Engineered peptide-based nanobiomaterials for electrochemical cell chip
© The Author(s) 2016
Received: 22 May 2016
Accepted: 11 June 2016
Published: 25 July 2016
Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly-l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.
Recently, cell chip based electrochemical sensing has been proved to be a potential tool for bio sensing , environmental monitoring [2–4], and in vitro drug effect studies [5, 6]. This label free detection method provides accurate, in situ monitoring of analytes avoiding photo bleaching effect of the traditional colorimetric spectrochemical assays . This important tool requires mammalian cell immobilized platform on which the analytes were exposed prior to electrochemical recoding of the cellular responses. The treatment of analytes and recording cellular response required several washing steps that might cause cell eruption from the electrode surface. To avoid such possibility, farm cell-electrode attachment should be ensure for effective sensing of analytes. Therefore, bioengineers are still looking for suitable material with superior cell adhesion ability. Hence, this review focuses on the applications of engineered cell adhesion molecules at the cell electrode interfaces with special emphasis on the effects of RGD motif at various two- and three-dimensionally patterned nanostructures on the sensitivity of electrochemical measurements.
Materials of biological origin i.e. biomaterials have numerous applications in vitro biological or biomedical or tissue engineering research. In the recent decades, a number of researches have been carried out on the use of biomaterials with in vivo like functionality for tissue engineering applications [8–10]. The roles of biomaterials found to be depends on their two or three dimensional topography as well as their nano or microscale spatial arrangements [11–13]. The nanoscale arrangements with desired topography of biomaterials proved to be potential for specific function in advancing the field of biology and medicine. Like in vivo condition, the precise arrangement of nanostructured extra cellular matrix (ECM) materials allows their adhesion motif to the cellular receptors and found to posse significant influences on cell functions [12–14]. Hence, establishment of nanostructured biomaterials on the artificial surface is pivotal for the fabrication of living cell based bioplatform for tissue engineering and sensing application. The repeated unites of Arg-Gly-Asp (RGD) in ECM proteins are considered to be functional motif for cell-ECM interaction through RGD integrin linkage [15–17]. Recently, this RGD tripeptide sequences were synthesized at a various archistructural arrangements like RGD-Map-C and C(RGD)4 for the functionalization of artificial surface for establishing mammalian cells [10, 18]. The RGD motif organized on the artificial surfaces at a define nanoscale arrangement is essential for the sensing applications of fabricated cell based platform.
The nanoscale patterning of biomaterials has become an important topic in cell chip based research. Recently, RGD tripeptide sequences are patterned at various spatial or archistructural arrangements using the self-assembling (commonly known SA method) of biomaterials with several copolymers or guided assembling of the materials through size controlled porous masks (mask guided self-assembly, MGSA method) [10, 11]. The former SA method allows the definite spatial arrangement by controlling the ratio between the materials and copolymers . However, recently introduced modified self-assembly method i.e. MGSA has given an excellent opportunity for assembling biomaterials at a definite spatial as well as archistructural arrangements . This precise spatial and size controlled nanostructuring opportunity together with the excellent cell adhesion ability of RGD peptide has great significance in the rapidly expanding cell chip technologies .
Therefore, this review discusses on the use of several biomaterials at cell electrode interfaces of a living cell based chip together with their influence on cellular adhesion as well as on the electrochemical measurements. In addition, the detailed method of establishment of cell adhesion molecules with special emphasis on RGD nano-structuring protocol has been discussed here in this review. The performances of various materials modified surfaces have been discussed with special emphasis on their cellular adhesion and electrochemical measurements. Moreover, the effect of RGD nanostructures and their homogenous thin film like arrangement has been discussed here critically to suggest a suitable RGD nanostructure for cell chip. This article recommends that cell immobilized on RGD-Map-C nanostructures modified conductive cell chip is very effective tool for the electrochemical measurement.
2 Biomaterials used at cell electrode interface for cell chip fabrication
Cell in tissue produces extracellular materials on which it is strictly attached, proliferates and organized to attain a specific tissue structure. ECM materials or their component like collagen, PLL, RGD peptides have been extensively used for engineering of artificial bioplatform suitable for mammalian cell adhesion and proliferation with the aim of cell based sensing applications or tissue regenerations [20–23].
2.2 PLL peptide
2.3 RGD peptide
3 Establishment of biomaterials on the metal electrode surface
3.1 Nanoscale RGD peptide thin film preparation
3.2 Formation of Nanopattern RGD peptide layer on Au
Formation of RGD peptide thin film on the Au surface is relatively simple and can be performed using self-assembly method as discusses in the earlier [3, 4]. But, their defined nanoscale pattern on Au surface requires a relatively complex modified self-assembly method [10, 11]. Here in, the detailed steps for the formation of nanoscale RGD pattern using the modified MGSA protocol will be discussed sequentially [10, 11, 18].
3.3 Formation of 2D/3D RGD peptide nano-patterns
4 Role of biomaterials on cell adhesion on the metal electrode surface
The collagen forms thicker layer on the metal surfaces which provide excellent support for cell adhesion, spreading and proliferation, but acts a mechanical beerier for the electron exchange at the cell-electrode interfaces [28, 33]. On the other hand, RGD peptide sequences form homogenous thin layer over the electrode which provides sufficient attachment motif for integrin receptor available on the cell surface [4, 29, 43]. The nanopatterned RGD peptide modified surface provides better cell adhesion ability compared to their homogenous thin film like arrangement . This is because of the spatial arrangement of the RGD peptide specific with the receptor availability of the integrin motif on the cell surface [12, 15]. In addition, the various topographic arrangements of the RGD tripeptide sequences on the artificial surfaces found to affect cellular adhesion, proliferation and differentiation. In our previous research it was reveals that RGD nano pillar like arrangement provides the best possible effect on cell functions compared their RGD nano-rod and dot like arrangements . The nano-rod like arrangement proves better cell immolation ability than their nano dot like arrangement. These materials were proved successful for establishment of rat pheochromocytoma cell (PC-12), Human neuroblastoma cells (SH-SY5Y), Human epithelial carcinoma cells (HeLa), and Human embryonic kidney cells (HEK-293T) on the metal electrode surfaces [3, 4, 10, 11].
5 Role of biomaterials on electrochemical measurements
Electrochemical measurement of a cell based chip depends on the conductivity of the electrode material, proper cell electrode interaction and the actual electro-physiologic state of the cell. The electrode materials conductivity is a pre-requisite for any electrochemical devise. Therefore, conductivity of the material must be ensured during designing a chip. Cell-electrode interaction is also an important arena of cell chip based research that has significant impact on the sensitive measurement of a chip. Usually adhesion molecules, proteins and peptides are used for insuring the proper cell electrode interaction of a cell based chip. Several studies have been undertaken to search for a suitable material to maintain proper cell-electrode interaction for increasing cell adhesion as well as for enhancing electron transfer efficiencies [2, 3, 10, 28]. Our previous study reported that large proteins or peptides modified surfaces form a mechanical barrier at the cell electrode interface that impaired electron exchange [10, 28]. Therefore, this review focuses on the engineering of the short adhesion molecules on the electrode surface for increasing cell adhesion without affecting electrode exchange phenomenon [10, 11, 44].
The whole cell based chip has become a potential tool for electrochemical monitoring or sensing environmental toxicity and in vitro drug effect study. This important tool requires a living cell immobilized conductive surface and a transducer device. The suitability of the cell chip largely depends on cell-electrode interaction force and electron exchange phenomenon between them. The biomaterials having cell adhesion molecules/motif has great impact on the electron exchange mechanism as they are readily employed at the cell electrode interfaces. Therefore, this review focuses on the selection of suitable biomaterials and their engineering on the electrode surfaces to insure farm cell-electrode adhesion as well as to enhance electron exchange between them. The detail discussion reveals that biomaterials like whole ECM materials or its components proteins (laminin, collagen etc.) or small peptides (PLL, RGD etc.) can be used for adhesion and proliferation of cell on metal electrode surfaces. However, most of the large protein when immobilized on the electrode surface they form as thick layer which cause mechanical hindrance of electron transfer between cell and electrode. On the other hand, the small protein or their peptide like RGD, PLL etc. form a homogenous thin film over the electrode which result farm cellular attachment without hampering the electron exchange mechanism. Considering this benefit series of research has been performed on the engineering of the small peptide at the cell electrode interface. The major outcome of such researches include development of protocols for the formation of RGD peptide homogenous thin-film formation and spatially or vertically controlled 2D or 3D nanostructures for enhancing sensitivity of the chip. Most of the research reported that nanostructured RGD tripeptide modified electrode found to be more suitable than their homogenous thin-film like arrangement. Among the fabricated nanostructures RGD nano pillars were found to be more suitable than RGD nano dots. In addition to the spatial and archistructural arrangement, the several polymorphs of synthetic RGD (CRGD4, RGD-Map-C) peptides also have influence on the sensitivity of cell based chip. RGD-Map-C was suitably engineered on the electrode surface for the electrochemical monitoring of the cell cycle. Moreover, cell chip with RGD-Map-C nanostructured modified conductive surface was proved to be a suitable platform for onsite electrochemical monitoring of environmental analytes. Therefore, further miniaturization and automation of the chip can improve its application in environmental monitoring or drug effect study.
MAK, HYC and JWC wrote the manuscript. MAK designed the figures. All authors read and approved the final manuscript.
This research was supported by the Leading Foreign Research Institute Recruitment Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (MSIP) (2013K1A4A3055268).
The authors declare that they have no competing interests.
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