OTA stock solution (1 mM) was prepared by dissolving ochratoxin in absolute ethanol and stored at -20 °C. Changyu Rose Red Wine was produced by YanTai ChangYu Pioneer Wine Company Limited (Yantai, China). Tsingtao beer was produced by Tsingtao beer Company Limited (Tsingtao, China). All other chemicals were of analytical grade and used as received without further purification. Ultrapure water with an electrical resistivity of 18.2 MΩ cm was obtained from a Milli-Q ultra-high-purity water system (Millipore, Bedford, MA, USA).
2.2 Instrumentation Cary 500 Scan UV-vis Spectrophotometer (Varian, USA) was used to quantify the oligonucleotides. An Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a fluorescence detector was used to record HPLC chromatograms. A JASCO J-810 spectropolarimeter (Tokyo, Japan) was used to collect circular dichroism (CD) spectra in 10 mM Tris buffer (pH 8.5). A RF-5301PC fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) was used to record the fluorescence spectra with a 150 W Xenon lamp as the excitation source. Meanwhile, emission spectra were recorded within the wavelength range of 480-620 nm upon excitation at 495 nm. Slit widths for excitation and emission were set at 3 nm. All measurements were performed at room temperature unless stated otherwise. 2.3 Analysis of aptamer conformation with CD
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CD spectropolarimeter using solutions of DNA aptamer (1 µM) in an optical chamber (1 cm path length, 1 mL volume), which was deoxygenated with dry purified nitrogen (99.99%) before use and kept in nitrogen atmosphere during experiments. Each CD spectrum was the accumulation of three scans at 200 nm/min with a 1 nm band width and a time constant of 1 s. The data were collected within 230-340 nm at 0.1 nm intervals. The background of the buffer solution was subtracted from CD data. 2.4 Optimizing Exo I concentration Increasing concentrations of Exo I (0–10 units) were added to a constant concentration of ssDNA aptamer (100 nM) in binding buffer (final volume, 400 µL). After 30 min of incubation at 37 °C, 100 µL of 10 × SYBR Gold was mixed with the samples. After equilibrating the solution in the dark at room temperature for another 15 min, fluorescence intensity was measured. 2.5 Optimization of incubation time of Exo I with ssDNA aptamer on fluorescence intensities 5 units of Exo I were added to 100 nM ssDNA aptamer in binding buffer (final volume, 400 µL). The mixture was incubated at 37 °C for 0–40 min. Subsequently, 100 µL of 10 × SYBR Gold was added to the samples, and fluorescence intensities were recorded. 2.6 Fluorescent detection of OTA For quantitative measurement of OTA, 200 µL of solution containing 250 nM of aptamer was mixed with buffer solution of different OTA concentrations and allowed to settle for 15 min. Subsequently, 5 units of Exo I were added, and the total final
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volume was 400 µL. The mixtures were incubated at 37 °C for 30 min. Afterward, 100 µL of 10 × SYBR Gold was added to the samples. After equilibrating the solution in the dark at room temperature for another 15 min, fluorescence intensity was then measured. 2.7 Application To confirm the feasibility of this sensor for analysis of actual samples, three different concentrations of OTA (20, 100, and 500 nM) were spiked into red wine and beer, and fluorescent intensity was used to determine OTA concentrations.
3. Results and discussion 3.1 Design strategy for OTA detection The designed aptasensor is based on target-induced conformation of G-quadruplex structure, digestion of unfolded ssDNA by Exo I, and the ability of SYBR Gold as a fluorescent probe. A schematic representation of OTA detection is illustrated in Scheme 1.
In the presence of target molecules (OTA), the conformation of the aptamer specific for OTA was switched from a random coil to an antiparallel G-quadruplex. Subsequently, Exo I was added into the mixture to digest the unfolded aptamers selectively, which are the preferred substrates of Exo I. Finally, SYBR Gold was inserted into the G-quadruplex, which resulted in the significantly increased
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fluorescent intensities that are directly proportional to the OTA concentrations. As shown in Fig. 1A, when no nuclease digestion was involved, the sample with or without OTA (1 µM) exhibited similar fluorescent intensities. By contrast, when Exo I (5 U) digestion was included in the assay steps, the sample with 1 µM OTA showed a dramatically higher fluorescent intensity than the blank sample because of stronger resistance to nuclease digestion of the G-quadruplex structure formed by aptamer and OTA (Fig. 1B).
CD measurement was utilized to monitor the conformation change of aptamer in different cases (Fig. 2). Before the addition of OTA into the solution containing the OTA aptamer, the CD spectrum of aptamer showed the characteristic of a random coil DNA (curve a in Fig. 2). Upon adding OTA (1 µM), the CD spectrum of the aptamer had significantly enhanced positive and negative bands at 290 and 265 nm, respectively (curve b in Fig. 2). Such observation is the typical characteristic of antiparallel G-quadruplex structures [41]. This change in CD spectra should be attributed to the interaction of aptamer with OTA, which led to the increase ellipticity at 290 and 265 nm. Notably, only a small change of ellipticity at 245 nm was observed upon OTA addition.
3.2 Optimum concentration of Exo I To obtain the optimum concentration of Exo I, increasing concentrations of Exo I were added to a fixed ssDNA concentration. As shown in Fig. 3A, Exo I reached to its
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maximum activity at a final concentration of 12.5 U/ml (the added amount is 5 U), therefore, a higher Exo I concentration (>12.5 U/ml) is unnecessary. 3.3 Time optimization for maximum Exo I activity To obtain the optimum time for maximum Exo I activity, the profile of SYBR Gold emission in the presence of unfolded ssDNA and Exo I was monitored at 0, 5, 10, 20, 30 and 40 min. As shown in Fig. 3B, fluorescence intensity decreased rapidly with the increase of incubation time and closed to the minimum at 30 min. Time is an important factor that should be considered in biosensors, therefore, we choose 30 min as the optimum time for maximum Exo I activity. 3.4 Sensitivity of OTA detection For sensitivity studies, different concentrations of OTA solutions were investigated. As illustrated in Fig. 4A, fluorescence measurement showed that the fluorescence intensity in the solution increased along with the increasing OTA concentration. The calibration curve of fluorescence intensity as a function of concentration (0–5 µM) was plotted (Fig. 4B). The inset in Fig. 4B is the linear relationship between the fluorescence intensity and the logarithm of OTA concentration within the range of 20–500 nM (R2=0.990). Moreover, the limit of detection (LOD) was defined as the concentration corresponding to the fluorescence signal at three fold standard deviation of blank without OTA. The calculated LOD of this aptasensor was 16.5 nM. 3.5 Selectivity of OTA detection To determine the specificity of this method, we tested the sensing platform against various structure analogs (Ochratoxin B, OTB; Ochratoxin C, OTC) and other
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mycotoxins (AFB1 and ZEN). In the selectivity experiment, OTA concentration was 200 nM, whereas the concentrations of the other toxins were 1 µM. As shown in Fig.5, the presence of AFB1 or ZEN had a negligible impact on OTA detection, whereas the presence of OTB or OTC resulted in slightly increased fluorescence intensity. OTB and OTC were structural analogs of OTA, which to some extent, still possessed a weak combination ability with OTA aptamer. However, AFB1 and ZEN were distinct from OTA; thus, fluorescence responses were almost negligible. In addition, we tested the interference effect of structure analogues (OTB and OTC) and other mycotoxins (AFB1 and ZEN), cationic ions (Na+, K+, Mg2+, Ca2+) and cationic polymers (PAH, PDDA) on the detection of ochratoxin A. The presence of AFB1 and ZEN only exhibited negligible effect on the detection of ochratoxin A; the presence of OTB, and OTC merely resulted in a slight increase of fluorescence intensity (Fig.S1A). Meanwhile, fluorescence intensity change caused by cationic ions and cationic polymers were less than 10% when detecting ≤ 200 nM of OTA (Fig.S1B). The above results indicated that this fluorescent sensor for OTA detection had outstanding specificity and selectivity. The high selectivity of the developed sensing system was mainly caused by the high recognition ability of OTA aptamer and its binding constant to the target analyte.
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3.6 Method validation Reproducibility and stability are crucial for the aptasensor. To evaluate the reproducibility of the aptasensor, five different aptasensors were prepared independently and used to measure five different samples with the same OTA concentration of 100 nM (Fig.S2A). The relative standard deviation (RSD) of the tests for the five aptasensor was 4.3%, which indicated that the reproducibility of the aptasensor was quite good. The aptasensor stability was evaluated by measuring the fluorescence intensity of the same sample (100 nM OTA) five times (Fig. S2B), and an RSD of 2.5% was obtained, thereby showing that the measurements had good stability. 3.7 Analysis of OTA in real samples To evaluate the feasibility and reliability of the proposed sensing system for practical applications, the aptasensor was used for determining the recoveries of three different OTA concentrations via standard addition methods. We challenged our system with red wine and beer, which can be contaminated by OTA. The absence of OTA in red wine and beer samples purchased from the local supermarket was first confirmed through HPLC method. Different OTA concentrations (20, 100, and 500 nM) were spiked into the samples and detected directly without any pretreatment, except for a 100-fold dilution with buffer solution. The spiked samples were further quantified using the fluorescent aptasensor. As shown in Table 1, the recoveries of the spiked samples and RSDs were in the following ranges: 93.6%–101.98% and 2.31%–4.95% in red wine; and 98.26%–107.9% and
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3.25%–5.42% in beer. These results indicated that the label-free aptasensor is feasible for OTA analysis in a complex sample matrix. To further confirm the applicability of the proposed fluorometric aptasensor in real samples, the present methodology was validated using conventional technique HPLC [42]. Prior to use, all OTA samples were filtered using 0.4 μm filter. As shown in the Table 1, the observed values came closer to the added values, thereby further suggesting that the aptasensor can be easily employed for the sensitive detection of OTA in real samples.
4. Conclusions We designed a label-free aptasensor based on SYBR Gold and Exo I for a simple and sensitive OTA detection. Compared with other aptamer-based fluorescence assays, our proposed label-free method eliminates the laborious and expensive process of DNA modification. Therefore, our method is economical, simple, and convenient. Because of the good selectivity, excellent reproducibility, and acceptable stability of the proposed strategy, it has great potential to be used as a routine tool for the determination of OTA not only in wine and beer but also in other food commodities that are prone to OTA contamination, such as cereals, coffee, and dried fruits. The integration of nuclease, such as Exo I, with specific recognition molecules, such as nucleic acids, to recognize and bind target analytes in the sample matrix has shown a promising future for the detection of targets like toxins, ions, small molecules, and proteins. The simple approach demonstrated here could be modified and coupled with other various detection platforms as well as being useful in high-throughput and 14
paralleled analysis of multiple targets.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 31460423 and 31360384), the department of education of Jilin Province (2016252) and the department of Sciences & Technology of Jilin Province (20160520047JH).
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Author Biographies Name:
Zhijun Guo, Ph.D
Born:
7 December 1980
Email:
[email protected]
Education: 2010-2013
Co-Supervising Ph.D. students at the State Key Laboratory
of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences Dissertation: “Study on aptamer based G-quadruplex for selective determination of ochratoxin A” 2009-2013
Jilin Agricultural university, Changchun, China
Degree: Ph.D. Major: Food science 2004-2007
Jilin Agricultural university, Changchun, China
Degree: Master Major: Food science Thesis: “Cloning, sequence analysis and expression vector construction on proteinase inhibitor cDNA fragments of buckwheat” 2000-2004
Jilin Agricultural university, Changchun, China
Degree: B.A. Major: Applied chemistry
Work experience: 18
2007-2009
Assistant Professor, Yanbian University, China
2009-present
Lecturer, Yanbian University, China
Research interests: My research interests are in microfabrication and nanotechnology for food, biomedical, and environmental applications. Current researches focus on (1) Aptamer and G-quadruplex based biosensors; (2) Small molecule detection based on nanopore; (3) Gold nanoparticles for cell-surface recognition, cancer diagnosis, and delivery applications.
19
200
240 Aptamer + SYBR Gold Aptamer + OTA + SYBR Gold
A
200
180
Fluorescence Intensity (a.u.)
Fluorescence Intensity (a.u.)
220
180 160 140 120 100 80 60 40
B
Aptamer + Exo I +SYBR Gold Aptamer + OTA + Exo I +SYBR Gold
160 140 120 100 80 60 40 20
20 0
0 520
540
560
580
600
620
Wavelength (nm)
520
540
560
580
Wavelength (nm)
Fig. 1. The fluorescence spectra of SYBR Gold/aptamer mixture with or without 1 μM OTA, without (A) or with (B) the Exo I digestion involved. 8
Ellipticity (mdeg)
6 4 2
a
0 -2 -4
b
-6 -8
240
260
280 300 Wavelength (nm)
320
340
Fig. 2. CD spectra of aptamer (1 μM) at different conditions: (a) aptamer (1 μM) in 10 mM Tris buffer (pH 8.5); (b) aptamer (1 μM) + OTA (1 μM) in 10 mM Tris buffer (pH 8.5).
20
600
620
Fluorescence Intensity (a.u.)
220
240
A
220
0U 1U 2U 5U 10 U
200 180 160 140
Fluorescence Intensity (a.u.)
240
120 100 80 60 40 20
0 min 5 min 10 min 20 min 30 min 40 min
B
200 180 160 140 120 100 80 60 40 20
0
0 520
540
560
580
600
620
520
540
560
580
600
620
Wavelength (nm)
Wavelength (nm)
Fig. 3. Factors involved in fluorescence emission intensity. (A) Fluorescence intensity in the presence of various concentrations of Exo I (0, 1, 2, 5 and 10 units were added from top to bottom). (B) Fluorescence intensity as a function of Exo I incubation time (0, 5, 10, 20, 30 and 40 min from top to bottom).
200
200 180
5000 nM 2000 nM 1000 nM 500 nM 200 nM 100 nM 50 nM 20 nM Blank
160 140 120 100 80 60 40
160 140
100 80 60 40 20
0
0 540
560
580
600
620
Wavelength (nm)
120
120
20
520
B
Fluorescence Intensity (a.u.) (a.u.) Intensity Fluorescence
A
Fluorescence Intensity (a.u.)
Fluorescence Intensity (a.u.)
180
R2 = 0.990 2
R =0.990
R
100
80
60
40
20 1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Log C COTA(nM) Log (nM) OTA
0
1000
2000
3000
4000
5000
OTA Concentration (nM)
Fig. 4. (A) Fluorescence results for the label-free detection strategy corresponding to various concentrations of OTA. (B) The maximum peak of fluorescence spectra in (A) were plotted as a function of OTA concentration. Inset in B: linear part of the plot in B. Error bars were obtained from three experiments.
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3.0
Fluorescence Intensity (a.u.)
100
80
60
40
20
0 Blank
AFB1
ZEN
OTC
OTB
OTA
Fig. 5. Selectivity of the sensor toward OTA (200 nM) against other structure analogues and mycotoxins (1 μM). OTA (ochratoxin A), OTB (ochratoxin B), OTC (ochratoxin C), AFB1 (aflatoxin B1), ZEN (zearalenone). Error bars were obtained from three experiments.
O TA
Strong Fluorescence
N
o
O TA
Weak Fluorescence
Aptamer
OTA
Exo I
SYBR Gold
Scheme 1. Schematic description of OTA detection based on label-free fluorescence assay.
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Table 1 Application of aptasensor for OTA determination in red wine and beer samplesa. Sample
Added ( nM )
Red wine
20 100 500
Beer
20 100 500
a
Detected ( nM ) Present method HPLC 20.3 93.6 509.9 21.1 107.9 491.3
Recovery (%) Present method HPLC
RSD (%) Present method HPLC
19.6 102.3 492.9
101.5 93.6 101.98
98 102.3 98.58
3.92 2.31 4.95
4.12 2.61 3.25
18.6 102.3 512.9
105.5 107.9 98.26
93 102.3 106.58
3.25 5.42 4.69
4.91 3.96 2.33
The data reported in the table represents the average of four measurements.
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S0925-4005(17)30370-2 http://dx.doi.org/doi:10.1016/j.snb.2017.02.143 SNB 21874
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
12-10-2016 22-2-2017 23-2-2017
Please cite this article as: Lei Lv, Donghao Li, Renjie Liu, Chengbi Cui, Zhijun Guo, Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.02.143 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe Lei Lva,b, Donghao Lia, Renjie Liuc, Chengbi Cui a,b*, Zhijun Guoa,b* a
Key Laboratory of Natural Resources of Changbai Mountain and Functional
Molecules, Yanbian University, Ministry of Education, YanJi, 133002, China. b
Department of Food Science and Engineering, Yanbian University, YanJi, 133002,
China. c
Institute of food science and engineering, Jilin agricultural university, Changchun,
130118, China. E-mail: [email protected]; [email protected] Fax: (+86) 433-2435549; Tel: (+86) 433-2435549
1
Highlights 1. A label-free fluorescent aptasensor for OTA detection was designed. 2. The aptasensor shows high selectivity toward OTA with a low limit of detection 3. The proposed sensing system can be applied to the analysis of Ochratoxin A in real samples. 4. The principle can be extended to the detection of other targets.
Abstract: A label-free sensing strategy employing aptamers, SYBR Gold, and exonuclease I (Exo I) for ochratoxin A (OTA) detection was designed. In the presence of target molecules (OTA), the conformation of the aptamer specific for OTA is switched from a random coil to an antiparallel G-quadruplex. Subsequently, Exo I is added into the mixture to digest the unfolded aptamers selectively, which are the preferred substrates of Exo I. Following the addition of SYBR Gold as probe, a strong fluorescence intensity is obtained. This aptasensor shows high selectivity toward OTA with a low limit of detection (16.5 nM). The validity of the procedure and applicability of the aptasensor are successfully assessed through the detection of OTA in spiked red wine and beer without interference from the sample matrix. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples indicates its usefulness as a tool for verifying the effectiveness of mycotoxin control strategies.
Keywords: Ochratoxin A, Label-free, SYBR Gold, Aptamer, G-quadruplex
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1. Introduction Mycotoxins are toxic metabolites produced by fungi, mostly by saprophytic molds growing on various foodstuffs, including that of animal feeds, and by many plant pathogens [1]. Ochratoxin A (OTA) is one of the mycotoxins (a natural, toxic secondary metabolite) produced by several species of the fungi Penicillium and Aspergillus. OTA has been widely detected in cereal-derived products, dried fruits, spices, beer, and wine. OTA is potentially carcinogenic to human beings. This mycotoxin is also weakly mutagenic and can cause immunosuppression and immunotoxicity [2]. To avoid the risk of OTA consumption, the detection and quantification of OTA level in contaminated raw materials are considerably useful. Various analytical methods have been established for the determination of OTA. OTA analysis is typically performed via conventional chromatographic methods, such as thin layer chromatography, high-performance liquid chromatography (HPLC), or gas chromatography coupled to ultraviolet visible, fluorescence, or mass spectrometry [3-8]. However, these methods generally require multiple steps, including extraction, extensive sample cleaning, preconcentration, and analyte derivatization, prior to detection. Such sample treatment not only makes the analysis time consuming and costly but also requires trained personnel. Immunoassays provide a simple and economical alternative to instrumental methods for OTA analysis. A common alternative for OTA detection is the immunoassay based on antigen–antibody interactions, such as enzyme-linked immunosorbent assay (ELISA). Several groups have previously established immunoassays for OTA [9-13]. Nevertheless, ELISA is a
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heterogeneous method and involves multiple washing steps. In addition, the storage and application conditions of the antibody, such as temperature, pH, and ionic strength, are strictly defined. All these conditions have limited the ability of the existing antibody-based methods to satisfy the current detection requirements. Aptamers are single-stranded DNA or RNA molecules that can bind specifically to their targets and selected via an in vitro process called systematic evolution of ligands by exponential enrichment [14]. Aptamers have been considered a good candidate alternatives to replace antibodies. Furthermore, aptamers can be selected for a broad range of targets, including small molecules, proteins, nucleic acids, cells, tissues, and organisms. The binding ability of aptamers is as good as that of antibodies, but their synthesis, maintenance, and delivery are easier. Thus, aptamers are promising molecular receptors for bioanalytical applications [15, 16]. Recently, a series of assays using aptamers against OTA have been developed. These assays mainly involved fluorescence [17, 18], electrochemical [19, 20], and colorimetric transducers [21, 22], etc. Among these assays, electrode processing is troublesome for the electrochemical method, and the sample color may interfere with the test results for the colorimetric method. Fluorescence methods have received increasing attention because of their distinct advantages, such as high sensitivity, rapid analysis, and little damage to sample [23-27]. Biosensing via fluorescence is one of the well known methods for the design of aptamer-based assays, largely because of the ease of detection, good sensitivity, and potential for high-throughput analysis. However, frequently in these assays, the aptamer needs to be labeled with
4
fluorophores. The process of DNA modification is laborious and expensive. [28-30]. Moreover, these fluorescent and/or quenching molecules may even alter the binding properties of aptamers [31-33]. In addition, the majority of current labeled dyes, particularly near-infrared dyes, suffer from poor photostability and low emission intensities, ultimately limiting the further improvement of fluorescence methods [34, 35]. Therefore, the development of label-free methods for fluorescent detection is highly desirable. A few label-free fluorescent aptasensors for OTA detection have been reported. For example, Zhang et al. have reported a fluorescent aptasensor based on Tb3+ and structure-switching aptamer for label-free detection of ochratoxin A [36]. However, the Tb3+ is poisonous, and the performance is tedious. Lv et al. have reported a simple and sensitive label-free fluorescent aptasensor for ochratoxin A detection [37]. However, the sensing systems are in turn-off mode, and such turn-off assays might compromise specificity because other quenchers or environmental stimuli might lead to fluorescence quenching, thereby leading to “false positive” results . Considering these problems, we designed a label-free turn-on fluorescent aptasensor for OTA detection based on exonuclease I (Exo I) enzyme and SYBR Gold dye. SYBR Gold was applied as fluorescent probe because of its high sensitivity and specificity for nucleic acids, as well as its low toxicity and good stability [38]. The aptamer specific for OTA can fold to form antiparallel G-quadruplex structure upon exposure to OTA [39]. The formation of antiparallel G-quadruplex structure is resistant to Exo I digestion. The amount of aptamer left after nuclease reaction is proportional to OTA
5
concentrations, thereby making it proportional to the fluorescence intensities from SYBR Gold that can only stain nucleic acids but not their digestion products, nucleoside monophosphates. The results of our experiment also confirmed that our design strategy was very successful. The major advantage of combining Exo I enzyme and SYBR Gold with aptamer is the elimination of the need for DNA modification, thereby significantly reducing the cost. This fluorometric method is low cost, highly sensitivity, and involves a simple performance. The elements used in the assays are cheap and commercially available. Furthermore, the method is a nearly universal one because of the exceptional structure selectivity of Exo I. The principle can be extended to the detection of other targets, such as ions, small molecules, and proteins.
2. Materials and methods 2.1 Materials The OTA aptamers (5’-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3’) [40] were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The fluorescent dye SYBR Gold (10 000× concentrated) was purchased from Invitrogen (CA, USA). Exo I was bought from TaKaRa Biotechnology Inc. (Dalian, China). OTA, ochratoxin B (OTB), ochratoxin C (OTC), aflatoxin B1 (AFB1), and zearalenone (ZEN) were purchased from Fermentek (Jerusalem,
Israel).
Poly
(allylamine)
hydrochloride
(PAH),
poly
(diallyldimethylammonium chloride) (PDDA) were purchased from Sigma Aldrich (Shanghai, China). Binding buffer (10 mM Tris, pH 8.5, 120 mM NaCl, 5 mM KCl,
6
10 mM MgCl2, and 20 mM CaCl2) was used for the binding reaction between the aptamer and OTA. The OTA stock solution (1 mM) was prepared by dissolving ochratoxin in absolute ethanol and stored at -20 °C. Changyu Rose Red Wine was produced by YanTai ChangYu Pioneer Wine Company Limited (Yantai, China). Tsingtao beer was produced by Tsingtao beer Company Limited (Tsingtao, China). All other chemicals were of analytical grade and used as received without further purification. Ultrapure water with an electrical resistivity of 18.2 MΩ cm was obtained from a Milli-Q ultra-high-purity water system (Millipore, Bedford, MA, USA).
2.2 Instrumentation Cary 500 Scan UV-vis Spectrophotometer (Varian, USA) was used to quantify the oligonucleotides. An Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a fluorescence detector was used to record HPLC chromatograms. A JASCO J-810 spectropolarimeter (Tokyo, Japan) was used to collect circular dichroism (CD) spectra in 10 mM Tris buffer (pH 8.5). A RF-5301PC fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) was used to record the fluorescence spectra with a 150 W Xenon lamp as the excitation source. Meanwhile, emission spectra were recorded within the wavelength range of 480-620 nm upon excitation at 495 nm. Slit widths for excitation and emission were set at 3 nm. All measurements were performed at room temperature unless stated otherwise. 2.3 Analysis of aptamer conformation with CD
7
CD spectropolarimeter using solutions of DNA aptamer (1 µM) in an optical chamber (1 cm path length, 1 mL volume), which was deoxygenated with dry purified nitrogen (99.99%) before use and kept in nitrogen atmosphere during experiments. Each CD spectrum was the accumulation of three scans at 200 nm/min with a 1 nm band width and a time constant of 1 s. The data were collected within 230-340 nm at 0.1 nm intervals. The background of the buffer solution was subtracted from CD data. 2.4 Optimizing Exo I concentration Increasing concentrations of Exo I (0–10 units) were added to a constant concentration of ssDNA aptamer (100 nM) in binding buffer (final volume, 400 µL). After 30 min of incubation at 37 °C, 100 µL of 10 × SYBR Gold was mixed with the samples. After equilibrating the solution in the dark at room temperature for another 15 min, fluorescence intensity was measured. 2.5 Optimization of incubation time of Exo I with ssDNA aptamer on fluorescence intensities 5 units of Exo I were added to 100 nM ssDNA aptamer in binding buffer (final volume, 400 µL). The mixture was incubated at 37 °C for 0–40 min. Subsequently, 100 µL of 10 × SYBR Gold was added to the samples, and fluorescence intensities were recorded. 2.6 Fluorescent detection of OTA For quantitative measurement of OTA, 200 µL of solution containing 250 nM of aptamer was mixed with buffer solution of different OTA concentrations and allowed to settle for 15 min. Subsequently, 5 units of Exo I were added, and the total final
8
volume was 400 µL. The mixtures were incubated at 37 °C for 30 min. Afterward, 100 µL of 10 × SYBR Gold was added to the samples. After equilibrating the solution in the dark at room temperature for another 15 min, fluorescence intensity was then measured. 2.7 Application To confirm the feasibility of this sensor for analysis of actual samples, three different concentrations of OTA (20, 100, and 500 nM) were spiked into red wine and beer, and fluorescent intensity was used to determine OTA concentrations.
3. Results and discussion 3.1 Design strategy for OTA detection The designed aptasensor is based on target-induced conformation of G-quadruplex structure, digestion of unfolded ssDNA by Exo I, and the ability of SYBR Gold as a fluorescent probe. A schematic representation of OTA detection is illustrated in Scheme 1.
In the presence of target molecules (OTA), the conformation of the aptamer specific for OTA was switched from a random coil to an antiparallel G-quadruplex. Subsequently, Exo I was added into the mixture to digest the unfolded aptamers selectively, which are the preferred substrates of Exo I. Finally, SYBR Gold was inserted into the G-quadruplex, which resulted in the significantly increased
9
fluorescent intensities that are directly proportional to the OTA concentrations. As shown in Fig. 1A, when no nuclease digestion was involved, the sample with or without OTA (1 µM) exhibited similar fluorescent intensities. By contrast, when Exo I (5 U) digestion was included in the assay steps, the sample with 1 µM OTA showed a dramatically higher fluorescent intensity than the blank sample because of stronger resistance to nuclease digestion of the G-quadruplex structure formed by aptamer and OTA (Fig. 1B).
CD measurement was utilized to monitor the conformation change of aptamer in different cases (Fig. 2). Before the addition of OTA into the solution containing the OTA aptamer, the CD spectrum of aptamer showed the characteristic of a random coil DNA (curve a in Fig. 2). Upon adding OTA (1 µM), the CD spectrum of the aptamer had significantly enhanced positive and negative bands at 290 and 265 nm, respectively (curve b in Fig. 2). Such observation is the typical characteristic of antiparallel G-quadruplex structures [41]. This change in CD spectra should be attributed to the interaction of aptamer with OTA, which led to the increase ellipticity at 290 and 265 nm. Notably, only a small change of ellipticity at 245 nm was observed upon OTA addition.
3.2 Optimum concentration of Exo I To obtain the optimum concentration of Exo I, increasing concentrations of Exo I were added to a fixed ssDNA concentration. As shown in Fig. 3A, Exo I reached to its
10
maximum activity at a final concentration of 12.5 U/ml (the added amount is 5 U), therefore, a higher Exo I concentration (>12.5 U/ml) is unnecessary. 3.3 Time optimization for maximum Exo I activity To obtain the optimum time for maximum Exo I activity, the profile of SYBR Gold emission in the presence of unfolded ssDNA and Exo I was monitored at 0, 5, 10, 20, 30 and 40 min. As shown in Fig. 3B, fluorescence intensity decreased rapidly with the increase of incubation time and closed to the minimum at 30 min. Time is an important factor that should be considered in biosensors, therefore, we choose 30 min as the optimum time for maximum Exo I activity. 3.4 Sensitivity of OTA detection For sensitivity studies, different concentrations of OTA solutions were investigated. As illustrated in Fig. 4A, fluorescence measurement showed that the fluorescence intensity in the solution increased along with the increasing OTA concentration. The calibration curve of fluorescence intensity as a function of concentration (0–5 µM) was plotted (Fig. 4B). The inset in Fig. 4B is the linear relationship between the fluorescence intensity and the logarithm of OTA concentration within the range of 20–500 nM (R2=0.990). Moreover, the limit of detection (LOD) was defined as the concentration corresponding to the fluorescence signal at three fold standard deviation of blank without OTA. The calculated LOD of this aptasensor was 16.5 nM. 3.5 Selectivity of OTA detection To determine the specificity of this method, we tested the sensing platform against various structure analogs (Ochratoxin B, OTB; Ochratoxin C, OTC) and other
11
mycotoxins (AFB1 and ZEN). In the selectivity experiment, OTA concentration was 200 nM, whereas the concentrations of the other toxins were 1 µM. As shown in Fig.5, the presence of AFB1 or ZEN had a negligible impact on OTA detection, whereas the presence of OTB or OTC resulted in slightly increased fluorescence intensity. OTB and OTC were structural analogs of OTA, which to some extent, still possessed a weak combination ability with OTA aptamer. However, AFB1 and ZEN were distinct from OTA; thus, fluorescence responses were almost negligible. In addition, we tested the interference effect of structure analogues (OTB and OTC) and other mycotoxins (AFB1 and ZEN), cationic ions (Na+, K+, Mg2+, Ca2+) and cationic polymers (PAH, PDDA) on the detection of ochratoxin A. The presence of AFB1 and ZEN only exhibited negligible effect on the detection of ochratoxin A; the presence of OTB, and OTC merely resulted in a slight increase of fluorescence intensity (Fig.S1A). Meanwhile, fluorescence intensity change caused by cationic ions and cationic polymers were less than 10% when detecting ≤ 200 nM of OTA (Fig.S1B). The above results indicated that this fluorescent sensor for OTA detection had outstanding specificity and selectivity. The high selectivity of the developed sensing system was mainly caused by the high recognition ability of OTA aptamer and its binding constant to the target analyte.
12
3.6 Method validation Reproducibility and stability are crucial for the aptasensor. To evaluate the reproducibility of the aptasensor, five different aptasensors were prepared independently and used to measure five different samples with the same OTA concentration of 100 nM (Fig.S2A). The relative standard deviation (RSD) of the tests for the five aptasensor was 4.3%, which indicated that the reproducibility of the aptasensor was quite good. The aptasensor stability was evaluated by measuring the fluorescence intensity of the same sample (100 nM OTA) five times (Fig. S2B), and an RSD of 2.5% was obtained, thereby showing that the measurements had good stability. 3.7 Analysis of OTA in real samples To evaluate the feasibility and reliability of the proposed sensing system for practical applications, the aptasensor was used for determining the recoveries of three different OTA concentrations via standard addition methods. We challenged our system with red wine and beer, which can be contaminated by OTA. The absence of OTA in red wine and beer samples purchased from the local supermarket was first confirmed through HPLC method. Different OTA concentrations (20, 100, and 500 nM) were spiked into the samples and detected directly without any pretreatment, except for a 100-fold dilution with buffer solution. The spiked samples were further quantified using the fluorescent aptasensor. As shown in Table 1, the recoveries of the spiked samples and RSDs were in the following ranges: 93.6%–101.98% and 2.31%–4.95% in red wine; and 98.26%–107.9% and
13
3.25%–5.42% in beer. These results indicated that the label-free aptasensor is feasible for OTA analysis in a complex sample matrix. To further confirm the applicability of the proposed fluorometric aptasensor in real samples, the present methodology was validated using conventional technique HPLC [42]. Prior to use, all OTA samples were filtered using 0.4 μm filter. As shown in the Table 1, the observed values came closer to the added values, thereby further suggesting that the aptasensor can be easily employed for the sensitive detection of OTA in real samples.
4. Conclusions We designed a label-free aptasensor based on SYBR Gold and Exo I for a simple and sensitive OTA detection. Compared with other aptamer-based fluorescence assays, our proposed label-free method eliminates the laborious and expensive process of DNA modification. Therefore, our method is economical, simple, and convenient. Because of the good selectivity, excellent reproducibility, and acceptable stability of the proposed strategy, it has great potential to be used as a routine tool for the determination of OTA not only in wine and beer but also in other food commodities that are prone to OTA contamination, such as cereals, coffee, and dried fruits. The integration of nuclease, such as Exo I, with specific recognition molecules, such as nucleic acids, to recognize and bind target analytes in the sample matrix has shown a promising future for the detection of targets like toxins, ions, small molecules, and proteins. The simple approach demonstrated here could be modified and coupled with other various detection platforms as well as being useful in high-throughput and 14
paralleled analysis of multiple targets.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 31460423 and 31360384), the department of education of Jilin Province (2016252) and the department of Sciences & Technology of Jilin Province (20160520047JH).
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17
Author Biographies Name:
Zhijun Guo, Ph.D
Born:
7 December 1980
Email:
[email protected]
Education: 2010-2013
Co-Supervising Ph.D. students at the State Key Laboratory
of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences Dissertation: “Study on aptamer based G-quadruplex for selective determination of ochratoxin A” 2009-2013
Jilin Agricultural university, Changchun, China
Degree: Ph.D. Major: Food science 2004-2007
Jilin Agricultural university, Changchun, China
Degree: Master Major: Food science Thesis: “Cloning, sequence analysis and expression vector construction on proteinase inhibitor cDNA fragments of buckwheat” 2000-2004
Jilin Agricultural university, Changchun, China
Degree: B.A. Major: Applied chemistry
Work experience: 18
2007-2009
Assistant Professor, Yanbian University, China
2009-present
Lecturer, Yanbian University, China
Research interests: My research interests are in microfabrication and nanotechnology for food, biomedical, and environmental applications. Current researches focus on (1) Aptamer and G-quadruplex based biosensors; (2) Small molecule detection based on nanopore; (3) Gold nanoparticles for cell-surface recognition, cancer diagnosis, and delivery applications.
19
200
240 Aptamer + SYBR Gold Aptamer + OTA + SYBR Gold
A
200
180
Fluorescence Intensity (a.u.)
Fluorescence Intensity (a.u.)
220
180 160 140 120 100 80 60 40
B
Aptamer + Exo I +SYBR Gold Aptamer + OTA + Exo I +SYBR Gold
160 140 120 100 80 60 40 20
20 0
0 520
540
560
580
600
620
Wavelength (nm)
520
540
560
580
Wavelength (nm)
Fig. 1. The fluorescence spectra of SYBR Gold/aptamer mixture with or without 1 μM OTA, without (A) or with (B) the Exo I digestion involved. 8
Ellipticity (mdeg)
6 4 2
a
0 -2 -4
b
-6 -8
240
260
280 300 Wavelength (nm)
320
340
Fig. 2. CD spectra of aptamer (1 μM) at different conditions: (a) aptamer (1 μM) in 10 mM Tris buffer (pH 8.5); (b) aptamer (1 μM) + OTA (1 μM) in 10 mM Tris buffer (pH 8.5).
20
600
620
Fluorescence Intensity (a.u.)
220
240
A
220
0U 1U 2U 5U 10 U
200 180 160 140
Fluorescence Intensity (a.u.)
240
120 100 80 60 40 20
0 min 5 min 10 min 20 min 30 min 40 min
B
200 180 160 140 120 100 80 60 40 20
0
0 520
540
560
580
600
620
520
540
560
580
600
620
Wavelength (nm)
Wavelength (nm)
Fig. 3. Factors involved in fluorescence emission intensity. (A) Fluorescence intensity in the presence of various concentrations of Exo I (0, 1, 2, 5 and 10 units were added from top to bottom). (B) Fluorescence intensity as a function of Exo I incubation time (0, 5, 10, 20, 30 and 40 min from top to bottom).
200
200 180
5000 nM 2000 nM 1000 nM 500 nM 200 nM 100 nM 50 nM 20 nM Blank
160 140 120 100 80 60 40
160 140
100 80 60 40 20
0
0 540
560
580
600
620
Wavelength (nm)
120
120
20
520
B
Fluorescence Intensity (a.u.) (a.u.) Intensity Fluorescence
A
Fluorescence Intensity (a.u.)
Fluorescence Intensity (a.u.)
180
R2 = 0.990 2
R =0.990
R
100
80
60
40
20 1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Log C COTA(nM) Log (nM) OTA
0
1000
2000
3000
4000
5000
OTA Concentration (nM)
Fig. 4. (A) Fluorescence results for the label-free detection strategy corresponding to various concentrations of OTA. (B) The maximum peak of fluorescence spectra in (A) were plotted as a function of OTA concentration. Inset in B: linear part of the plot in B. Error bars were obtained from three experiments.
21
3.0
Fluorescence Intensity (a.u.)
100
80
60
40
20
0 Blank
AFB1
ZEN
OTC
OTB
OTA
Fig. 5. Selectivity of the sensor toward OTA (200 nM) against other structure analogues and mycotoxins (1 μM). OTA (ochratoxin A), OTB (ochratoxin B), OTC (ochratoxin C), AFB1 (aflatoxin B1), ZEN (zearalenone). Error bars were obtained from three experiments.
O TA
Strong Fluorescence
N
o
O TA
Weak Fluorescence
Aptamer
OTA
Exo I
SYBR Gold
Scheme 1. Schematic description of OTA detection based on label-free fluorescence assay.
22
Table 1 Application of aptasensor for OTA determination in red wine and beer samplesa. Sample
Added ( nM )
Red wine
20 100 500
Beer
20 100 500
a
Detected ( nM ) Present method HPLC 20.3 93.6 509.9 21.1 107.9 491.3
Recovery (%) Present method HPLC
RSD (%) Present method HPLC
19.6 102.3 492.9
101.5 93.6 101.98
98 102.3 98.58
3.92 2.31 4.95
4.12 2.61 3.25
18.6 102.3 512.9
105.5 107.9 98.26
93 102.3 106.58
3.25 5.42 4.69
4.91 3.96 2.33
The data reported in the table represents the average of four measurements.
23