Aptamer-Controlled Reversible Inhibition of Gold Nanozyme Activity

This study addresses the need for rapid pesticide (acetamiprid) detection by reporting a new colorimetric biosensing assay. Our approach combines the ...
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Aptamer-controlled reversible inhibition of gold nanozyme activity for pesticide sensing Pabudi Weerathunge, Rajesh Ramanathan, Ravi Shukla, Tarun Kumar Sharma, and Vipul Bansal Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5028726 • Publication Date (Web): 23 Oct 2014 Downloaded from http://pubs.acs.org on October 25, 2014

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Analytical Chemistry

Pabudi Weerathunge,† Rajesh Ramanathan,† Ravi Shukla,† Tarun Kumar Sharma†,§,* and Vipul Bansal†,* †

Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Applied Science, RMIT University, GPO Box 2476V, Melbourne VIC 3001, Australia. § Centre for Biodesign and Diagnostics, Translational Health Science and Technology Research Institute, Gurgaon, Haryana 247667, India. ABSTRACT. This study addresses the need for rapid pesticide (acetamiprid) detection by reporting a new colorimetric biosensing assay. Our approach combines the inherent peroxidase-like nanozyme activity of gold nanoparticles (GNPs) with high affinity and specificity of an acetamiprid-specific S-18 aptamer to detect this neurotoxic pesticide in a highly rapid, specific and sensitive manner. It is shown that the nanozyme activity of GNPs can be inhibited by its surface passivation with target-specific aptamer molecules. Similar to an enzymatic competitive inhibition process, in the presence of a cognate target, these aptamer molecules leave the GNP surface in a target concentration-dependent manner, reactivating GNP nanozyme activity. This reversible inhibition of the GNP nanozyme activity can either be directly visualized in the form of color change of the peroxidase reaction product or can be quantified using UV-visible absorbance spectroscopy. This approach allowed detection of 0.1 ppm acetamiprid within an assay time of 10 min. This reversible nanozyme activation/inhibition strategy may in principle, be universally applicable for the detection of a range of environmental or biomedical molecules of interest.

Pesticides are indispensable component of modern crop management practices as they minimize the loss in agricultural productivity caused by insects and pests. To protect plants from insect infestation, a myriad of pesticides belonging to diverse classes of organophosphates, organochlorines and neonicotinoids are used. In particular, acetamiprid, a neonicotinoid has recently replaced organophosphates for the control of sucking insects and has become one of the most routinely used pesticides globally.1-3 Although invaluable for crop protection, acetamiprid acts as a neurotoxin by causing agonistic effects against nicotinic acetylcholine receptors (nAchRs). 3 Since the injudicious use of acetamiprid has resulted in contamination of surface/ground water and food products alike, the sensitive and selective detection of this nerve poison has become critical for human and environmental health. Contemporary pesticide detection methods such as high pressure liquid chromatography (HPLC),4 gas chromatography (GC),5 mass spectrometry (MS),6 flow7 and antibody-based immunoassays (AIA)1,8 rely upon sophisticated instruments and skilled manpower, making such approaches impractical for on-site and regular environmental monitoring. To address these issues, nanoparticle-based colorimetric approaches have recently emerged for the detection of pesticides.3,9-21 Most of these assays are based on gold nanoparticles (GNPs), predominantly because of the ease in synthesis and surface modification of GNPs9-12,14,22-24 coupled with their unique surface plasmon resonance (SPR) properties that assist in attaining a visual readout of the sensing event.22-26 However, these GNP-based assays frequently encounter certain challenges, the most common being the uncontrolled aggregation of GNPs in complex environmental samples, resulting in poor specificity. 23, 27

The recent discovery of the inherent peroxidase-like activity of GNPs may offer new opportunities for biosensor development, as this nanozyme activity (nanoparticles’ enzyme mimicking activity) is independent of the change in SPR caused by nanoparticle aggregation.26-30 This concept has recently been utilized for non-specific detection of glucose and H2O2,27 however due to the lack of molecular recognition elements (MREs) in such studies, the potential of nanozyme activity to develop highly specific sensing platforms remains untapped. Over the last decade, non-stereotypical synthetic nucleic acids have emerged as outstanding MREs, as these acquire target-responsive secondary and tertiary structures to bind to their targets with high affinity and specificity.31-33 Due to their outstanding target recognition ability, these functional nucleic acids (FNAs) have become chemical rivals to antibodies by offering additional advantages such as high stability, low invitro synthesis cost and ability to be easily functionalized with flourophores and nanomaterials.34-36 Such single stranded DNA or RNA molecules that are generated through an in-vitro selection process called Systemic Evolution of Ligands by EXponential enrichment (SELEX) are known as aptamers. 34-36 In contrast to an antibody, the most remarkable feature of an aptamer is that it can be generated against a myriad of targets, including small molecules such as antibiotics, pesticides, amino acids and nucleotides; biomacromolecules such as carbohydrates, proteins and nucleic acids; as well as against an intact prokaryotic or eukaryotic cell.12,24,32,34,37-39 Since acetamiprid pesticide is a small molecule, our study employed an acetamiprid-specific S-18 aptamer that was reported for its specificity to acetamiprid (supporting information, Figure S1).2

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Figure 1. Schematic representation of the reversible inhibition of the nanonozyme activity of GNPs using an acetamiprid-specific S-18 ssDNA aptamer. Step A shows intrinsic peroxidase-like activity of GNPs that gets inhibited after shielding of GNP surface through conjugation of S-18 aptamer molecules (Step B). In the presence of acetamiprid target, aptamer undergoes targetresponsive structural changes and forms a supramolecular complex with acetamiprid resulting in free GNP to resume its peroxidase like activity (Step C).

The current study is built around the hypothesis (Figure 1) that the peroxidase-like activity of a GNP results from interaction of its surface with the peroxidase substrate (Step A). Therefore, this peroxidase-like activity can be inhibited by shielding the GNP surface through the adsorption of targetspecific ssDNA aptamer molecules (Step B). While in the absence of a target analyte such as acetamiprid, the nanozyme activity of the aptamer-conjugated GNPs remains masked; in the presence of a cognate target, the aptamer undergoes targetresponsive structural changes followed by desorption from the GNP surface to allow an aptamer-target binding event. This subsequently allows the GNP to reverse in its original form and resume target-specific peroxidase-like activity (Step C). This strategy is similar to the competitive inhibition process shown by natural enzymes that in the presence of an inhibitor lose their activity, whereas when that inhibitor is removed, in the presence of a substrate with higher binding affinity, the enzymatic activity is resumed. While this new biosensing concept should, in principle, be universally applicable for any target analyte, the current study demonstrates proof-of-concept detection of acetamiprid pesticide by applying this simple, rapid, sensitive and specific colorimetric assay. Notably, so far, peroxidase-like activity of GNPs has not been employed either independently or in conjunction with aptamers for sensing of environmental contaminants such as acetamiprid pesticide. Specific to the current study, exposure of pristine tyrosine-reduced GNPs18,19 to a colorless peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2 results in TMB oxidation to a purplish-blue product (step A). After adsorption of acetamiprid-specific S-18 ssDNA aptamer on GNP surface, no color change of the TMB substrate is observed (step B). Conversely, the presence of acetamiprid causes target responsive structural changes in S-18 aptamer, promoting its desorption from the GNP surface and leading to the recovery of peroxidase-like activity of bare GNPs to oxidize colorless TMB into a purplish-blue product (step C). While the physico-chemical characteristics of GNPs and aptamer-conjugated GNPs (GNP-S18) are discussed in supporting information (Figure S2), this new biosensing strategy offers an opportunity for quantitative detection of acetamiprid by simply measuring the change in absorbance of oxidized TMB using a UV-visible absorbance spectrophotometer. The sensitivity of acetamiprid detection using nanozyme-aptamer strategy is evident from Figure 2, which shows that within 10 min, GNP-S18 system is able to detect as low as 0.1 ppm concentration of acetamiprid with a dynamic linear range of 0.1 to

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Figure 2. (a) Relative peroxidase-like activity of GNP-S18 nanoconjugate as a function of acetamiprid concentration monitored at A650 nm for oxidized TMB after 10 min of reaction. (b) Linear fit of the activity obtained for 0.1-10 ppm acetamiprid.

10 ppm. Figure S3 in the supporting information shows wellresolved concentrations when the same data is plotted on a log x-axis. The linear regression of 0.996 evinces the desirable linearity of the curve, supporting the quantitative detection ability of this approach. It is apparent that at acetamiprid concentration above 10 ppm, the curve loses its linearity due to the saturation of the GNP nanozyme activity post-aptamer removal. Therefore, the concentration of GNP-S18 test reagent was doubled (from originally X to 2X) in the next set of experiments to evaluate whether this limitation of the saturation response could be overcome. Figure S4 in the supporting information shows the ability of this sensor to also operate in the dynamic linear range of 2.5 to 25 ppm by simply doubling the amount of test reagent employed during sensing. This shows the flexibility of the proposed biosensing platform to directly operate in different environments that may require operational sensitivity in different dynamic ranges. Nonetheless, acetamiprid detection limit obtained in the current study falls within the range of lowest tolerance limit (0.1 ppm) of acetamiprid set by United State Environmental Protection Agency (USEPA).40 Notably, this detection limit is five time more sensitive than a previously reported SERS-based acetamiprid detection assay.41 Further, in comparison to a recently reported aptamer-based electrochemical acetamiprid assay, the current method omits the need for thiol modification of aptamers and electrochemical deposition of aptamer-GNP complex on a bare gold electrode, which reduces the cost, complexity and detection time for the proposed assay.42 To evaluate the performance of these nanozyme biosensor, important parameters such as limit of detection (LoD), limit of quantification (LoQ), precision and accuracy were also determined for the GNP-S18 biosensors involving X concentration of the test reagent. The biosensor showed outstanding performance in all these aspects with LoD of 1.8 ppm (3.4 ppm for 2X concentration), LoQ of 5.5 ppm and precision of 97.34 %, while operating with an accuracy of 100 % at 10 % confidence level (20 replicates). Inter-batch variations from 10 different batches of nanoparticle synthesis were also calculated, which revealed the precision of 95.44 % and an accuracy of 90 % at 10% confidence level. Figure 3 shows that it is also possible to obtain a visual read-out of the acetamiprid detection using this strategy within 10 min. The GNPs and GNP-S18 utilized in this study are ruby-red in color (Amax 520 nm, Figure S2) whereas the oxidized TMB typically shows a purplish-blue color (Amax 650 nm). Therefore, in the presence of ruby-red GNPs, the oxidized TMB appears bluish. However, after conjugation of S-

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Figure 3. Peroxidase-like activity of pristine GNP and GNP-S18 nanoconjugate in the presence and absence of 5 ppm different analytes [acetamiprid (acet), agritone (agrit), endothal (end) and imidacloprid (imid)] after 10 min of reaction. % activity is calculated from the A650 nm of the oxidized TMB, while considering the activity of pristine GNPs as 100%.

Figure 4. Time-dependent kinetics showing peroxidase-like activity of pristine GNP and GNP-S18 nanoconjugate in the absence and presence of 5 ppm acetamiprid [acet], agritone [agrit], endothal [end] and imidacloprid [imid].

18 aptamer on GNP surface, the reaction product remains ruby-red due to the blocking of GNP surface and its inability to oxidize TMB. The addition of aptamer-specific acetamiprid analyte to GNP-S18 solution allows removal of S-18 aptamer from GNP surface, leading to a visual detection of sensing event in the form of a purplish-blue product. When the GNPS18 nanoconjugate is exposed to similar concentration of other non-specific pesticides including agritone, imidacloprid and endothal, no visual change in GNP color from ruby-red is observed (structures of pesticides are shown in supporting information, Figure S5). Further validation of this visual read-out is evident from spectroscopic monitoring of the corresponding TMB oxidation product at 650 nm in Figure 3. This shows that compared to pristine GNP, the test reagent GNP-S18 shows less than 20% of the original peroxidase activity, which changes only marginally in the presence of non-specific analytes including agritone (26%), imidacloprid (22.4%) and endothal (23.6%), but resumes to over 60% in the presence of aptamer-specific acetamiprid. This cross-reactivity study provides substantial evidence on the ability of the S-18 aptamer to detect acetamiprid with high specificity by employing the proposed approach. A fundamental difference between the output of the current biosensing approach and previously reported GNP-aptamer mediated methods is that the current approach provides a colorimetric read-out of an oxidized peroxidase substrate, whereas other approaches have relied upon change in GNPs SPR as a consequence of salt-induced aggregation.26,30 Since efficient salt-induced aggregation of GNPs requires almost complete removal of aptamers from the GNP surface, the previous strategies relied upon high concentrations of the target analyte to allow complete aptamer removal, thereby limiting the detection sensitivity. Additionally, the long incubation time (typically 30-60 min) required during salt-induced aggregation also limited the effectiveness of such approaches.10,22,23 Conversely, in the proposed approach; the use of the inherent nanozyme activity of GNPs for biosensing eliminated the need for saltinduced aggregation, leading to improved sensitivity and faster readouts (10 min). Since time taken for detection of analyte molecules is a critical parameter for an efficient biosensor, the real-time monitoring of GNP nanozyme activity towards TMB oxidation in

the presence of different analytes was also performed (Figure 4). While pristine GNPs as well as GNP-S18 nanoconjugates showed almost completion of peroxidase reaction within 5 min either in the absence of target analyte (acetamiprid) or in the presence of non-specific analyte (agritone, endothal and imidacloprid); GNP-S18 nanoconjugates, in the presence of target analyte, did not show a saturation of activity at least until 25 min. Notably, the experiments discussed in Figures 2 and 3, which showed sensitive and selective detection of acetamiprid were performed at 10 min. Considering that an increase in the difference between the activity of GNP-S18 nanoconjugate in the absence and presence of acetamiprid was observed from 10-25 min, it should be possible to further improve the limit of detection (LoD) of the proposed approach by performing the reaction at 25 min. However, since 10 min detection time was found suitable to detect 0.1 ppm acetamiprid that is equivalent to the lowest tolerance limit set by EPA,40 no efforts were made to further improve the LoD of the proposed sensing platform. To validate the fundamental aspects of proposed biosensor, a number of control experiments were also performed, which are detailed in the supporting information. Briefly, to confirm that the spectroscopic outputs were indeed due to oxidized TMB, and not due to aggregation of GNPs, the stability of GNPs post-reaction was assessed using TEM (Figure S6). The mode of interaction of pristine GNPs with different analytes in the absence of aptamer was studied via nanozyme activity assays (Figure S7), while the potential peroxidase-like activity of S-18 aptamer molecules was invalidated through timedependent kinetic assays (Figure S8). Target concentrationdependent desorption of S-18 aptamer from the surface of GNPs was validated by studying the FRET (fluorescence resonance energy transfer)-mediated quenching of a FAM-tagged fluorescent S-18 aptamer (Figure S9). Further, the structural changes in the S-18 aptamer after desorption from the GNP surface in the presence of cognate target acetamiprid were confirmed via circular dichroism (CD), which revealed targetresponsive folding of the S-18 aptamer in the ‘stem-loop’ conformational state (Figure S10). In summary, we demonstrate a new aptamer-nanozyme based reversible biosensing strategy for fast, highly selective and sensitive detection of acetamiprid, an important pesticide,

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yet a potent neurotoxin. This strategy exploits the inherent peroxidase-like activity of GNPs by combining it with high affinity and specificity of S-18 ssDNA aptamer towards acetamiprid. The specific features of this novel colorimetric biosensing assay for acetamiprid include a simple visual read-out for qualitative monitoring, highly sensitive detection of as low as 0.1 ppm acetamiprid to satisfy EPA guidelines, high specificity in the presence of other non-specific pesticide, and assay results within 10 min without the need for expensive instrument and cumbersome data analysis. These aspects make the proposed platform at least five fold more sensitive and three times faster than the previously published methods.41, 42 Further, the current biosensing platform not only offers the opportunity to improve the LoD by making some potential compromise with the low detection time, it may also offer a generic approach for the detection of a range of environmental contaminants by employing other target-specific aptamers, antibodies or other MREs. Similarly, this approach may also be extended to other nanomaterials that show intrinsic peroxidase-like or other nanozyme activities.

Detailed experimental section, nanomaterial characterization, secondary structure of the S-18 aptamer, structures of pesticides, control experiments, FRET assay and CD measurements are presented in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org

*Email: [email protected] (V. B.); [email protected] (T. K. S.); Fax: +61 3 99253747 (V. B.); Tel: +61 3 99252121 (V. B.)

This work was supported through an Australian Research Council Future Fellowship to V. B. (FT140101285) and an Endeavour Research Award to T. K. S. by the Commonwealth of Australia. V. B. acknowledges the generous support of the Ian Potter Foundation for establishing an Ian Potter NanoBioSensing Facility at RMIT University. Authors acknowledge the support from the RMIT Microscopy and Microanalysis Facility (RMMF) for technical assistance and providing access to characterization facilities.

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