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Biocompatibility of semiconducting AlGaN/GaN material with living cells

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Abstract

With the aim of developing a highly sensitive, mass producible biosensor, we have investigated the growth of human embryonic kidney (HEK) 293 cells on the surface of semiconductor grade AlGaN/GaN heterostructures. Our results demonstrate that, even without specialised surface treatment, a substantial amount of attachment and proliferation of cells is observed. Growth and mortality rates on the AlGaN surface were comparable to standard control culture plates. Quantitative studies of mortality measured by flow cytometry correlate well with qualitative monitoring of biocompatibility. The percentage of dead cells increases marginally with increasing Al concentration. Cell attachment was investigated qualitatively using focused ion beam/scanning electron microscopy (FIB/SEM) and transmission electron microscopy (TEM). Imaging showed strong attachment at the cell/semiconductor interface at the nanometre level. These measurements are the first study of live cell/semiconductor interactions using complementary methods for proliferation, mortality, and attachment, and confirm that the combination of live cells as the biosensing element and AlGaN/GaN heterostructures as the transducer has significant potential for biosensor applications.

Introduction

The range of technologies being investigated for biosensors is as wide as the application space. One approach of increasing interest utilises sensors based on AlGaN/GaN high electron mobility transistor (HEMT) structures, due to high gate charge sensitivity, stability in harsh environments and low toxicity [1], [2], [3]. Use of AlGaN/GaN HEMTs as sensors relies on modulation of the conducting channel, a two-dimensional electron gas, via changes in the surface potential of the HEMT. One such modulation can be achieved by exposing the active area of gateless AlGaN/GaN HEMTs to charged particles such as ions [4]. When live cells are grown directly on the active area, ion transport through the cell membranes can therefore trigger changes in source–drain current which represents the sensor signal. Due to the exponential relationship between surface potential and channel charge, not only can these devices directly sense charged particles adsorbed onto the exposed gate area, but the measured source–drain current is amplified compared to the charge controlling the gate. Although the devices are naturally sensitive to any ionic charge [4], appropriate modifications can ensure selectivity to particular analytes of interest. Living cells by their nature are capable of complex differentiation between processes and ions. Their microstructure incorporates enzymes, nucleic acids, ions, proteins and small organic molecules. Under the influence of any stimulus, they process multiple incoming signals by both parallel and integrated activation of different pathways, generating a distinct response dependent upon the type of stimulus. The use of living components capable of a direct response to incoming information therefore enables investigations of external physical or chemical stimulus effects on a living system [5]. However, working with whole cells provides additional challenges to functionalisation, due to the need for biocompatibility. Although studies of cell viability on other wide bandgap semiconductors, such as ZnO can be found in the literature [6], biocompatibility of AlGaN/GaN and living cells is of particular interest due to potential biosensor applications for cell activity analysis and electrophysiological monitoring, crucial for fields such as drug discovery and environmental screening of chemical and biological pollutants. This paper focuses on toxicity as well as on cell morphology and attachment. Together these incorporate a biocompatibility study to contribute to the fundamental understanding of the nature of the signals (source–drain current modulation) occurring in AlGaN/GaN heterostructure field effect transistors due to the changes in flow of ion current through adherent membranes of biological cells [7], [8]. HEK293 cells were chosen as the biocompatibility test vehicle because they demonstrated relatively higher sensitivity compared to other cells in work by Cimalla et al. [9]. Only limited studies of the biocompatibility of AlGaN/GaN HEMT structures and HEK or other cells have been published previously [9], [10], with no broad investigations of AlGaN/GaN HEMT and HEK293 cell biocompatibility using complementary methods. Therefore we present for the first time a comprehensive study of biocompatibility, incorporating qualitative and quantitative assessment of HEK293 cell growth and mortality as a function of Al composition of the AlxGa1−xN. This includes studies of cell proliferation and mortality using both optical and flow cytometry techniques, as well as one of the first examples of electron microscopy imaging of the cell/semiconductor interface to obtain insights into cell attachment. All of these aspects are likely to be crucial for future biosensor applications.

Section snippets

Proliferation and mortality studies

The composition of the AlGaN (Al concentration) in an AlGaN/GaN HEMT will affect both the channel charge and the reactivity of the surface. Therefore the dependence of cell viability on Al concentration in the AlGaN layer of HEMT heterostructures was investigated. Moreover many of the previous publications on AlGaN/GaN HEMTs use a thin GaN cap to enhance the electrochemical stability for devices operating in electrolytes [7]. Therefore, the biocompatibility of GaN (effectively an Al

Proliferation and mortality studies

Previous qualitative studies of cell growth and proliferation on AlGaN/GaN substrates were carried out over 2 weeks using optical microscopy, demonstrating substantial attachment and subsequent cell growth, even without specialised surface treatment [13].

In the current study, we sought to quantify cell mortality as a measure of biocompatibility. Flow cytometry measurements of mortality rate over 3 days are shown in Fig. 1 where day 1 corresponded to 24 h after cell seeding. There are two

Conclusions

This study investigating multiple aspects of biocompatibility has provided qualitative and quantitative data for the mortality and proliferation of living cells on AlGaN/GaN semiconductor material. Quantitative flow cytometry data together with optical monitoring of cell growth indicated that the number of dead cells seems to increase and proliferation speed seems to decrease with increasing Al concentration. Importantly, cells survived on the entire range of AlxGa1−xN/GaN compositions, from x = 

Acknowledgements

We acknowledge partial funding of the work by the Australian Research Council (ARC) Discovery Project scheme (DP0988241). William and Marlene Schrader Postgraduate Scholarship for Biomedical Engineering. KDGP is an ARC Future Fellow (FT100100271). We also acknowledge the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, and the Curtin University

Anna Podolska received her BE degree in physical and biomedical electronics from the National Technical University of Ukraine “Kiev Polytechnic Institute” in 2007. She has worked as a research associate with the Microelectronic Research Group at The University of Western Australia and is currently study toward her PhD degree at The University of Western Australia with the same research group, funded by the prestigious William and Marlene Schrader Postgraduate Scholarship (Biomedical

References (14)

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    Citation Excerpt :

    Gallium nitride (GaN)-related materials have shown promise in research and commercial fields such as power electronics, [1,2] communication, [3] lighting [4] and sensing [5,6]. Specifically, research on portable chemical sensors based on AlGaN/GaN heterostructures is appealing because of a series of advantages of the structures and the material: AlGaN/GaN is relatively inert, [7] making the sensors resistant to harsh chemicals; the conductive two-dimensional electron gas (2DEG) channel [8–10] in the structures makes AlGaN/GaN-based sensors normally on, and no reference electrodes are required as a result; [11–14] AlGaN/GaN is biocompatible, which enables the development of AlGaN/GaN biosensors. [15] Numerous studies have demonstrated the ability of AlGaN/GaN-based portable sensors for ions in aqueous solutions, many in a reference-electrode-free configuration, as summarized in a recent review article. [6]

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Anna Podolska received her BE degree in physical and biomedical electronics from the National Technical University of Ukraine “Kiev Polytechnic Institute” in 2007. She has worked as a research associate with the Microelectronic Research Group at The University of Western Australia and is currently study toward her PhD degree at The University of Western Australia with the same research group, funded by the prestigious William and Marlene Schrader Postgraduate Scholarship (Biomedical Engineering).

Stephanie Tham will be completing a BE/LLB combined degree in 2012 at the University of Western Australia (UWA). During her engineering studies she worked with the Microelectronics Research Group at UWA on development of a III-N biosensor and since worked at leading IP specialist firm, Griffith Hack.

Robert D. Hart received his BSc degree in 1989 and PhD degree in 1996 from The University of Western Australia. He is a senior research fellow with The Centre for Materials Research at Curtin University. His current areas of research interest are industrial applications and uses of scanning and transmission electron microscopy, and advanced X-ray and electron diffraction techniques. This includes research into new materials such as ceramics for solid oxide fuel cells, and old materials such as Australian geological/regolith minerals.

Ruth Seeber received her BSc (Hons) degree in 1999 from The University of Western Australia. She is currently a research officer in the Molecular Endocrinology Laboratory at the Western Australian Institute for Medical Research (WAIMR) and the Centre for Medical Research, The University of Western Australia.

Martin Kocan received his Master of Science (MSc) degree in electronics and microelectronics at the Slovak University of Technology, Slovakia in 2000 and a PhD in electronic engineering sciences at RWTH University Aachen, Germany in 2003. During his PhD studies he worked at the Institute of Thin Films and Interfaces, which is part of the Research Centre Juelich in Germany. For the period from 2003 to 2005 he was working as scientific researcher at Georg-August-University Goettingen in Germany and between 2005 and 2008 joined the Microelectronics Research Group at The University of Western Australia as a research fellow. Since January 2009 he has been working as a research cell/module scientist at Solar Systems Pty Ltd in Australia.

Martina Kocan obtained her PhD in 2005 in natural sciences (biochemistry) at the Heinrich-Heine University, Duesseldorf in Germany. As a PhD student she worked at the Institute of Biotechnology, which is part of the Research Centre Juelich in Germany. On completion of her PhD, she moved to Australia and was awarded a postdoctoral position at the Western Australian Institute for Medical Research (WAIMR) and the Centre for Medical Research, University of Western Australia in the research team of A/Prof Karin Eidne and A/Prof Kevin Pfleger. In February 2009, she joined Monash Institute of Pharmaceutical Sciences and Department of Pharmacology at Monash University as a postdoctoral research fellow under the leadership of Prof Roger Summers.

Umesh Mishra received the MS degree in electrical engineering from Lehigh University, Bethlehem, PA, in 1981 and the PhD degree in electrical engineering from Cornell University, Ithaca, NY, in 1984. He is a professor with the Department of Electrical and Computer Engineering, University of California, Santa Barbara. He has worked at various laboratories and academic institutions including North Carolina State University, Raleigh, Hughes Research Laboratories, Malibu, CA, University of Michigan, Ann Arbor, and General Electric, Syracuse, NY. He was the recipient of the Presidential Young Investigator Award from the National Science Foundation, the Hyland Patent Award presented by Hughes Aircraft, the Young Scientist Award presented at the International Symposium on GaAs and Related Compounds, and the 2007 David Sarnoff Award from the IEEE.

Kevin Pfleger received his PhD degree in 2003 in molecular endocrinology/pharmacology from Edinburgh University, having obtained an MA from Cambridge University. He was a National Health and Medical Research Council (NHMRC) of Australia Peter Doherty research fellow from 2005 to 2008, and is currently associate professor and head of Molecular Endocrinology at the Western Australian Institute for Medical Research (WAIMR) and the Centre for Medical Research, University of Western Australia. He is also chief scientific officer of Dimerix Bioscience, a spin-out company of the University of Western Australia. In 2009, he was awarded Western Australian Young Scientist of the Year. In 2011, he was awarded the Australian Museum 3M Eureka Prize for Emerging Leader in Science.

Giacinta Parish received the BS degree in Chemistry in 1995, and the BE and MEngSc degrees in electronic engineering in 1995 and 1997, respectively, all from The University of Western Australia, Perth, and the PhD degree in electrical engineering in 2001, from the University of California, Santa Barbara. She joined The University of Western Australia as an Australian postdoctoral fellow in 2001, and is now an associate professor at the same institution. Her main research interests are III–V nitride materials and devices, and more recently porous silicon. Specific interests within these areas currently include development of processing technology, transport studies, and development of novel chem- and bio-sensors.

Brett D. Nener received his BE degree in 1977 and PhD degree in 1987 from The University of Western Australia, and the MESc (1980) from the University of Tokyo. He is professor and head of Electrical, Electronic and Computer Engineering at The University of Western Australia. His current areas of research interest are Biosensors; III-N devices; modeling of atmospheric effects such as refraction, scintillation and aerosol scattering; electro-optic systems; infrared and UV photodetector devices and models; the measurement of semiconductor deep-level traps. He is a senior member of IEEE.

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Present address: Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia.

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