Short Communication 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 DOI: 10.1002/elan.201700734 Voltammetric Detection of Thrombin by Labeling with Osmium Tetroxide Bipyridine and Binding with Aptamers on a Gold Electrode Sarasi K K Galagedera,[a] Loan Huynh,[a] Falko Wachholz,[b] Martin Jacobsen,[b] Phensinee Haruehanroengra,[a] Jia Sheng,[a] Alan A Chen,[a] and Gerd-Uwe Flechsig*[a, b] Abstract: This communication reports on electrochemical detection of thrombin based on labeling with osmium tetroxide bipyridine [OsO4(bipy)] Tryptophan amino acids can be labeled at the CÀC-double bond, and at least some tryptophan moieties are accessible for labeling in thrombin Using the catalytic hydrogen signal from adsorptive stripping voltammetry performed on hanging mercury drop electrode, we could detect as little as 1.47 nM [OsO4(bipy)]-modified thrombin We also tested the binding of [OsO4(bipy)]-modified thrombin with the classic thrombin binding aptamer (TBA) on gold electrodes This preliminary study revealed that even after modification, a major part of the affinity was conserved, and that the aptamer self-assembled monolayer (SAM) could be regenerated several times Molecular simulations confirm that [OsO4(bipy)]-modified thrombin largely preserves the high binding affinity also of the alternative HD22 aptamer to thrombin, albeit at slightly reduced affinities due to steric hindrance when tryptophans 96 and 237 are labelled Based on these simulations, compensatory modifications in the aptamer should result in significantly improved binding with labelled thrombin This combined experimental-computational approach lays the groundwork for the rational design of improved aptamer sensors for analytical applications Keywords: Hanging mercury drop electrode · Osmium tetroxide-2,2 -bipyridyl · Thrombin binding aptamer · Gold electrodes · Adsorptive stripping voltammetry Thrombin as a central protease of hemostasis represents a major analytical challenge No methods for direct detection are available in routine molecular-diagnostic laboratories Recent methods for international normalized ratio (INR) blood testing include indirect techniques such as enzyme-immuno-assays for the detection of prothrombinfragments + (i e side products formed during biosynthesis of thrombin) The CoaguChek INR test system from Roche is based on such an indirect electrochemical thrombin assay [1] The thrombin binding aptamer (TBA) system is a widely used model to test new approaches for bioanalytical assays [2, 3] Recent reviews on aptamers for thrombin or other clinical target molecules revealed that routine diagnostic tests are still not available [4, 5] Chemical modification of amino acids is being used extensively to characterize proteins and their reaction mechanisms in biological systems Tryptophan can be specifically modified with an electrochemically active label [OsO4(bipy)] at its CÀC-double bond [6–8] This was used by Palecek et al for electrochemical detection of peptides [9] The osmium complex, however, despite the CÀC-double bond, did not attack histidine At least, no signal could be attributed to this amino acid The reaction was also studied by CE and MALDI-TOF MS [10] Fojta et al have applied this method to investigate accessibility of tryptophan in streptavidin and avidin It turned out that in the comwww.electroanalysis.wiley-vch.de plexes of biotin with streptavidin or avidin, the tryptophan moieties were not accessible for labeling with [OsO4 (bipy)], and that the avidin and streptavidin tetrameric structures would be affected by the labeling [11] Multilabelling capability is a clear advantage of [OsO4(bipy)] as a label for both nucleic acids and proteins, and we have used this label to detect DNA hybridization on gold electrodes [12, 13] Here, we modified thrombin with [OsO4(bipy)] for detection at both mercury drop and gold electrodes The aptamer immobilized on the gold electrode serves as a capture probe for the modified thrombin molecule It is expected that the binding constant is affected by multiple labeling of the analyte, making it weakly bind to the aptamer It is therefore intended to predict an optimized aptamer sequence taking the known TBA-thrombin cocrystal as a starting point [a] S K K Galagedera, L Huynh, P Haruehanroengra, J Sheng, A A Chen, G.-U Flechsig Department of Chemistry, University at Albany-SUNY, 1400 Washington Ave, Albany, NY 12222, United States E-mail: gflechsig@albany.edu [b] F Wachholz, M Jacobsen, G.-U Flechsig Department of Chemistry, University of Rostock, A.-EinsteinStr 3a, D-18055 Rostock, Germany E-mail: gflechsig@albany.edu 2018 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Electroanalysis 2018, 30, – These are not the final page numbers! ÞÞ Short Communication 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Fig A) Electrocatalytic response in adsorptive stripping voltammetric determination of [OsO4(bipy)]-modified thrombin on HMDE in 0.5 M Acetate buffer, pH 4.5; deposition potential À0.8 V, pulse amplitude 50 mV, pulse time 50 ms & sweep rate 20 mV/s B) Calibration plot of [OsO4(bpy)]-modified thrombin; The LOD of 1.47 nM has been obtained using the prediction band (95 %) of the calibration plot Fig A) Voltammetric response after repetitive hour hybridization, 120 nM Os-labelled thrombin (a, b & c) and de-hybridization (d, e & f) of [OsO4(bipy)]-modified thrombin at gold electrode with the immobilized TBA in 0.1 M Britton-Robinson buffer at pH 3.8 B) Response obtained upon repetitive hybridization and dehybridization with three different gold disk electrodes, with error bars indicating the standard deviation Preliminary results with both adsorptive stripping voltammetry (AdSV) on the classic hanging mercury drop electrode (HMDE), as well as gold electrode modified with self-assembled monolayers (SAM) of the TBA are shown in Figure and 2, respectively The electrocatalytic signal at À1.25 V using the HMDE allows for trace thrombin detection, with 1.5 nM limit of detection (LOD) This result demonstrated that at least some tryptophan moieties in thrombin are unprotected and accessible for labeling with [OsO4(bipy)] However, www.electroanalysis.wiley-vch.de adsorption on the Hg surface is not specific We also performed preliminary experiments with the TBA as capture probe layer on gold electrodes as illustrated in Scheme As it may be difficult to perform SELEX with osmium tetroxide-labeled aptamers, a very promising approach seems to be computer-aided re-design of the existing aptamers Figure depicts the detection of [OsO4(bipy)]-labeled thrombin at a TBA-SAM modified gold electrode Three repetitive hybridization-dehybridization cycles are exhib- 2018 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Electroanalysis 2018, 30, – These are not the final page numbers! ÞÞ Short Communication 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Scheme [OsO4(bipy)]-modified thrombin bound to surfaceconfined aptamer containing spacer nucleotides (green color) coupled to a thiol-linker “S” Structure reproduced from [18] with permission of the International Union of Crystallography (CCBY License), DOI: 10.1107/S0907444913022269 ited The voltammetric scan has been performed after hybridization and dehybridization, respectively On gold electrodes, the catalytic Os-signal cannot be used due to the large hydrogen background Instead, we use for the first time the reversible Os(VI/IV) signal to detect a protein after hybridization on gold The square wavevoltammetric (SWV) signal appears at À0.42 V (Figure 2A) Good reproducibility with significant differences of signals was obtained after hybridization and dehybridization of the surface-confined TBA with Os-labeled thrombin using three different gold electrodes, and three repetitive cycles as illustrated in Figure 2B These preliminary results indicate, that osmium tetroxide-labeled thrombin can be voltammetrically detected on gold electrodes following a hybridization step with immobilized thiol-linked aptamers The question is now, whether the common thrombin aptamers are still the optimal fit for osmium-modified thrombin Fojta et al had observed that [OsO4(bipy)]-labeled avidin and streptavidin both lost their ability to bind biotin, and the tetrameric structure of these proteins was affected On the other hand, the presence of biotin in the binding site protected the involved tryptophan moieties from reacting with [OsO4 (bipy)] [11] In that case, the binding site was a bit hidden and somewhat narrow Thrombin obviously, does not completely lose its affinity to the aptamer after the labeling with [OsO4(bipy)] However, we expected at least a slight decline in binding affinity Since the HD22 sequence has been identified as a better aptamer for thrombin than the classical TBA (KD smaller by a factor of 20 or more [14]), we performed www.electroanalysis.wiley-vch.de simulations on the new HD22 sequence to investigate the effect of osmium-labeling upon the binding free energy Using molecular simulations, we were able to identify which specific [OsO4(bipy)] label sites most negatively affect the binding of the thrombin-HD22 complex In prior work [15], we have demonstrated that this method is capable of folding non-canonical RNAs to sub-angstrom resolution [16], as well as achieve quantitative agreement with mass-spectrometry binding experiments [17] Using all-atom molecular dynamics simulation, the free energy for dissociating the thrombin-HD22 complex was calculated both with and without the [OsO4(bipy)] labels at different sites The simulations found that only labels at tryptophans 96 and 237 interfered with aptamer binding, confirming the partial loss of binding affinity (Table 1) It is significant that even if [OsO4(bipy)] labels at the thrombin binding interface occur at both sites simultaneously, a majority of the HD22 binding affinity is retained, thus explaining why even a fully modified thrombin is electrochemically detectable at nanomolar concentrations This agreement hints that it may be possible to recover the lost binding affinity via compensatory aptamer mutations identified via a combined in-silico and experimental approach, which is the subject of ongoing studies Such computer-optimized aptamer design may be a more general approach to obtain strongly and specifically binding aptamers, even in cases of labelled targets and aptamers, where SELEX cannot be used Table Thrombin/DNA aptamer binding free energies System[a] DGbind (kcal/mol) Trelax/Arelax OTrelax/Arelax À41 Ỉ À33 Æ [a] (T) Thrombin and (A) aptamer (OT) [OsO4(bipy)]-conjugated thrombin Subscripts relax indicates without crystallographic restraints Experimental Thrombin from bovine plasma, osmium tetroxide, 2,2’bipyridyl and Tris(hydroxymethyl) amino-methane were purchased from Sigma All other chemicals were of analytical grade Solutions were prepared using megapure water The used thiol-linked aptamer was based on the TBA (the exosite I-binding aptamer), was delivered by FRIZ Biochem GmbH (Munich, Germany), and had the following sequence including a spacer of 10 adenines and a thiol-linker with three dithian groups: 5’GGTTGGTGTGGTTGG aaaaaaaaaa[Dithio]3-3’ First, labelling of mM thrombin with mM [OsO4 (bipy)] solution was done in an unstirred 100 mM Tris buffer (pH adjusted with HCl to 7.0) at room temperature for hours Then the solutions containing labelled thrombin were dialyzed using “Slide-A-Lyzer Mini Dialysis units, with a first buffer exchange performed after hours and a second for overnight (19 h) dialysis The 2018 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Electroanalysis 2018, 30, – These are not the final page numbers! ÞÞ Short Communication 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 dialysis step was followed by buffer exchange (3 times) with 10 K filter tubes to further remove any free [OsO4 (bipy)] The electrochemical response of the labelled thrombin was measured by means of adsorptive stripping differential pulse (DPV) voltammetry at a hanging mercury drop electrode using a 663 VA stand controlled by an Autolab PGSTAT204 potentiostat and software NOVA 1.11 (Metrohm-Autolab, Switzerland) A threeelectrode system was used for the measurements with a glassy carbon electrode as the counter electrode and Ag/ AgCl in M KCl solution as the reference electrode Detection of labelled thrombin on gold electrodes has been performed by SWV after repetitive h hybridization and de-hybridization (1 at 50 8C) of osmium tetroxide-labelled thrombin at a gold electrode with immobilized aptamer in 0.1 M Britton-Robinson buffer at pH 3.8 The simulations are based on the HD22 sequence (the exosite II-binding aptamer): 5’-GTCCGTGGTAGGGCAGGTTGGGGTGAC-3’ as illustrated in Figure Fig Snapshot of (orange ribbons and multicolored nucleotide bases) HD22-27mer DNA aptamer interacting with (grey) thrombin, with modeled (yellow) osmium conjugated to tryptophan residues 96 and 237 NPT simulations at 300 K and atm were conducted using GROMACS 4.5.5, and included 11,800 TIP3P water molecules as well as 0.38 M NaCl The AMBER-99 parameters were used, including the Chen-Garcia [15] modifications for nucleic acids Dissociation free energies were calculated using virtual replica-exchange umbrella sampling [16], using 29 umbrellas spanning intermolecular www.electroanalysis.wiley-vch.de distances of 2.4–3.8 nm with a spring constant ku, of 2000 kJ/mol/nm2 Acknowledgement The authors are grateful to SUNY Albany for start-up support and to Deutsche Forschungsgemeinschaft (DFG Heisenberg Fellowship FL 384/7-1, FL 384/8-1, and FL 384/11-1) for financial support This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562 References [1] L R Bereznicki, S L Jackson, G M Peterson, E C Jeffrey, K A Marsden, D M Jupe, J Clin Pathol 2007, 60, 311– 315 [2] L C Bock, L C Griffin, J A Latham, E H Vermaas, J J Toole, Nature 1992, 355, 564–566 [3] B Deng, Y Lin, C Wang, F Li, Z Wang, H Zhang, X.-F Li, X C Le, Anal Chim Acta 2014, 837, 1–15 [4] G S Baird, Am J Clin Pathol 2010, 134, 529–531 [5] A V Lakhin, V Z Tarantul, L V Gening, Acta Naturae 2013, 5, 34–43 [6] J S Deetz, E J Behrman, J Org Chem 1980, 45, 135–140 [7] J S Deetz, E J Behrman, Int J Pept Protein Res 1981, 17, 495 [8] M Emerman, E J Behrman, J Histochem Cytochem 1982, 30, 395–397 [9] S Billova, R Kizek, E Palecek, Bioelectrochemistry 2002, 56, 63–66 [10] O Sedo, S Billova, E M Pena-Mendez, E Palecek, J Havel, Anal Chim Acta 2004, 515, 261–269 [11] M Fojta, S Billova, L Havran, H Pivonkova, H Cernocka, P Horakova, E Palecek, Anal Chem 2008, 80, 4598–4605 [12] G.-U Flechsig, T Reske, Anal Chem 2007, 79, 2125–2130 [13] K Biala, A Sedova, G.-U Flechsig, ACS Appl Mater Interfaces 2015, 7, 19948–19959 [14] D M Tasset, M F Kubik, W Steiner, J Mol Biol 1997, 272, 688–698 [15] W Stephenson, P N Asare-Okai, A A Chen, S Keller, R Santiago, S A Tenenbaum, A E Garcia, D Fabris, P T X Li, J Am Chem Soc 2013, 135, 5602–5611 [16] A A Chen, A E Garcı´a, Proc Natl Acad Sci USA 2013, 110, 16820–16825 [17] S Rauscher, C Neale, R Pomes, J Chem Theory Comput 2009, 5, 2640–2662 [18] I Russo Krauss, A Pica, A Merlino, L Mazzarellaa, F Sica, Acta Crystallogr 2013, D69, 2403–2411 Received: November 10, 2017 Accepted: December 20, 2017 Published online on &&&, &&&& 2018 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Electroanalysis 2018, 30, – These are not the final page numbers! ÞÞ 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 SHORT COMMUNICATION S K K Galagedera, L Huynh, F Wachholz, M Jacobsen, P Haruehanroengra, J Sheng, A A Chen, G.-U Flechsig* 1–5 Voltammetric Detection of Thrombin by Labeling with Osmium Tetroxide Bipyridine and Binding with Aptamers on a Gold Electrode ... approach to obtain strongly and specifically binding aptamers, even in cases of labelled targets and aptamers, where SELEX cannot be used Table Thrombin/ DNA aptamer binding free energies System [a] ... including a spacer of 10 adenines and a thiol-linker with three dithian groups: 5’GGTTGGTGTGGTTGG aaaaaaaaaa[Dithio]3-3’ First, labelling of mM thrombin with mM [OsO4 (bipy)] solution was done in an... (DPV) voltammetry at a hanging mercury drop electrode using a 663 VA stand controlled by an Autolab PGSTAT204 potentiostat and software NOVA 1.11 (Metrohm-Autolab, Switzerland) A threeelectrode