Placement of Nanospace on an Electrode for Biosensing€¦ · ANALYTICAL SCIENCES NOVEMBER 2012, VOL. 28 1037 1 Introduction Mozart has touched us with beautiful melodies, which were

ANALYTICAL SCIENCES NOVEMBER 2012, VOL. 28 1037

1 Introduction

Mozart has touched us with beautiful melodies, which were composed by playing a sequence of musical notes, and Beethoven arranged great symphonies for posterity through the effective placement of rests in sequences of notes. We can observe an effective array of notes and rests in the scores of symphonies they have composed. As well as these, an effective array of substances as notes can create functional space as rest. The development of new functional materials is achieved through effective structural control based on combinations of substances and spaces.

New strategies using a combination of nanomaterials and

nanofabrication techniques have been developed in the field of biosensors. Researchers have been developing nanostructures and protocols needed to apply these strategies to ultrasensitive biosensors. The performance of biologically specific events on the surface of nanostructured materials on an electrode results in unique and effective signal transduction.

Molecular recognition can be used in the development of substrate-specific sensors to bind complementary chemical or biological substrates, or for the recognition of structural features in biomolecules. The “lock and key” metaphor proposed by Fischer describes that a substrate fits like a key into a lock of an active site on the enzyme, which is highly structurally controlled as a function of its constituent amino acids.1 Numerous types of biological and artificial receptors with various binding affinities

2012 © The Japan Society for Analytical Chemistry

† To whom correspondence should be addressed.E-mail: [email protected]

1 Introduction 10372 Molecularly Imprinted Electrode 10393 Nanogap Electrode 1042

4 Summary and Future Outlook 10465 Acknowledgements 10466 References 1046

Placement of Nanospace on an Electrode for Biosensing

Hiroshi SHIIGI,*† Shiho TOKONAMI,** Yojiro YAMAMOTO,*,*** and Tsutomu NAGAOKA*

* Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-2 Gakuencho, Naka, Sakai, Osaka 599–8570, Japan

** Nanoscience and Nanotechnology Research Center, Osaka Prefecture University, 1-2 Gakuencho, Naka, Sakai 599–8570, Japan

*** GreenChem. Inc., 202, 930-1 Fukuda, Naka, Sakai 599–8241, Japan

Electrical and electrochemical methods are well established as very useful techniques in the field of biosensing because they can easily handle signals and devices. This paper provides an overview of biosensing using a nanometer-sized space functionally. Placed effectively on the electrode, the nanospace offers several advantages, such as increased sensitivity, improved selectivity, decreased response time, and the potential for instrument miniaturization. Given the impressive technological progress of nanospace biosensors and its growing impact on analytical science, this review offers an easy-to-understand presentation describing the history, recent advances, new methods, and future prospects of nanospace biosensors.

(Recieved August 23, 2012; Accepted September 5, 2012; Published November 10, 2012)

Hiroshi SHIIGI received his PhD degree from Yamaguchi University in 2000. He worked at Yamaguchi University as a Research Associate (2001 – 2003). He joined Osaka Prefecture University as an Assistant Professor in 2003, and became an Associate Professor in 2008. His current research interest is the development of nanobioelectronics and biosensors based on molecular recognition.

Shiho TOKONAMI received her PhD degree at Osaka Prefecture University in 2007. She was a postdoctoral fellow at Hiroshima University, Tokyo University of Science Yamaguchi, and Osaka Prefecture University from 2007 to 2009. She is currently a special lecturer at Nanoscience and Nanotechnology Research Center, Osaka Prefecture University. Her research area is nanotechnology and analytical chemistry, including the development of sensors for bio-associated materials.

Reviews

1038 ANALYTICAL SCIENCES NOVEMBER 2012, VOL. 28

or other interactions have been explored and developed, such as antibiotics, enzymes, supramoleculars, and molecularly imprinted polymers. Their receptors can recognize a target molecule at the recognition site through various mechanisms, including electrostatic, hydrogen binding, van der Waals, and donor-acceptor interactions. An array of functional groups within the chemical structures of the receptors forms selective sites for binding target molecules.

The application of biological principles to electroanalytical chemistry can be traced to the findings of Cremer, who showed the dependence of the membrane potential of a thin glass membrane on the concentration of hydrogen ions,2 and to the construction of a glass electrode by Haber and Klemensiewicz.3 These concepts have contributed to the development of molecular recognition, particularly the ion-selective electrode (ISE).4 Pressman examined in detail the ion-transport ability of antibiotic valinomycin,5 and Duax revealed an ion-recognition mechanism based on the complex formation of an internal cavity by potassium ions.6 Moreover, a single-channel current recording using antibiotics was also pioneered.7,8 After Pedersen successfully synthesized a crown-ether, apparently a salt-polyether complex formed by ion-dipole interactions between the cation and negatively charged oxygen atoms of the polyether ring,9 ISEs using complex-forming neutrally charged carriers were employed for the recognition of target ions.10 Cram and Lehn expanded Pedersen’s two-dimensional compounds into three dimensions to synthesize functional mimics of enzymes and other natural molecules based on their complementary three-dimensional structures, which led to the emergence of molecular recognition as a new domain of chemical research, which expanded over other areas and became supramolecular chemistry.11,12 Many subsequent efforts have been devoted to the isolation and synthesis of compounds

containing cavities with nanometer molecule-size dimension.Recently, research interests have focused on expression and its

application to new features by top-down and bottom-up structural control of materials. Structural control within a nanometer-sized space, such as a pore, molecular-shaped cavity, or nanogap, has been applied to biosensing, as reported in Table 1. New technologies for the application of science in nanospace are created from nanoscale technologies used to define the nanospace shape and dimension. The development of nanomaterials and features for the applications of technical and scientific phenomena is also needed. Nanomaterials will bridge the gap between cutting-edge science and technology materials. Brought together and applied across a broad spectrum of analytical science disciplines, such as biotechnology, medicine, and environmental science, the challenge will lie in extending basic techniques to nanospace functions. In this paper, we focus on efforts to develop electrical and electrochemical biosensing based on molecular recognition within a nanospace environment.

The development of a nanopore-based device allowing high-resolution detection and analysis of a single biomolecule without labeling or amplification has been based on the concept of the Coulter counter, which has been traditionally used to count and size particles.13 A voltage difference across pores arranged between two electrolytic solutions produces an electrical current. The pore size limits the free flow of ions, essentially determining the magnitude of the current. This technique creates a highly confined space that can be analyzed at high throughput, enabling the detection of single molecules, such as ions, nucleotides, nucleic acids, or proteins.14–24 Moreover, the signal, which is based on the native order of the nucleobases in a polynucleotide, can facilitate rapid sequencing of DNA or RNA.25–31

One attractive approach to synthetically mimic nature is

Yojiro YAMAMOTO received his PhD degree at Osaka Prefecture University in 2007. His main research interest is the fabrication of electroless plating by using metallic nanoparticles. He started his own company, GreenChem. Inc., in 2010. He is currently a guest investigator at Graduate School of Engineering, Osaka Prefecture University.

Tsutomu NAGAOKA received his D. Sc. Degree from Graduate School of Science, Kyoto University in 1982, and became a Research Associate of Department of Applied Chemistry, Yamaguchi University. He became Associate Professor in 1990 and Professor in 2000. He moved to Osaka Prefecture University as a Professor in 2003. At present, he is a Professor at Graduate School of Engineering, Osaka Prefecture University. His research area includes analytical applications of molecularly imprint polymers and metal nanoparticle material science.

Table 1　Bioanalysis and biosensing techniques using nanospace

Nanospace Nanopore14–31 Molecular imprinting32–63 Nanogap64–88

Analyte Antibiotics,16 proteins,17–20 nucleotides,21,22 peptides,23,24 DNAs,25–31 etc.

Amino acids,47–50 glucose,51,52 cholesterol,53–57 bile acids,58 nucleotides,59–61 etc.

DNAs,71,76,77,82–84 proteins,85–88 etc.

System

Aterial α-Hemolysin, SiN, SiO2, polymer, etc.

SiO2, SAM, organic polymer, etc.

Metal thin films, nanoparticles, carbon nanotube, etc.


“molecular imprinting,” which is used in the preparation of substrate-selective recognition sites within a matrix using a molecular template. Polyakov32 reported effects from the presence of organic solvents, such as benzene, toluene, and xylene, on a silica pore structure, and Pauling33,34 and Dickey35 later reported on silica gel polymerization using sodium silicate to selectively absorb various organic dyes. Recognition sites are typically constructed on polymeric material by the copolymerization of functional and cross-linking monomers in the presence of a target molecule (Fig. 1).36

The template molecule first interacts with functional elements, and it is sequentially locked onto a matrix formed by the polymerization of monomers. Subsequent removal of the target from the polymer matrix creates a shape-complementary cavity with excellent molecular recognition ability. Moreover, the molecularly imprinted polymer (MIP) technique offers the significant advantage of customization for various targets. Therefore, molecular imprinting can be described as a way of making an artificial “lock” for a molecular “key”.37–41 The construction of an MIP on an electrode combines the molecular imprinting method with electrical and electrochemical sensing techniques, resulting in effective measurement and signaling for a variety targets,42–46 amino acids,47–50 glucose,51,52 cholesterol,53–57 bile acids,58 and others,59–63 using an artificial component to mimic the biological receptor.

Pairs of electrodes with a nanometer-scale gap are the fundamental building blocks for the fabrication of nanometer-sized devices and circuits.64–81 They are also important tools for characterizing the properties of molecules and materials at the nanometer scale. During the past decade, many techniques for fabrication and potential applications of nanogap electrodes for the analysis of molecules and materials beyond the capability of traditional microfabrication technologies have been reported. In particular, the combination of top-down and bottom-up approaches has provided a desired configuration, placement of molecules into nanospace, and promising molecular-recognition results.76,77,79–88 Bottom-up fabrication is achieved through the binding, interaction, self-assembly, and self-organization characteristics of molecules and materials as building blocks.89

Nanogap devices provide a sufficient level of sensitivity, to as few as a single or small number of biomolecules, with direct transduction of biomolecule-specific binding events into electrical signals.

As mentioned previously, the nanospace is an important key to the development of a new field within analytical science, and has become a prominent and continuously expanding area of research. Therefore, we will discuss promising strategies for performance enhancement and key challenges of biosensors, especially with respect to the application of molecular imprinting and nanogap technologies to electrical and electrochemical biosensing.

2 Molecularly Imprinted Electrode

The fabrication of nanospace on an electrode has been achieved using film modification techniques: self-assembly,53,55,56,90 SiO2 sol-gel,50,61 cross-linking,62 electrochemical polymeriza-tion,47–49,51,52,54,57–60 and others.91 The formation of a nanospace cavity to a target molecule essentially relies on the binding and interaction between constituents and targets. Therefore, a cavity formed in a matrix with a complementary shape and sites for target interaction and binding, provides excellent molecular recognition.

The self-assembled monolayer (SAM), created by spontaneous Au–S binding, is the simplest technique for the formation of molecular-recognition sites on an electrode. To perform this procedure, a planar gold electrode was immersed in an ethanol solution containing cholesterol as a template and stearylmercaptan as a matrix.55–57 Voltammograms from each step of the preparation and sensing are shown in Fig. 2A. The cholesterol molecule is usually handled as an electroinactive species, and therefore the redox active species potassium ferrocyanide (K4Fe(CN)6) is used as a redox marker for its electrochemical measurement. A negligible response was obtained using a modified gold electrode because its surface is completely enclosed by electroinactive species, namely stearylmercaptan and cholesterol, as represented by curve b. The SAM electrode with an attached cholesterol template was immersed in ethanol to extract the cholesterol molecules. The extraction of cholesterol molecules restored the electrochemical activity of the SAM electrode, and completed creation of complementarily shaped cavities resulting from van der Waals interactions between adjacent stearylmercaptan molecules in the tightly-packed monolayer as represented by curve c. Before the cavity binds a cholesterol molecule, the redox marker does not diffuse to the electrode surface, and the electrochemical signal is decreased, as shown in curve d. In contrast, when the cavity binds cholesterol, the redox marker diffuses to the electrode, and the signal increases as shown in curve c. Therefore, the response (ΔI) was normalized using the following formula:

ΔI = I0 – Ir, (1)

where I0 and Ir are the oxidation peak currents of ferrocyanide at the molecularly imprinted SAM electrode before and after the recognition of cholesterol, respectively. The current is related to the cavity concentration for the mass-transport of the redox marker on the molecularly imprinted SAM. Before the SAM electrode bound cholesterol, the current was decreased owing to the absence of marker diffusion to the electrode surface. In contrast, when the SAM electrode bound cholesterol, the marker diffused to the electrode surface and the current increased.

Figure 2B shows a plot of ΔI vs. the concentration of

Fig. 1　Conceptual illustration of the procedure for preparing a molecularly imprinted polymer (MIP).


cholesterol, which exhibits a linear relationship even at low concentrations, and the coefficient of correlation was excellent (0.9983). Moreover, this sensor had a rapid response, with a complete response time under 30 s. The length of the alkylthiol used for SAM formation was optimized for sensitivity and selectivity, as shown in Figs. 2C and 2D. Although a greater number of carbon atoms increased the response (ΔI), the maximum response was obtained at around 16 carbon atoms, and ΔI decreased with increasing number of carbon atoms greater than 18. Conversely, the SAM-modified electrode, which was prepared using shorter alkylthiols with fewer than 10 carbon atoms, represented oxidation peak currents of ferrocyanide without extracting cholesterol molecules. This indicates that the formation of the SAM using shorter thiols allowed the diffusion of a redox marker to the Au electrode surface as a result of the thickness and the low density of thiol molecules on the Au electrode. The selectivity increased with greater numbers of carbon atoms, with maximum selectivity obtained at around 18 carbon atoms. This is attributable to intermolecular van der Waals interactions, where longer alkyl chains are stronger than the shorter ones. Alternatively, the longer chain has a greater molecular length than cholesterol, and may impose greater control over the extraction of cholesterol, keeping it in the SAM as a result of its larger size and stronger hydrophobic interactions, thereby decreasing the ΔI and selectivity.

Conducting polymers are also useful for the formation of molecular recognition sites on electrodes, through the formation of a polymer film using a template molecule as a dopant. A polypyrrole (PPy) film was deposited electrochemically on a Au-evaporated quartz crystal electrode (surface area: 0.16 cm2)

in an aqueous solution containing pyrrole as a monomer and target molecule sodium 1-naphthalenesulfonate (1-NS) as a dopant anion.46,92 Afterward, potential cycling was used to dedope the 1-NS leads, and to form complementary cavities on the overoxidized PPy (OPPy) film. The OPPy-film-modified electrode provides excellent molecular-recognition ability to 1-NS, as shown in Fig. 3. An electrochemical microbalance observation started at an applied voltage of +0.3 V (vs. Ag | AgCl) immediately after the electrode was immersed in an aqueous NS solution. The frequency of the OPPy film electrode decreased for 1-NS; however, a change in the frequency was not

0 0.2 0.4 0.6 0.8 1Concentration of cholesterol/mM

0 0.4 0.8E/V vs. Ag|AgCl

2*10-6AI0

Ir

a

b

c

d

∆I/µ

A

1.2

0.8

0.4

Number of C atoms

C

Sel

ectiv

ity/-

0.8

0.6

0.4

0.2

D

5 10 15 20 0

0.5

1

1.5

∆I/µ

A

R2 = 0.9983

B

A

5 10 15 20 Number of C atoms

Fig. 2　(A) Voltammograms for (a) a bare gold electrode, (b) a SAM-modified electrode, and a molecularly imprinted SAM electrode (c) before and (d) after immersion in ethanolic 3 mM cholesterol solution. (B) Dependence of ΔI on the cholesterol concentration. (C) Dependence of the sensitivity, and (D) the selectivity of the molecularly imprinted SAM on the number of carbon atoms in the alkylthiol. The selectivity was calculated from the ΔI (cholesterol)/[ΔI (cholesterol) + ΔI (cholesterol acetate)].

Fig. 3　Resonance frequency response of a 1-NS-imprinted OPPy film to a target (1-NS) and its structural isomer (2-NS).46


observed for 2-NS. This technique, which achieves excellent molecular-recognition ability, is very useful and can be applied to various biomolecules, such as bile acids,58 amino acids,48,49 and nucleotides.61 The use of taurocholic acid (TC) as a dopant enabled the formation of a PPy film on the electrode, as shown in Fig. 4.58 The overoxidation process of PPy was characterized using cyclic voltammetry and an electrochemical quartz crystal microbalance (EQCM) technique. Figure 4 shows time profiles for the EQCM parameters for the overoxidation of a TC-doped PPy film. A broad anodic peak at 0.95 V (the inset) and at around 190 s (a), appearing only in the first sweep of the cyclic voltammetric experiment, indicates a response typical of PPy overoxidation.46,93–97 As shown in Fig. 4b, a slight increase (700 Hz) at 190 s and a subsequent large increase (2700 Hz) at 230 s were observed in the resonance frequency, while the resonance resistance sharply dropped at 230 s, suggesting curing of the film (Fig. 4c). The slight change in frequency with no change in resistance at the first step was attributed entirely to the amount of TC dedoped from the PPy film. Conversely, a 2700-Hz increase at the second step included not only the amount of dedoped TC, but also the viscoelastic change in the PPy film. A 930-Hz frequency change, corresponding with the viscoelastic change in the PPy film, was estimated from the respective value resulting from the EQCM measurement.98 The

total 2470 Hz (= 3400 – 930 Hz) increase corresponds to 4.9 nmol of TC extracted from the film through overoxidation. The frequency increases in the first and second steps are associated with the PPy overoxidation process at 190 s (1.4 nmol = 4.9 nmol × 700 Hz/2470 Hz) and the decrease in the resonance resistance at 230 s (3.5 nmol = 4.9 nmol × 1770 Hz/2470 Hz), respectively. It is noteworthy that major frequency and resistance changes occurred simultaneously after completion of the faradaic overoxidation process. Electrostatic dedoping on the removal of the positive charge in the polymer backbone caused the first step, whereas the second was triggered by a physical factor on film hardening. The large decrease in resistance (curing), occurring concomitantly with dedoping, ensured the retention of the complementary cavity shape. An estimated 1.3 × 1016 TC-cavities cm–2 were generated on the OPPy film.

Figure 5 shows the resonance frequency vs. time profile of a TC-imprinted OPPy film in 3.0 μM dehydrocholate (DhC) solution for repeated extraction (–0.4 V) and uptake (+0.3 V). The potential values were optimized to achieve both the maximum variation (ΔF) and stability in the resonance frequency, which is reflected in the uptake of bile acid by the film’s cavities. Accordingly, an uptake potential of +0.3 V was selected to optimize the reproducibility and stability. Effective extraction of the DhC molecules bound to the OPPy film requires the application of –0.4 V. When the extraction voltage was not applied, the film frequency recovered at a much slower rate as a result of electrostatic repulsion between DhC molecules

OPPyTC-doped PPyoveroxidation

CONH(CH2)2SO3-

OHHO

HO

NH

NNH

H

+ n nNH

NN

OOH

c

0 200 400 600 800 1000Time/s

a

0 0.4 0.8 1.2E/V vs. Ag|AgCl

1st

2nd

0.050 mA

b

0.05

0

500

600

700

0.10

0.15

8.982

8.980

8.978

Cur

rent

/mA

Res

ista

nce/Ω

Freq

uenc

y/M

Hz

0.95 V

0.95 V

Fig. 4　Time profiles of EQCM characteristics for overoxidation of a TC-doped PPy film during cyclic voltammetric activation for (a) the current, (b) resonance frequency, and (c) resonance resistance. The arrows in the figure indicate reversal of the potential sweep direction.58 Copyright (2005), The Electrochemical Society.

0

100

200

300

∆F/

Hz

Concentration of bile acids/M

DhC - -DC - -

CDC - -C - -

TC - -

0 500 1000 1500

Time/s

100 Hz

+0.3 V

-0.4 V

∆F

A

B

10-8 10-6 10-4

Fig. 5　(A) The resonance frequency vs. time profile of an OPPy (TC) film in 3.0 μM DhC solution for repeated uptake at +0.30 V and cleaning at –0.40 V. (B) Dependence of the resonance frequency of an OPPy film on the concentration of bile acids.58 Copyright (2005), The Electrochemical Society.


and the negatively charged OPPy backbone with carboxyl and carbonyl groups introduced by overoxidation. The application of a potential to facilitate both the uptake and extraction processes produced more rapid responses, as shown in Fig. 5A. Upon the application of +0.3 V, a sharp decrease in the resonance frequency of ~220 Hz was observed within 75 s. This observed frequency change indicated a 0.55 nmol uptake of DhC. In a subsequent extraction process, a rapid frequency rise was observed within 100 s indicating mass ejection of TC from the film. In each of these cases, ΔF was linearly related across a wide range of logarithmic bulk bile acid concentrations from 10–8 to 10–5 M with a detection limit of ~25 nM, as shown in Fig. 5B. The sensitivity, with an uptake slope of ΔF/log C, decreased across the sequence of molecular volumes, and the bile acid uptake effective complementary cavity for TC was 4.4 nmol cm–2. The estimated rate of cavity utilization was ~20% based on the maximum amount of adsorbed bile acid (0.7 nmol of DhC) and the amount of TC extracted from the film (3.5 nmol) through overoxidation. The sensitivity of the TC-imprinted OPPy film depended not only on the molecular volume of bile acid, but also on the binding constant (KB) of the cavity, as listed in Table 2. In these cases, the OPPy film demonstrated good reproducibility, stability, and sensitivity under iterative measurements, and should supply similar sensing characteristics for other bile acids.

Figure 6A shows the effect of the potential applied in an acidic solution (pH 1.7) on the uptake of L- and D-glutamic acids (Glu), as monitored by fluorescence spectrometry.48 Significant L-Glu uptake was observed before application of the electrical potential, while the uptake of the D-isomer was negligible. This L-Glu uptake was attributed to the insertion of cationic L-Glu molecules (pKa 2.2) into the film. Based on these results, it is evident that the electrical potential plays an important role in controlling the uptake/ejection of amino acids. Glutamic acid is inserted, when its chirality matches the template cavity, through electrostatic interaction with the negative charge accumulated to OPPy. Figure 6B shows the results for the exposure of an L-Glu-imprinted OPPy film to varying concentrations of L- and D-Glu. The number of L-Glu molecules (molecular volume = 91.2 × 10–3 nm3) taken up by the film was proportional to the bulk concentration over 10 mM, and the uptake amounts for 20 mM L- and D-Glu were 80 and 3.0 nmol cm–2, respectively (L/D uptake ratio of ~30). The capacity of L-Glu-imprinted OPPy for uptake of 10 mM L-Glu was not affected by the presence of D-Glu at a concentration of less than 15 mM. It was observed that the L-Glu uptake capacity of the L-Glu-imprinted OPPy film, at 80 nmol cm–2, was greater than the estimated TC uptake capacity of the TC-imprinted

OPPy film (4.4 nmol cm–2).58 This suggests that the molecular recognition of a molecularly-imprinted OPPy film is based not only on the density of the effective complementary cavity and the molecular volume of the template molecule, but also on the binding constant of the cavity.

3 Nanogap Electrode

Precise control in fabricating electrodes with a suitable spacing for specific molecules allows ultrasensitive biosensing. Several effective methods for fabricating nanogap electrodes have been reported to provide promising results, including mechanical break junctions,68 electromigration,72,99 electron-beam lithography,100 electrochemical plating,99,101,102 focused ion beam lithography,103 shadow mask evaporation,104,105 and nanoparticle array.74,76,77,106–115

A thin film of Au nanoparticles deposited onto microgap electrodes could be connected covalently in a series by organic thiol molecules, as shown in Fig. 7. Therefore, a film formed from repeated sequences of a two-dimensional (2D) Au nanoparticle-organic thiol molecule-Au nanoparticle arrangement, which have been fabricated by a self-assembling technique based on spontaneous Au–S binding, has an electrical nanogap between adjacent nanoparticles and is useful for ultrasensitive detection without the need for fluorescent labeling.76,77,106–108,116–121 A decanedithiol was adsorbed onto the Pt microelectrode by (1) dipping it into a binder solution and by

Table 2　Molecular volume and binding constants for bile acids58

Bile acidMolecular volumea/

×10–3 nm3 ΔF/log C KB × 105b

TCCCDCDCDhC

345.0285.7278.9278.8276.2

28.872.773.873.782.0

0.530.900.861.031.09

a. Molecular volume was calculated by the Butch FREE WHEEL software version 0.57T using the results from the MP3 calculation.b. The binding constant was equal to the adsorption coefficient.Copyright (2005), The Electrochemical Society.

E/V vs. Ag|AgCl

Upt

ake/

nmol

cm

-2

0.2 0.40-0.2 0.6

0

4

8OO

HO OH

N+H3

OO

-O OH

N+H3

pKa 2.2

Concentration of Glu/mM

Upt

ake/

nmol

cm

-2

100 20

0

40

80

L-Glu

D-Glu

10 mM L-Glu

10 mM D-Glu

A

B

at 0 V vs. Ag|AgCl

Fig. 6　Effect of (A) applied potential and (B) bulk concentration on uptake of L- and D-Glu by a L-Glu-imprinted OPPy film at pH 1.7, as determined by spectrofluorometry.


(2) self-assembly of the Au particles with the sulfur group of the binder molecule. This dip-coating procedure was repeated, producing a film with an equally spaced nanogap interval between each particle created by the binder molecule (ca. 1.3 nm), which can be adjusted through the length of the dithiol alkylchain.74,76,107,119 The resistance of the prepared film was consistent with the value reported by Han et al. (nonanedithiol, ca. 1.1 nm).116

The 2D nanoparticle array was modified with thiolated probe DNA by immersing the electrode in a TE buffer containing 500 pmol of a probe single-strand (ss) DNA (12-bases; 5′-HS-TCT CAA CTC gTA-3′) for 30 min. The amount of probe modification was estimated at approximately 5.4 pmol using an electrochemical intercalator.120 When the resistance stabilized, the film was exposed to a TE buffer containing sample ssDNA (500 pmol) for hybridization, as shown in Fig. 8A. Upon sample addition, a resistance decrease was immediately observed for S/N ratios above 40, followed by a steady state within 2 min. The magnitude of the response depended on the number of mismatched base pairs (bp) in the double-strand (ds) DNA, with the largest amongst the samples being the complementary strand (0.19 Ω). Increasing numbers of mismatches led to a decrease in the magnitude, with the 11-bp mismatched DNA showing the smallest response (0.05 Ω). The resistance change, nonlinear with respect to the number of mismatches, was observed for the complementary DNA concentrations within a range of 5 – 100 μM (25 – 500 pmol).120,121 Details in the impedance analysis indicate that ionic migration had only minor effects on the DNA sensing. Therefore, the resistance changes likely

result from the conductivity of the dsDNA chain, which can be explained in terms of π overlapping between adjacent base pairs.71,122–124 The presence of a mismatch would produce a defect at the localization of electrons, reducing the electron transfer rate.

The dependence of the sensor response (ΔR) on the film resistance is represented in Fig. 8C. The ΔR value increased gradually up to a film resistance of 600 Ω, increasing sharply over 600 Ω. When the DNA was hybridized onto the nanoparticle film, the total resistance (Rfilm+DNA) of the hybridized DNA/film circuit was regarded as being equivalent to the parallel resistances of the film (Rfilm) and DNA (RDNA). Therefore, Rfilm+DNA is defined using Rfilm and RDNA as follows:

R R RR R Rfilm+DNA

film DNA

film DNA film+DNA= ∗

+ =

1 1RR Rfilm DNA

+ 1 , (2)

Additionally, because ΔR represents the difference in resistance before and after the addition of target DNA, it should be represented as

∆R R R RR R

= − = +film film+DNAfilm

film DNA

2

. (3)

The result of the curve fitted against Eq. (3) using the least-squares method is shown as the dashed line. The estimated value of RDNA was 1.5 × 106 Ω, which was the total resistance of the dsDNA connected to the nanoparticle network film. The calculated curve increased steadily, and correlated well with the

Fig. 7　(A) Schematic illustration of the nanogap electrode based on a two-dimensional nanoparticle array. (B) Preparation procedure, (a) side, and (b) top views of the array. (C) SEM and AFM images of the array.


observed experimental values at Rfilm < 600; however, it deviated from the experimental data at Rfilm > 600 Ω, and ΔR increased considerably. Although a higher sensor output was achieved (Table 3), the stability and reproducibility of the sensor response were reduced at film resistances of Rfilm > 600 Ω because of the lower S/N ratio (less than 3).

To achieve highly sensitive DNA detection, an open bridge-structured electrode was fabricated using a repeated Au nanoparticle-DNA-Au nanoparticle sequence, where the gap between the particles is precisely controlled using the probe sequence. Using the combination of this electrode and the conductive DNA, we were able to measure very small changes in the electrical properties of DNA with a high S/N (>30) and a detection limit of 5.0 fmol; a remarkable difference was observed between even the 1-bp mismatched DNA and the complementary DNA. We were able to amplify the detection limit of the 1-base mismatch in the DNA sequence using the described system. The inset of Fig. 9 shows the time course of the array resistance after applying the target 24-mer DNA, which carries two complementary sections for probes A and B (in this case, the target is poly(A)24, and both the probes are poly(T)12). After reaching a steady state, an aliquot of the target DNA (500 pmol) dissolved in the TE buffer was applied to the array. The resistance immediately decreased, but became constant within 60 s. The ΔR value, defined as the difference in resistance before and after the hybridization, was 100 mΩ with an S/N ratio of >30. The sensor exhibited responses over a wide range of concentrations with a detection limit of 5.0 fmol, as shown in Fig. 9. Electrical measurements were conducted using the described array and others, having equivalent electrical resistance (~320 Ω), and repeated in triplicate. According to the observed results, Au nanoparticles (AuNPs) separated by a 1.3-nm gap were effective for detecting the hybridization event; however, their effectual concentration range was limited (detection limit of ~25 pmol) because not every probe could hybridize with a target DNA strand in the gap between the particles.76,120,121 Thus, the probes were not entirely responsible for the formation of conductive paths on the surfaces of AuNPs. Opening bridge hybridization also occurred between adjacent AuNPs; therefore, the altered conductivity can be explained not only by the molecular conductivity of DNA,71,76,120–122,124,125 but also by the reduced structural distance between the 46 nm parent particles with attached 12 nm probes owing to this bridging by hybridization.

Fig. 8　(A) Resistance changes from hybridization of the probe with (a) 11-base (3′-CCC CCC CCC CCC-5′), (b) 4-base (3′-AgA gTT AAC TCT-5′), and (c) 1-base (3′-AgA gTT gAg CCT-5′) mismatched sample strands, and (d) complementary oligonucleotide (3′-AgA gTT gAg CAT-5′); sample addition is indicated by the arrow. (B) Model illustrations for electron transfer with complementary and 1-bp mismatched strands. (C) Dependence of the sensor response on film resistance. The inset shows the circuit model after hybridization in the nanoparticle film. Probe and target DNA sequences were 5′-SH-TCT CAA CTC GTA-3′ and 3′-AgA gTT gAg CAT-5′, respectively.76 Copyright (2005), American Chemical Society.

Table 3　Responses to various DNA sequences121

Sample Probe DNAa Target DNAb ΔR/Ωc

Full matchd 5′-HS-AAA ggg ggg ggg-3′ 3′-TTT CCC CCC CCC-5′ 0.255′-HS-ggg ggg ggg ggg-3′ 3′-CCC CCC CCC CCC-5′ 0.27

1-Base mismatchd 3′-ACC CCC CCC CCC-5′ 0.133′-CCC CCC CCC CAC-5′ 0.13

Full matche 5′-HS-TCT CAA CTC gTA-3′ 3′-AgA gTT gAg CAT-5′ 0.191-Base mismatche 3′-AgA gTT gAg CCT-5′ 0.084-Base mismatche 3′-AgA gTT AAC TCT-5′ 0.0611-Base mismatche 3′-CCC CCC CCC CCC-5′ 0.05

Full matchf 5′-HS-TCT CAA CTC gTA-3′ 3′-AgA gTT gAg CAT-5′ 0.641-Base mismatchf 3′-AgA gTT gAg CCT-5′ 0.444-Base mismatchf 3′-AgA gTT AAC TCT-5′ 0.3611-Base mismatchf 3′-CCC CCC CCC CCC-5′ 0.33

a. Probe concentration was 100 μM, of which 27 pmol cm–2 was adhered to the film. b. Target DNA of 100 μM/5 μL was applied to the probe-modified film. c. The change in film resistance caused by hybridization. d. The film resistance was 275 Ω. e. The film resistance was 173 Ω. f. The film resistance was 768 Ω. Copyright (2008), The Electrochemical Society.


The binder decanedithiol (~1.3 nm) was used to attach child AuNPs (12 nm), which were arrowed in the TEM image (Fig. 10 inset), to parent AuNPs (46 nm), anchoring them to the parent and at the same time leaving a flexible tail free for target binding. When one side of the target DNA (24-mer) is hybridized to the probe DNA (12-mer), for example probe A, the hybridization of the other half of the target DNA sequence with the other probe DNA (probe B) leads to a decrease in the electrical resistance. The distance between the arrowed child AuNPs, shown in the inset of Fig. 10, was decreased compared to the distance before hybridization. A greater distance between the child AuNPs make it impossible to hybridize both halves of the target DNA, preventing formation of the conducting pathway at the gap. These results suggest that the efficient formation of the conductive path increases the detection limit by 103 over detection achieved using the single-AuNP array system.120,121 Moreover, the response to target DNA was consistent with the percolation theory. The AuNP film, consisting of bound parent and child AuNPs, had an electrical resistance of ~320 Ω, well below the percolation threshold level, which resulted in high reproducibility. To verify the effectiveness of this system for the identification of DNA mismatches, we performed experiments using the targeted DNA along with complementary, 1-bp, 2-bp, and 24-bp mismatched (fully mismatched) DNA sequences (Table 4). The results for the poly(T)-poly(A) hybridization, represented in Fig. 11 (circle), indicate that the response was highest for the complementary DNA, and decreased with increasing numbers of mismatched bases.

Target DNA concentration/µM

40

80

100

100 102

∆R

/mΩ

60

R2 = 0.9934

10-2

200 40 80

∆R

Time/s

100 mΩ

Fig. 9　Dependence of the sensor response on target DNA concentration. Target sequence, 3′-poly(A)24-5′; probe A, 5′-SH-poly(T)12-3′; and probe B, 5′-poly(T)12-HS-3′. The array resistance was 320 Ω. The inset is a sensorgram showing the addition of a 500-pmol 24-mer complementary DNA, poly(A)24. The arrow indicates the sample spike.77 Copyright (2008), American Chemical Society.

Fig. 10　Conceptual illustrations of the open-bridge array consisting of DNA-capped AuNP probes immobilized on the parent AuNP. Probe configuration before and after the addition of 24-mer target.77 Copyright (2008), American Chemical Society.

Table 4　Response to various DNA sequences77

Sample Target 24-mer DNAa ΔR/Ωb

Full matchc 5′-poly(T)24-3′ 0.111-Base mismatchc 5′-AAA AAA AAA AAA TAA AAA AAA AAA-3′ 0.062-Base mismatchc 5′-AAA AAA TAA AAA AAA AAT AAA AAA-3′ 0.0224-Base mismatchc 5′-TTT TTT TTT TTT TTT TTT TTT TTT-3′ 0

Full matchd 5′-AgA gTT gAg CAT TAC gAg TTg AgA-3′ 0.121-Base mismatchd 5′-AgA gTT gAg CAA TAC gAg TTg AgA-3′ 0.052-Base mismatchd 5′-AgA gTT CAg CAT TAC gAC TTg AgA-3′ 0.0424-Base mismatchd 5′-TCT CAA CTC gTA gTA ATg CTC AAC TCT-3′ 0

a. Target DNA of 100 μM/5 μL was applied to the probe-modified film. b. The change in film resistance caused by hybridization. c. Probe A and probe B were 5′-SH-poly(T)12-3′ and 5′-poly(T)12-SH-3′, respectively. d. Probe A and probe B were 5′-SH-TCT CAA CTC gTA-3′ and 5′-ATg CTC AAC TCT-HS-3′, respectively. Copyright (2008), American Chemical Society.


Finally, the smallest ΔR was observed with the fully mismatched sequence (ΔR < 10 mΩ). Furthermore, the response to each target sequence was consistent, regardless of whether the measurements were performed using the complementary or fully mismatched sequence. In this series of experiments, with repeated hybridizing and denaturing, the measured response to the complementary strand was stable, and no morphological changes were observed in SEM images of the array surfaces. The resistance was accurate within ±4.2% (less than ±5 mΩ, same sample run (n) = 3) in a given series of experiments, and the S/N remained constant. The response was nonlinear with respect to the number of mismatches, and the clear difference (> 50 mΩ) observed between the complementary DNA and the 1-bp mismatch suggests that this system can be effectively used for SNP diagnosis. A similar trend in the mismatch detection of randomly sequenced fragments suggests that this method could be used for practical DNA identification.

Using this approach, a molecular electronic device could be fabricated with an electrode spacing accuracy of nanometers. The use of a nanogap electrode has enabled the detection of very small electrical signals; therefore, we attempted to use these electrodes in highly sensitive and label-free DNA sensing applications. Moreover, one- or two-dimensional arrangements of nanoparticles have also evolved with respect to their pathognomonic optical characteristics, since metal nanoparticles have distinctive optical characteristics.126–128 Some promising results have suggested that arrangement and nanospace may be useful in optical biosensing.

4 Summary and Future Outlook

This review describes recent development of electrical biosensing methods based on the arrangement of nanospace. Molecular recognition using the nanospace was based on the “lock and key” metaphor, with binding and interactions between a target molecule and a recognition site. There are three important points related to nanospace formation for molecular recognition. First, the chemical structure and reactivity of the target molecule must be defined. Second, the nanospace

recognition site must be formed to exploit potential reaction and interaction based on the chemical properties of the target. Finally, the formation of nanospace on the electrode facilitates effective signaling of target recognition.

These developments may enable the realization of a high-density and high-throughput sensing platform for bio-associated materials, such as DNA, antigens, and enzymatic reactions. The findings of these studies may hold the keys to future development of the nanobioscience field and associated technologies. We anticipate the formation of a beautiful array of nanospaces and direct translation of the activity of a single molecule into an electrical signal.

5 Acknowledgements

We gratefully acknowledge the financial support provided by the Japan Society through a Grant-in-Aid for Young Scientists (A) (21680041, 24685013).

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Placement of Nanospace on an Electrode for Biosensing€¦ · ANALYTICAL SCIENCES NOVEMBER 2012, VOL. 28 1037 1 Introduction Mozart has touched us with beautiful melodies, which were