A simple, effective NADH sensor constructed with phenothiazine via AFM tip-induced oxidative polymerization

Journal of Electroanalytical Chemistry 677–680 (2012) 78–82

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A simple, effective NADH sensor constructed with phenothiazine via AFM tip-induced oxidative polymerization Yu Ming Chiang, Hsiang Ying Huang, Chong Mou Wang ⇑ Department of Chemistry, National Taiwan Normal University, Taipei 116, Taiwan

a r t i c l e

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Article history: Received 28 February 2012 Received in revised form 13 April 2012 Accepted 23 April 2012 Available online 29 May 2012 Keywords: AFM tip (field)-induced local oxidation Poly(phenothiazine) Microlithography Chemical sensor NADH

a b s t r a c t In this work, we demonstrate a simple, effective NADH sensor developed with phenothiazine compounds via atomic force microscope (AFM) tip (field)-induced local oxidation (ALO). When voltage pulses were applied with an AFM tip that was stationary above thionine (TH) on an indium-doped tin oxide (ITO) glass square, the monomer transformed into nanodomes immediately on the site. The resulting polymer (denoted poly(TH)) was highly symmetrical in shape and varied indifferently with the pulse width from 0.01 to 1 s, indicating that the field-induced polymerization was fast in kinetics, reaching completion in less than 0.01 s. Water was essential to the formation of poly(TH). However, hot electrons rather than oxyanions were the oxidants responsible for the polymerization. The ALO-induced polymerization showed potential for application in microlithography. In addition, when poly(TH) nanoline was positioned via ALO with a moving tip between a pair of source and drain electrodes prefabricated on ITO, separated by a 200 nm-wide microfluidic channel, the resulting device showed responses to NADH when NADH was injected through the channel. The sensitivity varied with the voltage applied to the drain (relative to the source), reaching the optimum condition near 0.5 V. Under this condition, the lowest detection limit for NADH reached a level around 1 lM. Toluindine blue and methylene blue also showed similar effects with NADH when substituted for TH. This simple device shows that ALO and phenothiazines are a promising approach for constructing NADH sensors. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Since its success in patterning non-conducting metal oxides on metal surfaces [1], atomic force microscope (AFM) tip (field)induced local oxidation (ALO) has been recognized as a promising approach for the development of nanoelectronic devices [2–4]. With multi-functional molecules as the ink [5], the application potential of ALO has been more diversified than expected [6,7]. Herein, we report that a simple, effective chemical sensor can be constructed based on ALO and phenothiazines for the transduction of nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH). NAD+ and NADH are essential electron-transfer mediators in biological systems [8,9]. However, they suffer from electrontransfer barriers at most electrodes as applied in biofuel cells [10]. Phenothiazines, such as thionine (TH), toluidine blue (TB) and methylene blue (MB), can reduce the overvoltage for NAD+/ NADH in solution or when surface-bound [5,11]. In addition, they can also serve as ink molecules for ALO-based lithography. An NADH transducer was thus developed. Experimental results

showed that when TH was positioned between a pair of source and drain electrodes, it could function as a chemical gate, transducing NADH and NAD+. The detection limit for NADH could reach a level around 1 lM. These results highlight that ALO is a simple, effective means for the construction of biocompatible nanoelectronic devices. 2. Experimental 2.1. Chemicals Thionine (TH, acetate salt), methylene blue (MB, chloride salt), toluidine blue O (TB, chloride salt), NAD+, and NADH were purchased from Aldrich, and used as received without further purification. ITO glass squares (0.7 mm thick, 20 X/square; nominal coating thickness, 150–300 Å) were supplied by Delta Technologies. Before use, the ITO squares were thoroughly rinsed with 1 M sulfuric acid, chloroform, and deionized water. 2.2. ALO modifications

⇑ Corresponding author. Tel.: +886 2 77346127; fax: +886 2 29324249. E-mail address: [email protected] (C.M. Wang). 1572-6657/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2012.04.031

Prior to ALO, TH, TB and MB (0.1 mM in water) were first spin-coated on ITO squares (0.5  0.5 cm2) and then allowed to

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dry under nitrogen. The deposited monomers were determined to be 10–20 nm in thickness by AFM shoveling experiments; the roughness was around 7 nm. For ALO lithography, voltage pulses (Vtip: 0 to 12 V) with various widths (0.01–10 s) were applied with the AFM tips (conductive mode) stationary above the resultant ITO squares (grounded). Before each pulse, the tip was moved to a new position. Unless otherwise stated, most ALO experiments were performed at Vtip = 6 V due to the fact that the tips corroded easily upon ALO, especially under the conditions |Vtip| > 7 V. After ALO, the ITO squares were rinsed with water and alcohol to remove the unreacted monomer. For the phenothiazine-based microfludic devices, polyphenothiazine nanolines (0.2  1 lm2) were prepared in a similar manner except that the AFM tips were allowed to move at a constant speed (2 nm s1) under a designated bias (typically, 6 V). The ITO squares employed in this case were pre-segregated into two parts with a 0.2 lm-wide, 0.1 lm-deep groove achieved by removing the upper ITO coating using an electron-beam lithographic technique to function as the source and drain electrodes, respectively. Ohmic contact was made with a silver paint (Alfa Products). The resulting ITO squares were then covered with polyacrylate sheets (5 mm thick) and sealed with epoxy adhesive (Georgia Tech.). The polyacrylate covers were pre-drawn a groove (1  1 mm2) on the side facing the ITO to form a microfluidic channel with the groove underneath, as shown in Fig. 1 (not to scale). The entire assembly was then sawed off to be 5 mm in length and locked into a stainless steel holder equipped with a home-built microfludic pump (Max. pressure: 2000 kPa; Max. flow rate: 0.1 lL/min) linked with stainless steel tubing, swageloks and valves. For NAD+ and NADH analyses, samples (5 lL) were spiked into the system through an injection valve (dead volume: 20 lL) with a 10-lL microsyringe. 2.3. Apparatus An AFM microscope (Nanoscope III E, Digital Instrument Corp.) with a 10-lm scanner was employed for surface imaging and ALO experiments. The experiments were implemented with a TiNcoated conductive tip (AIST/fpC10/TiN; spring constant: 0.1 N/m; width: 10 nm: contact mode). A home-built preamplifier, with a sensitivity of 1 nA/V and operational range from 1 pA to 10 nA, was used for current–voltage (I–V curves) measurements. Typically, the voltage was scanned from 3 V to 3 V against the grounded substrate at a constant speed (0.5 Hz s1, equivalent to 2.6 V s1). Microfluidic devices were manufactured using the National Taiwan Normal University microfabrication facility. Humidity was controlled by a home-built humidity controller. 3. Results and discussion 3.1. ALO-induced polymerization When voltage pulses (6 V) were applied with an AFM tip stationary over TH on ITO squares, nanodomes (denoted

Fig. 1. Schematic illustrations for an NADH transducer fabricated based on poly(TH) via ALO.

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poly(TH)) appeared on the sites, as shown in Fig. 2. According to the inset section profiles, the resultant formation is highly symmetrical in shape (height: 20 nm and width: 180 nm). We ascribe the formation of the nanostructure to the oxidative polymerization of TH and the symmetrical shape to an electric field uniformly distributed between the tip and the substrate. Notably, the shape did not vary with the pulse width (tALO) from 10 ms to 1 s at a constant humidity (89%). This result implies that the polymerization is fast in kinetics, that is, reaches completion in less than 10 ms, compatible with the kinetics observed for silicon during ALO [2]. Fig. 3 shows that increasing humidity and voltage could promote the formation of poly(TH), similar to the those observed for toluidine blue [5], silicon [2] and gallium arsenide [3]. The humidity effect indicates that water is essential to the polymerization. The voltage effect, however, implies that the poly(TH) may involve a polymerization mechanism different from that for silicon and gallium arsenide, because the poly(TH) particles tended to a limiting height as |Vtip| > 10 V and a threshold existed for their formation (onset voltage, ca 4 V). For silicon and gallium arsenide, the oxides caused by ALO increased in height linearly with Vtip with no clear thresholds. Reactive oxygen species, such as oxyanions, are likely the oxygenation sources [3], because these species generated in the water bridge beneath the AFM tip increase with the applied voltage in number. In fact, hot electrons can also be generated and participate in ALO. Once accelerated, the electrons can function as a second oxidation channel by injecting secondary electrons from the substrate. This mechanism depends on the energetics of the substrate. Voltage threshold is thus expected, for instance, the energy threshold for silicon is around 10 V [2,12]. Considering these mechanisms and the results obtained in our case, the polymerization of TH is less likely to be induced by oxyanions, and more likely, by hot electrons. The size indifference of poly(TH) to tALO suggests that the associated polymerization is facile in kinetics, which signals that the mass transport of TH (solid state) on ITO is a slower process, explaining why poly(TH) tended to a limiting volume as Vtip ? 1. Thanks to the ALO-induced polymerization, a pattern with letters of NTNU CHEM CYM (Vtip = 6 V and tALO = 1 s) is demonstrated in Fig. 4 to feature the application potential of TH in ALO-based microlithography. 3.2. Phenothiazine-based chemical field-effect transistor Polymeric phenothiazines had been shown as effective electron-transfer medaitors for NADH [5]. A TH-based chemical fieldeffect transistor was thus inspired for the analysis of NADH. The device is schematically illustrated in Fig. 1. A poly(TH) nanoline (0.2  1 lm2) was positioned on an ITO square via ALO with a moving AFM tip (Vtip = 6 V) at a speed of 2 nm s1. The ITO square (Fig. 5) was pre-segregated into two parts to be the source and drain with a 0.2 lm-wide, 0.1 lm-deep microfluidic channel. Before testing the device with NADH, the source was examined with conductive-mode AFM (CM-AFM) to ensure that both electrodes are electrically isolated. Fig. 6 shows that the currents tunneling from the source to the AFM tip were insignificant before the installation of the poly(TH) nanoline, regardless of whether the drain was biased (VD vs. the source) or NADH (0–0.1 lM) was present in the channel (traces (i)–(iii)). Notably, the tunneling currents started to boost and showed responses to VD and [NADH] (traces (iv)–(viii)) after poly(TH) was installed. TH and its structural analogues, such as TB and MB, are electrochemically active in solution or as polymers [5]. The current jump is apparently the result of electrons able to flow from the source through the poly(TH) nanoline to the drain. Because TH+/0 is also capable of mediating electron transfer for NADH, NADH can thus donate electrons to the

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t ALO 0.01s 0.1s 1s

Fig. 2. AFM images and height profiles (inset) for the poly(TH) generated via ALO (Vtip, 6 V; relative humidity, 89%).

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Humidity 89 % Height/ nm

30

71 %

20

10

0 0

-2

-4

-6

-8

-10

Vtip / V Fig. 3. Dependences of poly(TH) in terms of height on the applied voltage and humidity.

TH+ state at the junction between the poly(TH) and the drain (positively biased). The tunneling currents thus showed responses to [NADH] at a constant VD. Fig. 7 shows that after the ITO square was assembled as a sensor device (Fig. 1), the drain current (ID), or more precisely, the net current (DID = ID total  ID background), increased as a function of [NADH] at a constant VD (0.5 V relative to the source) when NADH was injected through the channel, consistent with the results shown in Fig. 6. Here ID total and ID background stand for the ID measured in the presence and the absence of NADH, respectively. TB and MB were also characterized for their performance as gates (Fig. 8). Both showed similar effects with NADH, except that the sensitivity was lower. The lower sensitivity is tentatively ascribed to the poorer polymerizability during ALO. The TH-based transistor could also sense NAD+. Since TH can mediate the reduction of NAD+, a current with opposite sign resulted. According to cyclic voltammetry studies,

Fig. 4. An ALO-based lithography pattern created under the conditions: Vtip = 6 V, tALO = 1 s, and relative humidity = 89%.

electron transfer between NADH and NAD+ is not ideally symmetrical, even under the mediation of TH+/0. The cathodic wave for NAD+ is somewhat less significant than the anodic wave for NADH at electrodes such as ITO. For this reason, a lower sensitivity was observed. For NADH, the DID tended to saturation when the concentration approached 5 lM. We tentatively ascribe this phenomenon to a limited capacity of the nanobridge for electrons, supported by the fact that as the number of poly(TH) gates was increased, ID total increased as well. Increasing VD could also enhance ID total. However, ID background was also raised, presumably because of the direct oxidation of NADH by the ITO near the edges of the source and the drain. The sensitivity to NADH thus reached the optimum condition near 0.5 V in terms of VD (Fig. 9). Under this

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NADH

NADH

NADH

(i) (ii)

ID

(iii) (iv) (v) (vi) (vii) 1 nA

0

500

1000

1500

2000

Time/ s Fig. 7. Responses of the device in Fig. 1 to NADH of (i) 0; (ii) 1; (iii) 2; (iv) 3; (v) 4; (vi) 5; and, (vii) 6 lM recorded at VD = 0.5 V.

1.5

NADH, TH NADH, TB NADH, MB + NAD , TH

1.2

Δ ID / nA

0.9 0.6 0.3 0.0 -0.3 -0.6 Fig. 5. AFM images of the ITO square in Fig. 1 before (upper) and after (lower) being installed with a poly(TH) nanoline.

0

1

2

3

4

5

6

c/ μ M Fig. 8. DID recorded for NADH and NAD+ (VD = 0.5 V) with various gates.

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(viii) (vii)

(v) (iv)

(vi)

(iii) (ii) (i)

10

1.2 1.0

0

TH

0.8

-10

Δ ID / nA

Current/ nA

20

-20

0.6

TB

0.4 -30 3

2

1

0

-1

-2

-3

VD / V Fig. 6. Dependences of the current tunneling between an AFM tip (normal force loading, 5 nN) and the source electrode in Fig. 1 on VD and [NADH]: (i) 0; (ii) 1  106; (iii) 1  105; (iv) 0; (v) 1  107; (vi) 1  106; (vii) 1  105; and (viii) 1  104 M before (i) –(iii) and after the installation of the poly(TH) gate (iv)–(viii). Scan rate: 2.6 V s1.

condition, the lowest detection limit for NADH is estimated to be around 1 lM. Uric acid or ascorbic acid could interfere with the sensitivity. Studies on minimizing the interference by incorporating anionic membranes such as Nafion are under way.

0.2

MB 0.0 0

1

2

3

4

5

VD / V Fig. 9. Sensitivity assays for the devices with polymeric TH, TB and MB as chemical gates to 5 lM NADH.

4. Conclusions Phenothiazine compounds, such as thionine, are potential ink molecules for ALO-based microlithography. When negative biases

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are applied with an AFM tip above thionine, the monomer transforms into nanodots immediately on the sites. The polymerization is fast in kinetics, reaching completion in less 10 ms. Water is essential to this polymerization. However, the reaction is likely to be caused by hot electrons induced by the AFM field in the water bridge beneath the tip. When poly(TH) is positioned between a pair of source and drain electrodes spaced by a microfluidic channel, it can function as the gate to transduce NADH flowing through the channel. The drain current shows significant responses to NADH as well as NAD+ at ambient conditions. The AFM tip (field)-induced local oxidation technique is thus shown to be a promising approach for fabricating biocompatible nanoelectronic devices. Acknowledgements We acknowledge financial support from the National Science Council, Republic of China (Grant Number: 99-2113-M-003-008MY3). The authors are also gratified to Professor Wei-Hsiu Hung for rendering assistance in the e-beam work.

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