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Insight into the Molecular Mechanisms of AuNP-based Aptasensor for Colorimetric Detection: a Molecular Dynamics Approach Witthawat Phanchai, Unnop Srikulwong, Apiwat Chompoosor, Chadamas Sakonsinsiri, and Theerapong Puangmali Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00701 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Insight into the Molecular Mechanisms of AuNP-based Aptasensor for Colorimetric Detection: a Molecular Dynamics Approach Witthawat Phanchai,† Unnop Srikulwong,‡ Apiwat Chompoosor,¶ Chadamas Sakonsinsiri,§ and Theerapong Puangmali∗,†,‡ Materials Science and Nanotechnology Program, Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand, Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand, Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand, and Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand E-mail: [email protected]

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Abstract

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with Na+ , Mg2+ ions exerted a more effective attractive force between OTA and anti-OTA aptamer. Three different binding modes of citrate on AuNP surfaces were found. The kinetics of the adsorption of unfolded aptamers onto the citrate-capped AuNP was also elucidated. Most importantly, our MD simulation revealed an insightful analysis of the molecular mechanisms in the AuNP-based aptasensor and paved the way for the design of a novel colorimetric aptasensor for other target molecules, which is not limited to OTA detection.

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INTRODUCTION

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Colorimetric aptasensor based on assembly of salt-induced gold nanoparticles (AuNPs) is a promising biosensor. However, the molecular mechanism of the aptasensor is far from being fully understood. Herein, molecular dynamics (MD) simulation was used to investigate molecular interactions in the detection of ochratoxin A (OTA) including: (i) the molecular recognition of the anti-OTA aptamer, (ii) OTAaptamer interactions in monovalent (Na+ ) and divalent (Mg2+ ) electrolytes, (iii) the binding mode of citrate on the AuNP surface, (iv) interactions of the aptamer with citrate-capped AuNPs and (v) a detailed mechanism of the aptasensor. Our MD simulations revealed a specific binding of the OTA-aptamer complex, compared with OTB and warfarin. Compared ∗

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To whom correspondence should be addressed † Materials Science and Nanotechnology Program, Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand ‡ Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand ¶ Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand § Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

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Ochratoxin A (OTA), a type of mycotoxins, is produced by microorganisms such as Aspergillus and Penicillium. OTA is chemically stable and has a relatively long half-life (35.5 days in humans). Its producers grow easily in various grains like coffee, peanuts, and rice. Additionally, the commercialized food systems like wine, bread, and even meat products can also be contaminated. 1–3 Several studies have introduced OTA as a nephro-, hepato-, neuro-, immunotoxic, and also teratogenic agent. 2 Therefore, it attracts research attention as a new threat to public health and is required to be

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regulated. To avoid the risk of OTA consumption, it is vitally important to have an appropriate analytical technique for an ultrasensitive detection of OTA. Aptamer-based biosensor is a promising candidate for a highly sensitive detection of OTA. Aptamers are single-stranded oligonucleotides that are designed through an artificial process named systematic evolution of ligands by exponential enrichment (SELEX). 4 Aptamers exhibit high selectivity and affinity. In addition, they provide a broad range of targets and are easily produced, readily available and highly reproducible and stable. 5 These advantages promote the extensive use of aptamers as the recognition elements in biosensors. Applying aptamers as the recognition elements provides sensitive aptamer-based sensors (aptasensors) 6 which can be used to detect various analytes including antibiotics, 7 toxins, 8 metal ions, 9 and viruses. 10 In recent years, numerous aptasensors have been introduced for the detection of OTA 11,12 which function through various detection modes. 13–15 Compared with these techniques, colorimetric methods have a simpler sensing mode which allows detection to be made

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Figure 1: Schematic illustration of the sens113 ing mechanism of AuNP-based colorimetric ap114 tasensor for OTA detection. The colors of anti115 OTA aptamer represent different nitrogenous 116 bases. Adenine (A), thymine (T), cytosine (C) 117 and guanine (G) correspond to blue, yellow, 118 green and purple, respectively. 119 120

by naked eye. 16 Aptamer-based colorimetric analysis is a method of detection of a target molecule in a solution with the aid of aptamers. There are diverse sensing strategies, and among them colorimetric aptasensor based on analyteinduced aggregation 17 /disaggregation 18,19 of gold nanoparticles (AuNPs) has been frequently reported in the literature. The review of the strategies, mechanisms and recent developments for the use of AuNPs as colorimetric biosensors was recently reported. 20 This sensing strategy is based on the interparticle distance dependence of optical properties of AuNPs. The apparent color of dispersed AuNPs in the solution changes from red to blue after aggregation due to the shift of surface plasmon resonance to a higher wavelength. Therefore, the presence of a particular analyte can be monitored by observing the color change of the solution. AuNPs-based aptasensor can be typically divided into two categories: (i) unfunctionalized AuNPs aptasensors and (ii) DNAfunctionalized AuNPs aptasensors. In unfunctionalized AuNPs aptasensors, the unfolded aptamers can adsorb onto the negatively-charged AuNPs, thus preventing AuNPs from aggregating in saline solution as illustrated in Figure 1. When aptamers turn into a target-aptamer complex, they will be released from the AuNPs, so the addition of NaCl leads to the aggregation of the AuNPs. 21 For the DNA-functionalized AuNPs aptasensors, disassembly of preformed DNA-functionalized AuNP dimers by analyte induction is another successful method which proves superior to unmodified AuNPs aptasensors in terms of stability, sensitivity and detection range. 18 To obtain higher analytical figures of merit, deeper understanding on the sensing mechanisms of the aforementioned strategies is required. However, there have been only a limited number of theoretical works 22 examining features of interactions of an anti-OTA aptamer with OTA. In addition, deeper molecular-level analysis of the sensing mechanisms for OTA detection has not been revealed yet, despite a large volume of experimental reports 17–19 of the aptamer-based biosensor. In light of this, we performed a computa-

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tional modeling, based upon molecular dynam- 166 ics (MD) simulation, to gain an insight into 167 the molecular mechanisms of a colorimetric ap- 168 tasensor. MD simulation was used to (i) in- 169 vestigate recognition interactions between the 170 anti-OTA aptamers and OTA molecules. Our 171 study shows recognition sites of the anti-OTA 172 aptamer and provides a deeper understanding 173 of the binding interaction. The specificity of 174 the anti-OTA aptamer can be performed by 175 MD simulation. Besides, (ii) the influences of 176 monovalent (Na+ ) and divalent (Mg2+ ) elec- 177 trolytes on the binding interaction and stabili- 178 ties of the OTA-aptamer complexes have been 179 observed. Furthermore, (iii) a detailed picture 180 of the AuNPs and binding modes of citrate was 181 obtained by computational modeling. Elucidat- 182 ing the binding mode of carboxylate-containing 183 ligands to AuNPs is crucial to the understand- 184 ing of their stabilizing role as a sensor probe. 185 This is of great importance for the insightful 186 discussion of the assembly and disassembly of 187 AuNPs. (iv) The binding of anti-OTA aptamer 188 onto the AuNP surface was taken into account. 189 Finally, (v) the sensing mechanisms for OTA 190 detection, but not limited to, can be clearly un- 191 derstood by our in silico study which can also 192 be used to explain the experiment of Yang and 193 co-workers 21 and related works. 18 194

web server that offers a variety of simulation methods to obtain a dynamic view of nucleic acid structures. The sequence of anti-OTA aptamer is 5 -GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3 . 18,21 Each nucleobase is hereafter denoted as BX, where B and X are a nucleobase and the order of the nucleobase from the 5 end, respectively. It was experimentally found that the antiOTA aptamer has high binding affinity with OTA molecules and possesses the ability to induce the formation of antiparallel G-quadruplex structure. 21 The force field for DNA atomistic simulation is AMBER parmBSC1 27 which had been parametrized from high-level quantum mechanical data. 28 AMBER parmBSC1 was tested by Ivani and co-workers 27 for nearly 100 systems and evaluated for a total simulation time of 140 μs which covered most of the DNA structures. For the biomolecules used in the simulation, including ochratoxin A, ochratoxin B (OTB), warfarin and citrate, the HartreeFock theory from Gaussian09 software package using HF/631G∗ method was used to optimize the structures, whereas the partial charges were derived from using ANTECHAMBER. 29 All force field parameters can be found in the supplementary information (SI) in Table S1 and Table S2.

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METHOD

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Since the ssDNA aptamer contains 36 bp, the

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Force Fields Biomolecules

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The nanocrystalline structure of the AuNP is 200 characterized as a face-centered cubic (FCC) 201 lattice structure. The equilibrium shape of the 202 AuNP is a modified truncated octahedron. 23 A 203 2-nm-in-diameter AuNP, composed of 309 Au 204 atoms, was constructed by cutting the crys- 205 tal out of a bulk gold face-centered cubic lat- 206 tice. The oxidation state of Au atoms is 207 Au(0). 24 The transferable AMBER-compatible 208 force field parameters for AuNP was obtained 209 from the literature. 25 For the single-stranded 210 DNA (ssDNA) aptamer, the structure of the 211 DNA was generated by NAFlex, 26 an on-line 212

culation consumes expensive computational resources. The accurate simulation of such a biomolecular system depends upon the solvation effect. As the solute-solvent distance increases, the properties of water tend to become similar to those of the bulk liquid. The bulk area could therefore be represented by a coarsegrained (CG) solvent model whereas the water around the solute was considered in atomistic details. 30,31 In our hybrid model, the system consists of a ssDNA aptamer, target molecules and citrate-capped AuNPs, surrounded by a shell of atomistic waters (TIP3P), embedded in CG molecules, called WatFour (WT4), to represent bulk water. In the WT4 water model,

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eleven water molecules are grouped into four 251 tetrahedrally interconnected beads. 32 This can 252 effectively mimic the dynamics of molecular systems composed of pure all-atom water. Af253 ter the insertion of the ssDNA aptamer, counterions (Na+ and Cl− ) were added to the sol- 254 vents (both atomistic and CG shells) to en- 255 sure electroneutrality. Compared with all- 256 atom model having the same simulation box 257 size (16.5×16.5×16.5 nm3 ), the number of wa- 258 ter molecules was dramatically reduced from 259 444824 atoms to 90463 atoms. This hybrid sol- 260 vation provides a significant reduction of the 261 number of water molecules leading to signifi- 262 cantly faster simulation time. In addition to a 263 reduction in the computational cost, it opens 264 the door for its employment to study the bio- 265 logical processes which occur on space and time 266 scales that are often unreachable for fully atom- 267 istic simulation. 268 269

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All simulations were performed by the GROMACS 5.0.4 package 33 with a time step of 2 fs. The LINCS algorithm was applied to all bonds connected to hydrogen atoms. The LennardJones potential was applied to estimate van der Waals interaction with a cut-off radius of 1.2 nm for gold atoms, and σ = 2.629e−1 nm and ε = 22.133 kJ/mol. The system temperature was set at 300 K. Berendsen temperature coupling and Parrinello-Rahman pressure coupling were used to set the system in an NPT ensemble. Time constant for pressure coupling (1 bar) was 6 ps. The particle mesh ewald (PME) method was applied to calculate the long-range electrostatic interactions. All visualizations were done with Visual Molecular Dynamics (VMD). 34

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RESULTS AND DISCUSSION Molecular Recognition of AntiOTA Aptamer Anti-OTA aptamers are functional ssDNA obtained from a random oligonucleotide library through SELEX technique. 35 Although the anti-OTA aptamer, 18,21 which binds to OTA with high affinity and specificity, has been widely used and developed into a novel detection method for the toxin, the binding interaction between the unfolded anti-OTA aptamer and OTA is still not clear. To confirm the affinity and specificity between the aptamer and the target molecule, we carried out MD simulations to compare the molecular recognition among three different structures including OTA, OTB and warfarin. The chemical structures and the molecular recognition of these target molecules are illustrated in Figure 2.

Figure 2: Recognition interaction of anti-OTA aptamer. The minimum distance between target molecules and the aptamer and the number of hydrogen bonds between target molecules and the aptamer with the simulation time of 100 ns. The target molecules include (a) ochratoxin A, (b) ochratoxin B and (c) warfarin. The dashed line shows a cut-off distance of 0.45 nm to define non-covalent binding.

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OTA and structural analogues of OTA, i.e., 320 OTB and warfarin, were used to evaluate the 321 selectivity and specificity of the aptamer. The 322 molecular interaction between the aptamer and 323 these target molecules was observed by MD 324 simulations for 100 ns. Basically, the binding 325 interaction between the aptamer and the tar- 326 get molecule is a result of non-covalent bonding 327 consisting of van der Waals forces, hydrogen 328 bonds and electrostatic interactions. 36,37 van 329 der Waals interactions typically extend to a dis- 330 tance of 0.6 nm for large biomolecules. In the 331 present simulation systems, we conservatively 332 used a cut-off distance (dc ) of 0.45 nm as the 333 distance to define non-covalent OTA-aptamer 334 binding. Accordingly, the OTA-aptamer combi- 335 nation is considered to be in a bound configura- 336 tion provided that the distance between atoms 337 within the binding region is lower than dc for 338 a minimum of 20 ns. For the hydrogen bond- 339 ing, the hydrogen bond is considered to have 340 a maximum distance of 0.35 nm and a corre- 341 sponding angle of 30◦ or less between the donor 342 and acceptor atoms. 37 343 To perform the binding simulations, OTA, 344 OTB and warfarin were placed in the same sim- 345 ulation box. For the OTA, it was found that 346 there were two states of the binding complex. 347 As shown in Figure S1, at t = 16 ns, OTA 348 could bind with C17, G18, T19 and A20 of 349 the aptamer and the binding continued until 350 t = 36 ns. Subsequently, the OTA strongly 351 bound with three nubleobases including G16, 352 C17 and A21 and remained bound with these 353 bases until the end of the simulation. A bound 354 configuration was observed at t = 20 ns (see 355 Figure 2(a)). At this state, the distance be- 356 tween the OTA molecule and the binding sites 357 fell below dc and remained below the cut-off 358 distance for the remainder of the simulation. It 359 was noticeable that the OTA frequently bound 360 with three nucleobases including G16, C17 and 361 A21. The interactions between the 21st adenine 362 nucleotide (A21) were present throughout the 363 OTA-aptamer complex simulations. This sug- 364 gests that A21 plays an important role in the 365 molecular binding. 366 Hydrogen bond analysis could be used to 367 identify such a structural change, since the ap- 368

tamer tertiary structure was held together by hydrogen bonding. As can be seen in Figure 2, the formation of hydrogen bonds generally corresponded with the minimal contact distance. In other words, when OTA was close to one of the aptamer bases, hydrogen bonds formed simultaneously. This was in accordance with the number of hydrogen bonds of the OTAaptamer complex, as illustrated in Figure 2(a). A detailed analysis of hydrogen bond formation between atoms within an OTA-aptamer binding complex reveals that there are two states of complex formation. The atom indexes in OTA are shown in Figure S2. In the initial state, hydrogen bonds are formed between hydrogen atoms of OTA and oxygen atoms of C17, G18 and T19 of the aptamer. The indexes of each oxygen atom in C17, G18 and T19 are shown in Figure S3(a)-(c). The hydrogen bonds at the initial state are due to the interactions between H9-O2(C17), H16-O2P(G18) and H8-O4(T19). As can be seen, the number of hydrogen bonds formed at the initial state is less than those found in the bound state (Figure S4). In other words, in the initial state solely weak interactions are observed. The second state involving of stronger bonding of atoms is stronger than in the initial one. The favored interactions are due mainly to the hydrogen bonding between oxygen atoms of OTA and hydrogen atoms of A21. Key atoms that form the hydrogen bonds between OTA and the aptamer are O1-H62(A21), O2-H62(A21) and O3-H62(A21), as illustrated in Figure S4. During the bound state, the number of hydrogen bonds fluctuated with a maximum number of hydrogen bonds of three. During this time, the aptamer structure reorganized to a stable conformation. Towards the end of the simulations, the variation within the number of hydrogen bonds in the aptamer structure was found to be minimal. Apart from C17, G18, T19 and A21, other nucleobases could also serve as binding positions from time to time. To sum up, the binding sites of the anti-OTA aptamer at the stable state included the following nucleotide bases: G1, G16, C17, G18, T19, A20, A21, G27, C28, G33, and A34. To ensure the specificity of the anti-OTA aptamer, we then observed the molecular recogni-

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The structures and functions of nucleic acids are sensitively correlated with the “counterion atmosphere” owing to their polyelectrolyte nature. 38 DNA is negatively charged due to its sugar-phosphate backbone and the partial negative charge in the OTA molecule is a result of carboxyl, carbonyl, ester, and chloro groups. Consequently, the interaction between the ssDNA aptamer and OTA is primarily through electrostatic and van der Waals forces. The counterions around the ssDNA aptamer and OTA plays a key role in electrostatic screening which reduces the repulsion between these biomolecules. Divalent ions (Mg2+ ), compared with monovalent ions (Na+ ), have stronger interactions with nucleic acids. 38 Li and co4

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Figure 3: (a)-(c) The minimum distance between the aptamer and OTA versus time with different magnesium ion concentrations [Mg2+ ]. (d)-(f) The number of hydrogen bonds between OTA and the aptamer with the [Mg2+ ] = 0, 0.15 and 0.30 M, respectively. The salt concentration of all simulations is 0.15 M.

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tions between the anti-OTA aptamer and OTB 412 and warfarin. Hydrogen bond analysis and the 413 distance between the aptamer and the target 414 molecules could identify the molecular selectivity of the aptamer. Those molecules could 415 not bind with the aptamer and the significant 416 formation of hydrogen bonding was not found, 417 as illustrated in Figure 2(b)-(c). Our simu- 418 lation confirms the recognition interactions of 419 the anti-OTA aptamer and is in good agree- 420 ment with the experiment in which the aptamer 421 bound with a 100-fold less affinity to OTB and 422 did not form compounds with warfarin. 35 423 Besides, we also performed the simulations to 424 verify the molecular recognition of the widely- 425 used sequence by shortening the aptamer. The 426 binding between OTA and three different mod- 427 ified aptamer sequences was conducted. The 428 distance between OTA and aptamer, and the 429 number of H-bonds of three modified sequences 430 are illustrated in Figure S5. For the sequence (a), the anti-OTA aptamer was shortened by removing twelve nucleobases from the 3 end. In the case of the sequence (b), six nucleobases were removed from both 3 and 5 ends. The sequence (c) was modified by substituting G11 by a cytosine residue and three additional nucleobases (ACG) were added at the 3 end, highlighted in red. It was found that the modification of the anti-OTA aptamer sequences induces losses of affinity and specificity, as shown in Figure S5(a)-(c). Compared with OTA, the binding with OTB is stronger for the sequence (a) (Figure S5(a)). In other words, the specificity is lost once the sequence of anti-OTA aptamer is modified. To sum up, the binding ability is sequence dependent. This is in accordance with the experimental findings. 35 Since the binding interactions depend upon the environment of the system such as solvents, in the following section, the effect of monovalent and divalent electrolytes on the binding of OTA and aptamer will be taken into consideration.

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workers reported 38 that Mg2+ ions have stable 479 hydration shells and stronger interaction with 480 DNA. Cruz-Aguado and Penner 35 selected various aptamers that can bind OTA with various 481 affinities. In this work, the effect of the magne- 482 sium concentrations on the aptamer-OTA bind- 483 ing affinity was analyzed, and the binding was 484 found to depend on the presence of Mg2+ . Since 485 the counterions are highly dynamic, it is dif- 486 ficult for experimentalists to provide a micro- 487 scopic description. In addition, the detailed 488 atomistic interacting picture of the effect of di- 489 valent electrolytes on the OTA-aptamer inter- 490 491 action is scarcely documented. 2+ In the present study, the effects of Mg ions, 492 in comparison with those of Na+ , on OTA- 493 aptamer binding interaction were studied by 494 MD simulations. It was found that Mg2+ ions 495 prefer to bind to the oxygen atom in the phos- 496 phate backbone through electrostatic interac- 497 tion. When the anti-OTA aptamer was in close 498 proximity to the OTA molecule, Mg2+ ions ac- 499 cumulated at the interfacial area and formed 500 ion-dipole interaction and electrostatic interac- 501 tion with chloro group in the OTA molecule 502 and oxygen in the phosphate group, respec- 503 tively, like “bridging” them together (see Fig- 504 ure S6). These Mg2+ ions exerted an effec- 505 tive attractive force between the two molecules. 506 Both OTA and anti-OTA aptamer can “clamp” 507 onto the Mg2+ ion. The O to Mg2+ distance 508 is 3.91 ˚ A, whereas the distance between Cl 509 atom of the OTA molecule and Mg2+ is 5.67 510 ˚ A. It plays more important role than Na+ ions 511 as the monovalent cations bind to DNA in a 512 quite diffusive manner which is much weaker 513 than that of a divalent bridge. 38 Therefore, the 514 binding between OTA and the aptamer in the 515 Mg2+ electrolyte is faster than in monovalent 516 electrolyte as one can see from the distance 517 (Figure 3(a)-(c)) and hydrogen bonding (Fig- 518 ure 3(d)-(f)) between OTA and anti-OTA ap- 519 tamer. However, an increase in Mg2+ concen- 520 tration, as shown in Figure 3, has no signifi- 521 cant effect on the binding time of OTA-aptamer 522 complex. Our computational results are consis- 523 tent with the experimental data published by 524 525 Cruz-Aguado and Penner. 35 526

Binding Mode of Citrate in the Stabilization of AuNPs The binding of citrate ligands to AuNPs is crucial for the stabilization of AuNPs. 39 It is accepted that the region surrounding the citratecapped AuNP is negatively charged due to anionic carboxylate groups. A more detailed picture of the interactions between the gold surface and the citrate ligand was recently reported by Al-Johani and co-workers. 24 By combining solid-state NMR, TEM, XPS and DFT calculations, they proposed reasonable structures and modes of interaction for citrate-stabilized AuNP surfaces. Based on DFT calculations, they concluded that there were various binding geometries between carboxylate-containing ligands and Au model surfaces and presented evidence for three different modes: monocarboxylate monodentate (1κO1 ), monocarboxylate bridging (μ2 − 1κO1 : 2κO3 ), and dicar boxylate bridging (μ4 − 1κO1 : 2κO3 : 3κO1 :  4κO3 ). XPS experiments indicated that AuNP surface atoms were mainly in the zero oxidation state Au(0). 23 Na NMR and TEM/EDS experiments also suggested that Na+ ions were present near the gold surface. In the present work, the mechanism of citrate anions adsorbed onto the AuNP surface in an electrolyte solution was characterized in detail. Our computational results confirmed recent experimental findings 24 regarding the characteristic binding modes of citrate anions on the AuNP surface. The simulation was performed at a temperature of 373 K with the citrate:Au molar ratio being 0.2:1. All surface gold atoms were in the zero oxidation state as reported in the literature. 24,40 After the system was equilibrated for 35 ns, the number of adsorbed citrate anions on a 2-nm-in-diameter AuNP surface was 19 anions, corresponding to the surface density of 1.58 anions/nm2 . We found that the Au(111) surface was the most populated facet to which the citrate anions bound due to its lowest surface energy. 14 The formation of citrate on Au(110) and Au(100) surfaces is less favorable We then investigated the binding mode of the carboxylate-containing ligands to AuNP. The binding mode was examined by the dis-

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tance between the citrate anion and gold atoms on the surface. Guided by the MD simulations, we found various binding geometries between the carboxylate group and the AuNP surface and three different modes were present as shown in Figure 4. The geometry with only one carboxylate oxygen interacting with one Au is referred to monocarboxylate monodentate (κ), as shown in Figure 4(a). The distance between O1 and Au atom was 0.33 nm. However, the transformation of the binding could be observed at t = 82 ns. As a result, O2 could bind with Au atom instead of O1. Our Au-O distance was close to that obtained from a DFT calculation falling in the range of 0.22 − 0.24 nm. 24 Our simulations also suggested that Na+ ions were present near the gold surface due to the binding between Na+ ions and oxygen atoms in the carboxylate group (Figure 4). As illustrated in Figure S7, more Na+ , compared with Cl− , were present around the AuNP surface. This is in accordance with the 23 Na NMR experiments. 24 In addition to the κ coordination mode, monocarboxylate bridging (μ2 ) and dicarboxylate bridging (μ4 ) were also found, as one can see in Figure 4(b)-(c). In the μ2 fashion, two Au-O binding motifs were formed at the gold surface. The resonance of electrons was responsible for this binding mode where three 572 Na+ ions were also found at the surface. Addi573 tionally, the μ4 motif could also be observed in which two terminal carboxylates were involved 574 in the binding interactions. It is worth noting 575 that most citrates have μ2 motif (eight and two 576 citrates on Au(111) and Au(100), respectively). 577 Four citrates with the μ4 fashion have been ob- 578 served on Au(100) and two molecules have κ 579 coordination mode on Au(111). Three citrate 580 581 anions are in the secondary shell. Understanding the binding mode of the cit- 582 rate ligands on the AuNP surface leads to a 583 complete understanding of the fundamental ad- 584 sorption of ssDNA onto citrate-capped AuNP 585 surfaces which will be discussed in the follow- 586 587 ing section. 588 589 590 591

Figure 5: ssDNA adsorption through displacement of citrate on AuNP surface. The green color represents the Au(100) plane. The number of citrate anions on the adsorbed plane is decreased with increasing time due to the electrostatic repulsion between the negatively charged phosphate backbone and citrate anions. Solvents and ions were not shown for clarity. The simulation was performed for 100 ns.

Interactions of Anti-OTA Aptamer with Citrate-capped AuNPs ssDNA can be adsorbed by citrate-capped AuNPs, resulting in an increased AuNP stability, which forms the basis of a number of biochemical and analytical applications. The mechanism of nonthiolated ssDNA adsorption, however, remains unclear. AuNPs are capped with negatively charged citrate, posing an electrostatic barrier for adsorbing negatively charged ssDNA. ssDNA adsorption must, therefore, be achieved by non-ionic forces. It has been observed that different bases show different binding affinities, 41,42 highlighting the importance of specific nucleobase-Au interaction. While there is extensive literature about the adsorption of bases, nucleosides and nucleotides in thin films of different metals such as gold, 43 silver 44 and even copper, 45 in the case of NPs, particularly AuNPs, studies are rather scarce.

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As the mechanism is still unclear, different models have been suggested. Recently, a mechanism has been proposed whose key lies in the stabilizing force caused by the interaction between the nucleotides and the AuNP. 46 According to this model, the ssDNA needs to approach the particle in order to displace the citrate and is then adsorbed in a latter step. The neutralization of the negative charges by salt addition favors the process and provides screening long range electrostatic repulsions. Upon adsorption, the DNA may change conformation to maximize contact points with the surface. The adsorbed molecules of DNA create a zone where other biopolymer molecules cannot get near and subsequent saturation of the surface with those zones impedes adsorption of further DNA molecules. In this work, the binding interaction between DNA aptamer and a 2-nm-in-diameter AuNP has been investigated. The simulation was performed for 100 ns. Based upon our simulations, we suggest the following mechanism to describe the ssDNA adsorption. Since the backbone of the oligonucleotide is negatively charged, the electrostatic repulsion between the ssDNA and citrate anions results in the repulsion of citrate anions to neighbouring surfaces, as illustrated in Figure 5. The electrostatic interaction not exclusively determines adsorption kinetics but also influences the binding capacity. At t = 0 ns, three citrate anions were on Au(100) plane. Once adsorbed, the ssDNA aptamer changes its 641 conformation on the surface to maximize the 642 surface contact. Due to the electrostatic repul- 643 sion, citrate anions on the AuNP surface were 644 then repelled to the neighboring crystal planes 645 and at t = 5 ns, there was no citrate anion 646 on the Au(100) plane (green color), resulting 647 in the formation of an area electrostatically ex- 648 cluded to the ssDNA aptamer. The adsorption 649 of oligonucleotide on the AuNP surface can be 650 further examined by the distance between the 651 nucleotide and the gold surface. As presented in 652 Figure 6(a)-(b), the phosphates of G9, G15 and 653 G16 are in close proximity to the AuNP sur- 654 face thus repulsing citrate anions to the neighbouring planes. At t = 0 ns, the distances between the phosphates of G9, G15, G16 and

(a)

(b) G15 G16 G9

(c)

(d)

G12 G13

7 8 9

6 5 1 4 3 2

Figure 6: Adsorption of oligonucleotide on the AuNP surface. (a) Snapshot of backbone binding interaction at t = 100 ns, (b) distance between phosphate and AuNP versus time, (c) snapshot of nucleoside binding interaction at t = 100 ns and the proposed structure for the AuNP interaction with guanine 47 (The dashed lines represent non-bonded interactions.), and (d) the distance between N and O of the nitrogenous bases and AuNP versus time.

the Au(100) plane were more than 4 nm. Due to the presence of the oxygen in the carbonyl group and N(7) nitrogen atom, guanines have a strong tendency to bind to AuNP through non-bonded interaction. 48 G9 was attached on Au(111), whereas G15 and G16 were in close proximity to Au(100). The adsorption involves the oxygen of the C(6) carbonyl group and the N(7) atoms of G11, G12 and G13, as shown in Figure 6(c)-(d). The physisorption process of ssDNA on the AuNP surface is through guanine moiety which is in good agreement with the binding interaction proposed by Pergolese and co-workers. 47

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t = 70 ns

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Figure 7: Interparticle distance between two 690 aptamer-bound AuNPs as a function of time. 691 The salt concentration is 0.15 M. The insets 692 show the interactions between AuNPs at t = 0 693 and t = 70 ns. The interparticle distance re- 694 mained higher than the threshold distance (1 695 nm) for a 100 ns simulation, due to the electro- 696 static repulsion from ssDNA which prevents van 697 der Waals attractions and enhances the stabil- 698 ity of AuNPs against salt-induced aggregation. 699 700 701 702 655 656

Mechanism of Aptasensor for OTA 703 704 Detection 705

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The unique optical properties of AuNPs, due to 706 localized surface plasmons excited by incident 707 electromagnetic field of specific energy, have at- 708 tracted great interest in the past decades be- 709 cause of several associated applications such 710 as ultrasensitive biological and chemical sens- 711 ing. The localized surface plasmon resonance 712 (LSPR) can be understood as a collective reso- 713 nant oscillation of free electrons within a metal 714 nanoparticle in response to an incident electro- 715 magnetic radiation. When the NPs are in close 716 proximity, the individual surface plasmons can 717 couple, leading to a shift in the optical response. 718 Surface plasmon coupling depends on the way 719 the NPs are assembled. Theoretical studies by 720 Nordlander and co-workers revealed that quan- 721 tum tunneling effects begin to disturb the inter- 722 action between the surface plasmons when the 723 interparticle distance (d) is below 1 nm. 49–51 As 724 the gap distance continues to decrease below a 725

“threshold tunnel-distance” (dth ), the electron tunneling effect completely modifies the behavior of the plasmonic response in the quantum regime. 52 Therefore, variations in the assembly parameters, i.e., interparticle distance, configuration and the number of NPs, allow one to finely tune the resonance wavelength. Since the plasmonic response of AuNPs is interparticle distance dependent, the assembly of citratecapped AuNPs in the saline solution was observed. We initially investigated the aggregation of two citrate-capped AuNPs in the NaCl solution with the concentration of 0.15 M. The initial interparticle distance was 5 nm and fell below 1 nm within ∼ 5 ns, as illustrated in Figure S8. The addition of NaCl would screen the charge on the surface of AuNPs, resulting in an aggregation (d < dth ). To prevent the aggregation of AuNPs, citrate-capped AuNPs were modified with ssDNA aptamers. Subsequently, we performed an MD simulation for two aptamer-bound AuNPs in a saline solution ([NaCl]= 0.15 M). The interparticle distance was initially 3 nm and it remained higher than the threshold distance (d > dth ) for a 100 ns simulation, as shown in Figure 7. This is due to the electrostatic repulsion from ssDNA and steric hindrance which prevent van der Waals attractions and enhances the stability of AuNPs against salt-induced aggregation. Disassembly of such AuNPs can be induced by OTA, the target molecules of the anti-OTA aptamer. Upon the addition of OTA, the specific binding interaction between OTA and antiOTA aptamer resulted in the desorption of the aptamer from the AuNP surface, as shown in Figure 8. This prevented the exposure of the aptamer bases to AuNPs, and thus the ability to protect AuNPs at a high-salt concentration was lost. The initial interparticle distance between AuNPs was 2.5 nm. At t = 43 ns, the binding interactions of OTA/anti-OTA aptamer was found and then the interparticle distance of AuNP-AuNP slightly decreased. At t = 400 ns, the interparticle distance nearly reached 1 nm. The plot of the interparticle distance as a function of time is shown in Figure S9. It is computationally expensive to perform the simulation until the aptamer is com-

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pletely released from the AuNP surface. How- 772 ever, it is expected that after the complete re- 773 lease of the DNA aptamer, the AuNPs will be 774 aggregated at a high salt concentration, as pre- 775 viously discussed in Figure S8. From the ex- 776 perimental point of view, as the optical proper- 777 ties of the AuNPs depends upon the interpar- 778 ticle distance, the color of the solution changes 779 with the decrease in interparticle distance and can be observed by naked-eye. Owing to the interparticle-distance dependent optical prop- 780 erties, this simple colorimetric method can be applied to colorimetric biosensors which are not 781 exclusively limited to OTA detection. 782 783

740

741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771

CONCLUSIONS

784

The hybrid solvation model was used to ex785 amine the molecular recognition at atomistic level. The binding interactions of the antiOTA aptamer and OTA were clearly eluci- 786 dated. Our simulations revealed the strong 787 binding zones of the anti-OTA aptamer including G1, G16, C17, G18, T19, A20, A21, G27, 788 C28, G33, and A34. The binding was evaluated by the intermolecular distances and the 789 number of hydrogen bonds. The divalent electrolyte (Mg2+ ) played an essential role as a 790 bridging ion in the binding interactions. The anti-OTA aptamer is capable of binding with 791 OTA specifically, compared with OTB and war- 792 farin. The binding mode of citrate and gold atoms was elucidated. Three different binding modes were found on the AuNP surface including monocarboxylate monodentate (κ), monocarboxylate bridging (μ2 ), and dicarboxylate bridging (μ4 ). Au(111) was the favorite binding plane of citrate. Salt-induced aggregation of citrate-capped AuNPs was a result of electrostatic screening. The adsorption of 793 the anti-OTA aptamer onto the surface of the 794 citrate-capped AuNPs was due to nucleoside 795 and backbone binding motif. The unfolded 796 aptamers could adsorb onto the negatively- 797 charged AuNPs, thus preventing AuNPs from 798 aggregating in salt solution. Upon the addi- 799 tion of OTA, the specific binding interaction between OTA and anti-OTA aptamer resulted in

the desorption of the aptamer from the AuNP surface. This prevented the exposure of the aptamer bases to AuNPs, and thus the ability to protect AuNPs under high-salt conditions was lost. Our MD simulation revealed an insightful discussion of the molecular mechanisms in the AuNP-based aptasensor and paved the way for the design of a novel colorimetric aptasensor.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxx/acs.langmuir.xxxxxx.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

ORCID Theerapong Puangmali: 0000-0003-2529-7528

Notes The authors declare no competing financial interest. Acknowledgement We would like to express our appreciation to Bureau of Information Technology, Khon Kaen University, for the computational resource. WP would also like to express our gratitude to Science Achievement Scholarship of Thailand for financial support.

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Figure 4: Binding of citrate on AuNP surface and assignment of the various citrate binding modes. The calculation of the distance between oxygen atoms (red) and Au(111) surface are performed in 100 ns. (a) monocarboxylate monodentate 1κO1 (κ), (b) monocarboxylate bridging μ2 − 1κO1 :   2κO3 (μ2 ) and (c) dicarboxylate bridging μ4 − 1κO1 : 2κO3 : 3κO1 : 4κO3 (μ4 ).

Figure 8: Snapshots of assembly of citrated-capped AuNPs (D = 2 nm) obtained from a 400 ns MD simulation (without water or ions). Thirty OTA molecules were placed into the simulation box but only eleven molecules (light purple) are shown for clarity. OTA molecules induce the formation of a structure in the anti-OTA aptamer and form the OTA-aptamer complex structure. The aptamer cannot protect the AuNPs from salt-induced aggregation resulting in the assembly of AuNPs. The interparticle distance (d) is decreased with increasing simulation time. The color code of oligonucleotide represents different bases. Adenine (A), thymine (T), cytosine (C) and guanine (G) correspond to blue, yellow, green and purple, respectively. ACS Paragon Plus Environment

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The sensing mechanism of AuNP-based colorimetric aptasensor for OTA detection. 262x88mm (300 x 300 DPI)

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