Investigation of the Binding Affinity of C-terminal Domain of SARS Coronavirus Nucleocapsid Protein to Nucleotide Using AlGaN/GaN High Electron Mobility Transistors You-Ren Hsu(1), Geng-Yen Lee(2), Jen-Inn Chyi(2), Chung-ke Chang(3), Chih-Cheng Huang(1), Chen-Pin Hsu(1), Tai-huang Huang(3),Fan Ren(4), and Yu-Lin Wang(1) 1. Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, 300, Taiwan, R.O.C. 2. Department of Electrical engineering, National Central University, Jhongli City, Taoyuan County 32001, Taiwan, R.O.C. 3. Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan R.O.C. 4. Department of Chemical Engineering, University of Florida, Gainesville, FL 32611, USA Abstract — In this article, we used AlGaN/GaN high electron mobility transistors (HEMTs) to construct the first label-free semiconductor-sensor-based binding assay to our knowledge. Our results suggested that the nucleotide-c terminal domain of SARS coronavirus (SARS-CoV) nucleocapsid protein interaction is a two- step binding event with two dissociation constants (Kd1 = 0.052 nM, and Kd2 = 51.24nM) extracted by using the modified two-binding-site Langmuir isotherm equation proposed here. We found that there were at least two protein binding sites on the specific 41-base SARS-CoV double-stranded DNA (dsDNA) genome (29,580-29621) conjugated with a 20-mer poly-dT tail. This result presented a high binding affinity is comparable with the antibody-antigen reaction, and suggested this designed dsDNA could be treated as an aptamer for SARS-CoV N protein capture. Introduction In this paper, we demonstrated the use of AlGaN/GaN high electron mobility transistors (HEMTs) to measure dissociation constants of nucleotide–protein interaction between the specific segment of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) dsDNA genome and the C-terminal domain of SARS-CoV nucleocapsid protein (N protein). A dissociation constant is commonly used to describe the affinity between a ligand and a receptor in biochemistry. Measuring the dissociation constant may help to decipher the complex interaction network of biological system [1]. The Electrophoretic Mobility Shift Assay (EMSA) and filter binding assay are two common methods to determine the dissociation constant of nucleotide-protein interaction, and have been widely used in the last 30 years [2- 6]. However, in order to characterize the binding fraction of nucleotides and proteins, fluorescent probes or isotope elements are often utilized to label nucleotides to provide signals for the quantitative molecule detection, which is expensive and may alter the binding affinity of molecules [6]. Hence, developing a low-cost, high-sensitivity, and label-free binding assay is of paramount importance. Recently, both AlGaN/GaN HEMTs [7] and nanowire field-effect transistors (NWFET) [8, 9] with immobilized antibodies have been shown capable of detecting specific biomarkers without fluorescent or isotopic labeling. It has also been shown that NWFET with immobilized antibody- mimic protein is capable of determining the dissociation constant between two molecules using the Langmuir isotherm equation [8, 10]. However, AlGaN/GaN HEMTs have two main advantages over NWFETs. First, GaN-based materials are extremely chemical stable, and to date there is no good wet chemical etchant available [7]. Second, the dimension of AlGaN/GaN HEMTs for biomarker detection can be in the range of tens of micrometers [7, 11, 12]. Therefore, AlGaN/GaN HEMTs-based sensor can be fabricated using semiconductor compatible manufacturing technology in a high-yield fashion. 978-1-4577-1767-3/12/$26.00 ©2012 IEEE Here, we demonstrate the AlGaN/GaN HEMTs can be used to detect nucleotide-protein binding events. Our system is the first semiconductor-sensor-base binding assay for studying nucleotide–protein interactions. We chose a 41-base dsDNA sequence SARS-CoV genome (29,580-29621) [13] and conjugated a 20-mer poly-dT ssDNA tail to its 3’-end of one strand of the dsDNA as receptors, and the C-terminal domain of SARS-CoV N proteins (SARS-CoV CTDs) as ligands. A modified two-binding-site Langmuir isotherm equation was proposed for determining dissociation constants. Experiment AlGaN/GaN high electron mobility transistors (HEMTs) were used for detecting biomolecule binding such as antibody-antigen, and ligand-receptor. Here, we demonstrated that the AlGaN/GaN HEMTs also have the capability of detecting nucleic acid-protein interaction with high sensitivity. Figure 1(a) and (b) show the schematics of a DNA- immobilized AlGaN/GaN HEMT sensor and the plan-view microphotograph of the device, respectively. We used the metal-organic chemical vapor deposition (MOCVD) to develop the AlGaN/GaN layers. The structure of the AlGaN/GaN layer consisted of a 3 μm-thick undoped GaN buffer, 150 Å-thick undoped Al0.25Ga0.75N and 10 Å-thick undoped GaN cap layer. The Mesa isolation was performed with an Inductively Coupled Plasma (ICP) etching with Cl2/BCl3/Ar based discharges at –90 V dc self-bias, ICP power of 300 W. Two 10 × 50 µm2 Ohmic contacts separated by a 5-µm gap consisted of e-beam deposited Ti/Al/Pt/Au that was patterned by lift-off and was annealed at 850 ºC, 45 sec under flowing N2. Phosphate buffered saline (10 mM sodium phosphate pH 6.0, 50 mM NaCl, 1 mM EDTA) was initially dropped on the sensor. As the current was in equilibrium state, SARS-CoV CTDs was added in to the buffer solution, and increased the concentration of SARS CTDs at next equilibrium state. Modified Two-binding-site Langmuir Isotherm Equation The Langmuir isotherm equation for single binding site shown below has been used to extract the dissociation constant of the current change measured by using NWFET, and localized surface plasmon coupled fluorescence fiber- optic biosensor, [8-10, 14-16] max max ] [ ] [ I K I Ligand I Ligand d Δ + Δ = Δ where ΔI was the amount of current change, and ΔImax was the saturated current change, [Ligand] was ligand concentration, Kd was the dissociation constant. However, in our system, the length of dsDNA was 60 bps, and the size of SARS-CoV CTDs was about 3 nm. Here we proposed a two- binding-site model, and the chemical equations were described as below: �R� � �L� � � ���� Kd� �RL� � �L� � � �RL�� Kd� where the [R], and [L] were the concentrations of receptors, and the ligands, respectively. The Kd1 and Kd2 were the dissociation constants for the two binding sites on the receptors. According to the single binding site model, the amount of current change could be presented as equivalent formula. (1) (2) (3) Figure 1. (a) Schematic of the AlGaN/GaN HEMT sensor. (b) Plan view of microphotograph of a completed device. The three red rectangles indicated the gate area of each device. ∆I � ∆������� ������ The total current change could be presented in the form of equation (6), and equation (7) after rearrangement, ∆I � ∆�� � ∆�� The total current change presented in the form of equation (6), and equation (7).The current change in equation (5) was substituted by equation (4). ∆I � ∆�������� ������� + ∆�������� ������� ∆I � ��∆����� � ��∆����� where α1 and α2 are the surface coverage ratio of two binding sites, respectively. Results and Discussion There was no net current change until the target protein concentration of 0.003nM of the protein was added. A clear current change was observed as the system reached a steady state (Figure 2). Real-time current monitoring spanned the range of protein concentrations from 0.003 nM to 3000 nM, and showed saturation as the concentration of SARS-CoV CTD larger than 300 nM. A clear current change was observed as the system was reaching a steady state. Real-time current monitoring spanning the range of target concentrations was from 0.003nM to 3000nM of the protein sample. The surface coverage (ΔI/ΔImax) of experiment data points were cover four orders of protein concentration from 0.1 to 0.9. This result indicated a two-steps binding process. The Figure 3 was curve-fitting result of the data points. The amount of current change at each equilibrium state was described by the modified two-binding-site Langmuir isotherm equation, and provided the two dissociation constants (Kd1 = 0.052nM, and Kd2 = 51.24nM). Furthermore, this result suggested there were at least two SARS-CoV CTDs binding sites on the dsDNA segment tested. These dissociation constants presented a high binding affinity is comparable with the antibody-antigen reaction, and suggested this designed dsDNA could be treated as an aptamer for SARS-CoV N protein capture[10]. Conclusion In conclusion, AlGaN/GaN high electron mobility transistors (HEMTs) immobilized nucleotide can be applied to measure the dissociation constants of nucleotide–protein interaction at physiological condition. We believed that semiconductor-sensor-based binding assay would have a wide range of potential applications in investigating of nucleic acids-protein interaction, as well potentially for network biology investigation by using AlGaN/GaN high electron mobility transistors (HEMTs) array. Acknowledgment This work was partially supported by National Science Council grant (No.99B20495A) and by the research grant (100N2049E1) at National Tsing Hua University. (4) (5) (6) (7) 0 1000 2000 3000 4000 5000 6000 610 612 614 616 618 620 622 624 626 Current (uA) Time (s) 1 ul Buffer 0.003nM 0.03nM 0.3nM 3nM 30nM 300nM 3000nM 1E-3 0.01 0.1 1 10 100 1000 0 2 4 6 8 10 12 |ΔI| (uA) Log Scale Protein Concentration (nM) 0.00 0.09 0.18 0.27 0.36 0.45 0.54 0.63 0.72 0.81 0.90 0.99 1.08 Surface Coverage Figure 2. Real-time detection of the c-terminal domain of SARS-CoV N protein from 0.003nM to 3000nM at constant bias of 350 mV Figure 3. Current change of each different protein concentration condition can be fitted by two step binding model, where Kd1=0.138nM, and Kd2=33.52nM. References [1] R. B. Jones, A. Gordus, J. A. Krall, and G. MacBeath, "A quantitative protein interaction network for the ErbB receptors using protein microarrays," Nature, vol. 439, pp. 168-174, 2006. [2] L. S. Klig, I. P. Crawford, and C. Yanofsky, "Analysis of trp repressor-operator interaction by filter binding," Nucleic Acids Research, vol. 15, pp. 5339-5351, July 10, 1987 1987. [3] M. M. Garner and A. Revzin, "A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system," Nucleic Acids Research, vol. 9, pp. 3047- 3060, July 10, 1981 1981. [4] M. Fried and D. M. Crothers, "Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis," Nucleic Acids Research, vol. 9, pp. 6505-6525, December 11, 1981 1981. [5] H.-M. Wu and D. M. Crothers, "The locus of sequence-directed and protein-induced DNA bending," Nature, vol. 308, pp. 509-513, 1984. [6] L. M. Hellman and M. G. Fried, "Electrophoretic mobility shift assay (EMSA) for detecting protein- nucleic acid interactions," Nat. Protocols, vol. 2, pp. 1849-1861, 2007. [7] S. J. Pearton, F. Ren, Y.-L. Wang, B. H. Chu, K. H. Chen, C. Y. Chang, W. Lim, J. Lin, and D. P. Norton, "Recent advances in wide bandgap semiconductor biological and gas sensors," Progress in Materials Science, vol. 55, pp. 1-59, 2010. [8] M. Curreli, Z. Rui, F. N. Ishikawa, C. Hsiao-Kang, R. J. Cote, Z. Chongwu, and M. E. Thompson, "Real-Time, Label-Free Detection of Biological Entities Using Nanowire-Based FETs," Nanotechnology, IEEE Transactions on, vol. 7, pp. 651-667, 2008. [9] A. Qureshi, I. Roci, Y. Gurbuz, and J. H. Niazi, "An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays," Biosensors and Bioelectronics, vol. 34, pp. 165-170, 2012. [10] F. N. Ishikawa, H.-K. Chang, M. Curreli, H.-I. Liao, C. A. Olson, P.-C. Chen, R. Zhang, R. W. Roberts, R. Sun, R. J. Cote, M. E. Thompson, and C. Zhou, "Label-Free, Electrical Detection of the SARS Virus N-Protein with Nanowire Biosensors Utilizing Antibody Mimics as Capture Probes," ACS Nano, vol. 3, pp. 1219-1224, 2009. [11] S. Alur, R. Thapa, T. Gnaprakasa, Y. Wang, Y. Sharma, E. Javalosa, E. Smith, C. Ahyi, A. Simonian, M. Bozack, J. Williams, and M. Park, "DNA hybridization sensor based on AlGaN/GaN HEMT," physica status solidi (c), vol. 8, pp. 2483- 2485, 2011. [12] S. U. Schwarz, S. Linkohr, P. Lorenz, S. Krischok, T. Nakamura, V. Cimalla, C. E. Nebel, and O. Ambacher, "DNA-sensor based on AlGaN/GaN high electron mobility transistor," physica status solidi (a), vol. 208, pp. 1626-1629, 2011. [13] M. A. Marra, S. J. M. Jones, C. R. Astell, R. A. Holt, A. Brooks-Wilson, Y. S. N. Butterfield, J. Khattra, J. K. Asano, S. A. Barber, S. Y. Chan, A. Cloutier, S. M. Coughlin, D. Freeman, N. Girn, O. L. Griffith, S. R. Leach, M. Mayo, H. McDonald, S. B. Montgomery, P. K. Pandoh, A. S. Petrescu, A. G. Robertson, J. E. Schein, A. Siddiqui, D. E. Smailus, J. M. Stott, G. S. Yang, F. Plummer, A. Andonov, H. Artsob, N. Bastien, K. Bernard, T. F. Booth, D. Bowness, M. Czub, M. Drebot, L. Fernando, R. Flick, M. Garbutt, M. Gray, A. Grolla, S. Jones, H. Feldmann, A. Meyers, A. Kabani, Y. Li, S. Normand, U. Stroher, G. A. Tipples, S. Tyler, R. Vogrig, D. Ward, B. Watson, R. C. Brunham, M. Krajden, M. Petric, D. M. Skowronski, C. Upton, and R. L. Roper, "The Genome Sequence of the SARS-Associated Coronavirus," Science, vol. 300, pp. 1399-1404, May 30, 2003 2003. [14] K. Maehashi, K. Matsumoto, Y. Takamura, and E. Tamiya, "Aptamer-Based Label-Free Immunosensors Using Carbon Nanotube Field- Effect Transistors," Electroanalysis, vol. 21, pp. 1285-1290, Jun 2009. [15] H. Vedala, Y. Chen, S. Cecioni, A. Imberty, S. b. Vidal, and A. Star, "Nanoelectronic Detection of Lectin-Carbohydrate Interactions Using Carbon Nanotubes," Nano Letters, vol. 11, pp. 170-175, 2011/01/12 2010. [16] J. C. Huang, Y. F. Chang, K. H. Chen, L. C. Su, C. W. Lee, C. C. Chen, Y. M. A. Chen, and C. Chou, "Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in human serum using a localized surface plasmon coupled fluorescence fiber-optic biosensor," Biosensors & Bioelectronics, vol. 25, pp. 320-325, Oct 2009.