“Signal Off” Aptasensor Based on Enzyme Inhibition Induced by

Article pubs.acs.org/ac

“Signal Off” Aptasensor Based on Enzyme Inhibition Induced by Conformational Switch Beatriz Prieto-Simón*,†,‡,∥ and Josep Samitier†,‡,§ †

Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10-12 08028 Barcelona, Spain Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), María de Luna 11, 50018 Zaragoza, Spain § Departament d’Electrònica, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain ‡

S Supporting Information *

ABSTRACT: A novel sensing strategy for electrochemical aptamer-based sensors is presented. Nucleic acid aptamers are considered alternatives to antibodies. However, some of their intrinsic properties, such as that they can undergo conformational changes during the binding of the target, can be used to design novel sensing strategies. Unlike other electrochemical “signal off” aptamer-based sensors, we report a strategy based on enzymatic inhibition. Our approach shows the feasibility to detect small molecules based on the aptamer conformational change induced by the target that leads to the inhibition of the enzyme used as a label. Additionally, we prove the ability to regenerate the function of the aptasensor by simply applying a short potential pulse. As a proof-of-concept, the widely used aptamer for ochratoxin A (OTA) has been selected as a model. After self-assembling short oligonucleotides onto a gold electrode, complementary to the 3′ end of the aptamer, hybridization of the aptamer takes place. To investigate the mechanism induced by the OTA-binding, surface plasmon resonance assays were performed, which confirmed the conformational switch of the aptamer rather than the aptamer displacement by dehybridization from the DNA-modified sensor surface. The electrochemical sensor can successfully detect OTA in wine at the limits stipulated by the European Commission. Given its sensitivity, rapid and easy detection, and regeneration, it can be envisaged as screening tool for OTA detection. Moreover, this sensing strategy has the potential to be applied to other aptamer-based biochemical assays for the detection of small molecules in the fields of food safety, environmental monitoring, and medical diagnostics.

O

Aptamers are characterized by complex three-dimensional structures, including stem-loops, bulges, hairpins, pseudoknots, multiway junctions, or G-quadruplexes.4 Most of the aptamers show complex folded structures in solution that slightly change upon target binding.5,6 In the absence of significant conformational change, working conditions can be tailored to destabilize the native aptamer folding. Then, the presence of the target enables the proper folding of the aptamer, generating a signal event. Several electrochemical aptamer-based sensors have been developed relying on a conformational switch of the aptamer as a sensing principle.7,8 Those sensors have been mainly based on changes in electron transfer caused by (1) modulation of the distance from a redox moiety, used as a label, to the electrode surface9 or (2) sorption/desorption of electroactive intercalators from the aptamer bases.10 Depending on the resulting increase or decrease in the electrochemical signal, “signal-on” and “signal-off” approaches have been described, respectively.11,12

ver the past decade, nucleic acid aptamers have emerged as alternative tailor-designed entities to traditional bioreceptors.1 Some of their outstanding properties involve structural and functional robustness, ability to be readily sitespecifically modified, and easy and cost-effective synthesis. Aptamer-based assays can be conceived as analogues of immunoassays, but more interestingly, the knowledge of their properties can boost their practical applications.2,3 As single stranded DNA, they can be incorporated in assays that rely on principles of signal generation different from those commonly found for immuno-based assays, e.g., their secondary structure can undergo analyte-dependent conformational changes, which combined with the ability to specifically modify them with particular labels, opens up a wealth of possible sensing schemes, irrespective of the detection method employed. Herein, we present the design of a new aptamer-based assay that exploits target-induced aptamer conformational changes. To that purpose, it is essential to optimize the immobilization, assay, and detection protocols, without affecting the specific physicochemical properties of aptamers, specially the proper folding that provides the tertiary structure required for the binding. © 2013 American Chemical Society

Received: July 22, 2013 Accepted: December 30, 2013 Published: December 30, 2013 1437

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GTAAAGGGAGCATCGGACATTTTTTTTTT-3′; alkaline phosphatase (ALP)-labeled anti-OTA aptamer (ALP-apt): 5′ALP-(CH 2 ) 6 -GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACATTTTTTTTTT-3′; complementary thiolated oligonucleotide (SH-capture): 5′-SH-(CH2)6-AAAAAAAAAA-3′ and noncomplementary thiolated oligonucleotide (SH-control): 5′-SH-(CH2)6-CATGGTCGAA-3′. Mercaptoethanol, potassium ferrocyanide (K4[Fe(CN)6]), potassium ferricyanide (K3[Fe(CN)6]), 4-nitrophenyl phosphate (pNPP), α-naphthyl phosphate (α-NP), tris-(hydroxymethyl) aminomethane (Tris), diethanolamine (DEA), and other components of buffers were from Sigma-Aldrich. All solutions were prepared using Milli-Q water. Red wine and beer were purchased from local grocery stores. Reacti-Bind Maleimide-activated microtiter plates were obtained from Pierce. Surface plasmon resonance (SPR) untreated gold sensor chips were purchased from GE Healthcare. Gold electrodes were fabricated as described previously23 on a flexible transparent substrate, polyethylene naphthalate (PEN), purchased from Goodfellow Cambridge Limited (U.K.). A thin layer of gold (150 nm thickness) was directly deposited by sputtering on the plastic substrate showing a good adhesion.24 The mask used for the photolithographic process patterned electrodes of 3 mm diameter with a conductive pad. After gold etching, the conductive pad was partially covered with a nonconductive polymer layer. Apparatus. Colorimetric measurements were performed with a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories Inc.). SPR experiments were conducted with a SPR Biacore T100 system (GE Healthcare). Electrochemical measurements were performed on a SP-150 potentiostat that integrates electrochemical impedance spectroscopy (EIS) capability (Bio-Logic SAS), using a three-electrode electrochemical cell with a photolithographed gold disk working electrode, a platinum wire counter electrode, and a Ag/AgCl reference electrode, placed into a Faraday cage. Data acquisition and analysis were accomplished using EC-Lab software (BioLogic SAS). Enzyme-Linked Oligonucleotide Assay. Prior to their modification, maleimide activated plates (8-well strips) were washed three times with 0.1 M sodium phosphate, 0.15 M sodium chloride (pH 7.4) containing 0.05% Tween (PBSNaCl-T). A thiolated oligonucleotide (SH-capture or SHcontrol) was immobilized via the addition of 100 μL of a 2.5 μM solution in 0.1 M sodium phosphate, 0.15 M sodium chloride, 10 mM EDTA, pH 7.4, to each well and incubated for 3 h at room temperature. Unreacted maleimide groups were blocked for 1 h with 200 μL of a 1 mM aqueous solution of mercaptoethanol (prepared prior to use). After washing with PBS-NaCl-T, 100 μL of 250 nM ALP-apt in 0.1 M Tris buffer was added to the wells and incubated 1 h at room temperature. Alternatively, a one-incubation step protocol was followed by adding mixtures of 250 nM ALP-apt and OTA in 0.1 M Tris buffer to the wells and incubating them for different periods of time at room temperature. Then, the wells were washed 4 times with 0.1 M sodium phosphate (pH 7.4) containing 0.05% Tween (PBS-T). A volume of 100 μL of 4 g L−1 p-NPP in 10% DEA buffer, 1 mM MgCl2, pH 9.8, was added to each well, and the absorbance value was read after 1 h at a wavelength of 405 nm. After washing the wells with PBS-T, several concentrations of OTA in binding buffer (10 mM Tris, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, pH 7.4) were incubated for 1 h. After a final washing step with PBS-T, 100 μL of 4 g L−1 p-NPP was added

Nowadays, commercial application of aptamer-based assays for the quantitative determination of target molecule concentration shows still a scarce success. The design of new sensing schemes to convert aptamer−target interactions into measurable signals seems likely to be a key contributing factor to widen the potential applications of aptamer-based assays. We show a novel sensing strategy that aims to prove the potential breakthroughs of seeking for alternative sensing principles. We have selected ochratoxin A (OTA) as a target molecule to pave the way beyond the established state-of-theart in the detection of small molecules. We have focused on electrochemical aptamer-based sensors due to the high sensitivity of the electrochemical techniques. Moreover, the instrumentation is simple, cost-effective, and easily miniaturized. Here we describe a new sensing strategy using OTA aptamer as a model.13 OTA is a mycotoxin produced by several species of Aspergillus and Penicillium, fungi that grow on several types of food and feedstuff.14 Toxicological studies show that OTA is adsorbed from the gastrointestinal tract, causing both acute and chronic lesions to kidneys and liver. It can also affect the immune system of many mammalian species and it is a genotoxic, teratogenic, myelotoxic, carcinogenic (in mice and rats), and possibly neurotoxic agent to several animal species.15 The International Agency for Research on Cancer has classified OTA as a possible human carcinogen (group 2B).16 Traditional methods for OTA analysis are usually performed by chromatography, coupled to ultraviolet−visible, fluorescence, or mass spectrometry.17 Alternative analysis techniques have emerged in the past decade, mainly based on immunoassays. The identification of a DNA sequence showing affinity toward OTA 5 years ago11 has been followed by numerous studies about its use as a biorecognition element in a myriad of assays: some of them based on a strategies analogue of the immunoassays while others have been focused on novel sensing strategies.18−22 To the best of our knowledge, we report for the first time a “signal off” aptamer-based assay approach that relies on enzyme inhibition. Immobilized aptamer interacts with toxin in solution leading to a signal decrease. We hypothesize that the observed signal decrease is based on conformational changes induced by the aptamer’s ability to fold upon OTA binding triggering the blocking of the enzyme’s active site. Complementary techniques, such as surface plasmon resonance, enzyme-linked oligonucleotide assays, and electrochemical characterization, have been used to support our results and to elucidate the mechanism involved. We have demonstrated the potential of this new sensing approach by detecting OTA in wine and beer in an easy onestep sample incubation and fast measurement. Moreover, the sensor is regenerated after the application of a short pulse potential, what adds value to use this method in routine analysis controls. This new sensing strategy opens up the possibility to design alternative assays to those based on immunosensing that can add some advantages, such as in this case a rapid and easy regeneration.

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EXPERIMENTAL SECTION Reagents. OTA (from Aspergillus ochraceous) was purchased from Alexis Corporation. OTA standard solutions were first prepared in ethanol (25 g L−1). All oligonucleotides were synthesized by Eurogentec, and their base sequences were anti-OTA aptamer (apt): 5′-GATCGGGTGTGGGTGGC1438

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were prepared by exposing the clean gold electrodes to 20 μL of 0.75 μM SH-capture or SH-control in 50 mM Tris buffer overnight in a humidity chamber. After ssDNA adsorption, 1 h incubation with 1 mM mercaptoethanol helped to minimize the DNA physisorption phenomena by displacement of the nonspecific adsorptive contacts between nucleotides and gold and occupies the vacant places within the monolayer.26 Moreover, it forces the probe orientation to favor hybridization. Hybridization was performed at 4 °C for 1 h by incubating 20 μL of 0.9 μM ALP-apt or aptamer in binding buffer. Electrochemical Detection Protocol. Prior to OTA incubation, activity of ALP was measured electrochemically using differential pulse voltammetry (DPV) as previously described27 (1st measurement). Briefly, electrodes were exposed for 4 min to a 2 mM α-NP solution in 0.1 M DEA buffer, pH 9.5. Then, a conditioning potential of 0.175 V vs Ag/ AgCl was applied during 10 s. We observed that the application of a positive potential improved the repeatability of measurements. We suggest that the application of a positive potential can help to desorb negatively charged species that could have been attracted and adsorbed onto the electrode surface, e.g., the excess of aptamer present in solution during the hybridization step. Finally, the potential was scanned at 0.05 V/s from 0 to 0.4 V. DPV data was corrected by subtracting the voltammogram obtained in the absence of α-NP. Oxidation currents were measured at 0.225 V. The electrodes were rinsed with binding buffer, and then samples containing OTA at different concentrations were incubated for 1 h. After rinsing the electrodes with binding buffer, ALP activity was measured again using DPV (2nd measurement). Results were shown as % of remaining ALP activity vs OTA concentration. A 2 min pulse of −0.4 V was applied to regenerate the aptamer-based sensors. Sample Preparation. Aliquots of OTA-free wine and beer samples were spiked with the stock solution of OTA to obtain dilutions from 0.1 nM to 100 μM OTA. Wine samples were prepared as untreated or treated samples. Pretreatment followed a protocol previously described based on the addition of 0.1 g of PVP to a 10 mL wine aliquot.27 The wine/PVP mixture was shaken for 2 min at room temperature, and after filtration (0.45 μm), the pH was adjusted to 7.2. Beer samples, previously cooled at 4 °C to prevent foam formation, were degassed by sonication for 1 h and finally filtered.28,29 To perform electrochemical measurements, samples of 50 μL were incubated onto the electrodes, by mixing 25 μL of wine/beer samples with 25 μL of 2× binding buffer.

to each well and the absorbance was recorded at 405 nm. Results were shown as % of remaining ALP activity vs OTA concentration. Absorbance values were corrected by subtracting the absorbance for a control with nonlabeled OTA aptamer. Each value was the mean of three wells. SPR Studies. SPR measurements with anti-OTA aptamerand ALP-labeled anti-OTA aptamer-modified chips were performed to study the effect of the presence of ALP enzyme on the SPR angle changes. The SPR Biacore T100 is equipped with a continuous flow system in which four channels are coupled in series. Gold chips were cleaned by exposure to piranha solution (70% concentrated sulfuric acid, 30% peroxide solution (30%)) (warning: piranha solution reacts violently with organic solvents) for 30 min. Then, the electrodes were rinsed thoroughly with deionized water. Immobilization of the aptamer onto the gold chip was performed ensuring its proper quadruplex configuration. Aptamer or ALP-aptamer was immobilized on three channels of the gold chip. The fourth channel, modified with a single stranded oligonucleotide of random sequence and similar length to the aptamer but lacking the quadruplex secondary structure, was used as a control for nonspecific adsorptions. Initially, SH-capture or SH-control (1 μM in 50 mM Tris buffer) was attached through a gold−sulfur bond. To avoid the nonspecific attachment of some nucleobases through their nitrogen atoms, the remaining free gold surface was blocked with 1 mM mercaptoethanol. Then, the DNA-modified chip was ready to hybridize the OTA aptamer. Injection of different OTA concentrations (from 0.0165 nM to 8.25 μM), prepared in binding buffer, was performed. A 3 min-injection of 100 mM HCl solution was used for the regeneration of the sensor surface. To evaluate the regeneration effect, ALP-apt, ALP-apt/ OTA mixtures, and OTA solutions were injected and SPR signals were recorded. Triplicate injections were performed for each OTA concentration. Nonspecific SPR signals obtained from the channel used as reference were subtracted from the other sensorgrams. Electrode Preparation and Electrochemical Characterization. Gold electrodes were electrochemically cleaned in 0.5 M H2SO4 by scanning the potential between 0 and 1.6 V vs Ag/ AgCl at 100 mV s−1 until there was no further change in the voltammogram. The microscopic surface area was calculated from the reduction charge associated with the monolayer of chemisorbed oxygen.25 All electrochemical data were normalized based on the microscopic surface of each electrode. Electrodes were characterized at each modification step using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Electrochemical measurements were performed in an unstirred solution of 2 mM ferrocyanide and 2 mM ferricyanide in 100 mM phosphate buffer, pH 7.4. Cyclic voltammograms were obtained by scanning the potential at 0.1 V/s from −0.2 to 0.6 V. EIS measurements were performed under open circuit potential conditions. Frequencies from 10 kHz to 0.1 Hz in logarithmic spacing were applied. The ac amplitude was 10 mV. The recorded impedance spectra were fitted to a Randles circuit, corresponding to a basic equivalent circuit that includes the following parameters: the solution resistance (Rs), the charge transfer resistance (Rct), the Warburg impedance (W), and the double-layer capacitance (Cdl). Because of the effect of the surface roughness that leads to the dependence of the interfacial capacitance on the potential modulation frequency, a constant phase element (CPE) was used instead of an ideal Cdl. Modified electrodes

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RESULTS AND DISCUSSION Aiming to design new sensing principles for aptamer-based sensors, details about the dependence of an aptamer folded structure on working conditions are essential. To exploit the conformational switch of the aptamer upon target binding, aptamers involving significant changes in structure are preferred. However, the possibility to select certain working parameters able to destabilize the native aptamer folding in the absence of target analyte can play a key role in developing new sensing strategies. This work was performed using OTA aptamer. Circular dichroism spectroscopic studies previously revealed that the aptamer exhibits a random coil structure in solution that switches to a rigid antiparallel G-quadruplex structure upon OTA binding.30,31 The conformational change forces a significant change in the location of the 5′-end of the aptamer that can be used to design novel sensing strategies. Therefore, our first concern was to find the proper working 1439

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block unreacted maleimide groups, can both contribute to the low values of absorbance. Moreover, the low sensitivity is a sign of the slow reaction kinetics associated with the chosen microplate format. Binding capacity of maleimide plates limits the efficiency of the formation of stable thioether bonds. Long incubation times favor the extent of the reactions involved but at the expense of long assay times, not competitive with other assay methods. Nevertheless, the performed ELONA helped to set the optimum working conditions for further assays using other detection methods. Initially the absorbance decrease was attributed to a displacement of the hybridized aptamer after a target-induced conformational change. However, hybridization of the aptamer involved only a short sequence of 10 thymine (T) bases not included in the aptamer sequence. Hybridization between those 10 Ts and the 10 adenines (A) previously immobilized onto the electrode surface is considered to be very strong and it is not involved in the target−aptamer interaction. Therefore, we carried out further experiments to elucidate the mechanism involved in the decrease of the signal obtained from the ALP enzyme after OTA interaction. The mechanism involved upon OTA binding could be assigned either to the aptamer displacement by dehybridization or to conformational changes triggering the inhibition of the enzyme ALP. Although SPR measurements cannot directly determine the structure of DNA aptamers, several studies show their ability to prove structural folding changes upon target binding.33 Conformational changes, added to the binding of large proteins or even small molecules, can significantly change the refractive index and, as a consequence, the angle at which resonance occurs. To elucidate whether OTA binding involves an aptamer conformational switch or the aptamer release by dehybridization, two SPR assays were performed where ALP-aptamer was immobilized onto the first channel, nonmodified aptamer was immobilized using different hybridization times onto the second and third channels, and the fourth channel was used as a control. Two assays were performed differing on the modification of the control channel either with a noncomplementary short thiolated-ssDNA or with a random ssDNA sequence. We assume that if OTA aptamer is shown as a random coil in solution, as demonstrated by circular dichroism spectroscopy, this structure persists on the sensor chip under similar buffer conditions. Furthermore, special care was taken to minimize nonspecific binding and adsorption of the analyte to the sensor surface to avoid misinterpretation of the results. Nevertheless, nonspecific binding of OTA to the DNA-modified sensor chip regardless of sequence and folding was encountered and led to responses larger than expected. To compensate this effect, signal from the reference channel was subtracted from each sensorgram. Moreover, to be sure about the reliability of our SPR data and considering that the dissociation constant for OTA−aptamer interaction (50 nM)13 is sufficiently lower than the OTA concentration at which nonspecific binding becomes significant, we processed data at low OTA concentrations expected not to be affected by nonspecific binding. Figure 2 shows the SPR signal for channels modified with ALP-aptamer and nonmodified aptamer after reference channel subtraction. To compare them, linear regression at low OTA concentrations gave us the sensitivity of each channel (Table 1). Channel with ALP-aptamer involves the largest sensitivity. If we assume that conformational changes occur instead of dehybridization, the difference in the value of sensitivity compared to the equivalent

conditions to keep the random coil structure of the aptamer after its immobilization. First, the aptamer was immobilized onto a ssDNA-modified surface through hybridization. The 3′end of the aptamer was initially modified by adding some extra nucleobases, which were complementary to the ssDNAmodified surface. Second, the binding buffer was chosen to stabilize the unfolded structure in the absence of OTA and the quadruplex structure upon OTA addition. To that purpose, type and concentration of monovalent cations was carefully selected. Potassium and sodium cations support quadruplex formation, as thermodynamically and kinetically demonstrated by Hardin et al.32 Thus, both monovalent cations were included in the binding buffer. Furthermore, as previously reported,13 the presence of divalent cations, Ca2+ or Mg2+, is essential for the specific recognition of OTA by aptamer. We chose to work with Mg2+. Indeed, 10 mM Tris buffer supplemented with 120 mM NaCl, 5 mM KCl, and 5 mM MgCl2 was selected as binding buffer. In our initial attempt at sensing strategy development, we performed an enzyme-linked oligonucleotide assay (ELONA). The first approach, based on an indirect competitive assay, was conducted in a one-incubation step with a fixed ALP-apt concentration and varying the OTA concentration. Results did not show any significant absorbance-concentration dependence, even after increasing OTA incubation times up to 5 h. Alternatively, the assay was split in two incubation steps: first the aptamer hybridization and second the OTA binding. Results show the dependence of % of binding on OTA concentration (Figure 1). A limit of detection (LOD) of 59 nM

Figure 1. Calibration curve for OTA obtained by colorimetric detection. Percentage of absorbance was calculated as the remaining ALP activity upon OTA incubation according to the described ELONA protocol. Error bars are standard deviations of the mean with n = 3.

was found, considered as the concentration of OTA causing an intensity current equal to an 80% of the remaining ALP activity upon OTA binding (Abs (%) = 65 + (35/(1 + ([OTA]/ 157)1.45)), EC50= 157 nM, LOD (80%) = 59 nM). The narrow linear range and the LOD higher than previously found values following analogue ELONA strategies can be attributed to the low values of absorbance obtained. Stringent washing conditions required to eliminate the background originated from nonspecific binding of ALP-aptamer and/or ALP that can remain free in solution, and mercaptoethanol backfilling to 1440

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Figure 3. Comparison of SPR signal due to initial hybridization (A) and injection of aptamer (B), OTA (C), and OTA/aptamer mixture (D) after regeneration.

Figure 2. Calibration curves of SPR signal vs [OTA] obtained for channel 1 (circle), 2 (square), and 3 (triangle), respectively.

Table 1. Sensitivity Values Derived from the Linear Regression Fitting at Low Concentrations of the Plots Shown in Figure 2 channel (biorecognition element/ hybridization time)

sensitivity (RU/nM)

R2

1 (aptamer-ALP/900 s) 2 (aptamer/900 s) 3 (aptamer/450 s)

61 25 35

0.9996 0.9989 0.9900

activity, which was envisaged as a principle of a novel electrochemical sensing strategy (Table S-1 in the Supporting Information). The principle of the electrochemical OTA detection is depicted in Scheme 1. Cyclic voltammograms and impedance spectra obtained at each step of modification provide qualitative information about the electrode functionalization and performance. The recorded impedance spectra were fitted to a Randles circuit, corresponding to the basic equivalent circuit depicted in Figure 4A. Impedance spectra shown in Figure 4A clearly shows an increase of the Rct after hybridization of the aptamer due to the repulsion between the negatively charged aptamer and the redox pair in solution. Similarly, a decrease of intensity current for the corresponding cyclic voltammograms is expected. However, Figure 4B shows that the electrode current is only slightly reduced, probably indicating that the aptamer hybridization does not end in a highly compact layer, enabling the access of the electroactive species to the electrode surface. The amount of aptamer immobilized onto the transducer was carefully optimized to freely allow the switch between the aptamer conformations in the presence and absence of OTA. Impedance spectra obtained after incubation of the aptamerbased sensor in a solution of OTA further increases the Rct, confirming the interaction of OTA with the immobilized aptamer. We suggest that the increase of Rct after OTA incubation is not only the result of the repulsion induced by the negatively charged OTA at pH 7.434,35 but also to the switch of the aptamer toward a more bulky conformation. Results in Figure 4 show that it is possible to regenerate the biosensor by simply applying a short pulse potential and subsequently washing the electrode surface with deionized water. The Rct decreases after surface regeneration down to the initial value prior to OTA incubation. These results corroborate that the presence of OTA switches aptamer conformation rather than causing dehybridization. Therefore, a pulse potential successfully regenerates the biosensor by forcing the initial aptamer conformation and subsequent OTA release. As previously mentioned, it has been already reported that OTA interaction switches the aptamer into an antiparallel Gquadruplex, where both ends of the DNA sequence are very close. On the basis of DPV measurements of the ALP activity linked to the 5′-end of the aptamer after incubation of a wide range of OTA concentrations, we suggest that the OTA

channel with aptamer shows that the ALP enzyme is involved in the conformational changes, increasing the effect on the SPR signal. Additionally, as longer times of aptamer hybridization were used, and thus, larger amounts of aptamer were immobilized, lower SPR sensitivity was obtained. The difference observed between channels with different amounts of immobilized aptamer proves the fact that crowding and steric hindrance on the surface may influence interactions with analyte in solution. To confirm that the conformational switch of the aptamer rather than dehybridization of the aptamer causes changes in sensitivity, the chip modified surface was treated with solutions of increasing stringency, aiming to release OTA from the immobilized aptamer without causing dehybridization. Incubation with 100 mM HCl for 180 s leads to a maximum signal decrease of 19 RU, except for the channel with aptamer-ALP, where the decrease reaches a maximum of 70 RU (approximately 10% of the signal achieved after hybridization of aptamer-ALP). After that, the sensor chip was incubated with aptamer, OTA, or OTA/aptamer mixture. The small signal achieved for all channels by the injection of aptamer after regeneration proves that the aptamer has not been dehybridized after HCl injection (Figure 3). The signal due to OTA injection, similar to previous results, confirms that the aptamer is still hybridized after regeneration. The signal due to OTA/ aptamer mixture injection (lower than OTA but higher than aptamer) can be attributed to free OTA still in solution. We hypothesize that regeneration releases OTA without dehybridizing the aptamer and denaturates the ALP enzyme, causing conformational changes that end up in SPR signal changes. Combining ELONA and SPR results, the switch of the aptamer upon OTA binding was confirmed. Moreover, the conformational change triggered an effect on the ALP enzyme 1441

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Scheme 1. Aptasensor for the Detection of OTA by DPV Detectiona

a

An ALP-tagged OTA-binding DNA aptamer immobilised on a gold surface through hybridization undergoes a conformational switch upon OTA binding that triggers the enzyme inhibition. The enzymatic activity can be easily regenerated by simply applying a short potential pulse.

Figure 4. Nyquist impedance plots (A) and cyclic voltammograms (B) recorded at a gold electrode modified with a short thiolated oligonucleotide complementary to the aptamer (dotted line), after hybridization with the aptamer (dashed line), incubation with a 100 nM OTA solution (solid line), and renewed electrode after regeneration by short pulse potential application (dashed-dotted line). Inset in part A: equivalent circuit used to fit experimental data.

binding-induced conformational change causes a certain degree of ALP inhibition that could be due to steric effects that block the enzymatic active site. As shown in Figure 5, the optimized biosensor enables the detection of OTA at concentrations from 1 nM to 10 μM, with a LOD of 4 nM. Although lower LODs have been described for other electrochemical OTA aptasensors,18−22 it is interesting to highlight the reproducibility among measurements (6.4%) and the wide linear range obtained. It is also of utmost importance the excellent reproducibility between calibration curves obtained after regeneration by simply applying a short pulse potential. We observed a decrease in ALP activity of less than 8% after 5 regeneration cycles, proving the feasibility to use this simple regeneration protocol between successive measurements. The simplicity of the measurement and regeneration points out the suitability of the developed biosensing strategy for rapid, reliable, and in situ detection of analytes at low concentrations. To demonstrate the applicability of the developed aptamerbased sensor using samples with complex matrixes, OTA-spiked wine and beer samples were analyzed. Table 2 summarizes the results for the nonlinear four parameter logistic regression

Figure 5. Calibration curve for OTA obtained by DPV detection using an ALP-aptamer modified electrode. Error bars are standard deviations of the mean with n = 3.

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Table 2. Curve Parameters Derived from the Nonlinear Four-Parameter Logistic Regression Fitting of the Plots Obtained for OTA in Phosphate Buffer, Wine, Pre-Treated Wine and Beer Using DPV Detection of the ALP Activity sample

IC50 (nM)

phosphate buffer

207

LOD (nM)a

SD (%)b

4

6.4

sigmoidal logistic equation

I = − 7.3 +

(

1+ wine

pretreated wine

beer

730

205

701

10

5.8

2

4.8

334

7.1

[OTA] 207

0.45

)

77

I = 8.9 +

0.9985

1+

(

[OTA] 730

1+

(

[OTA] 205

1+

(

0.58

)

77

I = 5.2 +

0.9986 0.77

)

0.9984

56

I = 44 +

[OTA] 701

R 0.9967

103

0.87

)

a

LOD calculated as the concentration of OTA causing an intensity current equal to 80% of the maximum ALP activity achieved. bSD values refer to the maximum SD values found in the calibration curve.

fitting of the plots obtained using OTA-spiked samples prepared with phosphate buffer, wine, pretreated wine and beer. Nontreated wine samples showed some interfering effects (10% slope deviation between calibration curves in buffer and in nontreated wine samples). A simple pretreatment based on removing polyphenols with PVP and pH adjustment was enough to eliminate interfering compounds. Percentage of slope deviation between calibration curves in phosphate buffer and in pretreated wine samples was lower than 3% and, thus, no significant interfering effects could be observed. Beer samples offer a more complex matrix that strongly affects the sensitivity of the assay (19% slope deviation). Beer is a complex matrix that includes polyphenols and a high content of several proteins that could be considered as interfering compounds of the electrochemical detection. Remaining proteins from original cereal grains include albumins, enzymes, amylase inhibitors, and lipid binding proteins that can affect the configuration of the aptamer as well as the electrochemical detection once adsorbed on the electrode surface. Further experiments will be performed to determine the matrix effects that lead to a significant decrease of sensitivity and subsequent increase of the LOD. Currently, the presence of OTA in beer is not regulated by the European Commission. There are only few regulations around the world setting the maximum allowable limits of OTA in beer. Such is the case of the directive issued by the Italian Ministry of Health that fixes a maximum level of 0.2 mg L−1 of OTA in beer (500 nM). Although OTA is a common contaminant of beer due to the presence of ochratoxigenic fungi in barley and the failure of the fermentation process to eliminate the toxin, the concentration nearly ever exceeds 0.2 mg L−1. The biosensing strategy that we propose is able to detect OTA in nontreated beer at lower levels (LOD = 334 nM) than those considered safe. Therefore, the developed sensor could be used as a reliable screening tool for the presence of OTA in beer.

decrease of ALP activity after OTA binding. We suggest that presence of OTA induces a conformational change that partially blocks the active site of the enzyme. However, it is worth considering the fact that OTA is an analogue of phenylalanine, an amino acid known to inhibit alkaline phosphatase. The percentage of inhibition is commonly low enough to work with alkaline phosphatase as an enzyme label. Nevertheless, based on previous published results and on our immobilization strategy, we have to consider that the binding and the subsequent aptamer conformational change keep OTA in a close distance to the active site of ALP. The proximity between OTA and the active site of ALP can induce a certain percentage of enzymatic inhibition. Further studies will be carried out to elucidate the influence of OTA in close proximity as an inhibitor of ALP and the steric hindrance induced by the conformational change into the degree of enzyme inhibition. Additionally, on the basis of the easy regeneration of aptamers under certain conditions, we show the excellent regeneration of the aptamer-sensor surface by simply applying a short pulse potential. Combination of the proper aptamer immobilization strategy through hybridization with short complementary thiolated oligonucleotide attached onto the gold surface via a gold−sulfur bond, easy working protocol based on the enzyme inhibition caused by the presence of OTA, and the simplicity and reliability of surface regeneration ends up in sensitive and reproducible biosensors able to detect OTA in wine and beer at the levels set or recommended by the European Commission. This novel biosensing strategy can be extended to other aptamer-based biochemical assays with interest in the fields of food safety, environmental monitoring, and medical diagnostics.

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ASSOCIATED CONTENT

S Supporting Information *

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Experimental results to prove that presence of OTA cannot directly affect the enzymatic activity of the ALP linked to antiOTA aptamer in solution. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSIONS Here we present a proof of concept for a novel biosensing strategy based on the inhibitory effect that aptamer conformational changes can exert on enzymes used as labels. OTA aptamer has been used as a sensing element due to the switch of its structure from random coil to antiparallel G-quadruplex upon OTA binding. The proposed sensing strategy relies on the principles of “signal off” sensors, in our case showing a

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 1443

dx.doi.org/10.1021/ac402258x | Anal. Chem. 2014, 86, 1437−1444

Analytical Chemistry

Article

Present Address

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B.P.-S.: Mawson Institute, University of South Australia, Mawson Lakes, South Australia 5001, Australia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

Dr. B. Prieto-Simón acknowledges financial support from the Ministerio de Ciencia e Innovación through the Juan de la Cierva program. This research has been partially funded by the “Departament d′Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya” (Grants 2010 CTP 00032 and 2009 SGR 505) and by the Science Support ́ The authors kindly Program of the Fundación Botin. acknowledge Dr. Marta Taulés for her support in the SPR ́ assays. The authors also thank Sergio Martinez and David Izquierdo for the design and fabrication of gold electrodes.

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