Functionalized graphene sensors for real time monitoring fermentation processes Chinnathambi, Selvaraj
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2020
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Chinnathambi, S. (2020). Functionalized graphene sensors for real time monitoring fermentation processes: electrochemical and chemiresistive sensors. University of Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
1.1. Introduction
Fermentation is an important biological process that plays a vital role in several industrial applications, for example in the food industry and beverage production. The fermentation processes is usually performed batch-wise in large bioreactors. Bacteria, yeast or fungi grow in a liquid culture medium containing a carbon, nitrogen, and phosphorus source and some other macro- and micro-nutrients depending on the nutritional requirements of the organism. In addition to that, most biological processes in bioreactors are monitored and controlled using one or multiple sensors. Most frequently, sensors for pH, dissolved oxygen, redox potential, and temperature are used for general fermentation process monitoring. If there is a need to optimize a particular fermentation process, ideally this would be performed in the same reactors as the reactors that will be used in the final production process. However, large volume of culture media, additional chemicals, and energy are necessary to maintain optimal process parameters like pH, dissolved oxygen, and temperature are required. In this regard, downscaling of the reactor size is crucial for efficient and economical determination of the effect of variations in process parameters, media composition and organisms. Small-sized fermentors can be the basis for the development of a high throughput screening platform for optimal growth and production conditions. However, extensive validation need to be performed to ensure that the optimal fermentation conditions are the same optimal conditions in large scale reactors. Another challenge with mini-reactors is the necessity of small-sized sensors to monitor and control the process parameters. For an efficient and reasonable priced high throughput screening system, the sensors should be cheap, easy to maintain or replace, and are readout with simple electronics that can be easily multiplexed to avoid the use of expensive multichannel analog-to-digital converters. The system should be simple to build with as little complexity as possible. For instance every reactor contains at least a pH sensor, dissolved oxygen sensor, temperature sensor, a heating element, and some kind of biomass
sensor. With one ground and 5 signals, 6 electrical connections per fermentor are needed. In a 96-reactor system this is 6 x 96= 564 electrical connections. The system becomes even more complicated if the liquid and gas connections are also taken into account. The reactors contain a feed-in, effluent out, base-in, acid-in, gas-in, and gas out resulting in 6 x 96 = 564 small tubes in and out of the reactor. So, more than 1000 connections to the screening platform need to be established. Wireless sensors (Bluetooth, Near Field Connections (NFC)), and/or Wi-Fi for the electrical signals reduces these complexities. Though it increases the prize of the sensor, the wireless sensing system creates the possibility to develop smart process control systems.
For pH measurement glass electrode based electrochemical sensors are commonly used. It is very difficult to miniaturize the glass electrode due to its fragility of thin glass and reference electrode system. Because of this problem, other miniaturized pH sensors based on ISFET and fluorescence are used in miniaturized bioreactors. The ISFET and optical pH sensors also have some disadvantages. Long term stability of ISFET pH sensors is an issue due to drifting of the output signal and fluorescence-based optical pH sensors have a limited sensing range. Even though limitations exist, optical pH sensors are used in microtiter plate based mini bioreactors (Matrix-24, BioLector). In this thesis, we developed chemiresistive sensors for monitoring the pH and dissolved oxygen. We show that the sensors can be applied in an integrated sensing platform for microtiter plate based mini bioreactors potentially to be expanded into a high throughput fermentation screening platform.
1.2. Bacterial fermentation Process
Fermentation is a complex biological process that is dependent on the environment and also changes the environment. Fermentation is performed by the microorganism that belongs to all three kingdoms, Bacteria, Archaea and Eukarya. Fermentation is defined as the process in
which the microorganisms generate their own electron acceptors if an external electron acceptor is not available or cannot be used by the microorganisms. The electron acceptors are reduced by the protons and electrons derived from the electron donor. Usually, the electron donor is the carbon source but hydrogen can also be used as the electron donor. The products are excreted e.g., short chain volatile fatty acids (acetate, propionate, butyrate), lactate, ethanol, butanol or methane. The excretion of these fermentation products led to a large number of food, feed, and energy applications by adding value, like taste, structure, and energy conversion to the source material. Nowadays, fermentations are an important application area in the industry. Process control during the fermentation process is critical to obtain optimized growth and production conditions.
Figure 1: Illustration of process parameters affecting the fermentation process
There are several process parameters that influences the bacterial fermentation. Among them, pH, dissolved oxygen, biomass, redox potential, and temperature play a crucial role in determining the quality of the fermentation process. For lab and industrial scale bioreactors,
Bacterial Fermentation pH O2 2 (DO) Temperature CO2 Biomass concentration Redox potential
conventional electrochemical (pH, O2, and redox), optical (pH, O2, and biomass), and
capacitance sensors (biomass) are normally used to the satisfaction of the process engineers. However, when bioreactor research follows the current trend of miniaturization and parallel processing, the traditional sensors are too big to fit into the small reactors and too expensive to apply in high throughput screening systems. Besides the online sensors for continuous process monitoring, there are several analytical methods used to monitor and control the fermentation process. In the past, classical analytical techniques like liquid chromatography, gas chromatography and mass spectroscopy and other analytical devices were used off-line. The progress in automatization and robotics enable the researchers to integrate the analytical equipment into online monitoring tools of fermentation processes.
1.2.1. Downscaling of Bioreactors
Small scale bioreactors are excellent tools for the screening of optimal fermentation conditions. They also might be useful in the selection of suitable microorganisms for the development of a new fermentation process. They might even serve as production reactors in continuous flow systems analogues to continuous-flow parallel microreactors in the chemical industry.
Miniaturization of cultivation systems has several effects on the physicochemical property of microorganisms and the operation of bioreactors [1]. Though the dimensional change of suspension of culture in a miniaturised reactor, compared to standard conical flask, has minimum effect on the physiology of the microorganism, there are other effects that dominates the culture in miniaturised reactors. A higher specific oxygen transfer rates (OTRs) is obtained in miniaturised culture systems due to high gas-liquid exchange area to the volume of the bulk liquid. Another important factor dominating the miniaturization effect is the surface tension of the culture medium. Microtiter plates have been successfully used as
miniaturised bioreactors and they become one of the important devices in the process of downscaling bioreactors because they are cheap, widely available in a variety of formats and construction material is sterile and compatible with a growing number of downstream analytical methods (Microtiterplate (MTP) readers, auto samplers, robotic liquid handling equipment, centrifuges, DNA extraction methods, etc.). The microtiter plates are increasingly being used instead of standard conical flask as a high throughput miniaturised reactor.
1.2.2. Microtiter plate mini bioreactors:
Microtiter plates have been used extensively as a high throughput screening platform for strain, gene library selection, or simple growth condition optimization as an alternative to multiple shaken flasks for screening large numbers of fermentative/bioconversion processes. However, screening experiments with concomitated monitoring and control of fermentation parameters (pH, DO, and biomass) in microtiterplates or multiple individual microreactors are limited [1-3]. Some advantages of using microtiterplates as a screening platform feature simultaneous investigation of different medium composition, effect of physiological conditions like pH and oxygen transfer rate, mixing and diffusion of reactants in the media. In addition to that, the standard microtiter plate is compatible with existing automated system and uses a small footprint [4-6]. Some newly designed mini bioreactors also are equipped with small magnetic stirring bars or gas-induced impellers to improve mass transfer to the culture. The growth of Escherichia coli in such mini-reactors was compared and it was found to be equivalent to that in larger scale (up to 3 L) bioreactors [7-8]. Pharmaceutical companies and research laboratories are looking closely at small-scale bioreactors to meet the needs of higher throughput controlled cell cultivation [5, 9-12]
Figure 2: Image of a standard 96-well microtiter plate
To use microtiter plate as a high throughput screening platform with online monitoring and control of process parameters, the miniaturised pH, dissolved oxygen, biomass, and temperature sensors needs to be implemented within the microtiter plate. The real time online measurements of pH, dissolved oxygen and biomass provide overall quantitative measurement of medium composition and culture growth. The integrated sensor platform for microtiter plate is investigated in the past using conventional electrochemical sensing techniques, for example, amperometric dissolved oxygen sensor and ISFET pH sensor [13]. These conventional sensing methods use reference electrodes and need special equipment for signal readout. It makes this integrated sensing platform not viable for commercial MTP bio reactors.
1.3. Chemiresistor based sensors
Chemiresistor is a material that changes the resistance/conductivity to a large extent when the chemical environment changes. The chemiresistor consists of two electrodes separated by a distance. The electrodes are connected by chemiresistor material. The resistance or conductivity of the material is measured by applying a constant voltage between the two
electrodes and record the resulting current. The sensor response of the chemiresistor is obtained by measuring the change of the resistance or current of the chemiresistor material upon interaction with the analytes. The sensing mechanism of chemiresistor sensor can be explained by three phenomena which can occur synergistically or independently. The resistance or conductivity change of the chemiresistor device can occur due to a change of doping level, modulation of the Schottky barrier, or, modulation of the junction resistance between particles. When analytes interact with chemiresistor material, the doping level is changed due to change in the concentration of the charge carriers. The effect of doping depends on the nature of the semiconductor material.
Figure 3: Schematic representation of a chemiresistor device
The electron donating analytes, for example NH3 injects an electron and the resistance of the
p-type semiconductor increases while the resistance of the n-type material decreases. Similarly, the resistance of the p-type material decreases and n-type material increases if the analytes are electron withdrawing groups. When analytes interact with the surface, the work function of material changes which results in the modification of the Schottky barrier between
Au electrode
Reduced Graphene oxide
Polyaniline
Hole proton
H+ +
the electrode-material contact. When analytes adsorb at the grain boundaries or particle junction the hopping distance increases due to an increase in the distance between the grain boundaries. A good chemiresistive sensor has features like fast response time, low drift, fast recovery, high sensitivity, and long term stability.
Several materials are used as chemiresistor, mainly, for gas sensing applications. Carbon nanotubes (CNT) [14-16], graphene [16-17], metal nanoparticles [18-25] and conducting polymers [26-28] are widely studied as chemiresistive material in gas sensing applications. Recently, a CNT-conducting polymer composite and a gold nanoparticle film are used as chemiresistor material in sensors for applications in aqueous solution [29-31]. Only a few chemiresistor have been developed to detect analytes in aqueous solution. Hence, it is highly challenging and also important to develop chemiresistor devices for the detection of analytes dissolved in aqueous solutions.
One of the significant advantages of Graphene is that its properties can be tuned by functionalization, giving modified graphene a considerable potential in sensing applications. Responsive molecules can be attached covalently or non-covalently onto the graphene surface using (electro-)chemical methods. In addition to these properties, the chemical stability of graphene is advantageous in chemically harsh environments.
1.4. Graphene
Graphene is a two-dimensional single atom layer that consists of an sp2-bonded carbon
network. Graphene was discovered by A. K. Geim and K.S. Novesolo by mechanical exfoliation of graphite using the famous scotch tape method [32-33]. Graphene has excellent properties like high electron mobility, thermal conductivity and high mechanical strength [34]. Mechanical exfoliation of graphene is an easy way to prepare high quality graphene, however, the production of large quantities for commercial application is cumbersome .
Hence, there are several methods of graphene preparation in development. Chemical vapor deposition, reduction of graphene oxide, electrochemical exfoliation of graphite, thermal decomposition in the presence of volatile compounds are some of the methods that are explored for graphene synthesis at a larger scale . Among them, production of graphene by chemical graphene oxide reduction (RGO) is a promising method to produce graphene on a larger scale.
Graphene is produced chemically by reducing graphene oxide. Graphite oxide is a precursor for RGO preparation. Hummers and Offeman developed a method for graphite oxidation [35]. They used potassium permanganate and concentrated sulfuric acid as oxidizing agents. A similar approach was developed by L. Staudenmaier using a mixture of KClO3 and sulfuric
acid to oxidize graphite [36].
Chemical production of graphene involves oxidation of graphite into graphite oxide, exfoliation of graphite oxide into graphene oxide, and the reduction of graphene oxide to graphene (RGO). There are several ways in which graphene oxide can be reduced to graphene. Graphene oxide can be reduced by various reduction methods among others chemical, thermal, electrochemical and photo catalytic reduction [37-60]. In the chemical approach, reducing agents such as hydrogen sulphide, hydrazine, or sodium borohydride are used under hot alkaline conditions (pH 10 at 90 ºC) [44-51]. For thermal reduction, graphene oxide is heated to 200-300 °C for one hour. During this treatment, the functional groups are removed by thermal decomposition, hence, hazardous gaseous species are liberated [52-54]. Electrochemical reduction of graphene oxide is considered as a much more green and environmental friendly reduction process. Graphene oxide is deposited on a conducting substrate. The reduction occurs around -0.8 V (Ag / AgCl) and the majority of the functional groups are removed by this method [55-57]. UV-assisted photocatalytic reduction is also used to reduce graphene oxide. In this process, reduction occurred by charge transfer reaction
between TiO2 and graphene oxide. During irradiation, the surface charge of TiO2 is increased
which is used for reduction of functional groups that are present on the graphene oxide [58]. RGO is also prepared by a hydrothermal reduction method. A GO dispersion is placed in a Teflon lined autoclave and heated upto 120-180 °C. In this process super-heated water molecules are involved in the reduction of GO [59-60].
Reduced graphene oxide contains defective sites due to the removal of oxygen containing functional groups and the reduction of GO is not always complete. This means that there are always some oxygen functional groups left on the RGO. The defects and left-over oxygen functional groups renders RGO as a high reactive material for further functionalization to make it useful in sensing applications [66-66]. Several, RGO based sensing devices are fabricated as gas and molecular sensors. RGO can be deposited as thin film, flakes, or rippon form on a e.g., interdigitated gold electrode. Chemiresistive sensing applications have been reported for NH3, NO2, O2 and volatile organic compounds but not for aqueous applications
1.5. Contents of the thesis
Chapter 1 introduces the objective of this thesis and explains the importance of downscaling
the bioreactor and the role of a sensor platform in it. As the graphene is used as a sensor material, its properties and the available methods of synthesis also are introduced in this chapter.
Chapter 2 describes the process parameters affecting the bacterial fermentation process and
the fundamentals of the sensor used for process monitoring. An overview of micro sensors for the miniaturised micro titer plate bioreactor is presented in this chapter.
In chapter 3, the fabrication of a polyaniline functionalized electrochemically reduced graphene oxide pH sensor is reported. Potentiometric and chemiresistive sensing of this material is explored and the results are discussed. The feasibility of this pH sensor to monitor the pH during the bacterial fermentation process is shown.
In chapter 4, the pH sensing property of hydrothermally reduced graphene oxide is investigated. Potentiometric and chemiresistive pH sensing of hydrothermally reduced graphene oxide is explored and results are discussed. Cyclic voltammetry is used to characterize the nature of functional groups present on the reduced graphene oxide.
In chapter 5, a Chemiresistive dissolved oxygen sensor based on nitrogen and boron doped hydrothermally reduced graphene oxide is explored as a new approach towards dissolved oxygen sensing. Material preparation, characterization, amperometric and chemiresistive sensor properties results are presented and discussed in detail. Further, the sensor is tested in a bacterial fermentation process and the results are discussed in this chapter.
Chapter 6 presents an integrated sensing platform for miniaturised microtiterplates based
bioreactor is explored and optical biomass sensor is developed to measure the optical density of the bacterial growth. The fabrication of integrated sensor lid containing chemiresistive pH, dissolved oxgyen and the fabrication of optical biomass sensor results are discussed. The miniaturized reactor with sensor housing lid is fabricated using 3D printing technology. The growth of bacteria in the 3D printed miniaturised bio reactor is investigated and the results are discussed in this section.
In Chapter 7, the complete thesis work is summarized and the future direction to improve the sensor properties are discussed.
1.6. Reference
1. W. A. Duetz, Microtiter plates as mini-bioreactors: miniaturization of fermentation methods, TRENDS in Microbiology 15 (2007) 469-475.
2. M. Micheletti and Gary J Lye, Microscale bioprocess optimisation, Current Opinion in Biotechnology 17 (2006) 611–618
3. P. Fernandes, J.M.S. Cabral, Microlitre/millilitre shaken bioreactors in fermentative and biotransformation processes—a review Biocatal. Biotransform. 24 (2006) 237-252.
4. G. J. Lye, P. A. Dalby, J. M. Woodley, Better biocatalytic processes faster: New tools for the implementation of biocatalysis in organic synthesis. Org Process Res Dev, 6 (2002) 434–440.
6. R. Hermann, N. Walther, U. Maier, J. Bu¨chs, Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation. Biotechnol Bioeng 74 (2001) 355–363.
7. Puskeiler R, Kaufmann K, Weuster-Botz D, Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD). Biotechnol. and Bioeng. 89 (2005) 512–523.
8. Z. Zhang, N. Szita, P. Boccazzi, A.J. Sinskey, K.R. JensenA well-mixed polymer-based microbioreactor with integrated optical measurements, Biotechnol. Bioeng. 93 (2006) 286-296.
9. Boswell. C, Microreactors gain wider used as alternative to batch production, CMR, 11 (2004) 266.
10. M. Maharbiz, W. Holtz, R. Howe, J. Keasling, Microbioreactor arrays with parametric control for high throughput experimentation. Biotechnol. Bioeng. 85 (2004) 376-381. 11. G. J. Lye, P. Ayazi-Shamlou, F. Baganz, P. A. Dalby, J. Woodley, Trends Biotechnol.
21 (2003) 29-37.
12. R. S. Islam, D. Tisi, M. S. Levy, G. J. Lye, Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched kLa, Biotechnol. Bioeng. 99 (2008) 1128-1139.
13. Erik Krommenhoek, Integrated sensor array for online monitoring micro bioreactors, PhD thesis, Delft technology university, 2007
14. J. Kong, N. R. Franklin, C. Zhou, M. G. Chapline., S. Peng, K. Cho, H. Dai, Nanotube Molecular Wires as Chemical Sensors Science 287 (2000) 622– 625.
15. A. J. Robinson, E. S. Snow, C. S. Badescu, T. L. Reinecke, F. K. Perkins, Role of Defects in Single-Walled Carbon Nanotube Chemical Sensors, Nano Lett. 6 (2006) 1747– 1751.
16. D. R. Kauffman, C. M. Shade, H. Uh, S. Petoud and A. Star, Decorated carbon nanotubes with unique oxygen sensitivity, Nature Chemistry 1 (2009) 500–506.
17. W. Yuan and G. Shi, Graphene-based gas sensors, J. Mater. Chem. A 1 (2013) 10078– 10091.
18. J. T. Robinson, F. K. Perkins, E. S. Snow, Z. Wei and P. E. Sheehan, Reduced Graphene Oxide Molecular Sensors, Nano Lett., 8 (2008) 3137–3140.
19. K. Fu, S. Chen, J. Zhao, and B. G. Willis, Dielectrophoretic Assembly of Gold Nanoparticles in Nanoscale Junctions for Rapid, Miniature Chemiresistor Vapor Sensors, ACS Sens. 4 (2016) 444–450.
20. G. Peng, U. Tisch, O. Adams, M. Hakim, N. Shehada, Y. Broza, S. Billan, R. Abdah-Bortnyak, A. Kuten, H. Haick, Diagnosing lung cancer in exhaled breath using gold nanoparticles. Nat.Nanotechnol. 4 (2009) 669−673.
21. Y. Joseph, B. Guse, T. Vossmeyer, A. Yasuda Gold Nanoparticle/Organic Networks as Chemiresistor Coatings: The Effect of Film Morphology on Vapor Sensitivity. J. Phys. Chem. C 112 (2008) 12507−12514.
22. J. S. Cooper, B. Raguse, E. Chow, L. Hubble, K. H. Müller, L. Wieczorek, Gold Nanoparticle Chemiresistor Sensor Array that Differentiates between Hydrocarbon Fuels Dissolved in Artificial Seawater. Anal. Chem. 82 (2010) 3788−3795.
23. F. J. Ibañez, F. P. Zamborini, Chemiresistive Sensing of Volatile Organic Compounds with Films of Surfactant-Stabilized Gold and Gold−Silver Alloy Nanoparticles. ACS Nano 2 (2008) 1543−1552.
24. N. Nagraj, J. M. Slocik, D.M. Phillips, N. Kelley-Loughnane, R. R. Naik, R. A. Potyrailo, Selective sensing of vapors of similar dielectric constants using peptide-capped gold nanoparticles on individual multivariable transducers. Analyst 138 (2013) 4334−4339.
25. K. Fu, S. Li, X. Jiang, Y. Wang, B. G. Willis, DNA Gold Nanoparticle Nanocomposite Films for Chemiresistive Vapor Sensing. Langmuir 29 (2013) 14335−14343.
26. J. Janata, M. Josowicz, Conducting polymers in electronic chemical sensors, Nature Materials 2 (2003) 19–24.
27. C. M. Hangarter, M. Bangar, A. Mulchandani and N. V. Myung, J. Conducting polymer nanowires for chemiresistive and FET-based bio/chemical sensors,
Mater. Chem. 20 (2010) 3131-3140.
28. U. Langea, V. M. Mirsky, Chemiresistors based on conducting polymers: A review on measurement techniques, Analytica Chimica Acta 687 (2011) 105–113.
29. B. Raguse, E. chow, C. S. Barton, and L. wieczorek, Anal. Chem. 79 (2007) 7333-7339.
30. B. Raguse, C. S. Barton, K. H. Mueller, E. chow, and L. wieczorek, J. Phys. Chem. C, 113 (2009) 15390-15397.
31. P. Gou, N. D. Kraut, I. M. Feigel, H. Bai, G. J. Morgan, Y. Chen, Y. Tang, K. Bocan, J. Stachel, L. Berger, M. Mickle, E. Sejdic, A. Star, Scientific reports 4 (2014) 4468.
32. K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, A. A. Firsov, Science 306 (2004) 666–669.
33. K. S. Novoselov, D. Jiang, F. Schedin, T. J. Booth, V. V. Khotkevich, S. V. Morozov, and A. K. Geim, Two-dimensional atomic crystals. Proc. Natl Acad. Sci. USA, 102 (2005) 10451–10453.
34. A. K. Geim, K. S. Novoselov, The rise of graphene, Nature Materials 6 (2007) 183– 191.
35. W. S. Hummers, R. E. Offeman, Preparation of Graphitic Oxide, J. Am. Chem. Soc. 80 (1958) 1339.
36. L. Staudenmaier, Process for the preparation of graphitic acid, Ber. Dtsch. Chem. Ges., 31 (1898) 1481–1487.
37. D. Li, M. B. Müller, S. Gilje, R. B. Kaner, G. G. Wallace, Processable aqueous dispersions of graphene nanosheets, Nature Nanotechnology. 3 (2008) 101–105.
38. Cristina Go´ mez-Navarro, R. T. Weitz, A. M. Bittner, M. Scolari, A. Mews, M. Burghard, and K. Kern, Electronic Transport Properties of Individual Chemically Reduced Graphene Oxide Sheets Nano Lett., 7 (2007) 3499.
39. S. Stankovich, D. A. Dikin, R. D. Piner, K. A. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S. T. Nguyen, R. S. Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon. 45 (2007) 1558–1565.
40. H. A. Becerril, J. Mao, Z. Liu, R. M. Stoltenberg, Z. Bao, and Y. Chen, Evaluation of Solution-Processed Reduced Graphene Oxide Films as Transparent Conductors ACS Nano. 2 (2008) 463–470.
41. Y. Zhu , S. Murali , W. Cai , X. Li , J. Suk , J. R. Potts and R. S. Ruoff , Graphene and Graphene Oxide: Synthesis, Properties, and Applications, Adv. Mater. 22 (2010) 3906–3924
42. I. K. Moon, J. Lee, R. S. Ruoff, H. Lee, Reduced graphene oxide by chemical graphitization, Nature Communications. 1 (2010) 73.
43. Song feng Pei, H. M. Cheng, The reduction of graphene oxide, CARBON. 50 (2012) 3210 – 3228.
44. U. Hofmann, and A. Frenzel, The reduction of graphite oxide with hydrogen sulphide, Kolloid-Zeitschrift 68 (1934) 149–151.
45. S. Park and R. S. Ruoff, Chemical methods for the production of graphenes. Nat. Nanotech. 4 (2009) 217–224.
46. R. Muszynski, B. Seger, and P.V. Kamat, Decorating graphene sheets with gold nanoparticles. J. Phys. Chem. C. 112 (2008) 5263–5266.
47. S. Stankovich, Graphene-based composite materials. Nature 2006, 442, 282–286.
48. G. X. Wang, Facile synthesis and characterization of graphene nano sheets. J. Phys. Chem. C. 112 (2008) 8192–8195.
49. W. Gao, L. B. Alemany, L. Ci, and P. M. Ajayan, New insights into the structure and reduction of graphite oxide. Nat. Chem. 1 (2009) 403–408.
50. Z. Fan et al. An environmentally friendly and efficient route for the reduction of graphene oxide by aluminum powder, Carbon 48 (2010) 1686–1689.
51. H. J. Shin etal, Efficient reduction of graphite oxide by sodium borohydride and its effect on electrical conductance. Adv. Funct. Mater. 19 (2009) 1987–1992.
52. W. Chen, L. Yan, P. R. Bangal, Preparation of graphene by the rapid and mild thermal reduction of graphene oxide induced by microwaves CARBON, 48 (2010) 1146 – 1152.
53. R. Larciprete, S. Fabris, T. Sun, P. Lacovig, A. Baraldi, and S. Lizzit, Dual Path Mechanism in the Thermal Reduction of Graphene Oxide J. Am. Chem. Soc. 133 (2011) 17315–17321.
54. Z. Wang, D. Xu, Y. Huang, Z. Wu, L. Wang and X. Zhang, Facile, mild and fast thermal-decomposition reduction of graphene oxide in air and its application in high-performance lithium batteries, Chem. Commun. 48 (2012) 976–978.
55. G. K. Ramesha, and S. Sampath, Electrochemical reduction of oriented graphene oxide films: An in situ Raman spectroelectrochemical study. J. Phys. Chem. C. 113 (2009) 7985–7989.
56. S. J. An, et al. Thin film fabrication and simultaneous anodic reduction of deposited graphene oxide platelets by electrophoretic deposition. J. Phys. Chem. Lett. 1 (2010) 1259–1263.
57. Y. Shao, J. Wang, M. Engelhard, C. Wang and Y. Lin, Facile and controllable electrochemical reduction of graphene oxide and its applications J. Mater. Chem., 20 (2010) 743–748.
58. G. Williams, B. Seger, and P. V. Kamat, TiO2-Graphene Nanocomposites.
UV-Assisted Photocatalytic Reduction of Graphene oxide, ACS Nano 2 (2008) 1487– 1491.
59. D. Long, W. Li, L. Ling, J. Miyawaki, I. Mochida, and S. Yoon, Preparation of Nitrogen-Doped Graphene Sheets by a Combined Chemical and Hydrothermal Reduction of Graphene Oxide Langmuir 26 (2010) 16096–16102.
60. Y. Zhou, Q. Bao, L. L. Tang, Y. Zhong, K. P. Loh, Hydrothermal Dehydration for the “Green” Reduction of Exfoliated Graphene Oxide to Graphene and Demonstration of Tunable Optical Limiting Properties, Chem. Mater. 21 (2009) 2950–2956.
61. W. Yuan and G. Shi, Graphene-based gas sensors, J. Mater. Chem. A. 1 (2013) 10078–10091
62. J. T. Robinson, F. K. Perkins, E. S. Snow, Z. Wei and P. E. Sheehan, Reduced Graphene Oxide Molecular Sensors, Nano Lett., 8 (2008) 3137–3140.
63. F. Yavari and N. Koratkar, Graphene based-chemical sensors J. Phys. Chem. Lett., 3 (2012) 1746–1753.
64. G. Ko, Y. Jung, K.-Y. Lee, K. Lee and J. Kim, Improved sorption characteristics of NH3 molecules on the solution-processed graphene sheets J. Cryst. Growth, 326 (2011) 208–211.
65. N. T. Hu, Y. Y. Wang, J. Chai, R. G. Gao, Z. Yang, E. S.-W. Kong and Y. F. Zhang, Gas sensor based on p-phenylenediamine reduced graphene oxide, Sens. Actuators, B, 163 (2012) 107–114.
66. V. Dua, S. P. Surwade, S. Ammu, S. R. Agnihotra, S. Jain, K. E. Roberts, S. Park, R. S. Ruoff and S. K. Manohar, All‐Organic Vapor Sensor Using Inkjet‐Printed Reduced
67. L. Guo, H. B. Jiang, R. Q. Shao, Y. L. Zhang, S. Y. Xie, J. N. Wang, X. B. Li, F. Jiang, Q. D. Chen, T. Zhang and H. B. Sun, Two-beam-laser interference mediated reduction, patterning and nanostructuring of graphene oxide for the production of a flexible humidity sensing device, Carbon, 50 (2012) 1667–1673.
68. J. D. Fowler, M. J. Allen, V. C. Tung, Y. Yang, R. B. Kaner and B. H. Weiller, Practical Chemical Sensors from Chemically Derived Graphene, ACS Nano, 3 (2009) 301–306.
69. R. K. Paul, S. Badhulika, N. M. Saucedo, and A. Mulchandani, Graphene Nanomesh As Highly Sensitive Chemiresistor Gas Sensor Anal. Chem. 84 (2012) 8171−8178.
70. R. K. Joshi, H. Gomez, F. Alvi and Ashok Kumar, Graphene Films and Ribbons for Sensing of O2, and 100 ppm of CO and NO2 in Practical Conditions, J. Phys. Chem.
