Subscriber access provided by UNIV OF NEWCASTLE
Article
Electroactivity of Aptamer at Soft Microinterface Arrays Bren Mark B. Felisilda, and Damien W.M. Arrigan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01172 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 8 1 2 3 4 5 6 7 8 9 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 59 60
Analytical Chemistry
Electroactivity of Aptamer at Soft Microinterface Arrays Bren Mark B. Felisilda, Damien W.M. Arrigan* Curtin Institute for Functional Molecules and Interfaces, School of Molecular and Life Sciences, Curtin University, GPO Box U1987, Perth, Western Australia, 6845, Australia. ABSTRACT: The electrochemical behavior of a synthetic oligonucleotide, thrombin-binding aptamer (TBA, 15-mer), was explored at a liquid-organogel microinterface array. TBA did not display any response when only background electrolytes were present in both phases. Based on literature reports that surfactants can influence nucleic acid detection, the response in the presence of cetyltrimethylammonium (CTA+) was examined. With both TBA and CTA+ in the aqueous phase, the transfer current for CTA+ was diminished, signifying the interaction of CTA+ with TBA. Experiments with CTA+ spiked into the organic phase revealed a sharp current peak, consistent with the interfacial formation of a CTA+-TBA complex. However, use of CTA+ as the organic phase electrolyte cation, as the salt with tetrakis(4-chlorophenyl) borate, greatly improved the response to TBA. In this case, a distinctive peak response (at ca. -0.25 V) was attributed to the transfer of CTA+ across the soft interface to complex with aqueous phase TBA. Employing this process as a detection step enabled a detection limit of 0.11 µM TBA (by cyclic voltammetry). Furthermore, the presence of magnesium cations at physiological concentration resulted in the disappearance of the TBA response, due to Mg2+induced folding of TBA. Also, the current response of TBA was decreased by the addition of thrombin, indicating TBA interacted with this binding partner. Finally, the interfacial surfactant-aptamer interaction was explored in a synthetic urine matrix that afforded a detection limit of 0.29 µM TBA. These results suggest that aptamer binding interactions can be monitored by electrochemistry at aqueous-organic interfaces and opens up a new possibility for detection in aptamer-binding assays.
Since their discovery in the 1990s, aptamers have been extensively used in many applications.1 Aptamers, a name derived from the Latin aptus, “to fit”,2 are synthetic oligonucleotides (RNA or DNA) obtained through a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX)3 and are known to possess high affinity and specificity towards a wide array of target molecules, e.g. proteins, drugs, amino acids, etc.4 This affinity and specificity is enhanced by their ability to structurally fold upon binding with the target molecule,5 giving an advantage over antibodies, their natural counterparts. Over time, aptamers became vital molecular tools, especially in diagnostics and therapeutics,6 where they are considered as good reagents for target validation in various disease models.7,8 These synthetic oligonucleotides have been developed to bind with proteins that are affiliated with various disease states, so paving the way for powerful protein antagonists.9 Generally, aptamers can serve two roles in therapeutic applications, either as a targeting modality or as the therapeutic agent itself. Despite their slow translation to the clinic, inventive modifications are appearing that can improve performance.10 Moreover, based on their roles as molecular targeting ligands, aptamers are also studied to enhance molecular imaging,11 leading to intense interest in research to further understand aptamers and their conformational and ligand-binding properties.5,12,13 This has resulted in a range of bioassays that use aptamers as receptors or immobilized ligands14,15 which have been applied to investigate numerous target analytes and open up analytical applications of aptamers. One well-known target analyte is thrombin, an allosteric serine protease that is Na+-activated and is the main protease in the coagulation cascade.16 Thus it plays a vital role in patho-
logical and physiological coagulation, which is also utilized as an indicator of various diseases like Alzheimer’s and some cancers.17,18 In order to achieve detection of significantly low concentrations of thrombin, the thrombin binding aptamer (TBA) was the focus of several studies.19 The 15-base oligonucleotide with the sequence 5’-GGT-TGG-TGT-GGT-TGG3’, which binds with thrombin, was the first example of a nucleic acid as a potential therapeutic agent.20 Moreover, this 15mer sequence was found to bind more to alpha-thrombin than to gamma-thrombin21 and was reported to form a folded structure, a G-quadruplex.22 Subsequent studies23,24 aimed to improve its binding with thrombin. Several review articles have reported the growing number of studies that detect thrombin with TBA via different analytical approaches.25 Given TBA’s array of applications, a simple yet direct method for detection of TBA is advantageous. Wu et al.26 used surface enhanced Raman spectroscopy (SERS) to analyse TBA and noted specific spectral peaks corresponding to its constituents. Joseph and colleagues27 reported the binding of fluorescent dyes, YO-Pro-1 iodide and YOYO-1 iodide, with TBA, finding smaller binding constants with TBA than with double-stranded DNA. However, these reports did not mention limits of detection for the aptamer. Electrochemical detection techniques were also extensively used in TBA investigations since these promise fast, affordable and sensitive detection and are prominent in the development of aptasensors28 based on binding-induced detection. Potentiometry with polymeric membrane ion-selective electrodes (ISEs) containing selective receptors or ionophores have yielded measurements of DNA,29 DNA hybridization,30 and protein-binding aptamers.31 Durust and co-workers32 reported the detection of double-stranded DNA in the μg mL-1
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 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 59 60
range via a potentiometric polyion sensor, based on DNA interacting with cationic protamine. Meanwhile, Shishkanova et al.33 investigated the use of a single-stranded oligonucleotide as an active component in a polymeric membrane ISE, finding that membranes modified with cholesterol-oligonucleotides were sensitive to a complimentary oligonucleotide. In recent years, electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) has been examined as the foundation for novel analytical strategies.34,35 The promises of amenability to miniaturization and label-free detection36 have propelled the use of electrochemistry at the ITIES into the exploration of biological molecules e.g. proteins37,38 and oligonucleotides.39 Vagin et al.40 described the detection of DNA via a supported liquid-liquid interface, achieving a detection limit of 10 nM. The use of surfactants with DNA was also explored at the ITIES, following reports that protein-surfactant complex formation41 enabled the transfer of proteins at these soft interfaces. Osakai and colleagues42 examined the interaction between a cationic surfactant and DNA at the ITIES, finding that the transfer of the surfactant was affected by adsorbed DNA. In addition, Kivlehan et al.43 reported the use of an acridine-functionalised calix[4]arene at the ITIES which enabled the binding of DNA with the acridine moiety at the interface. In this report, the electrochemistry of an aptamer, TBA, at an array of microscale ITIES is presented. The 15-mer DNA oligonucleotide TBA, sequence 5’-GGT-TGG-TGT-GGTTGG-3’, was examined using an aqueous-organogel microinterface array for voltammetric characterization and detection. The findings reveal that the organic phase electrolyte cation aided the detection of the aptamer, in this case the surfactant cetyltrimethylammonium (CTA+), via formation of an interfacial complex. It was also found that magnesium ion or thrombin, added to the aqueous phase, changed the electrochemical response, while limits of detection of 0.11 μM (for TBA in 10 mM LiCl (pH 8.5)) and 0.29 μM (for TBA in a synthetic urine mixture) were obtained. These findings suggest that aptamer electrochemistry at the ITIES offers scope for the detection of aptamer-binding processes.
water to achieve 100 μM stock solution and incubated at room temperature for approx. 30 mins. The resulting solution was vortexed for 20 s before centrifuging (10,000 x g) for 1 min. Aliquots of this were stored at -20 °C. Tetrapropylammonium (TPrA+) chloride was prepared in 10 mM LiCl and the synthetic urine mixture47 used was composed of ammonium chloride (1.00 g L-1), creatinine (1.10 g L-1), calcium chloride dihydrate (1.103 g L-1), potassium dihydrogen phosphate (1.40 g L-1), potassium chloride (1.60 g L-1), sodium sulfate (2.25 g L1 ), sodium chloride (2.295 g L-1), and urea (25 g L-1). The pH of the aqueous phase was adjusted via dropwise addition of 10 mM NaOH. All aqueous solutions were prepared by using deionised water (USF Purelab Plus UV, resistivity of 18.2 MΩcm). Apparatus. Electrochemical experiments were conducted using an AUTOLAB PGSTAT302N electrochemical workstation (Metrohm, The Netherlands) with NOVA software. The µITIES array utilized in the study was formed via a 30micropore array silicon membrane,48 in a hexagonal pattern, with pore diameter 22.4 µm, pore centre-to-pore centre distance 200 µm, and membrane thickness 100 µm. The total geometric area (total cross-sectional area of the micropores) of the microinterface array was 1.18 x 10-4 cm2. The silicon membranes were attached onto the lower orifice of glass cylinders using a silicone rubber (acetic acid curing/Selley’s glass silicone). The gelled organic phase was introduced into the micropore array through the glass cylinder using the tip of a pre-warmed glass pasteur pipette. The completed set-up was then set aside for at least 1 hour before use. When ready, the organic reference solution, composed of saturated BTPPACl or CTAB in 10 mM LiCl, was introduced into the glass cylinder to sit on top of the organogel. This organogel/silicon membrane assembly was then inserted into the aqueous phase to proceed with the voltammetric experiments. Scheme 1 summarizes the compositions of the electrochemical cells employed.
EXPERIMENTAL SECTION Reagents. All reagents were purchased from Sigma-Aldrich Australia Ltd and were used as received unless stated otherwise. The organic phase was bis(triphenylphosphoranylidene)ammonium tetrakis(4chlorophenyl) borate (BTPPATPBCl) or cetyltrimethylammonium tetrakis(4-chlorophenyl) borate (CTATPBCl) in 1,6dichlorohexane (DCH). The resulting electrolyte solution (10 mM) was gelled via the addition of 10% w/v poly(vinyl) chloride (low molecular weight).44 The organic electrolyte salt, BTPPATPBCl, was produced via metathesis of bis(triphenylphosphoranylidene)ammonium chloride (BTPPACl) and potassium tetrakis(4-chlorophenyl)borate (KTPBCl).45 In a similar manner, the organic electrolyte salt CTATPBCl was prepared via metathesis of cetyltrimethylammonium bromide (CTAB) and potassium tetrakis(4chlorophenyl)borate (KTPBCl).46 Thrombin (bovine plasma, lyophilized powder) was purchased from Sigma Aldrich Australia Ltd. The unmodified thrombin-binding aptamer (TBA), sequence 5’-GGT-TGG-TGT-GGT-TGG-3’, was purchased (in lyophilized form) from Integrated DNA Technologies Pte. Ltd. (Singapore). The aptamer was resuspended in deionized
Scheme 1. Schematic illustration of the electrochemical cells utilized, where x represents the thrombin-binding aptamer (TBA) concentrations.
Electrochemical Measurements. Two Ag/AgCl electrodes were employed in all electrochemical measurements. Cyclic voltammetry (CV) was conducted at a 5 mV s-1 scan rate unless otherwise stated. Other experimental parameters such as TBA concentration were varied accordingly. The calculated detection limits were based on three times the standard
ACS Paragon Plus Environment
Page 2 of 8
Page 3 of 8 1 2 3 4 5 6 7 8 9 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 59 60
Analytical Chemistry deviation of the blank (n=3) divided by the slope of the best-fit linear calibration line. All potentials were adjusted to the Galvani potential scale based on the experimental mid-point transfer potential of TPrA+ and its formal transfer potential (0.08 V) in the water│1,6-dichlorohexane system.49 Compensation of the iR drop was carried out a posteriori50 via the NOVA software, which employs the formula . The uncompensated resistance ( ) was measured with a CHI900B potentiostat (CH Instruments Inc., USA) via positive feedback at the open circuit potential with a pulse of 50 mV amplitude. The values were 255, 875 and 832 kOhms for Cells 1, 2 and 3, respectively.
Organic phase surfactant. Following the demonstration of an aqueous phase CTAB-TBA interaction, the next step was to incorporate the surfactant into the organic phase. This was done by the addition of CTAB to the gelled organic phase during organogel preparation. Since the concentration of the organic electrolyte, BTPPATPBCl, was 10 mM, two different concentrations of CTAB were studied. Figure 2 shows CVs of 10-50 μM TBA in the aqueous phase in contact with organic phases containing CTAB with 10 mM BTPPATPBCl.
RESULTS AND DISCUSSION Aqueous phase surfactant. Cyclic voltammetry (CV) was used to examine the electrochemical behavior of TBA at the µITIES array. Figure 1A shows CVs recorded in the absence (grey line) and presence (black-dotted line) of 20 μM TBA (Cell 1, Scheme 1). Upon scanning from the positive to the more negative potentials (forward scan), a decrease in the current was observed due to the transfer of the background electrolytes across the liquid-organogel interface. At the more negative potentials, this process equates to the movement of the anions (Cl-) from the aqueous phase to the organic phase as well as the cations (BTPPA+) from the organic to the aqueous phase. The two CVs perfectly overlay each other - there was no additional peak or response that would suggest the electroactivity of TBA under these conditions. Previous reports of DNA detection at the ITIES39 indicated its interaction with organic cations, specifically the impact of DNA in decreasing ion transfer currents for aqueous phase methyl viologen (MV2+). Thus, the absence of a response to TBA may be attributed to it having very weak or no interaction with the organic phase cation, bis(triphenyl)phosphoranylideneammonium (BTPPA+). Structurally, the positive center of BTPPA+ is surrounded by phenyl rings (inset, Figure 1A) which may hinder its interaction with the negatively-charged TBA. A similar situation was observed for sulfated polysaccharides.51 Based on previous reports of DNA-facilitated transfer of surfactant across the ITIES,42 the influence of a surfactant on TBA behavior was explored. Cetyltrimethylammonium bromide (CTAB) was chosen for its known interaction with DNA.52 Initially, different concentrations (10-100 μM) of CTAB were added to the aqueous phase. The CVs in Figure 1B show a peak response at ca. -0.40 V on the forward (negative-going) scan which increases proportionately with increasing CTAB concentrations. Subsequently, a broad peak response was seen on the reverse (positive-going) scan at ca. 0.20 V, also proportional to CTAB concentration. These responses can be attributed to the transfer of CTA+ across the interface. With the highest CTAB concentration (100 µM) present in the aqueous phase, varying concentrations of TBA (5-25 µM) were spiked into that phase and the resulting voltammograms are displayed in Figure 1C. As the TBA concentration increased, there was a decrease in the peak currents, suggesting that TBA interacted with CTA+ to lower the concentration available to transfer across the ITIES. A similar observation was reported by Horrocks and Mirkin39 when the ion transfer current for MV2+ decreased in the presence of high-molecular weight DNA.
Figure 1. Cyclic voltammograms of 10 mM LiCl (pH 8.5) (A) without (grey line) and with (black-dotted line) 20 μM TBA using Cell 1; (B) with 0, 10, 20, 40, 60, 80, 100 μM of CTAB and (C) with 100 μM CTAB plus 0, 5, 10, 15, 20, 25 μM TBA. Scan rate was 5 mV s-1.
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 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 59 60
Interestingly, 5 mM CTAB in the organic phase (Figure 2A) did not promote the detection of TBA (only the transfer of background electrolytes was seen), but upon doubling the CTAB concentration in the organic phase, a distinct peak response was observed (ca. -0.5 V) (Figure 2B). This suggests an aptamer-surfactant interaction, as this feature was not seen when surfactant was absent (Figure 1A). The sharp peak response is suggestive of an adsorption/desorption process and indicates that CTA+ and TBA interact at or near the interface. Figure 2C shows background subtracted reverse scan voltammograms, showing clearly the desorption peak. The inset in Figure 2C shows the resulting calibration graph of peak current versus aptamer concentration in the aqueous phase. Similar observations were made with other anionic biomolecules interacting with organic phase cations53, as well as with surfactant-protein interactions, as reported elsewhere.54
Figure 2. Cyclic voltammograms of 10 mM LiCl (pH 8.5) with 0, 10, 20, 30, 40, 50 μM TBA in aq. phase while (A) 5 mM CTAB + 10 mM BTPPATPBCl and (B) 10 mM CTAB + 10 mM BTPPATPBCl in organic phase; Scan rate was 5 mV s-1. (C) Background subtracted reverse scans from B, Inset: Calibration plot of C, using peak currents measured above the baseline current between -0.4 V and -0.3 V.
To further examine the interaction of the surfactant with the aptamer and its role in the detection of the latter, the surfactant as a salt with TPBCl- (i.e. CTATPBCl) was employed as the only electrolyte in the organic phase. This removes any competition for binding from different cations present. Consequently, any electrochemistry observed in the presence of the patamer can be attributed to the interaction between the anionic aptamer and CTA+ cation in the organic phase. Figure 3 shows the CVs obtained when the organic phase contained 10 mM CTATPBCl was the organic phase. In the presence of 10 μM TBA (black line, Figure 3A), a distinct peak response was observed (ca. -0.25 V) which was clearly not observed in the blank scan (dashed line, Figure 3A). Only transfers of background electrolytes were observed when TBA was absent. When BTPPATPBCl was present together with CTA+ in the organic phase (Figure 2B), the peak current for 10 µM TBA was ca. 1 nA. This increased to ca. 5 nA (See Figure 3A) when only 10 mM CTATPBCl was in the organic phase, offering the possibility to detect lower concentrations of TBA (Figure 3B). As seen, a distinct peak response (ca. -0.25 V) appeared when 2 μM TBA was present which increased proportionally with TBA concentration.
Figure 3. Cyclic voltammograms of 10 mM LiCl (pH 8.5) using Cell 2 (see Scheme 1) (A) with (black line) and without (grey dashed line) 10 μM TBA and (B) with varying (0, 1, 2, 3, 4, 5
ACS Paragon Plus Environment
Page 4 of 8
Page 5 of 8 1 2 3 4 5 6 7 8 9 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 59 60
Analytical Chemistry μM) TBA concentration. Scan rate: 5 mV s-1. Inset: Calibration plot of (B).
However, following background-subtraction of these CVs, a peak response was observed even at 1 μM TBA. This arrangement with CTATPBCl in the organic phase afforded a calculated detection limit of 0.11 μM TBA in 10 mM LiCl from a linear calibration graph (Figure 3B, inset) for the range 1-5 µM TBA, with a sensitivity of 0.95 nA µM-1. The charges under the peaks in Figure 3 provide estimates of the surface coverages (using Faraday’s Law and the geometric area of the interfaces), in this case, of the surface coverage of CTA+ species that bind with TBA at the interface, since that is the species that the ion-transfer process measures. The charge from Figure 3A provides a surface coverage of 3.2 x 10-11 mol cm-2, whilst the charge from the highest concentration present in Figure 3B provides a surface coverage of 1.4 x 10-11 mol cm-2. Since the TBA aptamer is a 15-mer, it can be assumed that its electrical charge is -15 and hence the surface coverages of TBA at the interface could be estimated, if a 1:1 binding of CTA+ with each anionic site of the aptamer occurs. However, by taking the approach of Osakai et al.,42 the fraction of available sites that are bound by CTA+ may be estimated instead. In the study of DNA adsorption at the ITIES, Osakai et al. considered that a single base pair occupied 6.8 x 10-15 cm2, or 3.4 x 10-15 cm2 per base (equivalent to a single anionic site in single-strand DNA, as used here). If TBA is fully adsorbed in a close-packed arrangement at the ITIES, the surface coverage of anionic sites would be 2.9 x 1014 cm-2. If each such anionic site binds a single CTA+ cation, then the surface coverage of CTA+ would be 4.9 x 10-10 mol cm-2. This suggests that the total CTA+ bound at the ITIES (i.e. 3.2 x 10-11 mol cm-2, Figure 3A) is
