Biosensors and Bioelectronics 80 (2016) 574–581
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Hetero-enzyme-based two-round signal amplification strategy for trace detection of aflatoxin B1 using an electrochemical aptasensor Wanli Zheng a, Jun Teng a, Lin Cheng a, Yingwang Ye a, Daodong Pan a, Jingjing Wu a, Feng Xue b,n, Guodong Liu c, Wei Chen a,n a School of Biotechnology and Food Engineering, Anhui Provincial Key Lab of Functional Materials and Devices, Hefei University of Technology, Hefei 23009, China b Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing 200002, China c Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58102, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 1 December 2015 Received in revised form 29 January 2016 Accepted 31 January 2016 Available online 1 February 2016
An electrochemical aptasensor for trace detection of aflatoxin B1 (AFB1) was developed by using an aptamer as the recognition unit while adopting the telomerase and EXO III based two-round signal amplification strategy as the signal enhancement units. The telomerase amplification was used to elongate the ssDNA probes on the surface of gold nanoparticles, by which the signal response range of the signal-off model electrochemical aptasensor could be correspondingly enlarged. Then, the EXO III amplification was used to hydrolyze the 3′-end of the dsDNA after the recognition of target AFB1, which caused the release of bounded AFB1 into the sensing system, where it participated in the next recognition-sensing cycle. With this two-round signal amplified electrochemical aptasensor, target AFB1 was successfully measured at trace concentrations with excellent detection limit of 0.6*10 4 ppt and satisfied specificity due to the excellent affinity of the aptamer against AFB1. Based on this designed tworound signal amplification strategy, both the sensing range and detection limit were greatly improved. This proposed ultrasensitive electrochemical aptasensor method was also validated by comparison with the classic instrumental methods. Importantly, this hetero-enzyme based two-round signal amplified electrochemical aptasensor offers a great promising protocol for ultrasensitive detection of AFB1 and other mycotoxins by replacing the core recognition sequence of the aptamer. & 2016 Elsevier B.V. All rights reserved.
Keywords: Electrochemical Aptasensor Signal amplification Aflatoxin B1 Gold nanoparticle
1. Introduction Mycotoxins are common hazardous materials found in animal feed and food samples stored or processed under favorable temperature and humidity conditions for the growth of fungi (Deng et al., 2013). More seriously, once these mycotoxins enter the food chain of animals and humans, they can enter their bodies and severely threaten their health (Saremi and Okhovvat, 2006), thus affecting the food industry and world economy (Hussein and Brasel, 2001). Aflatoxins, produced by Aspergillus species, are a major class of mycotoxins, which have been the subject of tremendous research documenting their acute and chronic effects on the gastrointestinal, respiratory, cardiovascular and central nervous systems of both animals and humans (Cuccioloni et al., 2008; Stetinova et al., 1998). Among the aflatoxin family, aflatoxin B1 n
Corresponding authors. E-mail addresses: [email protected] (F. Xue), [email protected] (W. Chen). http://dx.doi.org/10.1016/j.bios.2016.01.091 0956-5663/& 2016 Elsevier B.V. All rights reserved.
(AFB1) is the most abundant and toxic one due to the “doublehazardous” ability to bind with both DNA and proteins (Madden et al., 2002; Mckean et al., 2006; Meki et al., 2004; Singh et al., 2005) and has been listed as the group I carcinogen by the International Agency for Research on Cancer (IARC) (1993) (Khlangwiset et al., 2011). Different countries have set different permitted action level of AFB1 in food samples and animal feed. For example, in Korea, the limit of AFB1 commonly required is 5 μg/kg while the European Commission requirements is as low as 2 μg/kg of AFB1 (Shim et al., 2012,2014; EC, 2006). Therefore, development of rapid and sensitive screening methods to effectively monitor AFB1 levels at all stages of food and feed production is urgently required in order to guarantee the identification of AFB1 contaminated ingredients before feeding them to animals or humans. Currently, conventional gold standard methods for AFB1 detection are the liquid phase chromatography-based methods including HPLC, LC-MS and LC-MS/MS (Blesa et al., 2003; Ediage et al., 2011; Pamel et al., 2011; Shim et al., 2008). Although these instrumental-based methods are highly sensitive and offer good
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repeatability, they cannot be used for rapid and on-site screening of AFB1 due to the extensive sample pretreatment requirements and high cost of detections (Thirumala-Devi et al., 2002). In view of these intrinsic disadvantages of instrumental methods, development of immunological methods or biosensors for rapid detection of AFB1 have attracted great attentions and a lot of achievements have been reported (Anfossi et al., 2008; Arduini et al., 2007; Micheli et al., 2005; Lee et al., 2004; Tang et al., 2009). Nevertheless, these methods also suffer disadvantages due to issues regarding the stability and harsh conditions of antibody in practical detections. Li et al. have reported the preparation of heavy chain single domain antibody to increase the thermostability of antibody and the performance of associated methods (Wang et al., 2013). However, the long period and high cost of antibody preparation are still the main bottlenecks which greatly hinder the wide applications of immunological methods for onsite detection of AFB1. With the emergence of the new recognition molecules, termed aptamer (Ellington and Szostak, 1990; Robertson and Joyce, 1990), extensive researches have been carried out aiming to replace the traditional immunological methods. Among reported achievements, one important category of the sensing strategy is the electrochemical aptasensor for its easy operation, high sensitivity and portable properties (Evtugyn et al., 2014; Nguyen et al., 2013; Huang et al., 2013; Wu et al., 2014, 2012; Xue et al., 2013). However, for the “signal-off” model electrochemical biosensor, it is generally recognized that the signal suppression range greatly limits the sensitivity and sensing range of the developed approaches (Yin et al., 2012). Previously, we have also developed the “signal-off” electrochemical aptasensor for ochratoxin A (OTA) detection without any signal amplification treatments achieving sensitivity down to ppb for OTA (Kuang et al., 2010). How to effectively avoid the intrinsic drawbacks of signal-off biosensor approaches by integrating the various signal amplification strategies have been the hot topics. Presently, numerous in vitro DNA amplification techniques were adopted for signal enhancement in development of electrochemical biosensors. To date, however, there have been no reports of aptamerbased signal amplified electrochemical aptasensor for ultrasensitive detection of AFB1.
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Here we report a hetero-enzyme based two-round signal amplification strategy to develop a “signal-off” electrochemical aptasensor for ultrasensitive detection of AFB1. There are two aspects which will be addressed in protocol being proposed here: for one thing, the inspiration for the first-round signal amplification step stemmed from previous studies demonstrating that the 100% signal suppression greatly limits the utilizations of the “signal-off” model sensor in practical applications; for the other, the secondround signal amplification by EXO III-based catalytic hydrolysis of ssDNA to make the single molecule signal response possible. Theoretically, as illustrated in Scheme 1, only one single AFB1 molecule could be enough to induce the conformational change of the dsDNA probes (step b in Scheme 1) and maintain the probes in the specific spatial state with the 3′ end in the double-strand structure, which furnishes the necessary conditions for EXO IIIbased hydrolysis and the release of AFB1. After the release of target AFB1 from the sensing interface, it will further induce the next reaction cycle. Repeatedly, the sensing signal of AFB1 could be greatly enhanced and this is defined as the second-round signal amplification of the developed electrochemical aptasensor. Based on this two-round signal amplification strategy, comparatively, excellent sensing performance of trace AFB1 is accomplished by our electrochemical aptasensor. Additionally, this sensing and signal amplification strategy provides a powerful method for sensitive detection of AFB1 and also a potential technique for accurate screening of trace amounts of AFB1 in various fields.
2. Experimental 2.1. Materials and reagents All oligonucleotides used in this study were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The sequences of thiol-modified oligonucleotides aptamer (A1) used for gold electrode immobilization were 5′- GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-(CH2)6-SH-3′. The sequences of thiol-modified telomerase primer (TS primer) were 5′-SH-(CH2)6-AAT CCG TCG AGC AGA
Scheme 1. The schematic process of signal amplified electrochemical aptasensor for AFB1 detection ((a) formation of stem-loop structure of the immobilized probe; (b) immobilization of TS-primer-AuNP-cDNA onto the sensing interface; and (c) telomerase based first-round amplification).
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GTT-3′. The thiol-modified complementary DNA (cDNA) were 5′CAC AGA GAG ACA ACA CGT GCC CAA C-(CH2)6-SH-3′. 6-mercaptohexanol (MCH), ethylene glycol tetracetic acid (EGTA), phenylmethanesulfonyl fluoride (PMSE), propanesulfonic acid (CHAPS), tris-(2-carboxyethyl) phosphine hydrochloride (TCEPHCl) and HAuCl4 were all purchased from Sigma Aldrich, USA. AFB1 standard reagent was purchased from J&K Chemical Co. Ltd. Other common chemicals were all obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China) and used directly without any further purification. Ultrapure water obtained from the Millipore water purification system (Milli-Q, Millipore) was used throughout the research. All electrochemical measurements, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV), were performed with an electrochemical workstation (CH Instruments 650D, TX). The classic three-electrode system used includes a gold electrode with 2 mm diameter as a working electrode, a platinum wire as a counter electrode, and the saturated calomel electrode (SCE) as a reference electrode. CV was performed in 1 M NaOH solution from 0.35 to 1.35 V at a scan rate of 2 mV/s. EIS was performed in 0.1 mM KCl solution containing 5.0 mM [Fe(CN)6]3 /4- and the frequency range from 0.5 to 105 Hz. SWV was performed in 10 mM PBS (pH 7.4, 10 mM NaH2PO4, 10 mM Na2HPO4 and 1 mM MgCl2) within a potential window from 0 to 0.6 V, a potential step of 0.001 V, amplitude of 0.05 V and a frequency of 50 Hz. 2.2. Synthesis of gold nanoparticles (AuNPs) Citrate-coated AuNPs (15 þ1 nm) were prepared according to classic Turkevich citrate reduction method (Elghanian et al., 1997; Mei et al., 2013). Typically, 1% sodium citrate was rapidly added to a vigorously stirred boiling aqueous solution of 0.01% HAuCl4 (50 mL). After a continuous boiling for several minutes, the color of the mixed solution changed to wine red. The mixture was stirred and boiled for additional 5 min to ensure the uniform particle size. Finally, the solution was cooled to room temperature (RT) and stored in a refrigerator at 4 °C until ready for use. 2.3. Preparation of telomerase extracts Telomerase extracts were prepared as previously reported (Duan et al., 2014). Typically, HePG2 cells, a perpetual cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15-year-old Caucasian male who had a well-differentiated hepatocellular carcinoma, were collected in the exponential phase of growth, harvested with trypsin, washed twice with ice-cold PBS (0.01 M, pH 7.4), and re-suspended at a concentration of 106 cells/mL in 200 mL of ice-cold CHAPS lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS, 10% glycerol). The re-suspended cells were incubated in ice for 30 min, and then centrifuged 20 min at 12000 rpm, 4 °C, and the supernatant was carefully collected. The supernatant was immediately frozen and stored at 80 °C until used for the telomerase assay. 2.4. Preparation of TS primer-AuNPs-c-DNA Two microliter acetic acid buffer (500 mM) and 1 μL TCEP (1 mM) were added to a mixture of 10 mL TS primer probe (10 μM) and 10 μL single stranded capture DNA (c-DNA) probe (1 μM) and incubated at RT for 1 h. Then the mixture was added to 200 μL AuNPs (10-fold concentration, with a diameter of 15 nm). After stirring at RT for 30 min, 17 mL of dATP (100 mM) were added to the solution and further stirred for 15 min to block the residual sites of AuNPs and avoid nonspecific adsorption. Next, 40 μL of NaCl
(0.1 M) were added to stabilize the DNA-AuNPs conjugates, the mixture was incubated at RT for 30 min and subsequently incubated at 4 °C for 6 h to further increases the stability of the conjugates. Excess reagents in the reaction mixture were removed by centrifugation at 9400 g for 7 min, and re-suspended in 200 μL phosphate buffer (PBS) (pH 7.4) containing 1 mM NaH2PO4, 1 mM Na2HPO4 and 0.1 mM MgCl2. Finally, the TS primer-AuNPs-c-DNA was stored at 4 °C for further use. 2.5. Preparation of the electrochemical aptasensor Typically, the gold electrode was treated by polishing successively with 1, 0.3 and 0.05 mm alumina, followed by sonication in alcohol and ultrapure water for 5 min. Then the electrode was immersed in piranha solution (H2SO4:H2O2, 3:1 volume ratio) for 5 min, followed by rinsing with ultrapure water and electrochemical cleaning with 1 M NaOH. Above operations were all carried out at RT. Following, 5 μL thiol-modified oligonucleotides aptamer probe (A1) (0.5 μM) was incubated with 1 μL of Tris(2carboxyethyl) phosphine hydrochloride (TECP, 1 mM) for 0.5 h to cleave the disulfide bonds of the A1 probe, and subsequently dropped on the surface of the pretreated electrode and incubated at 37 °C for another 2 h, which would maintain the immobilized A1 probe in the stem-loop structure. Afterwards, the electrode was rinsed with ultrapure water to remove the unbound A1 probe. The rinsed electrode was then immersed into a 2 mM 6-mercaptohexanol (MCH) solution for 3 h to block the unoccupied sites of the electrode and eliminate the nonspecific adsorption effect. This was followed by rinsing with ultrapure water to remove the unbound MCH. Next, 10 μL of TS-AuNPs-c-DNA was dropped onto the surface of the electrode and incubated at 37 °C for 2 h, followed by rinsing with ultrapure water. Subsequently, 5 μL of telomerase extracts and 5 μL extension solution (20 mM Tris–HCl, pH 8.3, 4 mM MgCl2, 1 mM EGTA, 63 mM KCl, 0.05% Tween 20 containing 0.5 mM dATP, dTTP, dGTP, dCTP) were dropped onto the surface of electrode and incubated at 37 °C for 1 h, followed by rinsing with ultrapure water. After the telomerase amplification step, the electrode was immersed in the methylene blue (MB) solution (8 10 5 M) for 10 min. Finally, the electrode was rinsed with ultrapure water again and stored in dark at 4 °C for further use. At this stage, the fabrication of the electrochemical aptasensor was now complete. Of note, due to the specific step-loop structure design and double strand structure for signal probe immobilization, the work temperature and pH of this developed electrochemical aptasensor was suggest to be room temperature or physiological temperature and pH 7.4 for keeping the stability of fabricated sensing interface. All following measurements were all carried out under the suggested conditions. 2.6. AFB1 measurement with the fabricated electrochemical aptasensor and real sample determinations Before the electrochemical measurement, AFB1 solution was added into the sensing system followed by the addition of 20 U/mL EXO III and cultured for a while. After the culture and EXO III catalyzed hydrolysis, the sensing system was measured by square wave voltammetry (SWV). By dropping an aliquot of AFB1 at different concentration onto the surface of the electrochemical aptasensor, the oxidation signal from MB was measured using SWV in PBS. The real spiked corn samples were ground by a high-speed disintegrator and accurately weighed into 1 g per sample. And various AFB1 standard solutions at different concentrations in 10 mL of methanol/water (volume ratio, 80:20) was added respectively followed by sonication for 60 sec assisting the extraction performance. Following, these spiked samples were further centrifuged for 20 min at 14,000 g. Finally, 5 mL supernatants
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were collected and transferred into the flasks and diluted with PBS (pH 7.4). Based on the fact that stringent pre-treatments of the food samples are required for practical application, the complicate matrix effect could be avoided effectively, which would guarantee the successful applications of this electrochemical aptasensor for detection of real food samples.
3. Results and discussions 3.1. Construction of electrochemical sensing interface and heteroenzyme based two-round signal amplification platform for AFB1 detection In order to resolve the intrinsic problems or technical bottleneck associated with antibody-based methods and further widen the practical application of aptasensor, in this study, the specific aptamer for AFB1 was adopted as the core recognition unit for detection of AFB1. Firstly, the aptamer probe was designed in the shape of stem-loop structure on the surface of the electrode (step a in Scheme 1). It is important to note that, the signal-off sensing model, adopted here for the development of AFB1 aptasensor, has the intrinsic disadvantage that only a maximum of 100% signal suppression can be attained under any experimental conditions and it is thus not ideal for most biosensing applications. In order to effectively circumvent this disadvantage, the telomerase based amplification strategy with TS-primer-AuNP-c-DNA conjugates, which is defined as “the first-round amplification” in Scheme 1, was adopted to improve the intensity of the starting current and the region of the 100% signal suppression for sensing. The other important and key signal improvement treatment introduced in this sensing protocol was the EXO III based hydrolysis of ssDNA. Based on this treatment, theoretically, only one target AFB1 molecule is sufficient to induce conformational change and hydrolysis of the probes repeatedly, which in turn could lead to the distinguished signal variations required for ultrasensitive AFB1 detection in the “signal-off” model. Following this approach, the sensitivity of the constructed electrochemical aptasensor could be dramatically improved and this is defined as the “second-round amplification” of our sensing strategy in Scheme 1. It is worthy noted that compared with other previous similar achievements of toxin detections (Evtugyn et al., 2014; Nguyen et al., 2013), this electrochemical aptasensor for toxin detection is focus on the invitro nucleic acid amplification based signal enhancement for sensitivity improvement besides the sensing interface modification.
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3.2. Surface functionalization of the sensing electrode As previously reported, the EIS measurements were carried out for characterization of the electrode modification process. Firstly, the stem-loop structured probes were attached to the surface of the gold electrode via the Au–S bond. The electron transfer resistance (Ret) value was found to be increased compared with that of the bare electrode (Fig. 1a). This increase of Ret could be attributed to the blockage of the electron transfer of negatively charged [Fe(CN)6]3-/4- to the electrode surface by the negatively charged phosphate skeleton of the modified probe. In addition, a further increase Ret was observed when the electrode surface was blocked with dATP. After immobilization of the TS primer-AuNP-cDNA conjugates onto the electrode by hybridization, this electron transfer blocking phenomenon was more conspicuous due to the much higher density of the various ssDNA on the electrode. After adding the target AFB1 sample into the sensing system, the TS primer-AuNP-c-DNA conjugates were released from the surface of electrode due to the stronger affinity between the aptamer and AFB1. Therefore, conversely, the Ret was dramatically decreased to the level of aptamer modification. All these EIS results presented in Fig. 1a, excellently document each modification and recognition step of the electrochemical aptasensor. The present research on first-round telomerase-based signal amplification was carried out to widen the 100% current suppression region. Two kinds of ssDNA probes were assembled onto the surface of the AuNPs forming the TS-primer-AuNP-cDNA conjugates: one is the single stranded capture (c-DNA) probe for immobilization of the AuNPs onto the interface of the electrode, while the other is the TS primer for telomerase-based ssDNA amplification. The intensity of the current signal is undoubtedly affected by the ratio of TS primer to c-DNA probe. Herein, the ratio of the current intensity after the amplification of telomerase (I0′) to the current intensity of the aptamer modified electrode (I0-original) was chosen as the index of the optimization. Comparison results at different modification ratios are shown in Fig. 1b. These comparisons revealed that, at the early stage, the index of I0′ /I0-original increased with increasing ratio of TS primer/c-DNA probe, indicating the amplification of the starting current. This phenomenon could be attributed to the high density of TS primer on the surface of AuNPs for current signal arising after the telomerase amplification. However, when the ratio of TS primer/cDNA probe was increased above 10, the index of I0′/I0-original is conversely decreased. This could be the result of severe spatial hindrance effect induced by the higher density of TS primer on AuNPs, which could further inhibit the telomerase amplification
Fig. 1. EIS results of electrode surface functionalization (a) and ratio optimization of telomerase primer/capture DNA on AuNPs (b).
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Fig. 2. Confirming results of the first-round telomerase-based signal amplification (The inserted image is the electrophoresis result of telomerase amplification: lane 1: pure AuNPs; lane 2: telomerase primer modified AuNPs; lane 3: telomerase amplified products on AuNPs). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
on the surface of AuNPs. It is evident that the 10:1 ratio was found to be the best ratio of TS primer/c-DNA for ssDNA probe immobilization on AuNPs. In order to confirm the first-round signal amplification effect achieved with telomerase, the starting current at each stage was measured and compared. The results shown in Fig. 2 clearly demonstrated that using the gold nanoparticle immobilized with both the TS primer and c-DNA probes could enhance the starting sensing current (red line in the Fig. 2) compared with that of the aptamer modified electrode (black line in Fig. 2). Moreover, as expected, the start sensing current was even more dramatically increased after the in situ amplification with telomerase (blue line in Fig. 2). This first-round signal amplification could be attributed to the elongated ssDNA amplified with telomerase on the interface of AuNPs, which could adsorb much more MB molecules and induce the stronger current signal. Additionally, the telomerase amplification of the ssDNA on the AuNPs surface was characterized by agarose gel electrophoresis (the inset image in Fig. 2). The pure unmodified AuNPs run in the first lane (lane 1), aggregated into the large diameter blue sediments which could not migrate into the gel due to the electrostatic screening effect under the condition of high salt concentrations. After modification with TS and c-DNA probes, the TS-AuNP-c-DNA conjugates could maintain their good stability and migrate into the gel even under the high
Fig. 3. Detection performance related parameters and optimization results of the electrochemical aptasensor.
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ionic strength (lane 2), indicating the successful assembly of both ssDNA probes on the AuNPs surface. Similarly, after the telomerase amplification, the amplified products could also maintain the original stability of the AuNPs (lane 3, in red color). However, the amplified products could not migrate into the gel and are detained in the sample pore. This is could be attributed to the increased length of the ssDNA on AuNPs and their inhibited migration in the gel, which is definitely caused by the telomerase-based amplification. Both the electrochemical and gel electrophoresis analysis results in Fig. 2 clearly proved the first-round telomerase amplification on the interface of AuNPs and the corresponding signal amplification effect for AFB1 sensing. Some additional telomerase amplification related factors including the concentration of utilized telomerase and the dark field results of gel electrophoresis were also carefully studied and shown in the supporting information (Figure S1 & S2) to further illustrate the best sensing performance of AFB1. In sensing research, many key steps are highly time-related. Therefore, in this study, the different time-related steps were all carefully optimized for optimal performance. The electrochemically active tag of MB was adopted for signal monitoring. The incubation time of MB labeling was studied to determine the optimal sensing current (Fig. 3a). The results revealed that, as shown in Fig. 3a, the sensing current signal increased with increasing incubation time, but after about 6 min, the sensing current reached a stable value with negligible variations. Accordingly, considering the detection efficiency, 6 min incubation time was chosen as the best MB labeling culture time for electrochemical sensing. Meanwhile, the first-round amplification is known to be dependent on the telomerase and corresponding ssDNA primer (TS probe) on the surface of AuNPs, which are immobilized by the hybridization between the c-DNA on AuNPs and stem-loop probes on the electrode. Therefore, the hybridization time was also evaluated in this study. The results indicated that the hybridization process could be completed in 1.5 h as shown in Fig. 3b, thus such time was adopted for further research. Since, as mentioned above, the “signal-off” biosensor is limited in application because of the maximum of 100% signal suppression, the original non-observable current signal response at trace concentration of target AFB1 should be amplified in order to improve the sensitivity and sensing range. Accordingly, the telomerase-based nucleic elongation method was adopted to amplify the signal probes immobilized on the surface of AuNPs. Undoubtedly, the reaction time of the telomerase-based amplification would determine the final sensing performances. As demonstrated by the results presented in Fig. 3c, it is evident that the current response is enhanced with increasing telomerase amplification time. In detail, in the first one hour, the current response is increased dramatically and afterword, the current is kept a stable level or a little decreased. Therefore, 1 h was selected as the optimal telomerase amplification time for subsequent AFB1 detection. Finally, as depicted in Scheme 1, following the signal treatment of telomerase amplification, EXO III enzymatic hydrolysis of the ssDNA on the interface of the electrode was applied to further enhance the current response in the sensing process. Theoretically, only one target AFB1 molecule could be sufficient to induce the distinguished current response through the repeated hydrolysis reactions. In this process, the final current response was determined to be highly dependent on the hydrolysis time. Explicitly, as shown in Fig. 3d, the current was decreased by the EXO III-assisted hydrolysis of the ssDNA on the interface of the electrode, which in turn induced the release of the MB attached on the ssDNA and decreased the current. With the ongoing hydrolysis, the current was decreased and reached a
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Fig. 4. AFB1 detection results with the two-round signal amplification electrochemical aptasensor. (a) The SWV sensing results of AFB1 at different trace concentrations; (b) comparison results of two-round signal amplified and the traditional electrochemical aptasensor; and (c) the selectivity of the signal amplified electrochemical aptasensor.
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lower plateau at 45 min, which was defined as the optimal hydrolysis time of EXO III for subsequent sensing research. Under the optimized conditions, this signal amplified electrochemical aptasensor was used to detect trace amount of AFB1. Results from these analyses, shown in Fig. 4a demonstrated that the sensing response could be evidently differentiated even with trace variations of AFB1 in the sensing system. Indeed, based on these sensing responses, the linear sensing range was clearly achieved from 0.0001 to 100 ppt, which is about 6-order magnitude of AFB1 concentrations. In addition, the detection limit of signal amplified electrochemical aptasensor was estimated, based on the 3s rule, to be 0.6*10 4 ppt, which is the extraordinary sensing performance for AFB1 detection. Comparatively, as shown in Fig. 4b, without the signal amplification treatments, the sensing signal response including the 100% signal suppression could be neglected when compared with that of signal amplified aptasensors. Actually, it is remarkable that while the whole sensing range of the traditional model is from 0.1 to 100 ppt, in comparison, by using the hetero-enzyme based two-round signal amplification protocol described here, both the 100% signal suppression and the detection range are dramatically improved. Thus, it goes without saying that the detection limit was also greatly improved. Additionally, the specificity of the two-round signal amplified electrochemical aptasensor was interrogated with analogs and other common toxins. The results revealed, as shown in Fig. 4c, that only the target AFB1 could induce an obvious signal response, whereas the analogs or other common toxins (OTA, OTB, DON et al.) could not induce a comparable distinguished signal change, indicating the excellent specificity of the two-round signal amplified electrochemical aptasensor approach. Meanwhile, the practical application of this signal amplified electrochemical aptasensor to measure spiked samples was confirmed with HPLC methods, which indicated that satisfactory recoveries (95–110%) were obtained with our developed electrochemical aptasensor compared with the instrumental methods (detailed results in Table S1). Essentially, from our point of view, both the specificity and the recovery achieved with the electrochemical aptasensor can be attributed to the excellent properties of the selected aptamers. In addition, the fabricated electrochemical aptasensor was stored at 4 °C and subsequently employed it for detections. No obvious change in the sensing performance was observed after the electrode was kept for up to 4 weeks. These results demonstrated that the signal amplified electrochemical aptasensor exhibits the acceptable stability. Considering the detection procedures of the electrochemical aptasensor, it could be used directly for target toxin detection with signal amplified treatments after the fabrication of sensing interface, which is better than traditional immuno methods and comparable to other reported aptasensor methods. Therefore, future practical application research of this signal amplification strategy should be paid great attention in the field of high throughput detections and some preliminary results have already been achieved based on SPE, which is the suitable platform for high throughput detections (see detailed methods and comparisons results in Fig. S3 in Supporting information). And the integration with the portable detection device for on-site sensitive screening of analyte including the further optimization of sensing conditions is also the key step of great significance.
4. Conclusions A new hetero-enzyme based two-round signal amplification electrochemical aptasensor approach for sensitive detection of AFB1 has been successfully developed. This strategy effectively resolved the technical bottleneck associated with the signal-off
model sensing methods. The telomerase-based first-round amplification led to the generation of a sufficiently high initial current signal to increase the detection range, while the EXO III-based second-round amplification led to an observable signal response at trace concentrations. Taking advantage of this two-round signal amplification strategy, both the sensing range and detection limit of the developed electrochemical aptasensor were greatly improved by a 3-order magnitude widening and about 1000-fold enhancement respectively. The bottlenecks of the traditional signal-off model biosensor are all resolved with the strategies for signal amplification designed by our team. Further research of integrating this signal amplification strategy with screening printing electrode (SPE) based array sensing platform for high throughput is still going on in our group. Finally, this developed signal-amplified electrochemical aptasensor should provide further means to guarantee satisfactory resolution to the toxin-related food safety issues and, more importantly, contribute to the widespread application of traditional signal-off biosensor in various fields.
Acknowledgments This work is financially supported by the NSFC Grant of 21475030 and 31301460, the Science and Technology Research Project of Anhui Province 15czz03109, National 10000 TalentsYouth Top-notch Talent Program, the National and Zhejiang Public Benefit Research Project (201313010, 2014C32051) and the Jiangsu Science and Technology Support Program of BE201373, 2012780.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.01.091.
References Anfossi, L., Calderara, M., Baggiani, C., Giovannoli, C., Arletti, E., Giraudi, G., 2008. J. Agric. Food Chem. 56, 1852–1857. Arduini, F., Errico, I., Amine, A., Micheli, L., Palleschi, G., Moscone, D., 2007. Anal. Chem. 79, 3409–3415. Blesa, J., Soriano, J.M., Molto, J.C., Marin, R., Manes, J., 2003. J. Chromatogr. 1011, 49–54. Cuccioloni, M., Mozzicafreddo, M., Barocci, S., Ciuti, F., Pecorelli, I., Eleuteri, A.M., Spina, M., Fioretti, E., Angeletti, M., 2008. Anal. Chem. 80, 9250–9256. Deng, G.Z., Xu, K., Sun, Y., Chen, Y., Zheng, T.S., Li, J.L., 2013. Anal. Chem. 85, 2833–2840. Duan, R.X., Wang, B.Y., Zhang, T.C., Zhang, Z.Y., Xu, S.F., Chen, Z.F., Lou, X.D., Xia, F., 2014. Anal. Chem 86, 9781–9785. EC, 2006. Setting Maximum Levels for Certain Contaminants in Foodstuffs; Commission Regulation (EC). European Commission Regulation, Brussels. Ediage, E.N., Mavungu, J.D.D., Monbaliu, S., Peteghem, C.V., Saeger, S.D., 2011. J. Agric. Food Chem. 59, 5173–5180. Elghanian, R., Storhoff, J.J., Mucic, R.C., Letsinger, R.L., Mirkin, C.A., 1997. Science 277, 1078–1081. Ellington, A.J., Szostak, W., 1990. Nature 346, 818–822. Evtugyn, G., Porfireva, A., Stepanova, V., Sitdikov, R., Stoikov, I., Nikolelis, D., Hianik, T., 2014. Electroanalysis 26, 2100–2109. Huang, L., Wu, J.J., Zheng, L., Qian, H.S., Xue, F., Wu, Y.C., Pan, D.D., Adeloju, S.B., Chen, W., 2013. Anal. Chem. 85, 10842–10849. Hussein, H.S., Brasel, J.M., 2001. Toxicology 167, 101–134. IARC, 1993. Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. 56. International Agency for Research on Cancer: Lyon, France, p. 489. Khlangwiset, P., Shephard, G., Wu, F., Crit, 2011. Rev. Toxicol. 41, 740–755. Kuang, H., Chen, W., Xu, D.H., Xu, L.G., Zhu, Y.Y., Liu, L.Q., Chu, H.Q., Peng, C.F., Xu, C. L., Zhu, S.F., 2010. Biosens. Bioelectron. 26, 710–716. Lee, N.A., Wang, S., Allan, R.D., Kennedy, I.R., 2004. J. Agric. Food Chem. 52, 2746–2755. Madden, C.R., Finegold, M.J., Slagle, B.L., 2002. J. Virol. 76, 11770–11774. McKean, C., Tang, L., Tang, M., Billam, M., Wang, Z., Theodorakis, C.W., Kendall, R.J.,
W. Zheng et al. / Biosensors and Bioelectronics 80 (2016) 574–581
Wang, H.S., 2006. J. Appl. Toxicol. 26, 139–147. Mei, Z.L., Chu, H.Q., Chen, W., Xue, F., Liu, J., Xu, H.N., Zhang, R., Zheng, L., 2013. Biosens. Bioelectron. 39, 26–30. Meki, A.R., Esmail Eel, D., Hussein, A.A., Hassanein, H.M., 2004. Toxicon 43, 93–100. Micheli, L., Greco, R., Badea, M., Moscone, D., Palleschi, G., 2005. Biosens. Bioelectron. 21, 588–596. Nguyen, B.H., Tran, L.D., Do, Q.P., Nguyen, H.L., Tran, N.H., Nguyen, P.X., 2013. Mater. Sci. Eng. C 33, 2229–2234. Pamel, E.V., Verbeken, A., Vlaemynck, G., Boever, J.D., Daeseleire, E., 2011. J. Agric. Food Chem. 59, 9747–9755. Robertson, D.L., Joyce, G.F., 1990. Nature 344, 467–468. Saremi, H., Okhovvat, S.M., 2006. Commun. Agric. Appl. Biol. Sci. 71, 1175–1185. Shim, W.B., Choi, J.G., Kim, J.Y., Yang, Z.Y., Lee, K.H., Kim, M.G., Ha, S.D., 2008. J. Food Prot. 71, 781–789. Shim, W.B., Kim, K., Ofori, J.A., Chuan, Y.C., Chuang, D.H., 2012. J. Food Protect. 75, 1991–1999. Shim, W.B., Mun, H.Y., Joung, H.A., Ofori, J.A., Chung, D.H., Kim, M.G., 2014. Food
581
Control 36, 30–35. Singh, J., Singh, S., Dani, H.M., Sharma, R., Steinberg, P., 2005. Cell Biochem. Funct. 23, 9–13. Stetinova, V., Grossmann, V., Kvetina, J., 1998. Pol. J. Pharmacol. 50, 135–141. Tang, D.P., Zhong, Z.Y., Niessner, R., Knopp, D., 2009. Analyst 134, 1509–1728. Thirumala- Devi, K., Mayo, M.A., Hall, A.J., Craufurd, P.Q., Wheeler, T.R., Waliyar, F., Subrahmanyam, A., Reddy, D.V., 2002. J. Agric. Food Chem. 50, 933–937. Wang, Y.R., Li, P.W., Majkova, Z., Bever, C.R.S., Kim, H.J., Zhang, Q., Dechant, J.E., Gee, S.J., Hammock, B.D., 2013. Anal. Chem. 85, 8298–8303. Wu, J.J., Chu, H.Q., Mei, Z.L., Deng, Y., Xue, F., Zheng, L., Chen, W., 2012. Anal. Chim. Acta 753, 27–31. Wu, J.J., Zhu, Y.Y., Xue, F., Mei, Z.L., Yao, L., Wang, X., Zheng, L., Liu, J., Liu, G.D., Peng, C.F., Chen, W., 2014. Microchim. Acta 181, 479–491. Xue, F., Wu, J.J., Chu, H.Q., Mei, Z.L., Ye, Y.K., Liu, J., Zhang, R., Peng, C.F., Zheng, L., Chen, W., 2013. Microchim. Acta 180, 109–115. Yin, H.S., Zhou, Y.L., Chen, C.X., Zhu, L.S., Ai, S.Y., 2012. Analyst 137, 1389–1395.
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Hetero-enzyme-based two-round signal amplification strategy for trace detection of aflatoxin B1 using an electrochemical aptasensor Wanli Zheng a, Jun Teng a, Lin Cheng a, Yingwang Ye a, Daodong Pan a, Jingjing Wu a, Feng Xue b,n, Guodong Liu c, Wei Chen a,n a School of Biotechnology and Food Engineering, Anhui Provincial Key Lab of Functional Materials and Devices, Hefei University of Technology, Hefei 23009, China b Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing 200002, China c Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58102, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 1 December 2015 Received in revised form 29 January 2016 Accepted 31 January 2016 Available online 1 February 2016
An electrochemical aptasensor for trace detection of aflatoxin B1 (AFB1) was developed by using an aptamer as the recognition unit while adopting the telomerase and EXO III based two-round signal amplification strategy as the signal enhancement units. The telomerase amplification was used to elongate the ssDNA probes on the surface of gold nanoparticles, by which the signal response range of the signal-off model electrochemical aptasensor could be correspondingly enlarged. Then, the EXO III amplification was used to hydrolyze the 3′-end of the dsDNA after the recognition of target AFB1, which caused the release of bounded AFB1 into the sensing system, where it participated in the next recognition-sensing cycle. With this two-round signal amplified electrochemical aptasensor, target AFB1 was successfully measured at trace concentrations with excellent detection limit of 0.6*10 4 ppt and satisfied specificity due to the excellent affinity of the aptamer against AFB1. Based on this designed tworound signal amplification strategy, both the sensing range and detection limit were greatly improved. This proposed ultrasensitive electrochemical aptasensor method was also validated by comparison with the classic instrumental methods. Importantly, this hetero-enzyme based two-round signal amplified electrochemical aptasensor offers a great promising protocol for ultrasensitive detection of AFB1 and other mycotoxins by replacing the core recognition sequence of the aptamer. & 2016 Elsevier B.V. All rights reserved.
Keywords: Electrochemical Aptasensor Signal amplification Aflatoxin B1 Gold nanoparticle
1. Introduction Mycotoxins are common hazardous materials found in animal feed and food samples stored or processed under favorable temperature and humidity conditions for the growth of fungi (Deng et al., 2013). More seriously, once these mycotoxins enter the food chain of animals and humans, they can enter their bodies and severely threaten their health (Saremi and Okhovvat, 2006), thus affecting the food industry and world economy (Hussein and Brasel, 2001). Aflatoxins, produced by Aspergillus species, are a major class of mycotoxins, which have been the subject of tremendous research documenting their acute and chronic effects on the gastrointestinal, respiratory, cardiovascular and central nervous systems of both animals and humans (Cuccioloni et al., 2008; Stetinova et al., 1998). Among the aflatoxin family, aflatoxin B1 n
Corresponding authors. E-mail addresses: [email protected] (F. Xue), [email protected] (W. Chen). http://dx.doi.org/10.1016/j.bios.2016.01.091 0956-5663/& 2016 Elsevier B.V. All rights reserved.
(AFB1) is the most abundant and toxic one due to the “doublehazardous” ability to bind with both DNA and proteins (Madden et al., 2002; Mckean et al., 2006; Meki et al., 2004; Singh et al., 2005) and has been listed as the group I carcinogen by the International Agency for Research on Cancer (IARC) (1993) (Khlangwiset et al., 2011). Different countries have set different permitted action level of AFB1 in food samples and animal feed. For example, in Korea, the limit of AFB1 commonly required is 5 μg/kg while the European Commission requirements is as low as 2 μg/kg of AFB1 (Shim et al., 2012,2014; EC, 2006). Therefore, development of rapid and sensitive screening methods to effectively monitor AFB1 levels at all stages of food and feed production is urgently required in order to guarantee the identification of AFB1 contaminated ingredients before feeding them to animals or humans. Currently, conventional gold standard methods for AFB1 detection are the liquid phase chromatography-based methods including HPLC, LC-MS and LC-MS/MS (Blesa et al., 2003; Ediage et al., 2011; Pamel et al., 2011; Shim et al., 2008). Although these instrumental-based methods are highly sensitive and offer good
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repeatability, they cannot be used for rapid and on-site screening of AFB1 due to the extensive sample pretreatment requirements and high cost of detections (Thirumala-Devi et al., 2002). In view of these intrinsic disadvantages of instrumental methods, development of immunological methods or biosensors for rapid detection of AFB1 have attracted great attentions and a lot of achievements have been reported (Anfossi et al., 2008; Arduini et al., 2007; Micheli et al., 2005; Lee et al., 2004; Tang et al., 2009). Nevertheless, these methods also suffer disadvantages due to issues regarding the stability and harsh conditions of antibody in practical detections. Li et al. have reported the preparation of heavy chain single domain antibody to increase the thermostability of antibody and the performance of associated methods (Wang et al., 2013). However, the long period and high cost of antibody preparation are still the main bottlenecks which greatly hinder the wide applications of immunological methods for onsite detection of AFB1. With the emergence of the new recognition molecules, termed aptamer (Ellington and Szostak, 1990; Robertson and Joyce, 1990), extensive researches have been carried out aiming to replace the traditional immunological methods. Among reported achievements, one important category of the sensing strategy is the electrochemical aptasensor for its easy operation, high sensitivity and portable properties (Evtugyn et al., 2014; Nguyen et al., 2013; Huang et al., 2013; Wu et al., 2014, 2012; Xue et al., 2013). However, for the “signal-off” model electrochemical biosensor, it is generally recognized that the signal suppression range greatly limits the sensitivity and sensing range of the developed approaches (Yin et al., 2012). Previously, we have also developed the “signal-off” electrochemical aptasensor for ochratoxin A (OTA) detection without any signal amplification treatments achieving sensitivity down to ppb for OTA (Kuang et al., 2010). How to effectively avoid the intrinsic drawbacks of signal-off biosensor approaches by integrating the various signal amplification strategies have been the hot topics. Presently, numerous in vitro DNA amplification techniques were adopted for signal enhancement in development of electrochemical biosensors. To date, however, there have been no reports of aptamerbased signal amplified electrochemical aptasensor for ultrasensitive detection of AFB1.
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Here we report a hetero-enzyme based two-round signal amplification strategy to develop a “signal-off” electrochemical aptasensor for ultrasensitive detection of AFB1. There are two aspects which will be addressed in protocol being proposed here: for one thing, the inspiration for the first-round signal amplification step stemmed from previous studies demonstrating that the 100% signal suppression greatly limits the utilizations of the “signal-off” model sensor in practical applications; for the other, the secondround signal amplification by EXO III-based catalytic hydrolysis of ssDNA to make the single molecule signal response possible. Theoretically, as illustrated in Scheme 1, only one single AFB1 molecule could be enough to induce the conformational change of the dsDNA probes (step b in Scheme 1) and maintain the probes in the specific spatial state with the 3′ end in the double-strand structure, which furnishes the necessary conditions for EXO IIIbased hydrolysis and the release of AFB1. After the release of target AFB1 from the sensing interface, it will further induce the next reaction cycle. Repeatedly, the sensing signal of AFB1 could be greatly enhanced and this is defined as the second-round signal amplification of the developed electrochemical aptasensor. Based on this two-round signal amplification strategy, comparatively, excellent sensing performance of trace AFB1 is accomplished by our electrochemical aptasensor. Additionally, this sensing and signal amplification strategy provides a powerful method for sensitive detection of AFB1 and also a potential technique for accurate screening of trace amounts of AFB1 in various fields.
2. Experimental 2.1. Materials and reagents All oligonucleotides used in this study were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The sequences of thiol-modified oligonucleotides aptamer (A1) used for gold electrode immobilization were 5′- GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-(CH2)6-SH-3′. The sequences of thiol-modified telomerase primer (TS primer) were 5′-SH-(CH2)6-AAT CCG TCG AGC AGA
Scheme 1. The schematic process of signal amplified electrochemical aptasensor for AFB1 detection ((a) formation of stem-loop structure of the immobilized probe; (b) immobilization of TS-primer-AuNP-cDNA onto the sensing interface; and (c) telomerase based first-round amplification).
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GTT-3′. The thiol-modified complementary DNA (cDNA) were 5′CAC AGA GAG ACA ACA CGT GCC CAA C-(CH2)6-SH-3′. 6-mercaptohexanol (MCH), ethylene glycol tetracetic acid (EGTA), phenylmethanesulfonyl fluoride (PMSE), propanesulfonic acid (CHAPS), tris-(2-carboxyethyl) phosphine hydrochloride (TCEPHCl) and HAuCl4 were all purchased from Sigma Aldrich, USA. AFB1 standard reagent was purchased from J&K Chemical Co. Ltd. Other common chemicals were all obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China) and used directly without any further purification. Ultrapure water obtained from the Millipore water purification system (Milli-Q, Millipore) was used throughout the research. All electrochemical measurements, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV), were performed with an electrochemical workstation (CH Instruments 650D, TX). The classic three-electrode system used includes a gold electrode with 2 mm diameter as a working electrode, a platinum wire as a counter electrode, and the saturated calomel electrode (SCE) as a reference electrode. CV was performed in 1 M NaOH solution from 0.35 to 1.35 V at a scan rate of 2 mV/s. EIS was performed in 0.1 mM KCl solution containing 5.0 mM [Fe(CN)6]3 /4- and the frequency range from 0.5 to 105 Hz. SWV was performed in 10 mM PBS (pH 7.4, 10 mM NaH2PO4, 10 mM Na2HPO4 and 1 mM MgCl2) within a potential window from 0 to 0.6 V, a potential step of 0.001 V, amplitude of 0.05 V and a frequency of 50 Hz. 2.2. Synthesis of gold nanoparticles (AuNPs) Citrate-coated AuNPs (15 þ1 nm) were prepared according to classic Turkevich citrate reduction method (Elghanian et al., 1997; Mei et al., 2013). Typically, 1% sodium citrate was rapidly added to a vigorously stirred boiling aqueous solution of 0.01% HAuCl4 (50 mL). After a continuous boiling for several minutes, the color of the mixed solution changed to wine red. The mixture was stirred and boiled for additional 5 min to ensure the uniform particle size. Finally, the solution was cooled to room temperature (RT) and stored in a refrigerator at 4 °C until ready for use. 2.3. Preparation of telomerase extracts Telomerase extracts were prepared as previously reported (Duan et al., 2014). Typically, HePG2 cells, a perpetual cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15-year-old Caucasian male who had a well-differentiated hepatocellular carcinoma, were collected in the exponential phase of growth, harvested with trypsin, washed twice with ice-cold PBS (0.01 M, pH 7.4), and re-suspended at a concentration of 106 cells/mL in 200 mL of ice-cold CHAPS lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS, 10% glycerol). The re-suspended cells were incubated in ice for 30 min, and then centrifuged 20 min at 12000 rpm, 4 °C, and the supernatant was carefully collected. The supernatant was immediately frozen and stored at 80 °C until used for the telomerase assay. 2.4. Preparation of TS primer-AuNPs-c-DNA Two microliter acetic acid buffer (500 mM) and 1 μL TCEP (1 mM) were added to a mixture of 10 mL TS primer probe (10 μM) and 10 μL single stranded capture DNA (c-DNA) probe (1 μM) and incubated at RT for 1 h. Then the mixture was added to 200 μL AuNPs (10-fold concentration, with a diameter of 15 nm). After stirring at RT for 30 min, 17 mL of dATP (100 mM) were added to the solution and further stirred for 15 min to block the residual sites of AuNPs and avoid nonspecific adsorption. Next, 40 μL of NaCl
(0.1 M) were added to stabilize the DNA-AuNPs conjugates, the mixture was incubated at RT for 30 min and subsequently incubated at 4 °C for 6 h to further increases the stability of the conjugates. Excess reagents in the reaction mixture were removed by centrifugation at 9400 g for 7 min, and re-suspended in 200 μL phosphate buffer (PBS) (pH 7.4) containing 1 mM NaH2PO4, 1 mM Na2HPO4 and 0.1 mM MgCl2. Finally, the TS primer-AuNPs-c-DNA was stored at 4 °C for further use. 2.5. Preparation of the electrochemical aptasensor Typically, the gold electrode was treated by polishing successively with 1, 0.3 and 0.05 mm alumina, followed by sonication in alcohol and ultrapure water for 5 min. Then the electrode was immersed in piranha solution (H2SO4:H2O2, 3:1 volume ratio) for 5 min, followed by rinsing with ultrapure water and electrochemical cleaning with 1 M NaOH. Above operations were all carried out at RT. Following, 5 μL thiol-modified oligonucleotides aptamer probe (A1) (0.5 μM) was incubated with 1 μL of Tris(2carboxyethyl) phosphine hydrochloride (TECP, 1 mM) for 0.5 h to cleave the disulfide bonds of the A1 probe, and subsequently dropped on the surface of the pretreated electrode and incubated at 37 °C for another 2 h, which would maintain the immobilized A1 probe in the stem-loop structure. Afterwards, the electrode was rinsed with ultrapure water to remove the unbound A1 probe. The rinsed electrode was then immersed into a 2 mM 6-mercaptohexanol (MCH) solution for 3 h to block the unoccupied sites of the electrode and eliminate the nonspecific adsorption effect. This was followed by rinsing with ultrapure water to remove the unbound MCH. Next, 10 μL of TS-AuNPs-c-DNA was dropped onto the surface of the electrode and incubated at 37 °C for 2 h, followed by rinsing with ultrapure water. Subsequently, 5 μL of telomerase extracts and 5 μL extension solution (20 mM Tris–HCl, pH 8.3, 4 mM MgCl2, 1 mM EGTA, 63 mM KCl, 0.05% Tween 20 containing 0.5 mM dATP, dTTP, dGTP, dCTP) were dropped onto the surface of electrode and incubated at 37 °C for 1 h, followed by rinsing with ultrapure water. After the telomerase amplification step, the electrode was immersed in the methylene blue (MB) solution (8 10 5 M) for 10 min. Finally, the electrode was rinsed with ultrapure water again and stored in dark at 4 °C for further use. At this stage, the fabrication of the electrochemical aptasensor was now complete. Of note, due to the specific step-loop structure design and double strand structure for signal probe immobilization, the work temperature and pH of this developed electrochemical aptasensor was suggest to be room temperature or physiological temperature and pH 7.4 for keeping the stability of fabricated sensing interface. All following measurements were all carried out under the suggested conditions. 2.6. AFB1 measurement with the fabricated electrochemical aptasensor and real sample determinations Before the electrochemical measurement, AFB1 solution was added into the sensing system followed by the addition of 20 U/mL EXO III and cultured for a while. After the culture and EXO III catalyzed hydrolysis, the sensing system was measured by square wave voltammetry (SWV). By dropping an aliquot of AFB1 at different concentration onto the surface of the electrochemical aptasensor, the oxidation signal from MB was measured using SWV in PBS. The real spiked corn samples were ground by a high-speed disintegrator and accurately weighed into 1 g per sample. And various AFB1 standard solutions at different concentrations in 10 mL of methanol/water (volume ratio, 80:20) was added respectively followed by sonication for 60 sec assisting the extraction performance. Following, these spiked samples were further centrifuged for 20 min at 14,000 g. Finally, 5 mL supernatants
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were collected and transferred into the flasks and diluted with PBS (pH 7.4). Based on the fact that stringent pre-treatments of the food samples are required for practical application, the complicate matrix effect could be avoided effectively, which would guarantee the successful applications of this electrochemical aptasensor for detection of real food samples.
3. Results and discussions 3.1. Construction of electrochemical sensing interface and heteroenzyme based two-round signal amplification platform for AFB1 detection In order to resolve the intrinsic problems or technical bottleneck associated with antibody-based methods and further widen the practical application of aptasensor, in this study, the specific aptamer for AFB1 was adopted as the core recognition unit for detection of AFB1. Firstly, the aptamer probe was designed in the shape of stem-loop structure on the surface of the electrode (step a in Scheme 1). It is important to note that, the signal-off sensing model, adopted here for the development of AFB1 aptasensor, has the intrinsic disadvantage that only a maximum of 100% signal suppression can be attained under any experimental conditions and it is thus not ideal for most biosensing applications. In order to effectively circumvent this disadvantage, the telomerase based amplification strategy with TS-primer-AuNP-c-DNA conjugates, which is defined as “the first-round amplification” in Scheme 1, was adopted to improve the intensity of the starting current and the region of the 100% signal suppression for sensing. The other important and key signal improvement treatment introduced in this sensing protocol was the EXO III based hydrolysis of ssDNA. Based on this treatment, theoretically, only one target AFB1 molecule is sufficient to induce conformational change and hydrolysis of the probes repeatedly, which in turn could lead to the distinguished signal variations required for ultrasensitive AFB1 detection in the “signal-off” model. Following this approach, the sensitivity of the constructed electrochemical aptasensor could be dramatically improved and this is defined as the “second-round amplification” of our sensing strategy in Scheme 1. It is worthy noted that compared with other previous similar achievements of toxin detections (Evtugyn et al., 2014; Nguyen et al., 2013), this electrochemical aptasensor for toxin detection is focus on the invitro nucleic acid amplification based signal enhancement for sensitivity improvement besides the sensing interface modification.
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3.2. Surface functionalization of the sensing electrode As previously reported, the EIS measurements were carried out for characterization of the electrode modification process. Firstly, the stem-loop structured probes were attached to the surface of the gold electrode via the Au–S bond. The electron transfer resistance (Ret) value was found to be increased compared with that of the bare electrode (Fig. 1a). This increase of Ret could be attributed to the blockage of the electron transfer of negatively charged [Fe(CN)6]3-/4- to the electrode surface by the negatively charged phosphate skeleton of the modified probe. In addition, a further increase Ret was observed when the electrode surface was blocked with dATP. After immobilization of the TS primer-AuNP-cDNA conjugates onto the electrode by hybridization, this electron transfer blocking phenomenon was more conspicuous due to the much higher density of the various ssDNA on the electrode. After adding the target AFB1 sample into the sensing system, the TS primer-AuNP-c-DNA conjugates were released from the surface of electrode due to the stronger affinity between the aptamer and AFB1. Therefore, conversely, the Ret was dramatically decreased to the level of aptamer modification. All these EIS results presented in Fig. 1a, excellently document each modification and recognition step of the electrochemical aptasensor. The present research on first-round telomerase-based signal amplification was carried out to widen the 100% current suppression region. Two kinds of ssDNA probes were assembled onto the surface of the AuNPs forming the TS-primer-AuNP-cDNA conjugates: one is the single stranded capture (c-DNA) probe for immobilization of the AuNPs onto the interface of the electrode, while the other is the TS primer for telomerase-based ssDNA amplification. The intensity of the current signal is undoubtedly affected by the ratio of TS primer to c-DNA probe. Herein, the ratio of the current intensity after the amplification of telomerase (I0′) to the current intensity of the aptamer modified electrode (I0-original) was chosen as the index of the optimization. Comparison results at different modification ratios are shown in Fig. 1b. These comparisons revealed that, at the early stage, the index of I0′ /I0-original increased with increasing ratio of TS primer/c-DNA probe, indicating the amplification of the starting current. This phenomenon could be attributed to the high density of TS primer on the surface of AuNPs for current signal arising after the telomerase amplification. However, when the ratio of TS primer/cDNA probe was increased above 10, the index of I0′/I0-original is conversely decreased. This could be the result of severe spatial hindrance effect induced by the higher density of TS primer on AuNPs, which could further inhibit the telomerase amplification
Fig. 1. EIS results of electrode surface functionalization (a) and ratio optimization of telomerase primer/capture DNA on AuNPs (b).
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Fig. 2. Confirming results of the first-round telomerase-based signal amplification (The inserted image is the electrophoresis result of telomerase amplification: lane 1: pure AuNPs; lane 2: telomerase primer modified AuNPs; lane 3: telomerase amplified products on AuNPs). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
on the surface of AuNPs. It is evident that the 10:1 ratio was found to be the best ratio of TS primer/c-DNA for ssDNA probe immobilization on AuNPs. In order to confirm the first-round signal amplification effect achieved with telomerase, the starting current at each stage was measured and compared. The results shown in Fig. 2 clearly demonstrated that using the gold nanoparticle immobilized with both the TS primer and c-DNA probes could enhance the starting sensing current (red line in the Fig. 2) compared with that of the aptamer modified electrode (black line in Fig. 2). Moreover, as expected, the start sensing current was even more dramatically increased after the in situ amplification with telomerase (blue line in Fig. 2). This first-round signal amplification could be attributed to the elongated ssDNA amplified with telomerase on the interface of AuNPs, which could adsorb much more MB molecules and induce the stronger current signal. Additionally, the telomerase amplification of the ssDNA on the AuNPs surface was characterized by agarose gel electrophoresis (the inset image in Fig. 2). The pure unmodified AuNPs run in the first lane (lane 1), aggregated into the large diameter blue sediments which could not migrate into the gel due to the electrostatic screening effect under the condition of high salt concentrations. After modification with TS and c-DNA probes, the TS-AuNP-c-DNA conjugates could maintain their good stability and migrate into the gel even under the high
Fig. 3. Detection performance related parameters and optimization results of the electrochemical aptasensor.
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ionic strength (lane 2), indicating the successful assembly of both ssDNA probes on the AuNPs surface. Similarly, after the telomerase amplification, the amplified products could also maintain the original stability of the AuNPs (lane 3, in red color). However, the amplified products could not migrate into the gel and are detained in the sample pore. This is could be attributed to the increased length of the ssDNA on AuNPs and their inhibited migration in the gel, which is definitely caused by the telomerase-based amplification. Both the electrochemical and gel electrophoresis analysis results in Fig. 2 clearly proved the first-round telomerase amplification on the interface of AuNPs and the corresponding signal amplification effect for AFB1 sensing. Some additional telomerase amplification related factors including the concentration of utilized telomerase and the dark field results of gel electrophoresis were also carefully studied and shown in the supporting information (Figure S1 & S2) to further illustrate the best sensing performance of AFB1. In sensing research, many key steps are highly time-related. Therefore, in this study, the different time-related steps were all carefully optimized for optimal performance. The electrochemically active tag of MB was adopted for signal monitoring. The incubation time of MB labeling was studied to determine the optimal sensing current (Fig. 3a). The results revealed that, as shown in Fig. 3a, the sensing current signal increased with increasing incubation time, but after about 6 min, the sensing current reached a stable value with negligible variations. Accordingly, considering the detection efficiency, 6 min incubation time was chosen as the best MB labeling culture time for electrochemical sensing. Meanwhile, the first-round amplification is known to be dependent on the telomerase and corresponding ssDNA primer (TS probe) on the surface of AuNPs, which are immobilized by the hybridization between the c-DNA on AuNPs and stem-loop probes on the electrode. Therefore, the hybridization time was also evaluated in this study. The results indicated that the hybridization process could be completed in 1.5 h as shown in Fig. 3b, thus such time was adopted for further research. Since, as mentioned above, the “signal-off” biosensor is limited in application because of the maximum of 100% signal suppression, the original non-observable current signal response at trace concentration of target AFB1 should be amplified in order to improve the sensitivity and sensing range. Accordingly, the telomerase-based nucleic elongation method was adopted to amplify the signal probes immobilized on the surface of AuNPs. Undoubtedly, the reaction time of the telomerase-based amplification would determine the final sensing performances. As demonstrated by the results presented in Fig. 3c, it is evident that the current response is enhanced with increasing telomerase amplification time. In detail, in the first one hour, the current response is increased dramatically and afterword, the current is kept a stable level or a little decreased. Therefore, 1 h was selected as the optimal telomerase amplification time for subsequent AFB1 detection. Finally, as depicted in Scheme 1, following the signal treatment of telomerase amplification, EXO III enzymatic hydrolysis of the ssDNA on the interface of the electrode was applied to further enhance the current response in the sensing process. Theoretically, only one target AFB1 molecule could be sufficient to induce the distinguished current response through the repeated hydrolysis reactions. In this process, the final current response was determined to be highly dependent on the hydrolysis time. Explicitly, as shown in Fig. 3d, the current was decreased by the EXO III-assisted hydrolysis of the ssDNA on the interface of the electrode, which in turn induced the release of the MB attached on the ssDNA and decreased the current. With the ongoing hydrolysis, the current was decreased and reached a
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Fig. 4. AFB1 detection results with the two-round signal amplification electrochemical aptasensor. (a) The SWV sensing results of AFB1 at different trace concentrations; (b) comparison results of two-round signal amplified and the traditional electrochemical aptasensor; and (c) the selectivity of the signal amplified electrochemical aptasensor.
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lower plateau at 45 min, which was defined as the optimal hydrolysis time of EXO III for subsequent sensing research. Under the optimized conditions, this signal amplified electrochemical aptasensor was used to detect trace amount of AFB1. Results from these analyses, shown in Fig. 4a demonstrated that the sensing response could be evidently differentiated even with trace variations of AFB1 in the sensing system. Indeed, based on these sensing responses, the linear sensing range was clearly achieved from 0.0001 to 100 ppt, which is about 6-order magnitude of AFB1 concentrations. In addition, the detection limit of signal amplified electrochemical aptasensor was estimated, based on the 3s rule, to be 0.6*10 4 ppt, which is the extraordinary sensing performance for AFB1 detection. Comparatively, as shown in Fig. 4b, without the signal amplification treatments, the sensing signal response including the 100% signal suppression could be neglected when compared with that of signal amplified aptasensors. Actually, it is remarkable that while the whole sensing range of the traditional model is from 0.1 to 100 ppt, in comparison, by using the hetero-enzyme based two-round signal amplification protocol described here, both the 100% signal suppression and the detection range are dramatically improved. Thus, it goes without saying that the detection limit was also greatly improved. Additionally, the specificity of the two-round signal amplified electrochemical aptasensor was interrogated with analogs and other common toxins. The results revealed, as shown in Fig. 4c, that only the target AFB1 could induce an obvious signal response, whereas the analogs or other common toxins (OTA, OTB, DON et al.) could not induce a comparable distinguished signal change, indicating the excellent specificity of the two-round signal amplified electrochemical aptasensor approach. Meanwhile, the practical application of this signal amplified electrochemical aptasensor to measure spiked samples was confirmed with HPLC methods, which indicated that satisfactory recoveries (95–110%) were obtained with our developed electrochemical aptasensor compared with the instrumental methods (detailed results in Table S1). Essentially, from our point of view, both the specificity and the recovery achieved with the electrochemical aptasensor can be attributed to the excellent properties of the selected aptamers. In addition, the fabricated electrochemical aptasensor was stored at 4 °C and subsequently employed it for detections. No obvious change in the sensing performance was observed after the electrode was kept for up to 4 weeks. These results demonstrated that the signal amplified electrochemical aptasensor exhibits the acceptable stability. Considering the detection procedures of the electrochemical aptasensor, it could be used directly for target toxin detection with signal amplified treatments after the fabrication of sensing interface, which is better than traditional immuno methods and comparable to other reported aptasensor methods. Therefore, future practical application research of this signal amplification strategy should be paid great attention in the field of high throughput detections and some preliminary results have already been achieved based on SPE, which is the suitable platform for high throughput detections (see detailed methods and comparisons results in Fig. S3 in Supporting information). And the integration with the portable detection device for on-site sensitive screening of analyte including the further optimization of sensing conditions is also the key step of great significance.
4. Conclusions A new hetero-enzyme based two-round signal amplification electrochemical aptasensor approach for sensitive detection of AFB1 has been successfully developed. This strategy effectively resolved the technical bottleneck associated with the signal-off
model sensing methods. The telomerase-based first-round amplification led to the generation of a sufficiently high initial current signal to increase the detection range, while the EXO III-based second-round amplification led to an observable signal response at trace concentrations. Taking advantage of this two-round signal amplification strategy, both the sensing range and detection limit of the developed electrochemical aptasensor were greatly improved by a 3-order magnitude widening and about 1000-fold enhancement respectively. The bottlenecks of the traditional signal-off model biosensor are all resolved with the strategies for signal amplification designed by our team. Further research of integrating this signal amplification strategy with screening printing electrode (SPE) based array sensing platform for high throughput is still going on in our group. Finally, this developed signal-amplified electrochemical aptasensor should provide further means to guarantee satisfactory resolution to the toxin-related food safety issues and, more importantly, contribute to the widespread application of traditional signal-off biosensor in various fields.
Acknowledgments This work is financially supported by the NSFC Grant of 21475030 and 31301460, the Science and Technology Research Project of Anhui Province 15czz03109, National 10000 TalentsYouth Top-notch Talent Program, the National and Zhejiang Public Benefit Research Project (201313010, 2014C32051) and the Jiangsu Science and Technology Support Program of BE201373, 2012780.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.01.091.
References Anfossi, L., Calderara, M., Baggiani, C., Giovannoli, C., Arletti, E., Giraudi, G., 2008. J. Agric. Food Chem. 56, 1852–1857. Arduini, F., Errico, I., Amine, A., Micheli, L., Palleschi, G., Moscone, D., 2007. Anal. Chem. 79, 3409–3415. Blesa, J., Soriano, J.M., Molto, J.C., Marin, R., Manes, J., 2003. J. Chromatogr. 1011, 49–54. Cuccioloni, M., Mozzicafreddo, M., Barocci, S., Ciuti, F., Pecorelli, I., Eleuteri, A.M., Spina, M., Fioretti, E., Angeletti, M., 2008. Anal. Chem. 80, 9250–9256. Deng, G.Z., Xu, K., Sun, Y., Chen, Y., Zheng, T.S., Li, J.L., 2013. Anal. Chem. 85, 2833–2840. Duan, R.X., Wang, B.Y., Zhang, T.C., Zhang, Z.Y., Xu, S.F., Chen, Z.F., Lou, X.D., Xia, F., 2014. Anal. Chem 86, 9781–9785. EC, 2006. Setting Maximum Levels for Certain Contaminants in Foodstuffs; Commission Regulation (EC). European Commission Regulation, Brussels. Ediage, E.N., Mavungu, J.D.D., Monbaliu, S., Peteghem, C.V., Saeger, S.D., 2011. J. Agric. Food Chem. 59, 5173–5180. Elghanian, R., Storhoff, J.J., Mucic, R.C., Letsinger, R.L., Mirkin, C.A., 1997. Science 277, 1078–1081. Ellington, A.J., Szostak, W., 1990. Nature 346, 818–822. Evtugyn, G., Porfireva, A., Stepanova, V., Sitdikov, R., Stoikov, I., Nikolelis, D., Hianik, T., 2014. Electroanalysis 26, 2100–2109. Huang, L., Wu, J.J., Zheng, L., Qian, H.S., Xue, F., Wu, Y.C., Pan, D.D., Adeloju, S.B., Chen, W., 2013. Anal. Chem. 85, 10842–10849. Hussein, H.S., Brasel, J.M., 2001. Toxicology 167, 101–134. IARC, 1993. Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. 56. International Agency for Research on Cancer: Lyon, France, p. 489. Khlangwiset, P., Shephard, G., Wu, F., Crit, 2011. Rev. Toxicol. 41, 740–755. Kuang, H., Chen, W., Xu, D.H., Xu, L.G., Zhu, Y.Y., Liu, L.Q., Chu, H.Q., Peng, C.F., Xu, C. L., Zhu, S.F., 2010. Biosens. Bioelectron. 26, 710–716. Lee, N.A., Wang, S., Allan, R.D., Kennedy, I.R., 2004. J. Agric. Food Chem. 52, 2746–2755. Madden, C.R., Finegold, M.J., Slagle, B.L., 2002. J. Virol. 76, 11770–11774. McKean, C., Tang, L., Tang, M., Billam, M., Wang, Z., Theodorakis, C.W., Kendall, R.J.,
W. Zheng et al. / Biosensors and Bioelectronics 80 (2016) 574–581
Wang, H.S., 2006. J. Appl. Toxicol. 26, 139–147. Mei, Z.L., Chu, H.Q., Chen, W., Xue, F., Liu, J., Xu, H.N., Zhang, R., Zheng, L., 2013. Biosens. Bioelectron. 39, 26–30. Meki, A.R., Esmail Eel, D., Hussein, A.A., Hassanein, H.M., 2004. Toxicon 43, 93–100. Micheli, L., Greco, R., Badea, M., Moscone, D., Palleschi, G., 2005. Biosens. Bioelectron. 21, 588–596. Nguyen, B.H., Tran, L.D., Do, Q.P., Nguyen, H.L., Tran, N.H., Nguyen, P.X., 2013. Mater. Sci. Eng. C 33, 2229–2234. Pamel, E.V., Verbeken, A., Vlaemynck, G., Boever, J.D., Daeseleire, E., 2011. J. Agric. Food Chem. 59, 9747–9755. Robertson, D.L., Joyce, G.F., 1990. Nature 344, 467–468. Saremi, H., Okhovvat, S.M., 2006. Commun. Agric. Appl. Biol. Sci. 71, 1175–1185. Shim, W.B., Choi, J.G., Kim, J.Y., Yang, Z.Y., Lee, K.H., Kim, M.G., Ha, S.D., 2008. J. Food Prot. 71, 781–789. Shim, W.B., Kim, K., Ofori, J.A., Chuan, Y.C., Chuang, D.H., 2012. J. Food Protect. 75, 1991–1999. Shim, W.B., Mun, H.Y., Joung, H.A., Ofori, J.A., Chung, D.H., Kim, M.G., 2014. Food
581
Control 36, 30–35. Singh, J., Singh, S., Dani, H.M., Sharma, R., Steinberg, P., 2005. Cell Biochem. Funct. 23, 9–13. Stetinova, V., Grossmann, V., Kvetina, J., 1998. Pol. J. Pharmacol. 50, 135–141. Tang, D.P., Zhong, Z.Y., Niessner, R., Knopp, D., 2009. Analyst 134, 1509–1728. Thirumala- Devi, K., Mayo, M.A., Hall, A.J., Craufurd, P.Q., Wheeler, T.R., Waliyar, F., Subrahmanyam, A., Reddy, D.V., 2002. J. Agric. Food Chem. 50, 933–937. Wang, Y.R., Li, P.W., Majkova, Z., Bever, C.R.S., Kim, H.J., Zhang, Q., Dechant, J.E., Gee, S.J., Hammock, B.D., 2013. Anal. Chem. 85, 8298–8303. Wu, J.J., Chu, H.Q., Mei, Z.L., Deng, Y., Xue, F., Zheng, L., Chen, W., 2012. Anal. Chim. Acta 753, 27–31. Wu, J.J., Zhu, Y.Y., Xue, F., Mei, Z.L., Yao, L., Wang, X., Zheng, L., Liu, J., Liu, G.D., Peng, C.F., Chen, W., 2014. Microchim. Acta 181, 479–491. Xue, F., Wu, J.J., Chu, H.Q., Mei, Z.L., Ye, Y.K., Liu, J., Zhang, R., Peng, C.F., Zheng, L., Chen, W., 2013. Microchim. Acta 180, 109–115. Yin, H.S., Zhou, Y.L., Chen, C.X., Zhu, L.S., Ai, S.Y., 2012. Analyst 137, 1389–1395.
