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1、 S-1 Supporting Information for Aptamer-controlled reversible inhibition of gold nanozyme activity for pesticide sensing Pabudi Weerathunge, Rajesh Ramanathan, Ravi Shukla, Tarun Kumar Sharma,* and Vipul Bansal,* Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Ap
2、plied 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. *Email: vipul.bansalrmit.edu.au (V. B.); tarunthsti.res.in (T. K. S.) Fax: +61 3 9925 37
3、47 (V. B.); Tel: +61 3 9925 2121 (V. B.) S-2 Experimental Details. Materials. Gold (III) chloride (HAuCl4.3H2O), L-tyrosine, acetamiprid, endothal, imidacloprid, ready to use 3,3,5,5-tetramethylbenzidine (TMB) and other reagents used in this study were procured from Sigma-Aldrich (St. Louis, USA). A
4、gritone was procured from Nufarm Australia Ltd. The sequence for acetamiprid aptamer S-18 5TGTAATTTGTCTGCAGCGGTTCTTGATCGCTGACACCATATTAT- GAAGA3 was obtained from a previous study1 and custom-synthesized aptamer with or without a 5FAM fluorescent tag were procured through Integrated DNA Technologies
5、(IDT, USA). Synthesis of gold nanoparticles (GNPs). GNPs were synthesized using tyrosine amino acid as a reducing and capping agent, as elaborated in our previous studies.2,3 Briefly, 300 mL aqueous solution comprising 0.1 mM L-tyrosine and 0.1 mM KOH was allowed to boil. Under alkaline boiling cond
6、itions, 0.2 mM equivalent of AuCl4- ions were added to the above solution with subsequent boiling for further 5 min. This resulted in a ruby-red colored solution consisting of GNPs. To prepare concentrated GNP solution, as-synthesized nanoparticles were boiled to reduce the volume to 30 mL. These co
7、lloidal solutions were found to be highly stable even after concentration, signifying the strong tyrosine capping. Further, concentrated solution of GNPs was dialyzed overnight against deionized MilliQ water using 12 kDa molecular weight cut-off cellulose dialysis membranes, followed by exchange of
8、water twice to remove the excess amount of KOH, potentially unreduced metal ions and unbound tyrosine, if any. The concentration of gold in GNPs was determined using atomic absorption spectroscopy (Varian) after digesting GNPs in aqua-regia, followed by preparation of an aqueous GNP stock solution w
9、ith 1 mM equivalent of gold. This GNP solution was further used for characterization and subsequent biosensing experiments. Characterisation of GNPs. The homogeneous colloidal solution obtained after removal of unbound amino acids and ions was characterized by UVvisible absorbance spectroscopy using
10、 Envision multilabel plate reader (Perkin Elmer). The samples for transmission electron microscopy (TEM) were prepared by drop-coating the solution on to a carbon-coated copper grid, followed by TEM measurements using a JEOL 1010 instrument operated at an accelerating voltage of 100 kV. Functionaliz
11、ation of GNPs with S-18 aptamer. It has been well-established that in the presence of GNPs, ssDNA can uncoil sufficiently due to its structural flexibility thereby exposing its nitrogenous bases to GNPs, whereas dsDNA presents its negatively charged phosphate backbone to the surface of GNPs due to i
12、ts stable double helical geometry.4 Therefore, the coordination interaction between the nitrogenous bases of the unfolded ssDNA and GNPs is stronger than the electrostatic repulsion between the negatively charged phosphate backbone of dsDNA and GNPs. This concept was utilized for S-3 efficient non-c
13、ovalent adsorption of ssDNA S-18 aptamer onto the GNP surface. For GNP-aptamer binding, before incubation of aptamer with GNPs, the appropriate secondary structure of aptamer was ascertained by its heat-treatment at 92 C for 10 min, followed by snap-chilling on ice for 5 min and bringing it back to
14、the room temperature. Following this, different concentrations of aptamers (100-600 nM) were incubated with a fixed concentration of GNPs (125 M) for 10 min. Biosensing of acetamiprid using GNPs. To achieve high sensitivity without compromising specificity during biosensing, a number of experiments
15、were performed to optimize experimental parameters such as aptamer and GNP concentration as well as reaction temperature. To determine the optimum temperature for peroxidase-like activity of GNPs, in a 200 L reaction volume, 125 M GNPs were incubated with components provided in the TMB kit as per th
16、e suppliers (Sigma Aldrich) protocol, and activity was assessed at three different temperatures (25, 37 and 55 C) after 10 min of reaction by measuring oxidation product of TMB through UV-visible absorbance spectroscopy at 650 nm. Among these, 37 C showed the highest degree of oxidation of TMB. Ther
17、efore all further experiments in the current study were performed at 37 C. The optimum concentration of S-18 aptamer was optimized by incubating a range of aptamer concentrations (100-600 nM) with fixed concentration of GNPs (125 M) for 10 min, followed by addition of TMB and H2O2 in 200 L reaction
18、volume and evaluation of peroxidase-like activity of aptamer-functionalized GNPs (GNP-S18) after 10 min. From the above experiments, 200 nM aptamer concentrations showed the highest inhibition of nanozyme activity of GNPs, beyond which no further inhibition of activity was observed. Therefore all fu
19、rther experiments employed 200 nM aptamer functionalized on to the surface of 125 M GNPs at 37 C in 200 L volume, either in the presence or absence of different analytes (acetamiprid, agritone, endothal and imidacloprid). The above experiment was also performed in a concentration- and time-dependent
20、 manner, wherein the influence of different concentrations of acetamiprid on recovery of peroxidase- like activity of GNPs was studied as a function of time. In addition to the spectroscopic examination, optical photographs of the reactions were also captured using a digital camera (Canon) to allow
21、a visual readout of the biosensing event. In a separate experiment, to study the influence of concentration of GNP-S18 conjugate on biosensing performance, the concentration of GNPs and S-18 aptamer were doubled (2X) in comparison to the original concentrations of 125 M and 200 nM, respectively in 2
22、00 L volume, and the nanozyme activity was monitored in the presence of different concentrations of acetamiprid. Performance evaluation of nanozyme biosensor: To determine the performance of nanozyme biosensor, important parameters such as limit of detection (LoD), limit of quantification (LoQ), pre
23、cision, accuracy, and dynamic linear range were determined. The accuracy and precision of S-4 acetamiprid detection (GNPs-S18 conjugate at 5ppm acetamiprid) were calculated by analyzing 20 independent biosensing events within a particular nanoparticle batch (intra-batch) and variation of these param
24、eters in different batches was calculated by using data obtained from 10 independent nanoparticle batches (inter-batches). Intra- and inter-batch % accuracy was calculated at 10% confidence intervals as (n/N)*100, where n and N denote to the number of sensing events within the target concentration r
25、ange (5 ppm) and the total number of sensing events, respectively. Precision was calculated using coefficient variation (CoV) method, wherein % Precision = 100 - %CoV. LoD and LoQ were determined by using quotient of 3.3*SD to S and 10*SD to S, wherein SD and S represent standard deviation of y- int
26、ercepts, and the slope of the linear range of the graph obtained from the corresponding data set. To determine the linear dynamic range, the data was fitted using linear regression analysis and the number of data points with R2 value better than 0.99 was reported as linear dynamic range of the senso
27、r. Fluorescence recovery assay: For studying desorption of S-18 aptamer from the GNP surface in the presence of cognate target acetamiprid, a modified fluorescent form of S-18 aptamer with 5 end labeled with FAM, was used. To achieve the optimum secondary structure of the aptamer, immediately before
28、 the fluorescent spectroscopy studies, FAM-S-18 aptamer was heated at 92 C for 3 min, followed by rapid cooling on ice for 5 min, before allowing reverting back to the room temperature. 200 nM of this FAM-S-18 aptamer was added to quartz cuvette in 1 ml volume and fluorescence emission spectrum was
29、recorded at the excitation wavelength of 485 nm. To determine the interaction of GNPs with FAM-S-18 aptamer, 125 M of GNPs were incubated with 200 nM FAM-S-18 for 10 min, followed by measurement of fluorescence spectrum. Further, to study the desorption of FAM-S-18 aptamer from the GNPs surface, GNP
30、-FAM-S18 conjugates were incubated with different concentrations of acetamiprid (5, 10, 15 ppm) for 10 min and the emission spectra were collected. Fluorescent spectroscopy studies were performed at 28 C using a Horiba Scientific Fluoromax-4 spectrophotometer fitted with a model 350B temperature con
31、troller. Circular dichroism (CD) studies: To study the target-specific structural changes in S-18 aptamer, CD studies were performed. CD measurements were carried out at room temperature using an Applied Photophysics Chirascan spectropolarimeter (Mullheim, Germany). A cylindrical fused quartz cell o
32、f 0.1 mm path length (Hellma, Mullheim, Germany) was used. The CD values were expressed in terms of millidegrees (mdeg). References 1. H. Jiang, Y. Liu, M. Fan and X. Liu, J. Agric. Food. Chem. 2011, 59, 1582-1586. S-5 2. H. K. Daima, P. R. Selvakannan, R. Shukla, S. K. Bhargava and V. Bansal, PLoS
33、ONE 2013, 8, e79676. 3. P. R. Selvakannan, R. Ramanathan, B. J. Plowman, Y. M. Sabri, H. K. Daima, A. P. OMullane, V. Bansal and S. K. Bhargava, Phys. Chem. Chem. Phys. 2013, 15, 12920-12929. 4. H. Li and L. Rothberg, Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14036-14039. S-6 Figure S1. Secondary s
34、tructure of S-18 ssDNA aptamer as predicted by M-Fold tool based on Zuker algorithm. S-7 Figure S2. (a) UV-visible absorbance spectra and (b-c) TEM images of (b) pristine GNPs and (c) aptamer-functionalized GNPs (GNP-S18 nanoconjugates). Scale bars in TEM images correspond to 50 nm. The UV-visible a
35、bsorbance spectra of both pristine GNPs and GNP-S18 nanoconjugates show a typical gold SPR peaks with maxima at ca. 520 nm, confirming the stability of GNPs on aptamer functionalization. This observation is further supported by TEM studies which show no aggregation of GNPs post-functionalization wit
36、h S-18 aptamer. S-8 Figure S3. Linear fitting profile of relative peroxidase-like activity of GNP-S18 in the presence of 0.1- 10 ppm acetamiprid. This data is the same as that shown in Figure 2 in the main manuscript, while the x-axis has been shown as log10 to allow better resolution between differ
37、ent analyte concentrations. S-9 Figure S4. (a) Relative peroxidase-like activity of 2X 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 5-25 ppm acetamiprid. Please note that 2
38、X corresponds to twice the concentration of GNP-S18 nanoconjugate than that shown in Figure 2 in the main manuscript. S-10 Figure S5. Chemical structures of different pesticides used in the current study. S-11 Figure S6. TEM image of GNPs obtained after exposure of GNP-S18 nanoconjugate to 5 ppm ace
39、tamiprid during peroxidase assay. Therefore, this TEM image demonstrates the stability of GNPs during the proposed biosensing approach. S-12 Figure S7. Peroxidase-like activity of pristine GNP in the absence and presence of 5 ppm concentrations of different analytes acetamiprid (acet), agritone (agr
40、it), 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%. This control experiment reveals that when different analytes are directly exposed to GNPs in the absence of S-
41、18 aptamer, no reduction of the nanozyme activity of pristine GNPs, except in the case of imidacloprid, is observed. This suggests that while acetamiprid, agritone and endothal do not effectively bind to GNPs, imidacloprid may somehow passivate the GNPs surface, reducing GNPs nanozyme activity. Neve
42、rtheless, since the test reagent in this study is GNP-S18, while the biosensing strategy is based on the ability of S-18 aptamer to desorb from the surface of GNP-S18 in the presence of cognate target, the basal level activity of imidacloprid does not influence the sensing strategy. S-13 Figure S8.
43、Time-dependent kinetics showing peroxidase-like activity of pristine GNP, pristine S-18 aptamer , S-18 aptamer in the presence of 5 ppm acetamiprid acet, and pristine GNPs in the presence of 5 ppm acetamiprid acet, agritone agrit, endothal end and imidacloprid imid. Similar to the previous Figure S7
44、, this data demonstrates that among various tested pesticides, only imidacloprid binds non-specifically to the surface of pristine GNPs. Additionally, the baseline activity of pristine S- 18 aptamer in the absence or presence of acetamiprid reveals that aptamer itself does not contribute to any pero
45、xidase-like activity. S-14 Figure S9. (a) Schematic representation of the fluorescent recovery assay that was used to monitor the desorption of FAM-labeled S-18 aptamer molecule from the surface of GNPs in the presence of acetamiprid. (b) shows the fluorescence emission spectra arising from FAM-S18
46、aptamer at different stages shown in the schematic. Briefly, a fluorescent tagged S-18 aptamer FAM is first bound to the surface of GNPs that results in the quenching of the fluorescence signal in comparison to the signal obtained from pristine FAM-S-18 aptamer. In the presence of increasing concent
47、rations of the cognate target acetamiprid acet; 5, 10 and 15 ppm, FAM-S-18 aptamer leaves the GNP surface to bind to acetamiprid. This desorption results in recovery of the fluorescence signal arising from FAM-S-18 in a target concentration dependent manner. S-15 Figure S10. Circular dichroism (CD)
48、spectra obtained from S-18 aptamer conjugated to GNPs in the absence and presence of cognate target acetamiprid acet. Both the CD spectra show two positive- signatures at ca. 224 nm and 279 nm and a negative peak at 254 nm. However, in the presence of cognate target, a sharp increase in the CD inten
49、sity at 279 nm is notable. This indicates that while S-18 aptamer is bound to the GNPs, its secondary structure is compromised. Conversely, in the presence of acetamiprid, when aptamer leaves the GNP surface, it undergoes target-responsive folding to its typical stem-loop conformational state, thereby leading to increased intensity at 279 n