Electrodepositing DNA Self-Assembled ... - ACS Publications

Subscriber access provided by EKU Libraries

Article

Electrodepositing DNA Self-Assembled Monolayers on Au: detailing the influence of electrical potential perturbation and surface crystallography Kaylyn K Leung, Hua-Zhong Yu, and Dan Bizzotto ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01695 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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 24 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

ACS Sensors

Electrodepositing DNA Self-Assembled Monolayers on Au: detailing the influence of electrical potential perturbation and surface crystallography Kaylyn K. Leung,†,‡ Hua-Zhong Yu,¶ and Dan Bizzotto∗,†,‡ †AMPEL, University of British Columbia, Vancouver, Canada ‡Department of Chemistry ¶Department of Chemistry, Simon Fraser University, Burnaby, Canada E-mail: [email protected] Abstract The preparation of DNA self-assembled monolayers (SAMs) on single crystal gold bead electrodes using an applied potential is evaluated with in-situ electrochemical fluorescence microscopy. Applying a constant deposition potential or a square-wave potential perturbation during the formation of DNA SAMs is compared for two different modification methods: DNA SAM formation on a clean gold surface followed by alkythiol backfilling (as is typically done in literature) or via thiol-exchange on an alkylthiol modified gold surface. DNA SAMs prepared from a chloride containing deposition buffer were not significantly different when using either square-wave potential perturbation or at a constant applied potential even when considering different surface crystallographies. Greater variations were observed when applying more positive potentials for both DNA thiol-exchange and DNA adsorption on clean Au. Our results

1

ACS Paragon Plus Environment

ACS Sensors 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

suggest that using either a constant potential or a square wave potential perturbation for 5 min both create defects by weakening the gold-thiol bond. When the deposition is performed with the adsorption of chloride ions from the electrolyte, the electrodeposition results in a similar increase in DNA coverage when compared to depositions performed at open circuit potentials.

Keywords DNA self-assembled monolayer, electrodeposition, fluorescence microscopy, surface crystallography, thiol-exchange, surface coverage

2


Page 2 of 24

Page 3 of 24 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

ACS Sensors

Self-assembled monolayers (SAMs) of thiol-modified DNA strands prepared on gold surfaces are often used as biosensors for sequencing or disease diagnostics. 1–3 Typically, these sensors are prepared on a polycrystalline gold surface (e.g., vapor deposited gold film on glass) with procedures detailed in literature so as to achieve consistent control over the DNA coverage 4 . This can be challenging as the optimal sensing of a target sequence or a biomolecule requires an optimal DNA coverage, as low DNA coverage leads to low sensitivity and high DNA coverage hinders probe-target interactions. 5–7 Improving the commercial manufacturing of DNA SAM based sensors will require methods that will reduce preparation time, create uniformly deposited DNA with control over surface coverage. This is further complicated by the polycrystalline substrates typically used which introduces variability in the DNA surface assembly. To understand this influence would require a careful surface analytical study. Typically, DNA SAMs on gold are prepared from solutions of thiol-modified oligonucleotides without control over the potential of the substrate. Applying a positive constant potential during self-assembly of alkylthiols on gold has been shown to reduce defects in the SAM and facilitate the kinetics of SAM formation. 8 This was also demonstrated for the self-assembly of thiol-modified DNA on gold. 5 Applying a square wave potential perturbation during self-assembly has been shown to further reduce the time required for DNA self-assembly 9,10 however, the mechanism for the increased DNA coverage in a short deposition time is still not well understood. Quan and colleagues were first to demonstrate this phenomena by assembling DNA in 50 mM triethyl ammonium acetate buffer for an hour, characterizing their layers using an electrochemical quartz-crystal microbalance. 10 They believed that the increased DNA coverage was due to the positive potentials enhancing the oxidative thiol adsorption process while the negative potentials electrostatically reoriented the DNA away from the surface, which created additional sites for the DNA adsorption. Jambrec and colleagues deposited DNA SAMs from a 10 mM phosphate buffer with 450 mM K2 SO4 using a 50Hz square wave potential perturbation for 15 min resulting in a high 3


ACS Sensors 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

DNA surface coverage with fewer defects, measured using impedance spectroscopy as a high charge transfer resistance towards Fe(CN)6

3 – /4 –

redox process. They proposed that in the

high ionic strength buffer, the potential only affects the region near the electrode surface, and if the potential limits used for the square wave were centered around the potential of zero charge (pzc) of the surface, there would be an exchange of cations and anions causing stirring effect which transports DNA to the surface increasing coverage. 9 We have previously examined the role of a constant applied potential (Edep ) on the thiolexchange process used to modify a pre-existing alkylthiol monolayer with thiol-modified DNA. The DNA coverage was strongly influenced by the underlying surface crystallography as well as anion adsorption. The applied potential was thought to create defects in the SAM, the density of which varied depending on the potential and surface crystallography. These defects become sites where the alkylthiol modified DNA easily thiol-exchanged to populate the surface. 11 Using Edep potentials close to either reductive or oxidative cleavage of the Au-thiol bond facilitated the thiol-exchange reaction. In addition, competitive adsorption of Cl – onto the surface at positive Edep was found to impact the deposition process. In this work, the surface modification that resulted from using either a constant deSW position potential (Edep ) or a square-wave deposition potential profile (Edep ) during the

formation of DNA SAMs was compared for the assembly of DNA SAMs via thiol-exchange and for the formation of DNA SAMs on a clean gold surface. This was accomplished using in-situ fluorescence electrochemical microscopy to characterize the DNA SAMs created on single crystal gold bead electrodes. 12,13 The electrodeposited DNA SAMs were analyzed in a consistent fashion so as to reveal the influence of surface crystallography, the concentration of thiol modified DNA in the deposition buffer, and deposition potentials. These results shed light on previous potential assisted deposition studies and on the mechanism by which potential influences DNA SAM formation for a variety of surface crystallographies.

4


Page 4 of 24

Page 5 of 24 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

ACS Sensors

Experimental Materials Spherical single crystal Au bead electrodes were prepared from melting and cooling a gold wire (1mm diameter, 99.999% Alfa Aesar) as described previously. 13 The gold bead electrodes were cleaned and conditioned by cycling in 1M H2 SO4 . Thiol-modified DNA (Integrated DNA Technologies) was composed of a 30 base sequence (5’-CTG TAT TGA GTT GTA TCG TGT GGT GTA TTT-3’) with a HO(CH2 )6 SS(CH2 )6 modification at the 5’end and the fluorophore AlexaFluor®488 attached to the 3’-end. The sequence was selected so as to be not self complementary and not form secondary structures (confirmed with Oligiocalc, DNA Melt and mfold). 13,14 DNA surface modification was done after reduction of the disulphide to a thiol (HS(CH2 )6 ) moiety using 10 mM TCEP-HCl tris(2carboxyethyl)phosphine-hydrochloride (>98% Sigma Aldrich) neutralized to pH 7.5 with 10 mM KOH(99.99% semiconductor grade Sigma Aldrich). The reaction mixture was filtered using a GE MicrospinTM G-50 column as described previously. 14 The DNA was then diluted to 10 μM solutions in 10 mM TRIS(containing TRIS Base and TRIS HCl, Bioperformance >99.0%, Sigma Aldrich for both) pH 7.5 buffer in a sealed eppendorf tube at -20 °C and used within 2 weeks. DNA deposition solutions were made over a range of concentrations (0.1 to 1µM ) prepared by dilution using a pH 7.5 immobilization buffer (IB) containing 10 mM TRIS, 100 mM NaCl (≥99.5% BioXtra, Sigma Aldrich) and 500 mM MgCl2 (>99% Sigma Aldrich). MCH (6-Mercapto-1-hexanol, 99% Sigma Aldrich) was used from a stock of 50 mM in MeOH and stored at -20 °C for up to a month. Solutions used for MCH deposition were 1 mM in either MeOH (HPLC grade, Fisher) or in the aforementioned immobilization buffer (IB). All water used was from a Millipore Integral 5 system (18.3 MM Ω cm).

5


ACS Sensors 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

Potential-assisted DNA SAM formation on Au electrodes Immediately prior to formation of DNA SAMs on Au electrodes, the bead electrodes were cleaned by flame annealing with a butane torch and rinsed with Millipore water. DNA SAMs were made using two different methods: via DNA-thiol exchange and the adsorption of thiolated DNA onto clean Au beads. For the DNA thiol-exchange method, the Au electrodes were first immersed in a 1 mM MCH solution in MeOH for 30 minutes then rinsed and temporarily stored in MeOH. The MCH SAM modified bead was rinsed with Millipore water and then immersed in the DNA deposition solution contained within the electrodeposition cell. The electrode did not rest in the DNA solution for more than 1 minute prior to application of potential. Application of a constant positive potential during DNA thiol-exchange was performed for 5 minutes at the selected Edep (either +0.4V or +0.5V) versus a saturated calomel electrode (SCE) connected via a pipette salt bridge in a 2-electrode arrangement as described previously. 11 SW For electrodeposition using square-wave potentials, a 50 Hz square wave (Edep ) was applied

for 5 minutes from +0.4V -0.3 V/SCE or +0.5V to -0.3V/SCE using a 3-electrode setup. Potential and current were both sampled at 100 Hz during the 5 minute deposition with the first and last 0.2 seconds sampled at 5000 Hz. To improve the temporal response of the system, a counter electrode in the form of a MCH modified Au wire was added into the deposition solution. MCH modification was used to prevent DNA adsorption onto its surface thereby not depleting DNA from the deposition solution. After potential-assisted DNA modification, the spherical Au electrodes were rinsed with Millipore water then stored in IB overnight. Adsorption of thiol modified DNA onto clean Au bead electrodes was accomplished by immersing the Au electrode in the DNA solution immediately after flame annealing and rinsing in Millipore water. Applying the potential was done using the same setup as for the DNA SW thiol-exchange method. Edep or Edep was applied in the same manner as mentioned above.

After DNA modification, the electrodes were rinsed with Millipore water and immersed in a 6


Page 6 of 24

Page 7 of 24 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

ACS Sensors

1 mM MCH solution in IB for 90 minutes and then rinsed with Millipore water again and stored in IB overnight.

In-situ Electrochemical Fluorescence Microscopy Imaging The electrochemically prepared DNA SAMs were characterized using in-situ electrochemical fluorescence microscopy (iSEFMI) as previously described 12 with an inverted epi-fluorescence Olympus IX70 microscope at 5× magnification (NA = 0.13). Electrochemical measurements in this arrangement were done using a SCE and platinum coil counter electrode. A 10 mM TRIS (Bioperformance >99.0%, Sigma), 10 mM KNO3 (≥99.0% BioXtra, Sigma) buffer, adjusted with HNO3 (60%-70% ACS grade, Fisher) to pH 7.5, was used as the electrolyte. Fluorescence images were taken using a Photometrics Evolve® 512 EMCCD digital camera (512x512 pixels) with illumination provided by a Hg Arc 200 W Lamp (Xcite® Exacte w/ EXFO closed loop feedback). Images were taken through a filter set appropriate for AlexaFluor488 12 with 3 second exposure time while applying -0.4 V/SCE to the electrode to achieve maximum fluorescence intensity by electrostatically repelling the DNA from the electrode surface. 11 This potential was not negative enough to disturb the DNA SAM, but was used so as to decrease the fluorescence quenching by the gold electrode. After analysis, the DNA SAMs were reductively desorbed by scanning to -1.40 V and repeating until no fluorescence was visible. A background image was obtained at the same 3 second exposure time in the same optical configuration and used for background subtraction. All images were processed using ImageJ, converted to 32 bit, despeckled and gaussian blurred (2 px. radius) and background corrected by subtracting the image of the electrode after reductive desorption. Images displayed in this work are false coloured. One stereographic triangle is selected per electrode with the 111 and 100 facets identified in bright field images as previously explained. 11,13 Other surface crystallographies (110, 311 and 210) were located with reference to the 111 and 100. Average fluorescence intensities were used from the selected regions of interest (ROI) for each surface crystallographic region. Fluorescence 7


ACS Sensors 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

intensity measurements were correlated to an estimated DNA coverage in a separate set of experiments as detailed in the SI.

Results and Discussion SW A comparison of the influence of Edep vs Edep is presented first for the DNA thiol-exchange

process, building upon previous observations. 11 Particularly we have shown the influence of different Edep on the extent and surface crystallographic specificity of the DNA modification after 1 hour of potential treatment as compared to a similar set of conditions in the absence of an applied potential (at open circuit potential, OCP). The DNA coverage was found to be substantially higher with both positive and negative Edep as compared to assembly at OCP. SW Surface modification via Edep is shown below for DNA-thiol exchange on a MCH SAM coated

gold surface , and then compared for DNA SAMs prepared on clean gold surfaces.

Potential-Assisted DNA SAM formation via Thiol-Exchange Thiol-exchange is a method that can be used to modify an existing SAM by exposing it to a solution containing another alkylthiol which exchanges with bound thiols or populates the defects in the original SAM. 15–17 This is achieved through either insertion into an empty surface site or substitution of the previously adsorbed alkylthiols. 16 Preparing a DNA SAM via thiol-exchange with a mercaptohexanol (MCH) SAM provides a method of forming DNA SAMs with low coverage and with less non-specifically adsorbed DNA (adsorption of the nitrogenous bases). 14 We showed previously that applying a constant Edep = +0.4V /SCE for 60 min during DNA thiol-exchange results in a higher coverage of DNA due to an increase in the number of defects with which the thiol-DNA interacts compared to OCP preparation. 11 Creating similar DNA coverage on gold surfaces with shorter deposition times maybe possible using square-wave deposition potential perturbations. 9,10,18 To test this, a comparison of the SW surfaces prepared during a brief 5 min application of either Edep (+0.4V / SCE) or Edep

8


Page 8 of 24

Page 9 of 24 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

ACS Sensors

(50 Hz square wave, +0.4V to -0.3V/ SCE), was performed on MCH SAM modified gold bead electrodes during DNA thiol-exchange. The resulting modified surfaces were analyzed using iSEFMI and the fluorescence images are shown in Figure 1a &b with the measured fluorescence intensities from regions containing low index planes (111, 100 and 110) and selected high index planes (311 and 210) in Figure 1c. The estimated DNA coverage that corresponds to the average fluorescence intensity measured here is about 0.2×10−10 mol/cm2 , or 1.2 × 1012 DNA/cm2 , which is 10-20% of its maximum packing density. 5 The fluorescence intensity is roughly proportional to coverage at low values (5 × 1012 DNA/cm2 ), but levels off at higher coverages (Figure S1). These measurements were done by comparing an average measurement of DNA coverage using Ru(NH3 )6 3+/2+ redox 19 to the average fluorescence intensity measured on one half of the bead surface.

Figure 1: DNA SAM formation via thiol-exchange: Fluorescence images obtained through iSEFMI of DNA SAMs prepared using potential-assisted DNA thiol-exchange (for SW 5 min) at (a) Edep = +0.4V /SCE and (b) Edep (50 Hz square wave, +0.4V to -0.3V/ SCE). A stereographic triangle is used to label five surface crystallographies. c) Measured averaged fluorescence intensities on select surface crystallographies. Error bars correspond to standard deviation of the intensities measured for two or more samples. SW The overall fluorescence intensity of the DNA SAMs made using Edep or Edep appear very

similar. The fluorescence intensities for 5 selected regions show that the underlying surface crystallography has a more significant influence on the extent of thiol exchange. The largest DNA surface coverage is on the 311 region. This is a result of this region having a MCH SAM of weakly adsorbed thiols or due to the presence of more defects. 11,13 This contrasts 9


ACS Sensors 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

with the 210 region, another high index region, which has significantly lower DNA coverage as a result of the strength of the adsorption of MCH to the 210 surface. 13 Interestingly, SW methods shows they result in surfaces with similar comparing the constant Edep and Edep

coverages for these five regions. The 111 facet showed a significant difference when using SW a Student’s t-test but only at an 80% confidence interval. This suggests that Edep does

not result in a substantially higher DNA coverage overall, but some decerniable differences were noted. The fluorescence intensity pattern in the regions between the 100 facet and 311 SW which may reveal differences in the nature of or the 210 regions are more defined for Edep

the created defects. The application of a negative potential step near the reduction of the thiol bonded to the gold surface should destabilize or create defects in the SAM enhancing thiol-exchange. This seems to be specific for particular surface crystallographies as revealled SW by the fluorophore labeled DNA. Using Edep does impact the extent of the surface that

experiences thiol-exchange, in particular the regions around the 111 facet. This suggests SW that using Edep results in accelerated thiol-exchange characteristics which can be explained

through the formation of defects, or weakening of the gold-S bonds. It may also be due to an increase in the local concentration of DNA in the surface region due to pulsing as described previously 9 which can be investigated by using a lower [DNA] in the deposition buffer. Furthermore, this will allow for control over the DNA coverage without decreasing the electrodeposition time. The evolution of the DNA surface coverage on the various regions of the electrode as a function of [DNA] in the deposition solution may also reveal surface specific thiol-exchange SW characteristics enabling a detailed comparison of Edep (+0.4V) or Edep (+0.4V to -0.3V/SCE,

50Hz) (Figure 2a and b, respectively). The fluorescence intensities from the same crystallographic regions for this range of [DNA] are shown in Figure 2c.. The DNA coverage increases as expected with increasing [DNA] for both deposition methods. The relative fluorescence intensities from each crystallographic region remain the same but the overall fluorescence intensity (i.e. DNA coverage) increases. The DNA coverage is slightly higher for the 0.25 10


Page 10 of 24

Page 11 of 24 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

ACS Sensors

SW and 0.5μM DNA for Edep especially on and around the 111 facet as described previously SW . While though the overall DNA coverage does not significantly increase when using Edep

control over the DNA coverage can be realized by choosing the appropriate [DNA] in the deposition solution, nevertheless, DNA coverage is more influenced by the choice of the applied potential or potential limits(vide infra). Positive Potential Limit and Potential-Assisted DNA Thiol-Exchange Previously, we have shown that using a potential more positive than +0.4V/SCE during a 60 min thiol-exchange increases the DNA coverage because of the creation of a higher density of defects in the MCH SAM. This is due both to being at a potential near oxidative desorption and the influence of Cl – adsorption at these potentials. 11 Using a higher positive potential SW and comparing Edep =+0.5V/SCE, 5 min and Edep ( +0.5V to -0.3V/ SCE, 50 Hz, 5 min)

is shown in Figure 3 with a comparison of the fluorescence intensities for the thiol-exchange deposition methods from the 5 regions of interest given in Figure S2. Comparing electrodeposition at Edep =+0.4V/SCE and +0.5V/SCE shows a modest increase in DNA coverage for most of the regions studied with a significant increase observed for the 110 region. The SW positive potential limit also resulted in a DNA coverage increase (comincrease in the Edep SW pared with Edep (0.4 V to -0.3V/SCE)) except for the 311 that has a lower coverage. When SW comparing the Edep treated samples, the largest increases were observed for the 111, 110, and

210 regions. Even though the variability between samples was large, significant differences are still observed including in the regions which were not specifically analyzed. For example, SW for the Edep (0.4 V to -0.3V) samples, low DNA coverage was observed for regions around the

100, specifically between the 100-110 and 100-111 crystallographic zone axes. Increasing the positive potential limit significantly increased the DNA coverage in these regions, specifically along the 100-210-110 zone. This is also the case when analyzing the regions that surround the 111 facet, which are similar to small 111 terraces separated by many steps. 20 In agreement with the previous study, an increase in the positive electrodeposition potentials results 11


ACS Sensors 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

Figure 2: Fluorescence images of DNA SAMs prepared via DNA thiol-exchange (for 5 min) SW at a) Edep = +0.4V /SCE and (b) Edep ( +0.4V to -0.3V/SCE, 50 Hz) for varying concentrations of DNA in the deposition solution, c) average fluorescence intensity for five selected regions representing low index planes (111, 100, 110) and high index planes (210, 311). Individual data points correspond to replicate measurements. 12


Page 12 of 24

Page 13 of 24 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

ACS Sensors

in greater MCH SAM instability and facilitated DNA thiol-exchange. When compared to SW does not dramatically improve the DNA coverage, the constant Edep condition, using Edep

and in some cases decreases the coverage. This can be explained since the time spent at negative potentials, where the stability of the MCH SAM is greater, limits the time spent at the positive potential where the MCH SAM is less stable resulting in a lower DNA coverage due to a lower number of defects created in the MCH SAM. SW The surface modifications using Edep and Edep for the thiol-exchange approach manipu-

lates the creation of defects in the MCH SAM which is strongly dependent on the underlying surface crystallography. These studies also demonstrate the significant role which the positive potential limit has on the creation of defects and the extent of thiol-exchange with a more positive electrodeposition potentials favoring an increase in the DNA coverage. Using SW either Edep or Edep conditions does not seem to significantly impact the DNA coverage and

therefore the thiol-exchange process.

Figure 3: Fluorescence images obtained through iSEFMI of DNA SAMs prepared with DNA SW thiol-exchange (for 5 min) at a) Edep = +0.5V /SCE and (b) Edep ( +0.5V to -0.3V/ SCE, 50 Hz) with a stereographic triangle to label five surface crystallographies. c) measured fluorescence intensities for these crystallographic surfaces. Error bars correspond to standard deviation of two or more samples. Interestingly, the surface modified at Edep =+0.5V/SCE for 5 min is similar to the surface created after modification at Edep =+0.4V/SCE for 30 min, shown in our previous work. 11 In both cases, the DNA diffusion to the electrode surface will be similar as the electrophoretic 13


ACS Sensors 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

effect is negligible in high ionic strength buffer, which suggests that the more positive potential destabilizes or weakens the gold-thiol interaction (due to Cl – adsorption, or to oxidative desorption), significantly increasing the extent of thiol exchange. This destabilization is supported by the influence of thiols on the anodic oxidation of gold in Cl – containing electrolyte with dissolution starting at +0.6V/AgAgCl. 21 DNA coverage is not enhanced with applied square-wave potentials as compared to constant potential, rather influenced by the positive potentials applied in Cl – containing electrolyte.

Potential-Assisted DNA Deposition on a Clean Au Bead DNA electrochemical biosensors are typically prepared via the adsorption of thiol-modified DNA onto a clean Au surface done at open circuit potential. This is followed by thiolexchange with MCH to remove non-specifically adsorbed DNA (e.g., those not adsorbed via thiol moiety). 22 Previously, the influence of a constant potential during DNA adsorption onto clean gold was shown to increase DNA coverage in a short period of time (30 min rather than hours). 5 More recently, the use of a square wave deposition potential was employed 9,18 and the resulting surfaces were characterized with electrochemical impedance spectroscopy using Fe(CN)6

3 – /4 –

faradaic redox which indirectly measures the extent of DNA adsorption

or more accurately, the defects in the DNA SAM. A higher DNA coverage was reported for the SAMs prepared using 15 min of potential pulsing in phosphate buffers with 0.45M K2 SO4 as compared to OCP. Here, a comparison of the modified gold bead surfaces made at OCP, or using either Edep SW = +0.4 V/SCE or Edep = +0.4 V to -0.3 V/SCE (50Hz) during DNA adsorption onto clean

gold for 5 minutes was performed in a TRIS buffer containing 1.1M Cl – . Fluorescence images of DNA SAM modified surfaces (followed by immersion in MCH for 90 min) are shown in Figure 4a-c with fluorescence intensities from select crystallographic features compared in Figure4d. Surfaces made using a constant potential (Edep = +0.4 V/SCE) reveals a slight increase in coverage when compared to those prepared at OCP. In contrast to the thiol14


Page 14 of 24

Page 15 of 24 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

ACS Sensors

exchange method, SAMs of thiol-DNA on clean gold surfaces results in a more uniform distribution of DNA across the bead surface, with a corresponding increase in the total SW methods does DNA coverage. The difference between surfaces prepared using Edep or Edep SW method results in slightly higher fluorescence not appear to be significant though the Edep

intensity ( DNA coverage). The 111 and 311 regions show a statistically significant increase in fluorescence intensity when analyzing the results using a Student’s t-test with a confidence SW level of 80%. DNA adsorption for surfaces prepared using Edep appear to prefer the regions

around the 100 facet and the stepped regions surrounding the 111 facet. These regions are like miscut 111 or high index planes, similar to the substrate defects (e.g., grain boundaries) that would be present in vapor deposited gold film on glass or polycrystalline gold electrodes. Potential assisted deposition could significantly increase the average DNA coverage on those conventional substrates. Interestingly, the 311 surface region has the lowest fluorescence intensity in contrast to the thiol-exchange method demonstrating that the 311 surface is labile and thiol-exchange readily occurs there. In this case, a non-fluorescent thiol (MCH) is replacing a fluorescently labeled DNA, resulting in the fluorescence intensity decrease. The coverage of DNA is close to the maximum expected on this surface, so that differences may be difficult to observe. Further measurements using a lower concentration of DNA in the deposition buffer were also performed to reveal any differences.

Figure 4: DNA SAM formation on clean gold: Fluorescence images of DNA SAMs SW prepared on bare Au (for 5 min) at (a) OCP, (b) Edep =+0.4 V/SCE and (c) Edep = +0.4 V to -0.3 V/SCE (50Hz). A stereographic triangle is shown labeling the five surface crystallographies. d) measured fluorescence intensities for these crystallographic features. Error bars correspond to standard deviation of two or more samples. 15


ACS Sensors 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

Fluorescence images of gold beads modified with DNA SAMs prepared using potentialassisted deposition onto a clean Au bead surface with increasing concentrations of thiol SW modified DNA in the deposition solution are shown in Figure 5a and b for Edep and Edep SW methods result in the same increase in DNA coverage with respectively. Both Edep and Edep

increasing [DNA] in the deposition buffer. No significant differences in fluorescence intensity were observed between the two deposition methods for the range of concentrations studied for the five surface crystallographic regions selected (Fig. S3).

Figure 5: Fluorescence images of DNA SAMs made with DNA adsorption onto a clean Au SW bead electrode surface (for 5 min) using a) Edep =+0.4 V/SCE and (b) Edep = +0.4 V to -0.3 V/SCE (50Hz) with increasing concentrations of DNA in the deposition buffer.

Positive Potential Limit and DNA Deposition onto Clean Au Also studied was the deposition of DNA onto a clean gold bead electrode using a more positive SW potential. Edep =+0.5 V/SCE and Edep = +0.5 V to -0.3 V/SCE (50Hz) were applied for 5

min to clean gold electrodes in a DNA deposition buffer. After MCH treatment, the resulting surfaces were compared based on the fluorescence images (shown in Figure S4). Deposition at Edep = +0.5V results in a generally uniform and high DNA coverage SAM (comparison is 16


Page 16 of 24

Page 17 of 24 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

ACS Sensors

given in Fig 6) with lower coverage on the 311, as seen for Edep =+0.4V depositions. While SW an increase in fluorescence can be observed depending on the positive potentials used, Edep

did not result in a substantial increase in the DNA coverage. This may be a consequence of the saturation of fluorescence intensity (Fig S1b) which decreases the ability to measure the influence of the deposition potential for regions with a high DNA coverage. Other groups used this type of pulsed potential assisted DNA deposition on planar polycrystalline gold substrates and demonstrated a higher coverage than compared to OCP deposition based on their electrochemical impedance spectroscopy (EIS) measurents. 9,18 This conclusion is not evident using our fluorescence imaging approach, illustrating that each characterization technique seems to be sensitive to different aspects of the modified surface. While EIS is sensitive to defects and pinholes in the adsorbed layer, the fluorescence imaging is powerful for examining the distribution of coverage, especially in the lower surface coverage range. Nevertheless, our results can be used to explore the mechanism proposed for this enhanced SW DNA deposition when using Edep which has been suggested to be due to ion stirring effect

(increasing the transport of DNA to the surface). 18

Figure 6: Comparison of fluorescence intensities for DNA SAM formation on a clean gold bead electrode for five selected regions prepared for 5 min at OCP, Edep (+0.4V or +0.5V) SW and Edep (+0.4 V to -0.3 V/SCE (50Hz) or +0.5 V to -0.3 V/SCE (50Hz)).

17


ACS Sensors 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

Charging of the interface during Potential-Assisted DNA deposition SW The application of Edep during DNA deposition appears to less influential in the assembly

of DNA on the gold bead surface compared to Edep whether performed on bare gold or SW modulates the surface thiol modified gold surfaces as measured using fluorescence. Edep

charge density which manipulates the movement of ions near the electrode surface. This was proposed to be important in explaining increased DNA coverage in addition to using potential limits that are on either side of the potential of zero charge. 9 Since the pzc for clean gold is a function of crystallography, significant differences should be observed on the gold bead electrodes used in the present study. DNA deposition onto clean gold surface did not show an obvious correlation with the pzc, which suggests a different mechanism is at play. Furthermore, this can be supported SW by examining the amount of charge that is passed during the Edep method for the bare gold

surface and a MCH SAM covered gold surface, given that they have significantly different capacitances (~40 and 2 µF/cm2 respectively). SW The current during Edep was measured (at 5kHz) at the beginning and end of the 5 min

deposition. The charge passed for deposition via thiol-exchange and direct assembly on a clean gold surface are shown in Figure 7. The deposition solution was not free of O2 and may result in a slow negative drift in the charge depending on the surface coverage. The total magnitude of charge that moves through the interface for the MCH modified surface is significantly smaller (about 5%) when compared to the modification of the clean gold surface (Fig S5), as expected since the capacitance is lower. If charge modulation and ion stirring SW is causing an enhancement of DNA deposition, then Edep vs Edep would be significantly

different for DNA SAM formation on bare gold. This is not what was observed using our fluorescence imaging method in a high [Cl – ] buffer.

18


Page 18 of 24

Page 19 of 24 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

ACS Sensors

SW Figure 7: Charge measured over 0.2 seconds at the start and end of Edep deposition method using a) DNA thiol-exchange and b) DNA adsorption onto clean Au bead electrode.

Conclusions Different potential-assisted methods for rapid preparation of DNA SAMs (5 min) on single crystal gold bead electrodes were examined with in-situ spectroelectrochemical fluorescence microscopy for a consistent and reliable comparison. The use of a constant Edep or squareSW wave Edep for both thiol-exchange and direct assembly of DNA SAMs on clean gold surfaces

were studied. The application of a positive potential significantly influenced the DNA SAM coverage as compared to not applying a potential (OCP) for the thiol-exchange process. The DNA coverage clearly depended on the surface crystallography and all regions except for the 311 were strongly influenced by the application of potential. No significant difference was observed when comparing constant and square-wave potential deposition methods for negative potentials limited to -0.3V/SCE and positive potentials limited to +0.4V/SCE. The largest changes were observed when using a more positive potential (for either the constant potential or the positive potential limit of the square wave), which resulted in higher DNA coverage on the surface, and obvious dependence on the underlying substrate crystallography. The mechanism proposed to explain an improvement in DNA coverage through ion stirring was not supported in these measurements which may be due, in part, to the use of a supporting electrolyte that contains a large [Cl – ]. A defect mediated mechanism can uniformly explain the formation of the high coverage DNA SAMs and the influence of 19


ACS Sensors 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

the applied potentials, providing insight into the merit of electrodeposition of DNA SAMs for a facilitated, better controlled biosensor surface preparation.

Acknowledgement The work was supported by NSERC (Canada) Discovery and RTI grants.

Supporting Information Available Fluorescence intensity with increased DNA surface coverage, comparison of the DNA coverage for the 5 different deposition methods for thiol-exchange; fluorescence intensities for DNA SAM formation on a clean gold surface for increasing [DNA] in the deposition buffer; fluorescence images of the surfaces that result from electrodeposition onto clean gold electrodes using different values for the constant and step potentials; comparison of the total charge magnitude for DNA SAM formation by thiol-exchange and on a clean gold surface.

References (1) Huang, Y.; Xu, J.; Liu, J.; Wang, X.; Chen, B. Disease-Related Detection with Electrochemical Biosensors: A Review. Sensors 2017, 17, 2375–30. (2) Labib, M.; Sargent, E. H.; Kelley, S. O. Electrochemical Methods for the Analysis of Clinically Relevant Biomolecules. Chem. Rev. 2016, 116, 9001–9090. (3) Chao, J.; Zhu, D.; Zhang, Y.; Wang, L.; Fan, C. DNA nanotechnology-enabled biosensors. Biosens. Bioelectron. 2016, 76, 68–79. (4) Xiao, Y.; Lai, R. Y.; Plaxco, K. W. Preparation of electrode-immobilized, redoxmodified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat. Protoc. 2007, 2, 2875–2880. 20


Page 20 of 24

Page 21 of 24 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

ACS Sensors

(5) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. The effect of surface probe density on DNA hybridization. Nucleic Acids Res 2001, 29, 5163–5168. (6) Gong, P.; Levicky, R. DNA surface hybridization regimes. Proc. Natl. Acad. Sci. USA 2008, 105, 5301–5306. (7) White, R. J.; Phares, N.; Lubin, A. A.; Xiao, Y.; Plaxco, K. W. Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry. Langmuir 2008, 24, 10513–10518. (8) Ma, F.; Lennox, R. B. Potential-Assisted Deposition of Alkanethiols on Au: Controlled Preparation of Single- and Mixed-Component SAMs. Langmuir 2000, 16, 6188–6190. (9) Jambrec, D.; Gębala, M.; La Mantia, F. M. F.; Schuhmann, W. Potential-Assisted DNA Immobilization as a Prerequisite for Fast and Controlled Formation of DNA Monolayers. Angew. Chem. Int. Ed. 2015, 54, 15064–15068. (10) Quan, X.; Heiskanen, A.; Tenje, M.; Boisen, A. In-situ monitoring of potential enhanced DNA related processes using electrochemical quartz crystal microbalance with dissipation (EQCM-D). Electrochem. Commun. 2014, 48, 111–114. (11) Leung, K. K.; Gaxiola, A. D.; Yu, H. Z.; Bizzotto, D. Tailoring the DNA SAM surface density on different surface crystallographic features using potential assisted thiol exchange. Electrochim. Acta 2018, 261, 188–197. (12) Casanova-Moreno, J.; Yu, Z. L.; Massey-Allard, J.; Ditchburn, B.; Young, J. F.; Bizzotto, D. Luminescence in Electrochemistry; Springer International Publishing: Cham, 2017; pp 21–77. (13) Yu, Z. L.; Casanova-Moreno, J.; Guryanov, I.; Maran, F.; Bizzotto, D. Influence of Surface Structure on Single or Mixed Component Self-Assembled Monolayers via in Situ

21


ACS Sensors 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

Spectroelectrochemical Fluorescence Imaging of the Complete Stereographic Triangle on a Single Crystal Au Bead Electrode. J. Am. Chem. Soc. 2015, 137, 276–288. (14) Murphy, J. N.; Cheng, A. K. H.; Yu, H. Z.; Bizzotto, D. On the Nature of DNA SelfAssembled Monolayers on Au: Measuring Surface Heterogeneity with Electrochemical in Situ Fluorescence Microscopy. J. Am. Chem. Soc. 2009, 131, 4042–4050. (15) Kassam, A.; Bremner, G.; Clark, B.; Ulibarri, G.; Lennox, R. B. Place Exchange Reactions of Alkyl Thiols on Gold Nanoparticles. J. Am. Chem. Soc. 2006, 128, 3476–3477. (16) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Dynamics of Place-Exchange Reactions on Monolayer-Protected Gold Cluster Molecules. Langmuir 1999, 15, 3782–3789. (17) Schlenoff, J. B.; Li, M.; Ly, H. Stability and Self-Exchange in Alkanethiol Monolayers. J. Am. Chem. Soc. 1995, 117, 12528–12536. (18) Jambrec, D.; Conzuelo, F.; Estrada-Vargas, A.; Schuhmann, W. Potential-PulseAssisted Formation of Thiol Monolayers within Minutes for Fast and Controlled Electrode Surface Modification. ChemElectroChem 2016, 3, 1484–1489. (19) Yu, H. Z.; Luo, C.-Y.; Sankar, C. G.; Sen, D. Voltammetric Procedure for Examining DNA-Modified Surfaces: Quantitation, Cationic Binding Activity, and ElectronTransfer Kinetics. Anal. Chem. 2003, 75, 3902–3907. (20) Fisher, E. A.; Leung, K. K.; Casanova-Moreno, J.; Masuda, T.; Young, J.; Bizzotto, D. Quantifying the Selective Modification of Au(111) Facets via Electrochemical and Electroless Treatments for Manipulating Gold Nanorod Surface Composition. Langmuir 2017, 33, 12887–12896. (21) Smith, S. R.; Guerra, E.; Siemann, S.; Shepherd, J. L. Au dissolution during the anodic response of short-chain alkylthiols with polycrystalline Au electrodes. Electrochim. Acta 2011, 56, 8291–8298. 22


Page 22 of 24

Page 23 of 24 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

ACS Sensors

(22) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. Using Self-Assembly To Control the Structure of DNA Monolayers on Gold: A Neutron Reflectivity Study. J. Am. Chem. Soc. 1998, 120, 9787–9792.

23


ACS Sensors 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

Graphical TOC Entry

24


Page 24 of 24

Electrodepositing DNA Self-Assembled ... - ACS Publications

Recommend Documents