Printed carbon based interface for protein immobilization
- 117 Downloads
The aim of the investigations was to find a method of protein immobilisation in screen printed graphite layers. Three commonly used graphite powders were used to prepare conductive layers via screen printing. Several printing pastes with different carbon to polymer resin (polymethylmethacrylate) ratios were tested and the composition with the best electrical properties was selected for further investigations related to green fluorescent protein immobilisation. Six different procedures of protein immobilisation were examined including physical adsorption, electrochemical generation of carboxylic groups and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide activation, graphite functionalisation with succinic anhydride and graphite functionalisation with 3-(triethoxysilyl)propylsuccinic anhydride. Some of the functionalisation procedures were done before printing (functionalisation of graphite powders) and the others were done on printed and cured coatings. Printed graphite layers with immobilised green fluorescent proteins were studied with confocal fluorescent microscopy to assess the efficiency of the immobilisation procedures. The best results were observed for graphite functionalisation with succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide activation of carboxylic groups.
Protein based interfaces are very important and promising in biosensor market and thus different immobilization solutions are under development. To realize such an interface, proteins need to be attached firmly to the surface. There are several methods reported in the literature, some are irreversible and other reversible. Among the irreversible we can distinguish covalent binding [1, 2, 3], cross-linking [4, 5, 6], and entrapment [7, 8, 9]. The reversible methods of immobilization includes adsorption, both physical binding [10, 11, 12] and ionic binding , bio-affinity [14, 15], chelation or metal binding [13, 16], or disulphide bonds . Proteins may be an active sensing component themselves or may be used as an interconnection for other molecules. The structure and method of operation of the sensor determine if the bioreceptors should be bound to the conductive or insulating surface. Sensors may work for example as dipole antennas where load impedance changes its resonant frequency, return loss and reflected signal , then conductive surface with bioreceptors may be implemented. It may be also possible to create sensitive capacitors, where one of plates is functionalised with bioreceptors. Printing methods are one of the low-cost and high yield process that may be used for protein based devices and surfaces. The deposition may be done on rigid (glass, ceramic [19, 20, 21]), flexible (PET [22, 23, 24], paper [25, 26, 27], textiles [28, 29, 30]) as well as stretchable (silicone rubber [31, 32, 33], polyurethane [34, 35, 36]) materials. Moreover patterning to desired shape is possible without development and etching. Thanks to printing deposition and avoiding noble metals it is possible to realize the disposable approach that allows to avoid the regeneration or sterilisation of the sensor surface. Moreover, intensive research around carbon-based materials has led to the synthesis of numerous multifunctional composites based on diverse carbon allotropes such as graphite and graphene [37, 38, 39]. Sensing layers in sensors may be printed with diverse printing techniques such as screen printing , ink-jet printing , flexography , gravure  or aerosol jet . This layers may be already fully functional after printing or may be functionalized to acquire desired properties in additional processes after printing .
In this paper we are presenting the results of investigations related to a universal protein interface that can be used for various sensors with different bioreceptors. Our goal was to achieve a conductive graphite-based printed layer functionalised in such a manner that diverse bioreceptors, such as antibodies, enzymes, or peptides, might be attached to achieve desired functionalities of the sensing layer. Moreover, our results may also be applied to surface modifications performed with the aim to control the wettability or other surface properties.
2 Materials and methods
Investigated layers were deposited by means of screen printing technique, therefore specially tailored materials in form of screen printing pastes needed to be prepared. Pastes consisted of polymer resin filled with graphite materials.
Firstly 8.0 wt% polymer resin was prepared by dissolving polymethylmethacrylate (PMMA, Mw = 350,000) granulate in carbitol butyl acetate acquired from Dow Chemical Company by means of magnetic stirrer (70 °C for 12 h).
Afterwards diverse dispersions with different graphite powders were prepared. Three common types of graphite powder were used in the experiments: Emperor 2000 (acquired from Cabot Corporation), Vulcan XC72 (acquired from Cabot Corporation), and Graphite powder (GP20), < 20 µm, synthetic (acquired from Aldrich). Graphite powders, depending of the type, needed diverse concentrations in the paste to achieve good printability. Pastes were printed with the semiautomatic screen printer (Aurel C920) and cured in a chamber dryer 120 °C for 30 min. Samples were printed on the polyethylene terephthalate (PET) 100 µm thick film (3 M, 9962 Diagnostic Microfluidic Hydrophilic Film).
After selecting the best material and composition for the printing pastes (26 wt% GP20 graphite in PMMA resin) several investigations were done to find the most suitable way of attachment of proteins to the printed graphite layers. To assess the ability of immobilisation eGFP-HisTag—green fluorescent protein (GFP) was used and samples were studied with confocal fluorescent microscopy (Nikon Ti Eclipse with confocal system A1R, ion laser IMA101040AL5 emitting light of wavelength of 488 nm, objective CFI Plan Fluor × 40, and NISElements AR 4.13 software). GFP was delivered to the functionalised graphite surfaces in form of solution 0.01 mg/mL in phosphate buffered saline (PBS). PBS consists of 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4 at 25 °C. Used GFP (EMD Millipore, Merck) has excitation maximum for 488 nm and emission maximum for 509 nm. To show the morphology of sample surfaces used for immobilisation, Scanning electron microscope (SEM) pictures were taken using FEI Nova NanoSEM 450. Energy dispersive X-ray analysis (EDX) data were obtained using an Octane Elect detector from EDAX with 20 kV beam energy. X-ray photoelectron spectroscopy (XPS) was performed by spectrometer (Microlab 350 Thermo VG Scientific) equipped with a non-monochromated excitation source of Al Kα (1486.6 eV; 300 W, pressure 1.0 × 10−9 mbar).
2.1 Physical adsorption of GFP to printed carbon surface
2.2 Electrochemical generation of carboxylic groups and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide activation
2.3 Graphite functionalisation with 3-(triethoxysilyl)propylsuccinic anhydride (TESPSA)
3-(Triethoxysilylpropyl)succinic anhydride (TESPSA) was weighted in various amounts, then graphite and on the end PMMA resin was added in a way to achieve 26 wt% graphite in polymer resin. Samples with 1 wt% TESPSA, 5 wt% TESPSA, 10 wt% TESPSA in relation to graphite weight were prepared. All compositions were mixed and homogenized in a agate mortar for 20 min then printed in the same way as described above. Sample with TESPSA used as the only resin was rejected from further studies due to the high autofluorescence.
Printed graphite surfaces with TESPSA were cured in 120 °C for 1 h in chamber dryer to dehydrate succinic anhydride groups and then the surface was immersed in GFP solution for 1 h.
2.4 Graphite functionalisation with TESPSA and EDC activation of carboxylic groups
Printed graphite surfaces with TESPSA were cured in 120 °C for 1 h in chamber dryer, then they were incubated in EDC (4 mg/mL) for 15 min and at the end GFP protein was delivered to the surface (1 h incubation).
2.5 Graphite functionalisation with succinic anhydride
Succinic anhydride was introduced into printed layers by incubating the graphite powders in succinic anhydride solution before preparing the paste. Three series of samples were prepared: 0.1 mM, 0.5 mM, 1.0 mM of succinic anhydride. After 24 h incubation, modified graphite powders were dried in the oven (80 °C). Functionalized graphite powders were used for printing pastes preparation therefore PMMA resin was added to achieve desired compositions (26 wt% of graphite in polymer resin).
Printed graphite surfaces with succinic anhydride were cured in 120 °C for 1 h in chamber dryer and then GFP protein was delivered to the surface (1 h incubation).
2.6 Graphite functionalisation with succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide activation of carboxylic groups
Printed graphite surfaces with succinic anhydride were cured in 120 °C for 1 h in chamber dryer, then activated with EDC, and then GFP protein was delivered to the surface (1 h incubation).
3 Results and discussion
Sheet resistance and its standard deviation of printed conductive layers with addition of TESPSA
1 wt % TESPSA + GP20 + PMMA
2.33 ± 0.79
5 wt % TESPSA + GP20 + PMMA
2.38 ± 0.75
10 wt % TESPSA + GP20 + PMMA
2.53 ± 0.81
15 wt % TESPSA + GP20 + PMMA
3.15 ± 0.85
Sheet resistance and its standard deviation of printed conductive layers with functionalised graphite powder (GP20)
0.1 mM succinic anhydride
1.00 ± 0.23
0.5 mM succinic anhydride
1.46 ± 0.31
1.0 mM succinic anhydride
1.76 ± 0.32
Elemental surface compositions determined by XPS (atom%) for samples taken for protein immobilisation
TESPSA 1 wt%
TESPSA 10 wt%
0.1 mM succinic anhydride
1.0 mM succinic anhydride
The XPS measurements results are consistent with EDX results since graphite was functionalised before printing, thus the functionalisation elements are present not only on the surface but also in the depth of the material. Significantly higher amount of silicon might be seen for TESPSA and higher amount of oxygen for succinic anhydrite. The slight amount of silicon in samples functionalized with succinic anhydride may come from the process of mixing of the printing paste in the mortar which is made from ceramics.
From the technical point of view, methods where graphite is functionalised prior to printing pastes preparation are easier to apply in large scale than methods where surface processing is performed after printing. It is easier to conduct chemical modifications on powders than on printed layers when masking may be required.
Investigations described in the present paper show that conductive graphite layers deposited by means of screen printing method may be successfully enriched with proteins. The best results are achieved for procedure where graphite powders are functionalised prior to screen printing paste preparation—procedure no 6 “Graphite functionalisation with succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide activation of carboxylic groups”. Successful chemical functionalisation effecting in presence of functionalised groups was proven via EDX and XPS analysis. Those functionalised groups are well known and described as a tool for covalent bonding of proteins . Results may be used in i.e., bioelectronic interfaces with active antennas or capacitors or may be applied for tuning of surface properties. Thanks to deposition with printing technique it is easy to coat extensive surfaces with high yield. Moreover it is possible to use diverse substrates, rigid like glass or ceramics as well as flexible like polymer films, papers or textiles.
The research was financially supported in 60% from funds for research activities of the Institute of Metrology and Biomedical Engineering, Faculty of Mechatronics, Warsaw University of Technology (Poland), and in 40% from the project 3/DOT/2016 funded by the City of Gdynia (Poland).
- 15.G.F. Bickerstaff, Immobilized Enzyme Cells (Humana Press, New Jersey, 1997), pp. 1–12Google Scholar
- 33.A.S. Kurian, T. Giffney, J. Lee, J. Travas-Sejdic, K.C. Aw, Electroact. Polym. Actuators Devices 9798, 97980 (2016)Google Scholar
- 36.S. Chen, X. Shan, W. L. R. Tang, B. M. Mohaime, M. H. Goh, Z. W. Zhong, J. Wei, Electronics Packaging Technology Conference (EPTC) 19th, 1 (2017)Google Scholar
- 42.D. Maddipatla, B.B. Narakathu, S.G.R. Avuthu, S. Emamian, A. Eshkeiti, A.A. Chlaihawi, B.J. Bazuin, M.K. Joyce, C.W. Barrett, M.Z. Atashbar, IEEE Sensors 2015, 1 (2015)Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.