Portable Aptasensor of Aflatoxin B1 in Bread Based on a Personal

Jun 26, 2018 - Despite some recent developments on the portable on-site sensor of Aflatoxin B1 (AFB1), the complex and expensive preparation of ...
0 downloads 0 Views 699KB Size
Download PDF
Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Portable Aptasensor of Aflatoxin B1 in Bread Based on a Personal Glucose Meter and DNA Walking Machine Xinsheng Yang, Dongmin Shi, Shengmei Zhu, Baojuan Wang, Xiaojun Zhang, and Guangfeng Wang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00304 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

ACS Sensors

Portable Aptasensor of Aflatoxin B1 in Bread Based on a Personal Glucose Meter and DNA Walking Machine Xinsheng Yang†, Dongmin Shi†, Shengmei Zhu†, BaojuanWang‡, Xiaojun Zhang†, Guangfeng Wang†* † Key Laboratory of Chem-Biosensing, Anhui province; Key Laboratory of Functional Molecular Solids, Anhui prov-ince; College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, P. R. China. ‡ Institute of Molecular Biology and Biotechnology and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu 241000, P. R. China. ABSTRACT: Despite of some recent development on the portable on-site sensor of Aflatoxin B1 (AFB1), the complex and expensive preparation of recognition elements still limited their wide applications. In this paper, by the fast, low-cost and stable recognition of aptamer DNA-AFB1, a portable aptasensor was constructed for the on-site detection of AFB1 in food matrixes, with the readout of personal glucose meter (PGM) and DNA walking machine for signal probe separation. In such an assay protocol, the target could trigger the DNA walker autonomously moving on the electrode surface, propelled by unidirectional Pb2+-specific DNAzyme digestion, which could amplify the signal and separate the signal probe as well for further quantification by the PGM. Under optimized conditions, the increase of PGM signal was relative with the concentration of AFB1 ranging from 0.02 to 10 nM and the low limit of detection (LOD) was 10 pM (S/N=3). With the features of portability, and cheapness, the presented userfriendly method could be extended to various other analytes for wide Point-of-care (POC) application. KEYWORDS: aflatoxin B1, personal glucose meter, DNA walker machine, detection, on-site An old Chinese saying goes, “hunger breeds discontentment, food to safety first.” With the rapid development of economy and improvement of living conditions, food safety has become an esp. important issue in the current society1. Aflatoxin B1 (AFB1) produced mainly by Aspergillus flavus and Aspergillus parasiticus,2,3 exists in various kinds of food (bread, pastry, cake) and feedstuff products (such as maize, groundnut, and peas) mainly as their exposing in wet environment at room temperature4-6. The most toxic and carcinogenic among the major species of concern aflatoxins is AFB1. The long-term exposure to AFB1 maybe lead severe liver-related disease in human beings and animals.7 Until now, a variety of detection approaches for the quantification of AFB1 were developed.8,9 Despite of their accuracy and sensitivity, chromatography methods for the quantification of AFB1 usually required expensive instruments, skilled operators and complicated sample pretreatment as well, which was difficult for on-site detection, and thus limited their wide application, especially in developing countries9-11. Recently, some immunoassay-based methods have been developed to detect AFB1 on-site. For example, fluorescent, electrochemical and colorimetric AFB1 sensors based on the immunoreactions were developed for the on-site detection of AFB112-14a. However, despite of their improved analytical merits, some of them still required heavy instrumental systems and esp. the involvement of antibodies as bio-recognition element with the limitations of high cost, limited stability and engendered from the origin of animal. Because of the high affinity and specificity for various of targets, thermal and chemical stability, and cheap preparation of aptamers14b-d, it is desirable to fabricate the portable aptasensors for the on-site detection of AFB1. However, according to our limited literatures, the research on the portable AFB1 aptasensors is still limited. Gratifyingly, Zhu’s group designed portable AFB1 aptasensors by distance-readout microfluidic chip and volumetric bar chart chip based on an AFB1-responsive aptamer DNA hydrogel15. It was then coupled with enzyme hydrolyze reaction by Zhao et al. to develop a portable AFB1 aptasensor using pH meter as the readout16. However,

although these methods were portable for on-site monitoring of AFB1, the expensive preparation of DNA hydrogel and the complex fabrication steps may limit their wide application. Thus, it is essential and significant to explore novel portable AFB1 aptasensors with simplicity, low-cost, and sensitivity. Recently, a lot of efforts have been devoted to exploring inhome/in-field tests, eg. the pregnancy test paper, glucose strips, alcohol detector, to monitor pathogen, biomarker, and pollutant, etc. on-site in modern food safety, healthcare and environmental evaluation 17. Among these portable devices, the personal glucose meter (PGM), is an ideal portable device widely applied to the clinical diagnosis of hyperglycemia due to its being easy to carry on and operate, inexpensiveness, reliability and wide acceptability in the whole world18-20. Besides, with the recent integration of PGM with other enzymes, PGM has been applied for the design of various other non-glucose sensor.21 For example, with the aid of an invertase, Yang and coworkers developed the on-site detection of disease biomarkers using PGM as readout.22 Wang’s group combined a PGM with the microfluidic chip for the point of care (POC) detection of nucleic acids .23 PGM was further integrated with alkaline phosphatase for the fabrication of sensor of galactose-1-phosphate uridyltransferase in clinical galactosemia diagnosis by Lu et al.24 With the maltopentaose and α-glucosidase and PGM, the detection of α-amylase by one-step in real samples were developed by Wang et al. 25 Although PGM has been applied widely, such as the detection of heavy metal ion for environmental monitoring and the evaluation of disease biomarkers for medical diagnosis by the general public, few report was involved with the on-site monitoring of pollutant of food by PGM. Therefore, it is especially desirable to explore the PGM based aptasensor for on-site detection of AFB1 in food. Artificial molecular machines have gained growing attention in recent years due to their roles in simulating some biological processes.26-28 Nevertheless, the study on the practical applications of artificial molecular machines are still rare. Recently, as special molecular machines, DNA walking machines have been applied

ACS Paragon Plus Environment

ACS Sensors 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

in the fabrication of various biosensors29,30. As for these DNA walking machines, under the external stimuli such as the ions31 or special DNA strands, the migration of the DNA walker could locomote autonomously which could lead the variations of DNA nanostructures27,32. With the stimuli related variations, the detection of the ions or DNA stands could be realized. Furthermore, by the combination with restriction enzymes, the cargo migration could occur with a multistep migration and the locomotion usually could be regulated accurately in the programmed way at the nanoscaled level, which has showed remarkable application prospect in biosensing33,34. For example, with the assistance of enzymatic recycling cleavage strategy, Wang et al. has developed a novel miRNA-responsive DNA walker biosensor.35 Fan’s group reported the autonomous movement of DNA walker on a nucleic acid modified Au nanoparticles initiated by exonuclease III, resulting in the cascade signal amplification, which was further explored for bioanalytical applications.36 Therefore, it is interesting to combine the locomotion and controllability of DNA walker with the on-site detection of AFB1. Herein, a simple, low cost, portable and sensitive aptasensor for on-site detection of AFB1 was fabricated by coupling PGM with DNA walking machine. In this strategy, the intense and selective conjugation of aptamers with the targes (AFB1) possessed distinct advantages including fast, reproducible synthesis and great stability. Pb2+-specific DNAzyme are functional DNA molecules that exhibit catalytic activity toward specific substrate,37,38 which was employed to realized the separation of signal molecules. By the invertase modified on the DNA signal probe transformed the sucrose into the glucose molecules, the target could be detected by an external PGM. The process avoided the requirement of multiple separation and washing steps.

EXPERIMENTAL SECTION Material and Reagent. AFB1, aflatoxin B2 (AFB2), deoxynivalenol (DON), zearalenone (ZON), aflatoxin M1 (AFM1), aflatoxin G2 (AFG2), aflatoxin G1 (AFG1), bovine serum albumin (BSA), invertase (β-fructose-dase, from baker’s yeast) were achieved from Sigma-Aldrich (St. Louis, MO, USA). N-(3(Dimethylamino) propyl)-N-ethylcarbodiimide hydride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Shanghai Medpep Co. Ltd. (Shanghai, China). The DNA strands in HPLC purified form were ordered by Sangon Biotech Ltd., (Shanghai, China) and the corresponding sequences were listed as below: Substrate DNA: 5'-SH-GGGCCTAGCGArAGGGCACGAGACACAGAGAGA CAACACGTGCCCAAC-NH2-3'. Aptamer DNA: 5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCT TCGCTAGGCCC-3’. Walker DNA: 5’-TGTCTTGTGCTCCGAGCGGTCGAAATCGCTAGGC-3’. 0.1 M phosphate buffer solution (PBS, pH 7.4) was prepared with 0.1M Na2HPO4 and 0.1M NaH2PO4. 0.1 M PBS (pH 7.4) with 500 mM NaCl was employed as the hybridization buffer and washing buffer for oligonucleotide. The working solution of DNA was obtained by diluting the stock solution with 10 mM PBS buffer (pH 7.4). A 10 mM PBS (pH 7.4) buffer containing 10 mM [Fe(CN)6]3-/[Fe(CN)6]4- and 1 M KCl was used as the electrochemical impedance spectroscopy buffer. All other chemicals involved were of analytical-reagent grade. Ultrapure water obtained by purification system of PSDK2-10-C (resistance 18.2 MΩ cm) (Beijing, China) was used throughout the experiments. Apparatus. Electrochemical impedance spectroscopy (EIS) and cyclic voltammogram (CV) were carried out with a CHI 660B electrochemical workstation (Shanghai Chenhua Instrument, China). A conventional three-electrode system was used with a satu-

rated calomel electrode (SCE) as reference electrode, a platinum wire as counter electrode and a modified gold electrode (GE, Φ=2 mm) as working electrode. The glucose meter, ACCU-CHEK Active, was from Roche Diagnostics GmbH (Mannheim, Germany). Preparation of Invertase Labeled DNA. The invertase labeled DNA was prepared by the following steps. Initially, 0.4 mL 20 mg mL−1 invertase in PBS was mixed with20 mM EDC and 5 mM NHS in 1 mL of PBS (pH 7.4) for 2 h at 4 °C. Then, the single-stranded substrate DNA (50 µL, 100 µM) modified with group of amino was injected into the above PBS mixed solution to react for 12 h at 4 °C. After that, the invertase conjugated DNA were purified by Amicon-100 K with PBS for 5 times. Finally, the invertase labeled DNA conjugates were dissolved in 100 µL PBS (pH 7.4) for further use. The Fabrication Process of the Aptasensor. Before the modification of the gold electrode (GE, 2 mm diameter), it was first carefully polished with alumina powder of 0.3 and 0.05 µm. Meanwhile, it was rinsed thoroughly with water after each step of polishment, and then it was sonicated for 5 min sequentially in water, ethanol and water again. After that, it was immersed into the freshly prepared piranha solution (98% H2SO4: 30% H2O2, 3:1 by volume) for 10 min and washed with ultrapure water thoroughly. Afterwards, it was scanned in 0.1 M H2SO4 between −0.2 V and 1.55 V at 0.1 V/s until the repeatable cyclic voltammogram was attained. Finally, the electrode was rinsed with ultrapure water and dried under nitrogen. The cleaned GE was immersed with 20 µL of the invertase conjugated DNA solution for 10 h at 4 °C to obtain the invertase conjugated DNA modified GE (Invertase-DNA/GE). Afterward, the resulting modified electrode was immersed in 20 µL of the aptamer DNA solution (1 µM) for 2 h at room temperature to form the duplex of Invertase-DNA and aptamer modified electrode (duplex/GE). Then, the resulting modified electrode was incubated with 20 µL of series concentration of AFB1 for 2 h at 37 °C to release some of the aptamer and partially recover the InvertaseDNA/GE (p-Invertase-DNA/GE). Finally, the obtaining modified electrode was incubated with 20 µL of the DNA walker solution (containing 1 µM walker DNA and 5 µM Pb2+) for 2 h at room temperature. The Invertase-DNA on the electrode was cleaved obtaining the remaining part of Invertase-DNA on the electrode (r-DNA/GE). Meanwhile, the resulting solution contained the cleaved Invertase-DNA fragments to be detected. Herein, all the PGM signals were recorded at 20 min after the addition of the sucrose. Procedures for AFB1 Detection. First, 1.0 M sucrose solution (20 µL) was mixed with the resulting solution and reacted for 20 min at room temperature to transform the sucrose into glucose as the sample to be detected. Then the test strip was inserted carefully into the personal glucose meter (arrows and green squares upward). When the droplet symbol appeared on the display screen and a click was heard, 5 µL of the samples solution was dropped on the green squares of the test strip and then the record the value was displayed and recorded. In the control experiment, the roles of AFB1 (0.3 nM), invertase, walker DNA and Pb2+ were investigated through the same assay process as the above described except in the absence of the AFB1, invertase, walker DNA or Pb2+. Real Sample Analysis. In this experiment, three kinds of bread were acted as real food samples. To obtain moldy food samples, we kept the clean food samples in a wet environment for about 10 days at room temperature. Then the clean and moldy samples were extracted in the same conditions. The extraction procedures were performed according to previous report.39,40 Typically, 2 g of the above food samples were immersed in 2 mL of methanol. After being shaken for 30 min on a laboratory shaker, followed by centrifuging at 1000 ×g for 5 min. With repeating the above extraction procedure for three times, all the extractant were collect-

ACS Paragon Plus Environment

Page 2 of 8

Page 3 of 8 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

ACS Sensors ed and transferred into the 5 mL polyethylene tube. Subsequently, the solution was diluted to the mark with methanol. Finally, a 100 µL of the extracted sample solution was analyzed by PGM measurements.

RESULTS AND DISCUSSION Principle of the Portable PGM based AFB1 Aptasensor Based on DNA Walker Machine. In this study, a simple, low cost, accurate aptasensor was developed for portable detection of AFB1 utilizing PGM with DNA walking machine, as shown in Scheme 1. First, the substrate DNA containing a cleavage point rAG with the thiol and invertase modified at 3’ and 5’ end was assembled on the surface of the cleaned GE through the covalent bond of Au-S. Then, the aptamer DNA were captured on the electrode through the hybridization between substrate DNA and aptamer DNA. Followed by the modified electrode was first incubated with the AFB1, the aptamer DNA would leave away from the electrode surface because of the specific conjugation of AFB1 and aptamer DNA. Afterwards, the resultant electrode was treated with the walker DNA solution in the presence of Pb2+. The cleavage point rAG of substrate DNA strand was cut by Pb2+, releasing a short DNA fragment labeled with invertase in the resulting solution. The invertase could further catalyze sucrose to glucose to be detected by the PGM. The PGM readout is closely related with the target AFB1, which is the base for the assay. Due to it avoided the requirement of complicated instruments and sophisticated operators with PGM as readout, the present simple method has potential for on-site detection of AFB1.

Scheme 1. Illustration of the present AFB1 aptasensor with PGM as readout and DNA walking machine for separation. Characterization and Feasibility Investigation of the Aptasensor. Due to the electrochemical impedance spectra is considered as a useful technique to investigate the surface character, the stepwised construction procedure of the present modified GE was characterized through EIS. In the Nyquist plots of EIS, the diameter of the semicircle part was corresponded with the electron transfer resistance, Ret. As shown in Figure 1A, compared with the Ret of the bare GE (curve a), an obvious increase Ret was observed after the modification of substrate DNA on the GE surface (curve b). As the aptamer DNA was immobilized onto the GE surface, the Ret continuously increased, which was ascribed to the DNA phosphate skeletons with negative charges blocking the arrival of the redox probe [Fe(CN)6]3−/4− to the electrode surface (curve c). However, with the introduction of AFB1, the Ret decreased obviously (curve d) due to the specific biding of AFB1 and the aptamer DNA causing the libration of the aptamer from the surface of the electrode. Thus the accessibility of the redox probes to the electrode interface was enhanced. As expected, Pb2+-specific DNAzyme was then used to cleave substrate DNA sites and a further decreased Ret was observed due to the resulting shorter DNA on electrode surface (curve e). In addition, CV in

PBS containing redox probe of [Fe(CN)6]3−/4− was used to confirm the modification process. As displayed in Figure 1B, a pair of well-defined and reversible redox peaks (curve a) was obtained on the bare GE. After the GE was modified with the duplex DNA (substrate DNA/aptamer DNA), a noticeable decrease of the redox peak currents and an obvious increase of the redox peak separation appeared (curve b). Upon the AFB1 was introduced, the redox currents showed an obvious increase and the peak separation decreased (curve c). Furthermore, the redox currents showed slight increases (curve d) after incubation with walker DNA and Pb2+, indicating that the substrate DNA was cleaved successfully. These results confirmed the successful fabrication of the aptasensor, which may provide beneficial supports for the portable detection of AFB1 utilizing PGM with DNA walking machine. Moreover, the EIS was also carried out at regular intervals after the addition of walker DNA and Pb2+ to demonstrate the process of the cleaving step by step, as displayed in Figure 1C. As time went on, the Ret decreased gradually, indicating that an increasing amount of substrate DNA was cut off, releasing the cleaved DNA fragments from the electrode gradually. The above characterization provided a validation of the successful fabrication of the designed AFB1 sensor with PGM and DNA walking machine.

Figure 1. (A) EIS diagram with the frequency from 100 kHz to 0.1 Hz and signal amplitude of 5 mV, (a) bare GE; (b) InvertaseDNA/GE; (c) duplex/GE; (d) p-Invertase-DNA/GE; (e) rDNA/GE. (B) CV responses at the scan rate of 100 mV s−1 in the potential window from 0.6 to −0.2 V in 5mM [Fe(CN)6]3−/4− containing 0.1 M KCl of different electrodes: (a) bare GE; (b) duplex/GE; (c) p-Invertase-DNA/GE; (d) r-DNA/GE. (C) EIS diagram of r-DNA/GE relative to different reaction time in solution containing walker DNA and Pb2+ (every 12 min). (D) PGM readout of the aptasensor at different reaction time with (a) or without (b) 0.3 nM target AFB1. (E) and (F) Different test strips and their values corresponding to (a) and (b) in the picture of D. Furthermore, the feasibility of the proposed PGM based AFB1 aptasensor was investigated. First, the resulting solution with or without the existence of AFB1 was detected by PGM readout which was recorded every 10 m in.According to the result of Figure 1D, with the existence of the AFB1, the PGM value enlarged with the elongation of incubation time until it arrived at 60 min. After that, the PGM signal kept almost unchanged (curve a,) Nevertheless, the PGM signal gave no obvious changes with the incubation time increased over 60 min without the addition of AFB1 (curve b). Additionally, apart from the values shown in the PGM, the color in the display round window varied progressively from yellow to green (Figure 1E) with the addition of AFB1. Otherwise, no color change was observed (Figure 1F). The results suggested that (i) target AFB1 could initiate the conversion of the sucrose to glucose, proving the feasibility of the biosensor; (ii) it took some time for the scission of the substrate DNA by Pb2+-specific DNAzyme, further confirming the process of DNA walker.

ACS Paragon Plus Environment

ACS Sensors 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

In order to confirm the dependability of the proposed aptasensor, control experiments were conducted under the same conditions except without the AFB1, invertase, walker DNA, or Pb2+. As displayed in column a of Figure 2, no significant PGM signals were obtained in the absence of AFB1 under the same conditions. Similarly, the glucose could not be generated from sucrose in the case of that without invertase (column b), suggesting that invertase was necessary in the present sensors. Moreover, in the absence of walker DNA or Pb2+, the substrate DNA were not able to be cleaved because of Pb2+-specific DNAzyme could not form. Generally, there were no evident PGM responses in the absence of the corresponding AFB1, invertase, walker DNA or Pb2+ (columns a, b, c, d) because in these cases no obvious amount of glucose could be generated and thus the yellow color had no obvious change (strips a, b, c and d, inset). Meanwhile, it also indicated a low background response of the present method. Comparatively, an obviously high PGM signal (column e) and green color (strip e, inset) was obtained through the present sensor in the presence of 0.3 nM AFB1, as shown in Figure 2, resulting from the conversion of sucrose to glucose by the invertase. Based on the above results, it could be concluded that the designed PGM-based sensor had the potential for the determination of AFB1.

tration of Pb2+, incubation time between AFB1 and aptamer DNA, and the pH) were optimized. First, different cleaving time of DNAzyme from 20 to 160 min was investigated. The result in Figure 3A showed that the PGM signal enlarged with the time extending at the initial stage but then kept no more obvious change after 120 min. Therefore, the optimal experimental cleavage time was chosen as 120 min. Then, the effect of Pb2+ concentration was checked, as Figure 3B showed. Similarly, as the concentration of Pb2+ increased from 1 to 8 µM, the PGM readout enlarged. And then it kept constant at 5 µM, but was nearly unchanged even Pb2+ concentration was further increased. Hence, 5 µM was chosen as the optimized concentration of Pb2+ in further experiment. Furthermore, different incubation time of specific conjugation of AFB1 and aptamer DNA was studied to for the optimization. As shown in Figure 3C, the PGM response increased along with the increasing time of the binding from 20 to 160 min and had a level off tendency after 120 min. Therefore, the optimal reaction time for AFB1 conjugation is selected as 120 min. Moreover, due to the existence of the enzyme, DNA and AFB1, which may be affected by the strong acid or alkaline, the pH value may has great effect on the response of the aptasensor. For this purpose, PBS with different pH value from 5.4 to 8.9 was tested. As shown in Figure 3D, it was obvious that the PGM signal enhanced at first but then arrived at maximum of pH 7.4. After that, the signal further decreases. Therefore, pH 7.4 as the optimum pH value was chosen for further experiment. Analytical Performance of PGM Based Aptasensor toward Target AFB1. For the exploration of assay performances, the developed aptasensor with different concentrations of AFB1 was investigated under optimal conditions. The relationship of PGM value with the concentration of AFB1 was shown in Figure 4A. According to the result, the PGM value enlarged with the increase of the AFB1 concentrations and a linear relationship between PGM signal (mM) and AFB1 level (nM) could be achieved in the dynamic range from 0.02 to 10 nM (Figure 4B). The regression equation was S (mM) = 9.1044×Log [AFB1] + 17.1851 (nM, R2 = 0.9984, n = 9). The detection limit was about low to 10 pM.

Figure 2. PGM value towards the resulting solution prepared under the same conditions with 0.3 nM AFB1 (e) except without the AFB1 (a), invertase (b), walker DNA (c), or Pb2+ (d). Inset: the corresponding test strip.

Figure 3. Effect of the cleavage time of DNAzyme (A), concentration of the Pb2+(B), incubation time of AFB1 (C), pH of the solution (D) on PGM signal towards 0.3 nM AFB1. Optimization of Assay Conditions. In order to obtain better sensitivity and efficiency of the aptasensor, some experimental conditions (eg. cleaving time of Pb2+-specific DNAzyme, concen-

Figure 4. (A) The relationship between the PGM signal and the AFB1 concentration. (B) Calibration curve of the sensor towards AFB1 standards with different concentrations. (C) Selectivity analysis of the biosensor towards 3 nM AFB1, 10 nM interferences and their mixture. (D) Stability of the assay processed by ten-days detection of 3 nM AFB1. Inset of (A, C, and D): the corresponding test strips and values. With such a lower detection limit, the portable PGM based sensor possessed excellent analytical features by the comparison with the reported AFB1 portable assay methods (Table 1).

ACS Paragon Plus Environment

Page 4 of 8

Page 5 of 8 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

ACS Sensors Furthermore, the specificity of PGM based aptasensor was appraised by the investigation of a series of solutions containing different kinds of interfering agents. Figure 4C shows the corresponding PGM signal of solutions containing different kinds of interferences. It showed that the interferences did not bring an obvious PGM signal. In contrast, the significant PGM signals could be obtained towards AFB1 and their mixed sample solutions, indicating the excellent specificity of the present aptasensor. Moreover, the fabricated sensor was performed every day in 10-day consecutive measurements to investigate the stability.

As shown in Figure 4D, it could arrive 91.83% of the original value implying the acceptable stability. Real Sample Analysis. In order to investigate the performance of the PGM based sensor in real sample applications, the present aptasensor was used to detect the AFB1 in bread (Figure 5). Three kinds of bread were applied for the evaluation. The concentrations were determined from response curves generated by the standard addition method. The recovery values of AFB1 content in moldy bread obtained in the range of 98.5−103% with RSDs of 0.53−0.86% using developed method are shown in Table 2. Comparison with the reference values obtained from the commercial high-performance liquid chromatography tandem mass spectrometry (HPLC-MS) method was shown in Table 3.It was obvious that the proposed portable AFB1 aptasensor possessed good accuracy for the quantification of AFB1 in the actual samples.

Table 1. Comparison of different methods for the detection of AFB1. Device Recognition Method aptamer bound screen printed carbon electrodes aptamer-AFB1 handheld pH meter aptamer-AFB1 distance-readout microfluidic chip aptamer-AFB1 portable impedance detector antigen-antibody portable glucometer antigen-antibody surface plasmon resonance chip antigen-antibody chromatographic time-resolved fluoroimmunoassay strip antigen-antibody portable fibre-optic spectrometer / personal glucose meter aptamer-AFB1

Figure 5. Real food samples. Left row: the moldy food samples (from a to c) and the corresponding the clean food samples of three kinds of bread (from d to f). Right row: the extraction solutions were obtained from the above food samples (1, 2, 3, 4, 5 and 6 are corresponding to a, b, c, d, e and f respectively).

CONCLUSION In this work, a simple method with rapid detection time based on the binding-induced DNA walker and a personal glucose meter for cheap, easy to operate, and on-site detection of AFB1 was developed. Compared with the conventional portable AFB1 sensor with immunoassay formats, the present aptasensor based on PGM readout realized stable, low-cost and rapid determination of AFB1. The autonomous DNA walker movement was achieved through the AFB1 triggered DNA hybridization, which was a simple process to realize the signal separation, avoiding the requirement of multiple separation and washing steps. Moreover, it could be extended to other specific targets, simply by changing the target recognition elements. Generally, the presented method has a great potential to be employed in simple and on-site detections of various kinds of analytes in many fields including food safety monitoring, clinical diagnosis, and environmental analysis, etc.

Limit of Detection 0.38nM 0.1µM 1.77nM 16nM 16pM 51.2nM / 32nM 10pM

Linear Range 0.4nM-51.2nM 0.2µM-20µM 0.25µM-40µM 16nM-128nM 0.032nM-48nM 51.2nM-640nM 0.64nM-192nM 64nM-192nM 20pM-10nM

Ref. 41 16 15 42 43 44 45 46 this work

Table 2. Results of the Determination of AFB1 in Bread Samples. Found Added Found RSD Sample Recovery (nM) (nM) (nM) (n=3) 1 0.52 2 2.49 98.5% 0.60% 3 3.54 100.6% 0.77% 5 5.61 101.8% 0.86% 2 0.47 2 2.53 103% 0.79% 3 3.43 98.6% 0.57% 5 5.51 100.8% 0.86% 3 0.21 2 2.24 101.5% 0.80% 3 3.17 98.6% 0.57% 5 5.25 100.8% 0.53% Table 3. Comparison with the HPLC-MS method Addded HPLC-MS proposed a a Sample (nM) method method (nM) (nM) 1 0.50 0.53 0.55 2 0.40 0.47 0.49 3 0.20 0.21 0.22 a Average of three measurements

Relative error +3.7% +4.2% -1.2%

AUTHOR INFORMATION Corresponding Author Corresponding Author: Guangfeng Wang E-mail: [email protected] Fax: +86-553-3869303; Tel: +86-553-3869302

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Grants 21675001), the Anhui Provincial Natural Science Foundation (1608085MB46, 1608085MC67),

ACS Paragon Plus Environment

ACS Sensors 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

the Anhui Provincial Education Department Natural Sciences Key Fund (KJ2016SD23), the Key Program in the Youth Elite Support Plan in Universities of Anhui Province (gxyqZD2016023), Provincial Project of Natural Science Research for Colleges and Universities of Anhui Province of China (KJ2016A274).

REFERENCES (1) Nascimento, C.; Santos, P.; Pereira-Filho, E.; Rocha, F. Recent advances on determination of milk adulterants. Food Chem. 2017, 221, 1232-1244. (2) Reddy, K. R. N.; Reddy, C. S.; Muralidharan, K. Potential of botanicals and biocontrol agents on growth and aflatoxin production by Aspergillus flavus infecting rice grains. Food Control. 2009, 20, 173-178. (3) Hontanaya, C.; Meca, G.; Luciano, F. B.; Mañes, J.; Font, G. Inhibition of aflatoxin B1, B2, G1 and G2 production by Aspergillus parasiticus in nuts using yellow and oriental mustard flours. Food Control. 2015, 47, 154-160. (4) Lu, Z.; Wang, Y.; Xu, D.; Pang, L. Aptamer-tagged DNA origami for spatially addressable detection of aflatoxin B1. Chem. Commun. 2017, 53, 941-944. (5) Piermarini, S.; Micheli, L.; Ammida, N.; Palleschi, G.; Moscone, D. Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection. Biosens. Bioelectron. 2007, 22, 1434-1440. (6) Fernandez-Iban ́ ̃ez, V.; Martínez-Fernandez, A.; RozaDelgado, B.; Soldado, A. Application of near infrared spectroscopy for rapid detection of aflatoxin B1 in maize and barley as analytical quality assessment. Food Chem. 2009, 113, 629-634. (7) Bruix, J.; Boix, L.; Sala, M.; Llovet, J. M. Focus on hepatocellular carcinoma. Cancer Cell. 2004, 5, 215-219. (8) (a) Tang, D.; Lin, Y.; Zhou, Q.; Lin, Y.; Li, P.; Niessner, R.; Knopp, D. Low-cost and highly sensitive immunosensing platform for Aflatoxins using one-step competitive displacement reaction mode and portable glucometer-based detection. Anal. Chem. 2014, 86, 11451-11458. (b) Lin, Y. X.; Zhou, Q.; Tang, D. P.; Niessner, R.; Knopp, D. Signal-on photoelectrochemical immunoassay for aflatoxin B1 based on enzymatic product-etching MnO2 nanosheets for dissociation of carbon dots. Anal. Chem. 2017, 89, 5637−5645. (c) Lai, W. Q.; Wei, Q. H.; Xu, M. D.; Zhuang, J. Y.; Tang, D. P. Enzymecontrolled dissolution of MnO2 nanoflakes with enzyme cascade amplification for colorimetric immunoassay. Biosens. Bioelectron. 2017, 89, 645–651. (d) Lin, Y. X.; Zhou, Q.; Tang, D. P. Dopamineloaded liposomes for in-situ amplified photoelectrochemical immunoassay of AFB1 to enhance photocurrent of Mn2+-doped Zn3(OH)2V2O7 nanobelts. Anal. Chem. 2017, 89, 11803−11810. (9) Kong, W.; Wei, R.; Logrieco, A. F.; Wei, J.; Wen, J.; Xiao, X.; Yang, M. Occurrence of toxigenic fungi and determination of mycotoxins by HPLC-FLD in functional foods and spices in China markets. Food Chem. 2014, 146, 320-326. (10) Shim, W. B.; Yang, Z. Y.; Kim, J. S.; Kim, J. Y.; Kang, S. J.; Woo, G. J.; Chung, Y. C.; Eremin, S. A.; Chung, D. H. Development of immunochromatography strip-test using nanocolloidal goldantibody probe for the rapid detection of Aflatoxin B1 in grain and feed samples. J. Microbiol. Biotechnol. 2007, 17, 1629-1637. (11) Togashi, M.; Terai, T.; Kojima, H.; Hanaoka, K.; Igarashi, K.; Hirata, Y.; Urano, Y.; Nagano, T. Practical fluorescence detection of acrolein in human plasma via a two-step tethering approach. Chem. Commun. 2014, 50, 14946-14948. (12) Mozaffari Nejad, A. S.; Sabouri Ghannad, M.; Kamkar, A. Determination of aflatoxin B1 levels in Iranian and Indian spices by ELISA method. Toxin Rev. 2014, 33, 151-154. (13) Ren, M.; Xu, H.; Huang, X.; Kuang, M.; Xiong, Y.; Xu, H.; Xu, Y.; Chen, H.; Wang, A. Immunochromatographic assay for ultrasensitive detection of Aflatoxin B1 in maize by highly luminescent quantum dot beads. ACS Appl. Mater. Interfaces. 2014, 6, 1421514222.

(14) (a) Xu, W.; Xiong, Y.; Lai, W.; Xu, Y.; Li, C.; Xie, M. A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots. Biosens. Bioelectron. 2014, 56, 144-150. (b) Qiu, Z. l.; Shu, J.; Tang, D. P. Near-Infrared-toultraviolet light-Mediated photoelectrochemical aptasensing platform for cancer biomarker based on core−shell NaYF4:Yb, Tm@TiO2 upconversion microrods. Anal. Chem. 2018, 90, 1021−1028. (c) Zhou, Q.; Lin, Y. X.; Zhang, K. Y.; Li, M. J.; Tang, D. P. Reduced graphene oxide/BiFeO3 nanohybrids-based signal-on photoelectrochemical sensing system for prostate-specific antigen detection coupling with magnetic microfluidic device. Biosens. Bioelectron. 2018, 101, 146–152. (d) Cai, G. N.; Yu, Z. Z.; Ren, R. R.; Tang, D. P. Exciton−plasmon interaction between AuNPs/Graphene nanohybrids and CdS quantum dots/TiO2 for photoelectrochemical aptasensing of prostate-specific antigen. ACS Sens. 2018, 3, 632−639. (15) Ma, Y. L; Mao, Y.; Huang, D.; He, Z.; Yan, J. M.; Tian, T.; Shi, Y. Z.; Song, Y. L.; Li, X. R.; Zhu, Z.; Zhou, L. J.; Yang, C. Y. Portable visual quantitative detection of aflatoxin B1 using a targetresponsive hydrogel and a distance-readout microfluidic. chip Lab Chip. 2016, 16, 3097-3104. (16) Zhaoa, M. M.; Wang, P. L.; Guo, Y. J.; Wang, L. X.; Luo, F.; Qiu, B.; Guo, L. H.; Su, X.; Lin, Z. Y.; Chen, G. N. Detection of aflatoxin B1 in food samples based on target-responsive aptamercross-linked hydrogel using a handheld pH meter as readout. Talanta. 2018,176, 34-39. (17) Xiang, Y., Lu, Y. Portable and quantitative detection of protein biomarkers and small molecular toxins using antibodies and ubiquitous personal glucose meters. Anal. Chem. 2012, 84, 41744178. (18) Clark, L. C.; Lyons, C. Ann. Electrode systems for continuous monitoring in cardiovascular surgery. N. Y. Acad. Sci. 1962, 102, 2945. (19) Heller, A.; Feldman, B. Electrochemical glucose sensors and their applications in diabetes management. Chem. Rev. 2008, 108, 2482-2505. (20) Montagnana, M.; Caputo, M.; Giavarina, D.; Lippi, G. Overview on self-monitoring of blood glucose. Clin. Chim. Acta. 2009, 402, 7-13. (21) Carroll, A. E.; Marrero, D. G.; Downs, S. M. The healthpia glucopack diabetes phone: A usability study. Diabetes Technol. Ther. 2007, 9, 158-164. (22) Lin, B. Q.; Liu, D.; Yan, J. M.; Qiao, Z.; Zhong, Y. X.; Yan, J. W.; Zhu, Z.; Ji, T. H.; Yang, C. Y. Enzyme-encapsulated liposomelinked immunosorbent assay enabling sensitive personal glucose meter readout for portable detection of disease biomarkers. ACS Appl. Mater. Interfaces. 2016, 8, 6890-6897. (23) Wang, Q.; Wang, H.; Yang, X.; Wang, K.; Liu, F.; Zhao, Q.; Liu, P.; Liu, R. Multiplex detection of nucleic acids using a low cost microfluidic chip and a personal glucose meter at the point-of-care. Chem. Commun. 2014, 50, 3824-3826. (24) Zhang, J.; Xiang, Y.; Novak, D. E.; Hoganson, G. E.; Zhu, J.; Lu, Y. Using a personal glucose meter and alkaline phosphatase for point‐of‐care quantification of galactose-1-phosphate uridyltransferase in clinical galactosemia diagnosis. Chem.-Asian J. 2015, 10, 2221-2227. (25) Wang, Q.; Wang, H.; Yang, X.; Wang, K.; Liu, R.; Li, Q.; Ou, J. A sensitive one-step method for quantitative detection of α-amylase in serum and urine using a personal glucose meter. Analyst. 2015, 140, 1161-1165. (26) Liu, X. Q.; Niazov-Elkan, A.; Wang, F.; Willner, I. Switching photonic and electrochemical functions of a DNAzyme by DNA machines. Nano Lett. 2013, 13, 219-225. (27) Omabegho, T.; Sha, R. J.; Seeman, N. C. A bipedal DNA brownian motor with coordinated legs. Science. 2009, 324, 67-71. (28) Delius, M.; Geertsema, E. M. Leigh, D. A.; Tang, D. D. Design, synthesis, and operation of small molecules that walk along tracks. J. Am. Chem. Soc. 2010, 132, 16134-16145. (29) C. Y. Wang, J. S. Ren and X. G. Qu, A stimuli responsive DNA walking device. Chem. Commun. 2011, 47, 1428-1430.

ACS Paragon Plus Environment

Page 6 of 8

Page 7 of 8 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

ACS Sensors (30) Q. F. Xu, Y. Zhang and C. Y. Zhang, A universal sensing platform based on the repair ligation-mediated light-producing DNA machine. Chem. Commun. 2015, 51, 5652-5655. (31) J. M. Thomas, H. Z. Yu and D. Sen, A mechano-electronic DNA switch. J. Am. Chem. Soc. 2012, 134, 13738-13748. (32) Liu, X. Q.; Niazov-Elkan, A.; Wang, F. A.; Willner, I. Switching photonic and electrochemical functions of a DNAzyme by DNA machines. Nano Lett. 2013, 13, 219-225. (33) Shin, J. S.; Pierce, N. A. A synthetic DNA walker for molecular transport. J. Am. Chem. Soc. 2014, 126, 10834-10835. (34) Watson, J. D.; Crick, F. H. Molecular structure of nucleic acids: A Structure for deoxyribose nucleic acid. Nature. 1953, 171, 737738. (35) Wang, L. D., Deng, R. J., Li, J. H. Target-fueled DNA walker for highly selective miRNA detection. Chem. Sci. 2015, 6, 67776782. (36) He, M. Q.; Wang, K.; Wang, W. J.; Yu, Y. L.; Wang, J. H. Smart DNA machine for carcinoembryonic antigen detection by Exonuclease III-assisted target recycling and DNA walker cascade amplification. Anal. Chem. 2017, 89, 9292-9298. (37) Lu, Y. New transition‐metal‐dependent DNAzymes as efficient endonucleases and as selective metal biosensors. Chem. Eur. J. 2002, 8, 4588-4596. (38) Gong, L.; Zhao, Z. L.; Lv, Y. F. Huan, S. Y.; Fu, T. Zhang, X. B. Shen, G. L.; Yu, R. Q. DNAzyme-based biosensors and nanodevices. Chem. Commun. 2015, 51, 979-995. (39) Wu. L., Chen. K., Lu. Z., Li. T., Shao. K., Shao. F., Han. H. Hydrogen-bonding recognition-induced aggregation of gold nanoparticles for the determination of the migration of melamine monomers using dynamic light scattering. Anal. Chim. Acta. 2014, 845, 92-97. (40) Ni. Y., Wang L., Kokot, S. Voltammetric determination of butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate and tert-butylhydroquinone by use of chemometric approaches. Anal. Chim. Acta. 2000, 412, 185-193. (41) Yugender Goud, K.; Catanante, G.; Hayat, A.; Satyanarayana M.; Gobi, K. V.; Louis Marty. J. Disposable and portable electrochemical aptasensor for label free detection of aflatoxin B1 in alcoholic beverages. Sens. Actuators, B. 2016, 235, 466-473. (42) Li, Z. M.; Ye, Z. Z.; Fu, Y. C.; Xiong Y. H.; Li. Y. B. A portable electrochemical immunosensor for rapid detection of trace aflatoxin B1 in rice. Anal. Methods. 2016, 8, 548-553. (43) Tang, D. P.; Lin, Y. X.; Zhou, Q.; Lin, Y. P.; Li, P. W.; Niessner, R.; Knopp, D. Low-cost and highly sensitive immunosensing platform for aflatoxins using one-step competitive displacement reaction mode and portable glucometer-based detection. Anal. Chem. 2014, 86, 11451-11458. (44) Moon, J.; Byun, J.; Kim, H.; Lim, E. K.; Jeong, J.; Jung, J.; Kang, T. On-site detection of aflatoxin B1 in grains by a palm-sized surface plasmon resonance sensor. Sensors. 2018, 18, 598-605. (45) Zhang, Z. W.; Tang, X. Q.; Wang, D.; Zhang, Q.; Li, P. W.; Ding, X. X. Rapid on-site sensing aflatoxin B1 in food and feed via a chromatographic time resolved fluoroimmunoassay. PLOS ONE| DOI: 10.1371/journal.pone.0123266. (46) Arduini, F.; Neagu, D.; Pagliarini, V.; Scognamiglio, V.; Leonardis, M. A.; Gatto, E.; Amine, A.; Palleschi, G.; Moscone, D. Rapid and label-free detection of ochratoxin A and aflatoxin B1 using an optical portable instrument. Talanta. 2016, 150, 440-448.

ACS Paragon Plus Environment

ACS Sensors 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

for TOC only Portable Aptasensor of Aflatoxin B1 Based on a Personal Glucose Meter and DNA Walking Machine

ACS Paragon Plus Environment

Page 8 of 8