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LIST OF CONTRIBUTORS
Shamima Afroz
Department of Electrical Engineering, University of South Florida, Tampa, FL, United States of America
R. Alinovi
Department of Clinical and Experimental Medicine, Parma University, Parma, Italy
Edwige Bano
IMEP-LAHC Minatec, Grenoble, France
E. Bedogni
Chemistry Department – Parma University, Parma, Italy
F. Bigi
Chemistry Department – Parma University, Parma, Italy
A. Cacchioli
Department of Veterinary Science, Unit of Normal Veterinary Anatomy – Parma University, Parma, Italy
Fabiola Araujo Cespedes
Department of Electrical Engineering, University of South Florida, Tampa, FL, United States of America
Hamid Charkhkar
Electrical and Computer Engineering Department, Volgenau School of Engineering, George Mason University, Fairfax, VA, United States of America
Ji-Hoon Choi
IMEP-LAHC; LTM-CNRS Minatec, Grenoble, France
Stuart F. Cogan
Department of Bioengineering, Erik Jonsson School of Engineering & Computer Science, University of Texas at Dallas, Richardson, TX, United States of America
C. Coletti
Center for Nanotechnology Innovation@NEST, Pisa, Italy
L. Cristofolini
Physics and Earth Science Department – Parma University, Parma, Italy
Pasquale D’angelo
IMEM-CNR Institute of Materials for Electronics and Magnetism – Italian National Research Council, Parma, Italy
F. Fabbri
IMEM-CNR Institute of Materials for Electronics and Magnetism – Italian National Research Council, Parma University, Parma, Italy
PREFACE TO SECOND EDITION
The 21st century has long been hailed as the century of biology, as the extremely complex nature of the biological system is only now beginning to be more fully understood through the use of technology and scientific tools, which allow mankind to observe biological processes at the nanoscale. This has resulted in the ever-pressing need to develop advanced “smart materials” for biomedical applications, and this important field of research has taken center stage worldwide as a new frontier of advanced scientific research. While biocompatible materials have been in use for decades, the quest to merge smart materials such as those used in modern computer integrated circuits with the biological system has proven to be much more difficult than originally imagined. If we take as one example the brain–machine interface, or BMI, which was first reported in the early 1970s and was based on silicon probes, we learn that mankind is not much closer to the prospect of long-term (multiple years or even decades) implantation of these devices. Why? Simply put, most “smart materials” such as most semiconductors are typically toxic to the biological environment, and silicon is no exception. What is needed to realize true nanoscale functionality therefore is a material with the “smart properties” of silicon and the “biocompatible” properties of polymers. In this book such a candidate material, silicon carbide, is presented and the evidence that has been amassed, mostly over the last decade, indicates that this material system may prove to be the dream material for biomedical devices that must do more than simply provide structural support.
Silicon carbide, abbreviated SiC, has a long history as a robust and hard material, which was first used as an abrasive and cutting material in the 19th century and later as a high-temperature semiconductor for advanced applications in the 20th century. Indeed, the first solid-state blue light emission was observed from SiC in 1907, and this material has been under study ever since. One can consult the various available literature to fully understand all of the many aspects of SiC, from how it is formed, to its myriad crystal properties, and finally to the large number and types of applications it is being used in. Fortunately, it is sufficient here to provide a brief overview of SiC so that the reader can understand why this material is so compelling for biomedical applications and may become one of the most used biomaterials in the future. Indeed, the purpose of this second
book is to lay the foundation for exactly this – to introduce silicon carbide to biomedical engineers, medical professionals, and scientists, thus bringing together technologists from across many disciplines to help enable the ultimate use of SiC as one of the next generation of smart biomaterials to realize advanced biomedical devices.
The first edition of Silicon Carbide Biotechnology focused mainly on in vitro material studies in order to introduce the reader to the possibility that SiC can be an important player in the biomedical device community. Indeed, biocompatibility and hemocompatibility data were presented, which either cleared up prior misconceptions of SiC in this arena, or reinforced the known excellent performance of certain SiC material types, called polytypes, to the biological world.While prototype devices were introduced and discussed, the present edition focuses mainly on the significant progress that has been made in the development of SiC biomedical devices, ranging from implants to sensors to in vitro DNA assay systems. The organization of this second edition starts with an updated introduction of SiC, with a particular emphasis on biomedical applications, and is then divided into four distinct sections. First, a fully comprehensive treatment of SiC in vitro performance is presented with a rigorous study of 3C–SiC biocompatibility, conducted in full compliance with ISO 10993, in chapter: Cytotoxicity of 3C–SiC Investigated Through Strict Adherence to ISO 10993. Next, amorphous SiC (a-SiC) is studied using the same in vitro methods; additionally, a study of hemocompatibility for both 3C–SiC and a-SiC is documented in this chapter: Study of the Hemocompatibility of 3C–SiC and a-SiC Films Using ISO 10993-4 where we see both materials have great potential suitability for use in the cardiovascular system.
Having completed the in vitro materials studies required by the FDA to allow for subsequent clinical trials, the second section focuses on the important topic of SiC biosensors. Extremely comprehensive studies on the use of graphene on SiC, as well as surface functionalization of SiC, both for biosensor applications, are presented in the next two chapters: Graphene Functionalization for Biosensor Applications and: SiC Biosensing and Electrochemical Sensing: State of the Art and Perspectives, respectively. We see that the use of epitaxial graphene on SiC is not only a powerful method to realize advanced sensors, it ensures that the device substrate is compatible with the biological system, thus improving device performance and reliability. We see that SiC, due to the fact that it is a compound semiconductor, provides more flexibility in terms of biosensor design simply by taking into account differences in how one can functionalize the Si and C
surfaces of the material. This extra degree in design freedom is not possible with the mainstay advanced biosensor material, silicon, which has proved to have issues with bio- and hemocompatibility. After these two chapters, very important work to realize a continuous glucose-monitoring (CGM) device in vivo is presented, whereby semiinsulating 4H–SiC is used as an implantable RF sensor to detect, in real time, glucose levels in vitro. Given the significant number of humans suffering from diabetes worldwide, this work is extremely important and significant progress is being made in the remote sensing of glucose via a “radio frequency identification (RFID)type” sensor mechanism.
Having completed the sensor portion of this edition, we then move on to the nervous system where developments to realize more robust, longterm performing SiC-based neural implants is discussed. Building on the work presented in the first edition that indicated that 3C–SiC appeared to be an ideal choice for this application, extremely compelling data documenting the in vivo performance of 3C–SiC intracortical implants in “wild-type” mice is presented in chapter: In Vivo Exploration of Robust Implantable Devices Constructed From Biocompatible 3C–SiC, whereby no evidence of neurological immune system response was observed. To further motivate the development of 3C–SiC for this application, results of a comprehensive study, also conducted under the auspices of ISO 10993, shed light on the reason why 3C–SiC performs so well in vitro: the material is highly stable and does not degrade in either normal physiological environments or physiological environments that are enhanced to enable accelerated aging studies to be properly conducted. For any fully functional neural implant using 3C–SiC a suitable electrical insulator is required and this material must have similar biological performance to the support material, 3C–SiC. Amorphous SiC (a-SiC) is just such a material and, being part of the “SiC family of biocompatible materials,” is an ideal companion material for this (and other) applications. The in vivo compatibility of commercial implantable neural interfaces (INI) coated with a-SiC is documented in rats and, once again, proves the incredible potential of SiC for this highly challenging application. This work, including functional in vitro recording data made using microelectrode arrays coated with a-SiC, is presented in chapter: Amorphous Silicon Carbide for Neural Interface Applications. With the neural implant potential of SiC well documented, we then move on to the final section of this edition – the use of SiC nanotechnology for advanced applications such as label-free DNA detection (chapter: SiC Nanowire Based Transistors for Electrical DNA Detection), cancer treatment, and
been extremely supportive of my research activity and group. I especially want to thank Dr G. De Erausquin, Dr K. Muffly, and Dr L. Hoyte, all of the USF Morsani Medical School, for our very fruitful collaboration in the area of biomedical engineering and devices. I am especially indebted to my good friends and colleagues from IMEM in Parma, Dr S. Iannotta, Dr G. Salviati, and Dr F. Rossi, and their colleagues, for our continuing scientific collaboration in the area of SiC biosensors and nanotechnology. I again thank Dr C. Frewin for always being there to support and share my vision of SiC biotechnology and for his ever-faithful friendship and dedication without which I doubt this book would have been possible. Finally, and most dear to my heart, is the ever present love and support of my wife, Vaine Angelo Saddow, with whom we have two amazing children, Luca Edward and Sophia Angelo, who provide all of the incentive that anyone needs to continue to strive for scientific and technological excellence – in some small way I hope that all of this work will allow their quality of life to be the best that man on earth can provide.
Prof.
Stephen E. Saddow, Editor Tampa, FL
CHAPTER 1
Silicon Carbide Materials for Biomedical Applications
Stephen E. Saddow
1.1 PREAMBLE
Considerable progress has been made since the publication of the first edition of Silicon Carbide Biotechnology: A Biocompatible Semiconductor for Advanced Biomedical Devices and Applications in 2011, mostly in the area of SiC nanoparticle and nanowire incorporation in various biomedical devices. In our research group we have made significant progress to translate research performed in the area of SiC materials development for biomedical application into prototype SiC biomedical devices. Examples include 3C–SiC neural implants, functionalized biosensors, and the demonstration of semiinsulating 4H–SiC as a suitable platform for an in vivo glucose sensor based on an RF antenna transducer. In this edition, we build upon the materialsbased discussion from the first edition placing the focus on SiC biotechnology at the device level. We begin by adding additional information to the in vitro biological performance story of 3C–SiC (see chapter: Cytotoxicity of 3C–SiC Investigated Through Strict Adherence to ISO 10993) and
Implantable Devices Constructed From Biocompatible 3C–SiC, where a null inflammatory response to 3C–SiC implanted within a murine model is demonstrated. The next student to complete her research in this area was Dr Alexandra Oliveros, who performed the first in vitro studies on the biocompatibility of graphene and the use of self-assembled monolayers (SAMs) to demonstrate that SiC could be biofunctionalized in a similar way to Si (see Chapters 3 and 12 in the first edition, respectively). She extended this work to demonstrate that myoglobin, a blood protein present at the onset of a heart attack, could be detected using an electrochemical sensor based on SAMs and antimyoglobin antibody functionalization [6]. Following this excellent work in the SiC biosensor arena, Dr Joseph Register developed 3C–SiC as a suitable platform for neural implants that could stimulate optically sensitive neurons with light [7]. These SiC optrodes, which possess greater chemical and physical resilience, may replace silicon optrodes utilized in long-term in vivo neuroscience applications. Finally, Dr Maysam Nezafati completed the critical study of SiC bio- and hemocompatibility by using in-house methods developed under a DARPA program to evaluate materials for use in neural interfaces by strict adherence to ISO-10993. What is important about this work is that, for the first time, 3C–SiC was tested fully in accordance with the standard, which calls for chemical stability evaluation, biocompatibility assessment via cytotoxicity assays, and finally hemocompatibility studies using platelet-rich plasma in a dynamic Chandler’s loop setup [8]. Clearly, since the publication of the first edition a significant amount of progress has been made in the author’s research group, spanning the range from materials studies to novel device fabrication and testing.
New studies that are currently under way involve the use of SiC nanowires, in this case 3C–SiC, for numerous biomedical device applications. Dr Paola Lagonegro from the University of Parma and the CNRIMEM, also in Parma, visited the group in the spring of 2014 with her work focusing on the bio- and hemocompatibility of 3C–SiC nanowires [9]. This work is ongoing with the qualitative outcome being that this material system is compatible with L929 fibroblasts, as per ISO 10993 evaluation, and platelets exposed to the nanowires from platelet-rich plasma. A new study on the interaction of osteoblasts with 3C–SiC has been initiated with Dr Carlos Galli of the University of Parma Hospital and preliminary results indicate that, as expected, planar 3C–SiC performs well in vitro with MC3T3 osteoblast precursor bone cells (Galli C. Private communication, University of Parma Medical Center). This is very
scientists around the world to investigate and construct the machinations of the nervous system. For instance, planar MEA systems are used to study neurons and neural tissue in vitro [27]. Indeed, the MEA is an excellent means to screen novel material compatibility with the brain, as testing cells or tissue slices in vitro is much more convenient, humane, and economical than implanting MEAs within an animal model, such as a mouse or rat. Research performed by the group of Dr J. Pancrazio has shown that functional recordings of neural activities using an MEA are an excellent means to study the biocompatibility of a material. Their group has indicated that excellent neural recordings were obtained through an MEA using a-SiC as an insulator, which was in intimate contact with the functional neural network [28]. Work done by the same group reviewed and combined multiple ISO 10993 methods into one protocol, which is the method we now use in our group to assess a material’s biocompatibility (see chapter: Cytotoxicity of 3C–SiC Investigated Through Strict Adherence to ISO 10993) and functional assays [29]. In our group, we have realized several generations of SiC-based MEAs, all utilizing 3C–SiC as the base material. Early versions of this technology were used to stimulate cortical neurons from wild-type mice [30], and more recent versions have incorporated a-SiC insulators to realize more robust electrically insulating films [31]. In closing the introduction to the second edition of Silicon Carbide Biotechnology: A Biocompatible Semiconductor for Advanced Biomedical Devices and Applications, I would like to mention the collaboration with Dr Mario Gazziro of the Universidade Federal do ABC, Santo André (Sao Paulo), Brazil. This collaboration, sponsored by the Brazilian government’s Science without Borders program, seeks to develop a wireless neural implant, based on the 3C–SiC technology developed by my team at the University of South Florida. To date, an application-specific integrated circuit has been designed, fabricated, and tested for its electrical performance [32]. The chip consists of four differential recording channels with 46-dB gain up to 10 kHz with very low total power consumption of only 144 mW. During this project we also undertook to study the compatibility of 3C–SiC neural probes with MRI fields and discovered that low-doped n-type 3C–SiC probes produced no image artifacts during scanning using a 2T magnetic field, which is markedly different than metal electrodes. This work, in collaboration with Dr Luciene Covolan of the Universidade Federal de São Paulo (UNIFESP) and Jackie Malherios of the Universidade de São Paulo (USP), São Carlos, Brazil, will be further discussed in chapter: In Vivo Exploration of Robust Implantable Devices Constructed From Biocompatible 3C–SiC.
1.3 SUMMARY TO THE SECOND EDITION
In summary, this edition both provides an update of important developments since the publication of Silicon Carbide Biotechnology: A Biocompatible Semiconductor for Advanced Biomedical Devices and Applications, published in late 2011, while initiating discussions on new applications of the biomedical material, SiC. The bulk of the biomedical device-related research in the SiC biotechnology area has utilized 3C–SiC and a-SiC, particularly in the field of neuroengineering and neuroscience although bulk hexagonal 4H- and 6H–SiC have also been examined. Continuous glucose monitoring using a semi-insulating 4H–SiC RF sensor has shown excellent in vitro performance, and is currently being refined for use in vivo at the University of South Florida. Numerous new works in the area of SiC nanobioMEMS have been reported covering a wide range of applications, from cancer detection and treatment to artificial retinas, to DNA assays and functionalized biosensors. None of this would be possible without the continuing study of SiC as a suitable biomaterial, and a complete study comparing 3C–SiC and a-SiC with traditional biomedical device materials has been completed by our group and is reported here in chapter: Cytotoxicity of 3C–SiC Investigated Through Strict Adherence to ISO 10993. In chapter: Study of the Hemocompatibility of 3C–SiC and a-SiC Films Using ISO 10993-4 a study of the hemocompatibility of these same materials is reported using a dynamic Chandler loop, with the results being that these materials are indeed hemocompatible insofar as blood platelet attachment and activation are concerned. In chapter: Graphene Functionalization for Biosensor Applications new results pertaining to graphene biosensors on SiC will be reported, followed by a review of some interesting work on SiC electrochemical sensors for biomedical applications (chapter: SiC Biosensing and Electrochemical Sensing: State of the Art and Perspectives), and semi-insulating 4H–SiC for in vivo continuous glucose monitoring (chapter: SiC RF Antennas for In Vivo Glucose Monitoring and WiFi Applications). Progress in the development of SiC neural interfaces is discussed in chapter: In Vivo Exploration of Robust Implantable Devices Constructed From Biocompatible 3C–SiC, where work currently under way to develop a wireless neural interface is presented along with image data from 3C–SiC neural implants exposed to 2T MRI fields in a rat model. Extremely promising work in the area of robust, biocompatible a-SiC coatings for neurological applications is then presented (chapter: Amorphous Silicon Carbide for Neural Interface Applications) to close out the neuroengineering portion of this edition. The final two chapters present some very novel research in the area of DNA
assays using SiC nanowires (chapter: SiC Nanowire Based Transistors for Electrical DNA Detection) and the use of the same material system for various biomedical device applications closes this edition in chapter: Silicon Carbide Based Nanowires for Biomedical Applications.
It is hoped that you, the reader, will find this “application-driven” approach to the organization of this book easy to follow as well as logical and useful in your work. In an effort to provide continuity with the first edition of Silicon Carbide Biotechnology: A Biocompatible Semiconductor for Advanced Biomedical Devices and Applications we have repeated the introductory chapter here so that important general information on SiC and its properties is close by for your edification. If you are already familiar with this chapter you are encouraged to move on to the next chapter now, but I hope that you will find a quick review of SiC for biotechnological applications worthwhile and that you will agree with this assessment after a quick review of the rest of this chapter.
1.4 INTR ODUCTION TO THE FIRST EDITION
Silicon carbide has a long history as a robust and hard material, first used as a cutting material in the 19th century and later as a high-temperature semiconductor for advanced applications in the 20th century. The history of silicon carbide (SiC) is quite interesting and the reader is referred to the first chapter in a book dedicated to this subject [33]. It is best to consult the literature to fully understand all of the many aspects of SiC, from how it is formed, to its myriad crystal properties, and finally to the large number and types of applications it is being used in. Fortunately, it is sufficient here to provide a brief overview of SiC so that the reader can understand why this material is so compelling for biomedical applications and may become one of the most used biomaterials in the 21st century. Indeed, the purpose of this book is to lay the foundation for exactly this – to introduce silicon carbide to biomedical engineers, medical professionals, and scientists, thus bringing together technologists from across many disciplines to help realize the ultimate use of SiC as one of the next generation of smart biomaterials to realize advanced biomedical devices.
1.5 SILICON CARBIDE – MATERIALS OVERVIEW
SiC is first and foremost a material that consists of the covalent bonding of Si and C atoms, typically in biatomic layers as shown in Fig. 1.1. These form tetrahedrally oriented molecules of SiC, with a very short bond length
Figure 1.2 Atomic stacking sequence of the three technologically relevant SiC polytypes viewed in the [9–10] plane. From left to right, 4H–SiC (ABCB), 6H–SiC (ABCACB), and 3C–SiC (ABC). k and h denote crystal symmetry points that are cubic and hexagonal, respectively [1]. Note that only 3C–SiC consists of purely cubic crystal symmetry.
is currently leading to dramatic reductions in power consumption worldwide [36]. A comparison of the properties of SiC relative to Si is shown in Table 1.1 for reference.
1.6 SILICON CARBIDE MATERIAL GROWTH AND PROCESSING
There is a long history of how to grow, process, and characterize SiC materials – the reader is referred to several of the excellent references if further details are desired [37,38]. For the purpose of this book it is sufficient to provide an overview of the technology as it pertains to future biomedical devices, hence a simple discussion follows with sufficient references to fill in the details. We will first review how bulk hexagonal crystals of SiC are grown and, more importantly, what the state of the art is and the key characteristics of these commercially available substrates. Next, the growth and synthesis of thin films, starting with homoepitaxy on hexagonal substrates, followed by heteroepitaxy on Si substrates, and finally polycrystalline growth on various surfaces, will be presented. One of the great benefits of SiC is that it can be deposited in a-SiC form at lower temperatures than for single-crystal film growth. This technology will be reviewed in this chapter and the final section will be an introduction to how SiC can be micromachined, and how this points to the potential of SiC as an excellent bioMEMS material.
1.6.1 Bulk Growth
Single-crystal substrates of SiC have been in commercial production for more than two decades, starting with Cree, Inc.’s first commercial 6H–SiC wafers, which were 25 mm in diameter, in 1990 [41]. Much progress has been made worldwide since then, with both the quality and diameter of the wafers improving over the years; the current commercially available wafers are 100 mm (4 in.) and are available in both n-, p-, and intrinsic (very high purity “semi-insulating”) form. Several manufacturers around the world sell SiC wafers and the crystal specifications are readily available [42]. Recently, larger 150-mm (6-in.) diameter wafers of quite high quality have been produced [43], which bodes well for the economic viability of SiC as an electronics technology (Fig. 1.3).
All SiC bulk wafers are grown in a high-temperature (>2000°C) furnace that is either gas fed (silicon and carbon containing precursors
Figure 1.3 Commercially available SiC wafers can be found in off- and on-axis orientations, also with a Si or C terminated face. They can be either heavily doped or semiinsulating, depending on the application of use. Current wafers are 4 in. (100 mm) in diameter with 6-in. (150-mm) wafers demonstrated and ready for production in 2012. (Photo courtesy of II-VI, Inc. [44].)
