Biosensors and Bioelectronics 56 (2014) 328–333

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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

A regenerating self-assembled gold nanoparticle-containing electrochemical impedance sensor Amr M. Mahmoud a, Thompson Tang b,1, D. Jed Harrison a,c, William E. Lee b, Abebaw B. Jemere c,n a

Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2 Defence Research & Development Canada-Suffield Research Centre, Medicine Hat, AB, Canada T1A 8K6 c National Institute for Nanotechnology, National Research Council Canada, 11421 Saskatchewan Drive, Edmonton, AB, Canada T6G 2M9 b

art ic l e i nf o

a b s t r a c t

Article history: Received 7 November 2013 Received in revised form 2 January 2014 Accepted 13 January 2014 Available online 23 January 2014

We report on the development of an electrochemical reductive desorption protocol for repeated regeneration of gold electrodes modified with multi-layers of self-assembled surfaces for use in electrochemical sensing. The gold electrodes were first modified with 1,6-hexanedithiol to which gold nanoparticles were attached in a subsequent modification step. Attachment of thiolated single-stranded nucleic acid oligomers to the gold nanoparticles completed the electrochemical sensor. The changes of electrode behavior after each assembly and desorption processes were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques. The self-assembled sensor showed a wide dynamic range (0.1–100 nM), a low detection limit (20 pM) and high reproducibility (4.4% RSD) for complementary nucleic acid target molecules, along with reusability. On a single gold electrode, the complete sensor–target structure could be assembled and disassembled at least four times with 90% of its original signal intact. & 2014 Elsevier B.V. All rights reserved.

Keywords: Self-assembly Electrochemical impedance spectroscopy Cyclic voltammetry Electrode regeneration Gold nanoparticles modified electrodes

1. Introduction Electrochemical impedance spectroscopy (EIS) provides a powerful yet simple method for measuring changes in bulk or interfacial properties of materials on an electrode surface, including surfaces sensitive to molecular recognition events (Barsoukov and Macdonald, 2005; Wang et al., 2012; K'Owino and Sadik, 2005; Drummond et al., 2003; Katz and Willner, 2003; Lisdat and Schafer, 2008; Chang and Park, 2010). A major advantage of EIS is that detection can be performed label-free; i.e., the changes in the electrical properties of the electrode surface arise from the interaction with the target molecule alone. However, electrode surfaces typically suffer degradation with use in complex samples that require cleaning procedures or electrode replacement. While these steps are convenient in a laboratory, they are less so in a standalone, field deployable unit. In this work, we develop a sequence of self-assembly steps designed to allow the sensor layer to be used once, cleaned from the electrode surface, and then reassembled for the subsequent measurement. A simple molecular recognition system, surface immobilized 20-mer polydeoxyadenine (dA20) with complementary 20-mer polydeoxythymine (dT20) as target, was employed for demonstration. Signal transduction

n

Corresponding author. Tel.: þ 1 780 641 1712; fax: þ1 780 641 1601. E-mail address: [email protected] (A.B. Jemere). 1 Present address: AirBoss-Defense, 28A, boul. de l’Aéroport, Bromont, QC, Canada J2L 1S6. 0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2014.01.019

was obtained from the electron-transfer resistance of the redox couple, [Fe(CN)6]3
/4
, through the modified surface. Surface modification of electrodes is a critical area for electrochemical sensor development (Lisdat and Schafer, 2008; Chang and Park, 2010; Matharu et al., 2012; Oliveria et al., 2011; Love et al., 2005; Ding et al., 2005; Canaria et al., 2006; Qu et al., 2009; Prashar, 2012). It imparts insulation and orientation to the surface architecture of the electrodes, and can enhance reproducibility in the presence of fouling agents in a sample. The modification process herein was initiated by a self-assembled monolayer (SAM) of 1,6hexanedithiol (1,6-HDT) to which gold nanoparticles (Au NPs) were linked via the peripheral thiol group exposed to solution, followed by attachment of 30 thiol dA20. The Au NPs have the potential for enhancing the sensitivity of the sensor by increasing the surface area of an electrode (Wang et al., 2011; Zhang et al., 2008; Liu et al., 2010; Yamada et al., 2003; Jena and Raj, 2007). Long term stability of SAMs on gold electrodes has been shown to be a challenge in sensor design (Yan et al., 2006; Nishida et al., 1996; Shadnam and Amirfazli, 2005; Lee et al., 2004; Schoenfisch and Pemberton, 1998; Cooper and Legget, 1998; Cometto et al., 2012). For example, hexanethiol SAMs on Au surfaces were shown to be unstable above 30 1C (Cometto et al., 2012). Further, when molecular recognition via DNA hybridization is utilized for sensing on a SAM/Au assembly, the temperature required for melting the hybrid ( 80 1C) to regenerate an active sensing surface has been shown to cause loss of  20% of the SAM from the gold electrode (Cometto et al., 2012). In response to this problem, for many

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biochemical sensing applications the SAM modified electrode is discarded after use (Yan et al., 2006). In order to reuse the underlying electrode, a variety of SAM removal and surface cleaning methods have been employed. These include ion sputtering (Chenakin et al., 1999), UV–ozone cleaning (English et al., 2000), and chemical treatment (Schneider and Buttry, 1993) by either strong oxidant (e.g. piranha) or reducing agents (e.g. NaBH4). Electrochemical oxidation and reduction techniques have also been reported for removing SAMs from electrode surfaces (Canaria et al., 2006; Loglio et al., 2003; Bhalla et al., 2010; Jiang et al., 2003; Bullen et al., 1998; Sondag-Huethorst and Fokkink, 1995; Ferreira et al., 2009) and were selected here as they are well suited for cleaning electrodes embedded in microfluidic devices used for long term monitoring. Two related self-assembled sensor designs have been developed elsewhere to increase the amount of immobilized DNA probe and enhance the sensitivity of the sensor (Zhang et al., 2008; Liu et al., 2010). One (Liu et al., 2010) used a thiol/Au NP/thiolated DNA structure as similarly employed here, with high temperature dehybridization leading to fairly rapid electrode performance loss. The other approach (Zhang et al., 2008) eliminated the first thiol layer, and used NaOH to dehybridize the probe–target duplex, but reproducibility of the regenerated sensor performance was not described. Here we have taken a maximal approach with removal of the deposited materials using electrochemical reductive desorption to restore a bare Au electrode, with subsequent reassembly

329

of the entire sensor architecture. The effectiveness of the regeneration was studied electrochemically through cyclic voltammetry (CV), EIS and by X-ray photoelectron spectroscopy. The work presented here employs a number of characteristics of good sensor design in a single system such as self-assembly of the sensor, label-free all-electronic signal transduction, miniaturized electrodes, electrochemical regeneration of the sensor surface and high reproducibility. To our knowledge this is the first report wherein all these characteristics have been executed together to produce a complete regenerating sensor device based on molecular selfassembly and electrochemical detection. The overall challenge in sensor development is in good design, drawing together the technologies that will permit seamless function in the analytical process.

2. Experimental 2.1. Chemicals, solvents and materials K4[Fe(CN)6], K3[Fe(CN)6], KNO3, 1,6-hexanedithiol (1,6-HDT), and gold nanoparticles (diameter 10 nm, 0.75 A520 units/ml) were purchased from Sigma-Aldrich Canada (Oakville, ON). Anhydrous ethyl alcohol was obtained from Commercial Alcohols Inc. (Brampton, ON). Deionized water having a resistivity of 18 MΩ cm (Milli-Q UV Plus Ultra-Pure Millipore System) was used for aqueous

1

5 2 20

3

20

4

Scheme 1. Schematic representation of the regenerable self-assembled sensor fabrication process.

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solutions preparation and rinsing electrodes. Single-stranded DNA oligomers were purchased from Integrated DNA Technology Inc. (Coralville, IA): thiolated probe 50 -AAA AAA AAA AAA AAA AAA AA/ 3ThioMC3-D/-30 (dA20-thiol); target 50 -TTT TTT TTT TTT TTT TTT TT30 (dT20); non-complementary 50 -AAA AAA AAA AAA AAA AAA AA30 (dA20). The buffer solution for stock DNA was Tris–EDTA (10 mM Tris, 0.1 mM EDTA) pH 8.0; the hybridization buffer was 10 mM phosphate buffer/1 M NaCl, pH 7.4. 2.2. Gold electrode fabrication and biosensor preparation Au electrodes were fabricated on glass slide substrates (2.5  7.5  0.1 cm3) at the University of Alberta NanoFab (Edmonton, AB). Glass slides were first cleaned in piranha solution (a 3:1 ratio of H2SO4 and H2O2) for 30 min, then rinsed with ultrapure water and dried in a stream of nitrogen. The cleaned glass slides were coated with Ti/Au (5 nm/100 nm) using an electron beam evaporator (Kurt J. Lesker Co., Clairton, PA), evacuated to a base pressure of 4  10
7 Torr and a metal shadow mask to define the electrode geometric area (0.28 cm2). As described in Scheme 1, freshly deposited Au electrodes were immersed in 1 mM 1,6-HDT in ethanol for 2 h for self-assembly of the thiol layer. Au NPs were then self-assembled on the 1,6-HDT/ Au electrodes by immersing in a solution of Au NPs for 1 h. The Au/ 1,6-HDT/Au NP electrodes were further modified with 200 μl of 1 μM 30 -thiol-dA20, in Tris–EDTA buffer (pH 8.0), for 1 h to produce the sensor surface. Lastly, to the sensor surface, an aliquot of 200 μl of target dT20 was allowed to hybridize for 2 h. Electrodes were thoroughly rinsed with water and dried in a stream of nitrogen before electrochemical measurements. 2.3. Electrochemical measurements Cyclic voltammetry and electrochemical impedance measurements were made using a reference 600 Potentiostat (Gamry Instruments Inc., Warminster, PA). The working electrode was the Au electrode (Ti/Au) on glass slide. The reference electrode was an Ag/AgCl electrode (3 M NaCl saturated with AgCl) and Pt wire was used as the counter electrode. The redox couple was 1 mM K4[Fe(CN)6]/K3[Fe(CN)6] in 0.1 M KNO3 solution. In CV experiments, the scan rate was 100 mV s
1. For EIS experiments, the frequency was scanned from 0.1 Hz to 10 kHz, the ac amplitude was 10 mV at 0.28 V vs Ag/AgCl reference electrode. The electrochemical impedance data was modeled to a Randles equivalent circuit using Gamry-supplied software to estimate the value of circuit components.

NPs to complete the sensor surface. The target, dT20, was allowed to hybridize with the dA20 probe and the impedance of the sensor surface was monitored by EIS using [Fe(CN)6]3
/4
as the redox couple. Finally, the Au electrode surface was electrochemically regenerated by reductive desorption of the self-assembled monolayers and the steps of biosensor building were repeated. 3.1. Electrochemical characterization of the modification steps CV and EIS were used to characterize the self-assembled surfaces. Fig. 1 shows the CVs of three prepared electrode surfaces: bare Au electrode, 1,6-HDT SAM modified Au electrode and Au NP modified 1,6-HDT/Au electrode, in 1 mM [Fe(CN)6]3
/4
with 0.1 M KNO3 as the supporting electrolyte. The bare Au electrode shows redox response with peak-to-peak separation (ΔEp) of 0.11 V for the redox couple, indicating that the electron transfer reaction is quasi-reversible and diffusion controlled. In contrast, for the 1,6-HDT modified Au electrode, the CV exhibits a significantly inhibited redox current, indicating that a relatively compact monolayer of 1,6-HDT formed on the electrode surface preventing ready electron transfer of the redox couple to the electrode surface. Subsequent modification of the 1,6-HDT/Au electrode with Au NPs shows quasi-reversible voltammetry of [Fe(CN)6]3
/4
, with ΔEp of 0.12 V, similar to that of the bare gold electrode. We can thus infer that the Au NPs not only provided the necessary conduction pathways, but also acted as nanoscale electrodes in promoting the electron transfer between the electrode surface and the redox mediator. Fig. 2 shows the Nyquist plots of the same three electrode surfaces; the inset in Fig. 2 shows a standard Randles equivalent circuit to which the impedance data were fitted to obtain the values of circuit elements of the modified electrode surfaces. The Nyquist plot of a bare Au electrode exhibited an almost straight line, with a calculated solution resistance, Rs, of 101.3 Ω and electrode double layer capacitance, Cdl, of 9.1 μF, indicating a diffusion limited electrochemical process. The Nyquist plot of a 1,6-HDT SAM modified electrode shows a well-defined semicircle at higher frequencies, with a charge transfer resistance, Rct, of 3.5  103 Ω and Cdl of 4.5 μF. Assembly of the dithiol on the electrode surface inhibited electron transfer from the redox couple to the Au electrode surface (see Section 3.2). Subsequent modification of the 1,6-HDT SAM layer with Au NPs resulted in a straight line at lower frequency and a very small semicircle in the

1.5x10-4 1.0x10-4

Regeneration of the Au electrode surface was carried out by electrochemical reductive desorption of the self-assembled monolayers by applying a constant negative potential (
1.1 V) to the modified Au electrode vs Ag/AgCl reference electrode in 0.1 M KNO3. The negative bias was applied for three repeated cycles, 60 s each. Regenerated Au surfaces were characterized by CV, EIS and X-ray photoelectron spectroscopy (AXIS 165 Spectrometer, Kratos Analytical Inc., Spring Valley, NY).

5.0x10-5

3. Results and discussion

i (A)

2.4. Biosensor surface regeneration

Au

0.0 -5.0x10-5

Au/1,6-HDT -1.0x10-4 -1.5x10-4

-0.2 The steps used to fabricate the self-assembled surfaces are presented in Scheme 1. First, the Au electrode was modified by 1,6-HDT to yield a self-assembled monolayer. This modification with dithiol permits the self-assembly of Au NPs on the peripheral free thiol groups. The thiolated dA20 probe was then self-assembled on the Au

Au/1,6-HDT/AuNP

0.0

0.2

0.4

0.6

0.8

E (V) vs Ag/AgCl Fig. 1. Cyclic voltammograms of bare Au electrode, 1,6-hexanedithiol modified Au electrode, and Au/1,6-HDT/Au NP electrode, at a scan rate of 100 mVs
1 in 1 mM K4[Fe(CN)6]/K3[Fe(CN)6] containing 0.1 M KNO3 as supporting electrolyte using Ag/ AgCl as reference electrode and Pt wire as counter electrode.

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Cdl

331

4000

Rs ZW

3000

R ct

Z" (ohm)

5000 4000

Z" (ohm)

Au/1,6-HDT Au

3000

2000

Au/1,6-HDT/AuNP

1000

2000

Au/1,6-HDT/AuNP/dA20/dT20 Au/1,6-HDT/AuNP/dA20

1000

0 0

0 0

4000

8000

2000

4000

6000

8000

10000

Z' (ohm)

12000

Z' (ohm) Fig. 2. Nyquist plot of bare Au electrode, 1,6-hexanedithiol modified Au electrode, and Au/1,6-HDT/Au NP electrode. Frequency for impedance measurement was varied from 0.1 Hz to 10 kHz; all other experimental conditions are as in Fig. 1. Inset shows the Randles equivalent circuit employed for all fits. Rs is the solution resistance; Cdl is the electrode double layer capacitance; Zw is the Warburg impedance and Rct is the charge transfer resistance at the electrode interface.

Fig. 3. Nyquist plots of the self-assembled sensor (Au/1,6-HDT/AuNP/thiolated dA20) and after 2 h hybridization with 200 μl of 1 μM target dT20, All other experimental conditions are as in Fig. 2.

1.0x10-4

bare Au electrode

3.2. Desorption of thiol SAM and regeneration of Au electrode surfaces The potential required for the reductive desorption of thiol SAMs depends on the length of the alkyl chain and the type of terminal groups (Imabayyashi et al., 1997; Anderson and Baltzersen, 2003). In order to study the desorption of 1,6-HDT SAM on Au surfaces, the voltage applied to the modified electrode was scanned towards the negative potential vs Ag/AgCl electrode, Fig. 4. The large cathodic peak at 
0.94 V was attributed to the desorption of 1,6-HDT through the reduction of Au–S bond. The absence of an oxidation peak in the anodic scan implies the absence of re-adsorption of the desorbed product. The surface coverage (Γ) by the SAM was determined by integrating the charge (Q) associated with the reductive desorption peak in Fig. 4 where Γ ¼ Q =nFA

ð1Þ

0.0 -1.0x10-4

i (A)

higher frequency region (with Rct of 0.6  103 Ω and Cdl of 7.0 μF), indicating that self-assembly of Au nanoparticles on Au/1,6-HDT electrode facilitated the charge transfer between the electrode and redox couple in solution. This behavior is similar to what has been reported for gold nanoparticles modified electrodes and was attributed to formation of efficient charge transfer pathways formed by adsorption of Au NPs (Shein et al., 2009; Wang et al., 2006; Lu et al., 2002). Fig. 3 shows the Nyquist plot of the sensor surface, i.e., thiolated dA20 tethered to Au/1,6-HDT/AuNP. The attachment of thiolated dA20 to the NPs revealed a semicircle domain with an Rct of 2.4  103 Ω. The increase in Rct is probably due to the DNA inhibiting electron transfer. Hybridization of target dT20 to the sensor resulted in a further increase in Rct value (6.3  103 Ω). As a control EIS experiment, a non-complementary target (non-thiolated dA20) was incubated with the probe-modified electrode. There was essentially no change in Rct value for the control experiment compared to the probe-modified electrode. The Rct of the sensor surface also remained unchanged upon incubation with the hybridization buffer alone (Supplementary Fig. 1). These results indicated that the signal observed in Fig. 3 is due to the hybridization of the probe dA20 and the target dT20.

modified Au electrode

-2.0x10-4 -3.0x10-4 -4.0x10-4 -5.0x10-4 -1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

E (V) vs Ag/AgCl Fig. 4. Cyclic voltammograms of bare Au electrode and 1,6-HDT modified Au electrode at a scan rate of 100 mV s
1. All other experimental conditions are as in Fig. 1. Total charge was determined by the area under the redox peak.

and n is the number of electrons involved in the electrode reaction, F is Faraday's constant and A is the electrode surface area. The surface coverage calculated by Eq. (1) was 4  10
10 mol cm
2, an amount similar to literature values for compact n-alkane thiols and dithiols (Ferreira et al., 2009; Balasubramanian et al., 2006; Tkac and Davis, 2008; Walczak et al., 1991; Yang et al., 1997). A simple packing model for n-alkane thiol based on van der Waals radii gave a coverage of 3.7  10
10 mol cm
2 (mathematical calculation not shown). The similar CV experiment for the bare gold electrode, Fig. 4, showed a cathodic peak at a reductive potential o
1.1 V, which was attributed to evolution of hydrogen gas. An optimized protocol for total reductive desorption of 1,6HDT SAM from the Au electrode entailed the application of a fixed cathodic potential (
1.1 V) for three intervals of 60 s each (data not shown). The optimized reductive desorption protocol was able to regenerate the Au electrode and restore near original oxidation– reduction of the redox couple (Supplementary Fig. 2a). After 10 cycles of monolayer formation/desorption, 4 91% of both the current and total charge was recovered. These observations were supported by the EIS data whereby the impedance spectrum of the 10-cycle regenerated electrode was similar to that of pristine Au

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10000

Δ Rct(ohm)

8000

6000

6000 4000 2000 non-complemmentary target

0 -10

5000 4000

Z" (ohm)

-8

-6

-4

Log [dT20]

1 μM 0.1 μM 10 nM 1 nM 0.1 nM

3000 2000 1000 0 0

2000

4000

6000

8000

10000

12000

Z' (ohm) Fig. 5. Nyquist plots of the self-assembled sensor surface after hybridization (2 h) with various concentrations of the target dT20. Inset is a calibration plot of Rct of the sensor before and after hybridization vs logarithm of target concentration. All experimental conditions are as in Fig. 3.

electrode (Supplementary Fig. 2b). Together, the CV and EIS results indicated that reductive desorption was highly effective in removing 1,6-HDT SAM from Au electrodes and that the Au electrode could be used repeatedly, at least 10 times, for the self-assembly of thiols to fabricate sensor surfaces. In addition to electrochemical characterization of the recycled Au electrodes, high resolution XPS data was obtained for the selfassembled sensor, Au/1,6HDT/Au-NP/thiolated dA20, and for the same Au electrode after electrochemical reductive desorption. The absence of P2p signal from dA20 phosphate residues at 133 eV (Gong et al., 2006; Lee et al., 2007) in the regenerated Au electrode surface provided further evidence that the electrochemical reductive process was effective in removing the selfassembled thiol layers from the Au surface (Supplementary Fig. 3). 3.3. Analytical performance and reusability of the sensor surface Fig. 5 shows Nyquist plots of the sensor surfaces after hybridization with different concentrations of the target, dT20. The diameter of the semicircle increased upon hybridization with increasing concentration of dT20 before reaching saturation at 1 μM target. The plot of the analytical signal, ΔRct (ΔRct ¼Rct(dA20
dT20duplex)
Rct(dA20)), vs log[dT20] was linear over three decades concentration (0.1– 100 nM, inset Fig. 5). Three independent electrodes were processed in parallel for the acquisition of each data point in the calibration curve and for the baseline sample blank (dashed line inset of Fig. 5). The standard deviation was about 4.4%. The sample blank was 1 μM non-complementary dA20. The theoretical limit of detection, taken as the baseline (mean ΔRct(blank)) plus 3 standard deviations, was determined to be 20 pM. This limit of detection is comparable or superior to reported values for EIS DNA biosensors (0.5 nM (Liu et al., 2011), 1 nM (Keighley et al., 2008), 4.7 nM (Ben-Yoav et al., 2012), 10 pM (Kannan et al., 2011), 3.8 pM (Li et al., 2007), 1 pM (Wang et al., 2011; Liu et al., 2010), 0.67 pM (Zhang et al., 2008)). The reusability of the electrodes and reproducibility of the selfassembled sensor surfaces were measured following four cycles of reductive desorption and self-assembly (at the same cathodic potential as used to desorb SAM, 1.1 V). For hybridization experiments similar to that of Fig. 5, the regenerated electrode surfaces

retained about 90% of their initial ΔRct values for probe–target duplex (tested 0.1 nM and 1 nM dT20).

4. Conclusions The work presented here demonstrates the utility of selfassembly in sensors and electrode-based biomolecular research. Starting with a bare gold electrode we were able to construct electrochemical sensors from alkane dithiol SAMs topped with a layer of 10 nm gold nanoparticles. EIS analysis indicated high electrical insulation (i.e., electron transfer resistance) by the 1,6hexanedithiol monolayer and a subsequent decrease in electron transfer resistance when Au Nps were introduced. Molecular recognition elements were incorporated onto the NP layer through self-assembly to complete the sensor structure. The architecture of the sensor layering provided ease of construction, good electrochemical performance and a detection limit of 20 pM for complementary nucleic acid target. Regeneration of the bare gold electrode surface was accomplished in situ by reductive desorption of the dithiol layer followed by reconstruction of the surface modifications. The dithiol SAMmodified electrode surface could be assembled and disassembled at least 10 times with minimal degradation in electrochemical performance. A similar regeneration protocol for the complete sensor–target surface showed high reproducibility. Overall there was inherent robustness in the electrode fabrication and the physical chemical assembly and disassembly processes which allowed for the creation of a complete regenerating sensor device based on molecular self-assembly and electrochemical detection. This combination of molecular self-assembly and electrochemical impedance spectroscopy is an attractive concept for future chemical sensors research.

Acknowledgment The authors would like to thank Dimitre Karpuzov at the University of Alberta Center for Surface Engineering and Science

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