Effect of ITO surface properties on SAM modification: A review toward biosensor application

The aim of this review is to discuss the effect of physical and chemical properties of Indium tin oxide (ITO) thin film on its self-assembled monolayer (SAM) modification, as well as device performance toward biosensor applications. The emphasis is on surface morphology studies of the thin films in order to provide connecting points between surface properties with the broader field of material science of ITO. The morphology of SAM film deposited on it directly affects by the properties of these prepared ITO film. Thus, it is a topic of interest to study the influence of physiochemical properties of this film on device fabrication and applications as biosensor platform. Subjects: Chemistry; Coatings & Thin Films-Materials Science; Material Science; Materials Science; Surface Engineering-Materials Science


Introduction
Indium tin oxide (ITO) is a well-known electrode material and widely used for its unique optoelectronic properties. There has been a growing technological interest in materials with electrically conductive and optically transparent. ITO thin film is one of the most popular thin films and used in several applications including optoelectronic and electrochemical applications due to its very high transmittance and conductivity (Chopra, Major, & Pandya, 1983). The electrochemical and physical ABOUT THE AUTHOR The author Dr Md. Zaved Hossain Khan is working as an Assistant Professor in the Department of Chemical Engineering at Jessore University of Science and Technology, Bangladesh. Recently, he finished his Postdoc in Tohoku University, Japan and did his PhD from Waseda University, Japan on 2011. He has 7 years of experience in working with electrode chemistry and its SAM modification for biosensor application. He has several publications in electrochemistry and surface chemistry field.

PUBLIC INTEREST STATEMENT
Indium tin oxide (ITO) electrode over the past few years has become a very important material for use as a conducting substrate in sensor development due to its excellent optoelectronic properties. One of the key problems of such electrodes is their stability, since it affects their performance. The surface properties of ITO substrates are particularly critical for different fabrication. From that viewpoint, the relationship between the different properties of ITO treated by various surface treatments and their performance has been reported. Therefore, the relationship between the surface chemistry of ITO and the monolayer modification is of paramount importance for the development of sensors. For the detection of biomolecules, imparting specificity in molecular recognition to electrodes, in general by modification with certain molecules, is often carried out for the development of (electro) chemical sensors.
A highly degenerated semiconductor like ITO has so many dopants that the electron concentration in the conduction band is comparable with the density of states in the band. The hole concentration in the valence band is comparable in the case of P-type material. During the film growth, degeneracy caused with the creation of the oxygen vacancies and substitutional tin dopants.
Due to the prominent characteristics of ITO electrodes such as excellent optical transparency, high conductivity, these electrodes have attracted increasing interest recently and widely used for their excellent substrate adhesion and electrochemical properties. Because of those stable characteristics, ITO film has been widely used in industrial applications. Sputtering is the most common deposition technique and widely investigated for large-scale deposition setups for ITO thin film production. By this method, low-cost deposition system, good film performances, and high sputtering rate could be achieved.
For immobilization of biomolecules, it is necessary to modify the ITO surface with silane reagents under well condition. Basically in early stage, ITO films were used as electrodes for spectroelectrochemical measurements due to their conductivity and transparency. Nowadays, due to critical importance of electron transfer and surface properties, ITO electrodes have been employed in electroanalytical sensors. For making electro-optical devices like; LCD screens, CMOS imagers etc. ITO electrodes are widely used as transparent semiconductor.

Monolayer modification
Self-assembled monolayer (SAM) usually used to control the physical and chemical properties of solid surface (Niwa et al., 2004). Considering electrochemistry, the term SAM is very common in designing selective electrode for electroanalytical applications. SAM of organic and biological molecules on metal surfaces are important in many application fields, ranging from sensors (Mizutani, 2008;Wackerbarth et al., 2005) to nanostructured devices (Love, Estroff, Kriebel, Nuzzo, & Whitesides, 2005) and have been of interest for last few decades. SAM represents a promising strategy to functionalize noble metal surfaces by forming ordered organic films leading to molecular architectonic which continue to serve as major techniques for the fabrication of supramolecular structures (Madueno, Räisänen, & Silien, 2008;Theobald, Oxtoby, Phillips, Champness, & Beton, 2003). On the other hand, SAM-based techniques are of particular importance in molecular recognition system on electrode surface, and offer a vehicle for investigation of specific interactions at interfaces with their functional properties that bear promise for technological applications (Justin Gooding et al., 2003;Mizutani, 2008;Niwa et al., 2004;Wackerbarth et al., 2005;Love et al., 2005). Figure 1 represents the SAM-modified ITO electrode surface in light emitting diodes (OLEDs) and organic thin film transistors (OTFTs) applications.
Several studies have been done on attachment of silane molecule on ITO surface at different conditions (Asanov, Wilson, & Oldham, 1998;Jee, Kim, Ko, & Yoon, 2006;Madueno et al., 2008;Theobald et al., 2003). For fabricating biosensors, functionalization and patterning of silane SAMs is very popular method due to its simple preparation, reproducibility, and stability. The characterization of SAM of silane on ITO is important to study their interaction on the surface. The attachment of organic molecules (Moses, Wier, & Murray, 1975) and mixed SAMs of different functionality (Bain & Whitesides, 1988) shows formation of monolayer on ITO surface. Trialkoxysilanes has been used mostly by means of covalent attachment for activating ITO surfaces (Asanov et al., 1998;Jee et al., 2006;Madueno et al., 2008;Theobald et al., 2003). Other researchers have used SAM-modified ITO electrode for detecting nucleic acids (Napier & Thorp, 1997), heteropolyacid (Oh, Yun, Kim, & Han, 1999), nanometric components (Gardner, Frisbie, & Wrighton, 1995), etc. Nowadays, extensive studies are going on to elucidate the response mechanism of SAM on different inorganic surface and their electrochemistry (Vilan, Shanzer, & Cahen, 2000). Specially, the surface characterization is very important to examine the physiochemical properties of the surface before and after SAM modification. SAM of different silane molecules onto ITO surface is shown in Figure 2.
Several researchers used carboxylic and phosphonic acids onto ITO electrode instead of the adsorption of thiols on gold that effectively enable the controllable connection of nanometric components. For attaching organic and biological molecules onto ITO surface, functionalized alkyl silanes (aminopropyltrimethoxysilane; APTES) are widely used.  used APTES-modified ITO electrode for biosensor application as shown in Figure 3.
Due to high surface roughness of ITO electrode, small number of studies have been reported on silane chemistry for attaching alkyl chains onto its surface. Even though this conducting and transparent substrate is an attractive film for silane attachment, the characterization of SAMs on ITO surface and its reproducible preparation becomes difficult due to high surface roughness of the electrode. Several researchers have studied mixed-component SAMs with different functionality and chain length, of which covalently attaching organic molecules was also carried out. To minimize the possibility of polymer formation, many of them were careful to employ dry conditions during using tricholo and triethoxysilanes. To confirm monolayer formation, layer thickness measurement and its coverage test were done.
Due to the simple preparation method of silane SAM films, it is widely used to immobilize proteins, enzymes onto the semiconductor surfaces for fabricating biosensors. Moore, O'Connell, and Galvin (2006) had developed DNA sensor with silane-modified ITO electrode and described its covalent attachment chemistry which is shown in Figure 4. For the late immobilization of biomolecules such as antibodies, enzymes, DNA, etc., organic SAMs on the solid surface are one of the most suitable functional interlayers.  Both spectroscopic and microscopic techniques are widely used to characterize the SAMs on electrode surface. Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), Infrared (IR), Raman, Atomic force microscopy (AFM), Scanning electron microscopy (SEM), Scanning probe microscopy (SPM), etc. have been used for the mentioned purposes. Scanning tunneling microscopy (STM) and AFM study combined used for through description of the SAMs structure on the electrode surface.

Modified ITO electrode as a sensor
Detection of biomolecules that release from living cell is of great interest in the view of biological and medical science from last few decades. Different types of microelectrodes have been used as amperometric/potentiometric sensors for this purpose of molecule detection. Modification of ITO electrode surface is one of the efficient ways to increase its electroanalytical activity to work toward biosensor development. For the detection of transmitter released from single cells, different types of midwifed microelectrodes are used as amperometric/potentiometric sensors. Due to its good photopenetrability and high electrical conductivity, ITO electrode has been widely used for electrochemical detection of biomolecules. To increase the electroanalytical activity of ITO electrodes, usually surface modification is performed.
Wide range of surface can be created by immobilization of biomolecules onto semiconductor surface and used as a key step in biosensor applications (Armistead & Thorp, 2000;Fang, Ng, & Li, 2003;Ferreira, Fiorito, Oliveira, & Córdoba de Torresi, 2004;Prieto-Simon, Campas, & Marty, 2008;Ruan, Yang, & Li, 2002). Chemically modified ITO electrode has been widely used as working electrode for the detection of biomolecules. Osaka et al. observed enantioselective potential response for Note: A silane anchor was utilized to provide SAM coverage on the ITO film. PDITC stands for 1,4-phenylene diisothiocyanate. Source: Reproduced with permission of Elsevier (Moore et al., 2006). tryptophan with human serum albumin (HSA)-modified ITO electrode (Matsunaga, Ueno, Nakanishi, & Osaka, 2008) as shown in Figure 5. Usually, bio-components like enzyme, tissue, antigen-antibody, etc. attached to the electrode surface that functions as the biological recognition elements.
Numerous studies have been reported on the modified ITO electrode sensors by highlighting its electronic and optical properties. Chemical modification of ITO electrode is a prerequisite for biosensor application that usually done with organic anchor molecules. In most previous reports, silane molecules are commonly used for surface attachment on the hydroxylated ITO surface as anchor molecules through SAM process. The end part of the silane molecule needs to be activated for the immobilization of biomolecules or their ligands by chemical coupling reaction. Carboxylic and phosphonic acids and amines are also used for organic layer formation on ITO surface as other  (Matsunaga et al., 2008). modification procedures. Although the chemical modification of ITO electrode is an attractive approach, their functionality and characterization study is always in terms of further study. Table 1 represents different functional groups that are used to modify ITO electrode for the immobilization of biomolecules.
Gold, silicon, and ITO (Budnikov, Evtyugin, Budnikova, & Al'fonsov, 2008;Izake, 2007;Trojanowicz & Wcisło, 2005) electrodes are widely used due to easiest method of fabrication for chiral molecule detection with high sensitivity (Bakker & Qin, 2006;Horváth et al., 1997;Junxiang, Sato, Umemura, & Yamagishi, 2005;Yasaka, Yamamoto, Kimura, & Shono, 1980). But sometimes, the bulky molecules reduce this sensitivity (Grieshaber, MacKenzie, Vörös, & Reimhult, 2008). Attachment of various organic molecules on bare ITO electrode surfaces has demonstrated that it is rapid and directed electrochemically to precise positioning of adsorbent molecules. In biosensor applications, several functional molecules have primary amines; therefore, the attachment of primary amines seems very useful technique. Sometimes, primary amine groups attach on to bare ITO electrode surface by direct electrochemical modification. M.Z.H. Khan et al. have detected several biomolecules with SAMmodified ITO electrode shown in Figure 6. Ethylene glycol Preventing the non-specific adsorption of biomolecules CME has the distinguished feature of having quite thin film that can be formed by bonding of selected chemicals on the electrode surface which is rational, chemically designed manner comparing other electrodes. Biosensor can detect picomolar range analyte concentration by specific binding interactions. Protein or cells have a tendency to adsorb onto interfaces without specific receptorrecognition interaction between substrates and biological samples.
Due to surface properties of ITO electrode, the reproducibility of SAM preparation and its characterization is sometimes difficult task (Rasmusson, Bröms, Birgerson, Erlandsson, & Salaneck, 1996). For achieving reproducibility of conductive surface like ITO, surface characterization, and details, surface chemistry study is essential. Optimizations of surface controlling parameters are important factor for the development of biosensor.
For the reproducibility of the measurement, it's always important to examine the factors that can affect electrode-analyte interface performance. Changes in surface composition can easily affect its affinity and interaction properties. Controlling of electrochemical and physiochemical properties of surface is important task for designing nano-device electrode. During deposition process, care should have taken to achieve optimum properties and to reduce manipulation.

Surface roughness
Surface morphology including physical, chemical, mechanic properties plays an important role to control the efficiency of a device. Among them, surface roughness in an important parameter, since the behavior of an electrochemical device depends on surface morphology (Bourlange et al., 2008;Dulub, Diebold, & Kresse, 2003;Golovanov et al., 2005;Goniakowski, Finocchi, & Noguera, 2008;Milliron, Hill, Shen, Kahn, & Schwartz, 2000;Noguera, 2000;Park, Choong, Gao, Hsieh, & Tang, 1996;Zhou et al., 2008). Surface roughness plays an important role in controlling surface chemical composition. Surface chemical composition can be easily altered by changing roughness properties. Several techniques are used to determine the surface roughness properties. AFM is a method widely used to measure the surface roughness. Kelvin probe force microscope (KFM) is used to measure surface work function and is an important parameter to determine the electrode role in device performance and SAM modification. In particular, when ITO electrode used as electrochemical device, surface Source: Reported by . morphology and chemical properties plays important role and needs to optimize for achieving highest performance. Figure 7 shows AFM images of ITO surface with different roughness properties.
The measurement of finely spaced surface irregularities is usually termed as surface roughness (SR), which is a numerical value of the mean roughness height. The physical, mechanical, chemical behavior of the surface is often determined by the configurational characteristics of the surface, where roughness plays an important role. Considering the use of ITO films as electrochemical device, the behavior depends on the morphology of the rough surfaces and it is an important parameter to be considered.
AFM is a widely used instrument to provide details of surface roughness parameters. Specially, average surface roughness (S a ), surface skewness (S sk ), and surface kurtosis (S ku ) are the important parameters to describe the height distribution histogram of surface (Ballarin, Cassani, Gazzano, & Solinas, 2010;Kim et al., 2003;Stout et al., 1993). M.Z.H. Khan et al. observed several spikes on ITO surface that was reported as low roughness shown in Figure 8. The difference in near-surface composition is related with surface roughness differences and considered to affect the properties of ITO (Brumbach et al., 2007;Morales & Diebold, 2009;Morales, He, Vinnichenko, Delley, & Diebold, 2008;Walsh & Catlow, 2010). Therefore, the performance of electrochemical device like ITO depends on its surface morphology and can affect the characteristics of the thin film deposited on it. Details of surface roughness include not only average roughness but also height distribution coefficient. Average deviation of the measured z-values from the mean plane can describe the amplitude of roughness parameters. Gaussian height distribution also plays an important role for measuring local peak height distribution that is related with sharpness of the surface.
The Gaussian height distribution term is very common method used to determine the surface morphology and can be calculated by the surface kurtosis and skewness value. In Figure 9, surface height distribution of several types of ITO electrode was shown. Usually, AFM provides the statistical analysis of the surface roughness features. Considering the fabrication of electrochemical device, the control of surface morphology is very important term and widely studied problem in recent years. The device performance is strongly affected by the negative effects of uneven surface that affects the morphology of the thin film. Over the last few decades, many researchers have studied the relationship between the structural and optical properties of ITO electrode with its surface morphology.

Surface composition
The performance of ITO electrode is extremely sensitive to its surface condition. The study of the relationship between surface conditions and different properties of ITO electrode is subjected to various treatments. Several researchers reported the effect of surface chemical composition change on the electrochemical properties of ITO electrode (Morales & Diebold, 2009).

Figure 7. AFM images of ITO surface, (a) is the image of an ITO surface with large Rpv and small R rms and (b) is the surface with small R pv and Large R rms . In the upper right and lower right parts, 'spikes' can be seen.
Source: Reproduced with the permission of Japan Society of Applied Physics (Kim et al., 2003).
The surface composition of ITO electrode, especially In:Sn ratio effectively changes the electrochemical properties of the electrode due to surface Fermi level electronic properties by controlling the surface potential as well as its work function. ITO electrode with different surface roughness usually shows different surface composition as shown in Table 2. The concentration of Sn 4+ dopants of the near ITO surface can be effectively reduced by O 2 plasma treatment. The movement of the surface Fermi level toward the valence band maximum by physical treatment effectively increases ITO work function as shown in Figure 10. By replacing In 3+ atoms with Sn 4+ dopants, thereby  The surface chemistry of ITO electrode is complex due to the inherent heterogeneity of these films. The elucidation of the relationship between the electron transfer reactivity of ITO electrode and its microstructure, surface composition is complicated because the surface properties of these films strongly depends on the deposition process and parameters used. The performance of ITO electrode and its electron transfer reactivity strongly depends on its electronic density of states and surface functional groups, whereas sometimes, impurities also determine the performance.
Deposition process and parameters play important role in the structure and properties of ITO films and it is a complicated study due to the inherent heterogeneity of these films. The electron density of states and surface functional groups affects the electron-transfer reactivity of ITO electrodes.

Surface potential and work function
The potential due to the energy differences between bulk and the surface of the film can be defined as surface potential. KFM is a technique by which the surface potential can be measured with detailed analysis at different region on the interface. The changes of the electric surface potential on which the standard explanation of the sensor function is based due to adsorption/desorption can be done by the increase in surface coverage with negatively charged oxygen species. Comparing with other semiconducting oxides, the diffusivity of oxygen in ITO is higher because of particular crystal structure (bixbyite).
Several studies reported that the relationship between surface chemical composition of ITO film (In:Sn:O) and its different properties (Khan, Nakanishi, Kuroiwa, Hoshi, & Osaka, 2011;Mason et al., 1999;Sugiyama, Ishii, Ouchi, & Seki, 2000) to affect the electrochemical properties of the device. Physical treatment is one of the method to control the surface work function of the electrode (Lee, Jang, Kim, Tak, & Lee, 2004;You, Dong, & Fang, 2004). Oxygen plasma treatment is one of them and  is an effective method to increase the surface work function. Introduction of negative end on the surface by the formation of dipole layer using organics can increase ITO work function as reported by many researchers (Bruening, Moons, Cahen, & Shanzer, 1995;Bruening et al., 1994;Carrara, Nüesch, & Zuppiroli, 2001;Krüger, Bach, & Grätzel, 2000;Zehner, Parsons, Hsung, & Sita, 1999) as shown in Figure 11. Work function is known to be influenced by the electrostatic condition of the surface and usually controlled by using SAM (Jee et al., 2006).
The concentration of ITO surface states is very high due to the degenerate doping that lead to significant change of the electrostatic potential. As a manifestation of the physical part of the depletion layer, the surface Sn concentration found to be depended on the surface Fermi level position. Using photoelectron spectroscopy (PES), or KFM, the direct assessment of the Fermi level position is possible that can be used for straightforward determination of surface potential with respect to the valence band maximum as shown in Figure 12.
The difference between the electrochemical potential and the electrostatic potential of the metal's inside and outside, respectively, is termed as work function (WF) of that metal. The WF can be easily determined by KFM method, considering a fundamental parameter related to electronic structures. Due to the effect on energy barrier height at the heterojunction interface, the work function of ITO has a critical importance as device performance. The introduction of uncertainties due to interfacial layers can deduce the general values of ITO work function range from 4.1 to 5.1 eV. In n-type semiconductor, electrons can be easily excited into the conduction band by the addition of donor impurities contributes electron energy levels high. Whereas in p-type semiconductor, the addition of acceptor impurities contributes hole levels low in the semiconductor band gap, thereby leaving mobile holes in the valence band.  Due to interfacial layers and the uncertainties that creates all these work function values can be deduced from the tunneling characteristics of the electrode heterojunctions. To increase the ITO work function, introduction of negative end dipole organized furthest from the surface is always effective. Many researchers have used organic to introduce surface dipoles for increasing work function as shown in Figure 13.
Since substantial variations of the energy required removing an electron from the Fermi level, the work function is an extremely sensitive indicator. The ultraviolet photoelectron spectroscopy (UPS) measures the lowest work function patch of the surface, whereas KFM method measures the average work function difference under the probe. Therefore, the higher work function values can be obtained by KFM method. The electrostatic condition of ITO surface can change the work function; therefore, surface modification is usually done to increase the work function. Both plasma treatments and acid etching are used in this purpose. Several researchers reported about the SAMs modification to control the surface work function of ITO electrode. As reported by M.Z.H. Khan et al., successive modification with DSS can effectively increase ITO work function as shown in Figure 14.
During doping process, oxygen vacancies can create which plays important role in ultimate work function value determination. The surface morphology and crystallinity of ITO can affect by tin (Sn) that creates many amorphous domains during its addition. Moreover, introducing In and Sn impurities near the surface can easily lower the work function of ITO electrode by altering its surface chemistry.
It is well established that surface treatment of ITO electrode can affect its work function and surface potential. Not only to remove impurities, but also to change the surface chemical composition, surface treatment plays important role. To control the surface chemical composition and surface height distribution, chemical treatment is a very common method. Notes: The SAM materials were 4-chlorophenhyl trichlorosilane (4-CPTS), chloromethyl trichlorosilane (CMTS), 4-chlorophenyl phosphonic acid (4-CPPA), 3-nitrophenyl phosphonic acid (3-NPPA), and 2-chloroethyl phosphonic acid (2-CEPA) (Jee et al., 2006).

Concluding remarks
The effect of surface modification of ITO electrode on its response as sensing device was investigated by various researchers in the last few decades. Several types of polycrystalline ITO electrodes have been studied by employing experimental methods of surface characterization to investigate the effect of surface morphology and composition on its potential response in electrolyte solution. It was revealed that the surface roughness of ITO electrode has a strong effect on its potential stability in electrolyte. From an extensive investigation on the roughness parameters of different types of ITO electrodes, it was observed that in addition to the average roughness and root mean square roughness values, a combination of skewness and kurtosis values is useful for considering the utility of ITO electrode. An electrode with spiky surface often shows an unstable rest potential even at modified state. A slight difference in In:Sn ratio at the near surface of the ITO substrates is remarkable, and considered to relate with surface roughness. It was observed that a surface with Gaussian like distribution is desired for effective monolayer modification as well as stable rest potential. The difference in the magnitude of the change in work function after monolayer modification among samples was accounted for by the difference in the degree of orientation and coverage of surface-attached molecules.