Multiple MoS 2 Transistor -Integrated Microfluidic … MoS 2 Transistor -Integrated Microfluidic...
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Multiple MoS 2 Transistor-Integrated Microfluidic Biosensors for Quantifying Cancer-Related Biomarker Molecules with Femtomolar-Level Detection Limits Hongsuk Nam 1 , Bo-Ram Oh 1 , Pengyu Chen 1 , Mikai Chen 1 , Sungjin Wi 1 , Katsuo Kurabayashi 1 , and Xiaogan Liang 1 1 University of Michigan, Ann Arbor, USA ABSTRACT We developed multiple MoS 2 transistor biosensors and obtained calibrated sensor responses from the sensors for quantifying the kinetics of biomolecule interactions with detection limit as low as 60fM. The sensor response signals enabled us to create a standard curve and the equilibrium constant of the antibody-antigen pair was extracted to be K D =369±48fM. Additionally, we demonstrated the real time measurement and the association/dissociation constants of the antibody-antigen pair were extracted to be (5.03±0.16)×10 8 M -1 s -1 and (1.97±0.08)×10 -4 s -1 , respectively. This study advanced the biosensing applications with atomically layered transition metal dichalcogenides and paved the way to analyze the biomolecule interactions with fM-level detection limits. KEYWORDS: MoS 2 , Biosensor, Field-effect-transistor INTRODUCTION Atomically layered transition metal dichalcogenides (TMDCs), such as MoS 2 , have focused the spotlight of attention because of their excellent electronic and structural properties. Recently several works demonstrated that MoS 2 field-effect-transistor (FET) based biosensor could achieve 100-400 fM limit of detections for detecting specific biomarkers.[1][2] In order to attain the consistent sensor response behavior from multiple biosensors, a further device-oriented study is required for quantifying the different concentrations of biomarker molecules by calibrating the sensor response signals. Therefore, in this research, we fabricated multiple MoS 2 transistor biosensors and measured transport characteristics of those sensors with different concentrations of tumor necrosis factor – alpha (TNF-α), which is a cell secreted cytokine closely related to inflammatory diseases. In addition, we calculated calibrated sensor responses based on Langmuir isotherm model for generating the standard curve and obtained the equilibrium constant as well as association/dissociation constants of the antibody-(TNF-α) pair. EXPERIMENTAL Figure 1 illustrates the fabrication process of MoS 2 transistor biosensor. Firstly, a few-layer-MoS 2 flake is printed on a p + -Si substrate coated with 300nm SiO 2 layer with using our previous plasma nanoprinting method.[3] After printing, the drain and source contacts are patterned on the MoS 2 channel using photolithography followed with metal deposition (10 nm Ti/50 nm Au) and lift-off. In order to form insulating layer on the MoS 2 channel, 30nm HfO 2 layer is deposited using atomic layer deposition (ALD). Then 100nm SiO x layer is sputtered on top of the metal contacts for passivation. Before biodetection of such devices, we functionalized the MoS 2 FETs with anti TNF-α antibodies. First, back-gated MoS 2 FETs are immersed in 5% (3-Aminopropyl) triethoxysilane (APTES, Sigma- Aldrich Co. LLC.) in ethanol for 1 hour. After the incubation, the devices are carefully washed with phosphate buffered saline (PBS) and blown dry by nitrogen gas. Then the devices are submerged in 5% gluteraldehyde (Sigma-Aldrich Co. LLC.) in PBS for 2 hours performed in chemical fume hood followed by rinsing with PBS. Anti-TNF-α antibody (eBioscience, Inc.) of 50ug/ml concentration in DI water is dropped on the device and incubated for 45 min. Before static measurement, the anti-TNF-α solution is rinsed with DI water and gently blown dry by N 2 gas. The device characteristic curves of the antibody-attached FETs are measured using a probe station, and TNF-α solution with incremental concentrations (60/300/600/3000/6000fM) are incubated on 1601 978-0-9798064-8-3/μTAS 2015/$20©15CBMS-0001 19 th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
Transcript of Multiple MoS 2 Transistor -Integrated Microfluidic … MoS 2 Transistor -Integrated Microfluidic...
Multiple MoS2 Transistor-Integrated Microfluidic Biosensors for Quantifying Cancer-Related Biomarker Molecules with Femtomolar-Level Detection
Limits Hongsuk Nam1, Bo-Ram Oh1, Pengyu Chen1, Mikai Chen1, Sungjin Wi1, Katsuo
Kurabayashi1, and Xiaogan Liang1
1University of Michigan, Ann Arbor, USA
ABSTRACT We developed multiple MoS2 transistor biosensors and obtained calibrated sensor responses from the
sensors for quantifying the kinetics of biomolecule interactions with detection limit as low as 60fM. The sensor response signals enabled us to create a standard curve and the equilibrium constant of the antibody-antigen pair was extracted to be KD=369±48fM. Additionally, we demonstrated the real time measurement and the association/dissociation constants of the antibody-antigen pair were extracted to be (5.03±0.16)×108M-1s-1 and (1.97±0.08)×10-4s-1, respectively. This study advanced the biosensing applications with atomically layered transition metal dichalcogenides and paved the way to analyze the biomolecule interactions with fM-level detection limits. KEYWORDS: MoS2, Biosensor, Field-effect-transistor
INTRODUCTION
Atomically layered transition metal dichalcogenides (TMDCs), such as MoS2, have focused the spotlight of attention because of their excellent electronic and structural properties. Recently several works demonstrated that MoS2 field-effect-transistor (FET) based biosensor could achieve 100-400 fM limit of detections for detecting specific biomarkers.[1][2] In order to attain the consistent sensor response behavior from multiple biosensors, a further device-oriented study is required for quantifying the different concentrations of biomarker molecules by calibrating the sensor response signals. Therefore, in this research, we fabricated multiple MoS2 transistor biosensors and measured transport characteristics of those sensors with different concentrations of tumor necrosis factor – alpha (TNF-α), which is a cell secreted cytokine closely related to inflammatory diseases. In addition, we calculated calibrated sensor responses based on Langmuir isotherm model for generating the standard curve and obtained the equilibrium constant as well as association/dissociation constants of the antibody-(TNF-α) pair.
EXPERIMENTAL
Figure 1 illustrates the fabrication process of MoS2 transistor biosensor. Firstly, a few-layer-MoS2 flake is printed on a p+-Si substrate coated with 300nm SiO2 layer with using our previous plasma nanoprinting method.[3] After printing, the drain and source contacts are patterned on the MoS2 channel using photolithography followed with metal deposition (10 nm Ti/50 nm Au) and lift-off. In order to form insulating layer on the MoS2 channel, 30nm HfO2 layer is deposited using atomic layer deposition (ALD). Then 100nm SiOx layer is sputtered on top of the metal contacts for passivation.
Before biodetection of such devices, we functionalized the MoS2 FETs with anti TNF-α antibodies. First, back-gated MoS2 FETs are immersed in 5% (3-Aminopropyl) triethoxysilane (APTES, Sigma-Aldrich Co. LLC.) in ethanol for 1 hour. After the incubation, the devices are carefully washed with phosphate buffered saline (PBS) and blown dry by nitrogen gas. Then the devices are submerged in 5% gluteraldehyde (Sigma-Aldrich Co. LLC.) in PBS for 2 hours performed in chemical fume hood followed by rinsing with PBS. Anti-TNF-α antibody (eBioscience, Inc.) of 50ug/ml concentration in DI water is dropped on the device and incubated for 45 min.
Before static measurement, the anti-TNF-α solution is rinsed with DI water and gently blown dry by N2 gas. The device characteristic curves of the antibody-attached FETs are measured using a probe station, and TNF-α solution with incremental concentrations (60/300/600/3000/6000fM) are incubated on
1601978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
the sensors for specific antibody-antigen binding. Then the TNF-α solution is dropped on the device area and stabilized for 20 min followed with DI water rinse and N2 blow dry. Finally the device characteristic curves of the antibody-antigen-attached FETs are measured.
For real-time measurement, PDMS microfluidic channel with inlet/outlet tubing kit is integrated on top of the device and DI water is firstly inserted using motorized syringe pump for 20 min stabilization time under fixed VDS and VG. Afterwards, specific concentrations of TNF-α solution is injected into the sensor.
Figure 1: Flow chart for fabricating MoS2 FET biosensors: (a) nanoprinting of a few-layer MoS2 flake onto a p+ doped Si/SiO2 substrate; (b) fabrication of metal contacts; (c) ALD growth of the HfO2 insulating layer; (d) integration of a PDMS liquid reservoir on top of a MoS2 FET biosensor for obtaining equilibrium-state sensor responses with respect to TNF-α concentration; (e) integration of a microfluidic inlet/outlet tubing kit driven by a motorized syringe pump on top of a biosensor for real-time measurement; (f) functionalization of the HfO2 insulating layer with anti-TNF-α antibodies and following TNF-α detection. (g) An MoS2 FET biosensor and the measurement setup.
RESULTS AND DISCUSSION
Figure 2 (a) shows the transfer characteristics of the MoS2 FETs measured at different concentrations of TNF-α under fixed VDS. IDS values are plotted in the logarithm scale as a function of VG. The transfer characteristics of the sensor display a strong dependence upon TNF-α concentrations with detection limit as low as 60fM.
Figure 2: Sensor responses measured in the subthreshold regimes of MoS2 FET biosensors: (a) transfer characteristics of an MoS2 transistor sensor measured at different biodetection stages; (b) a set of calibrated sensor responses (S) measured from six different MoS2 transistor sensors with respect to TNF-α concentration (n).
Based on the curve, we choose a fixed VG within the subthreshold region and the IDS values from the
fixed gate voltage can be utilized to create sensor response signals. Calibrated sensor response quantities
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are obtained from the response signals based on Langmuir isotherm model and the equilibrium constant (KD) of the antibody-(TNF-α) pair is extracted to be 369 ± 48 fM. Figure 2 (b) illustrates a standard curve created from the calibrated sensor response quantity (S) as a function of TNF-α concentrations (n). Though different devices show various sensor response behavior, multiple sensors are consistent to each other and well fitted to the standard curve.
Figure 3: Time-dependent association/dissociation kinetics of the antibody-(TNF-α) pair: (a) time dependent association sensor responses under different TNF-α concentrations; (b) time dependent dissociation sensor responses under different TNF-α concentrations.
Additionally, we performed real-time measurement for the MoS2 biosensors. Figure 3 displays the
association/dissociation process and we measured the calibrated sensor response signal as a function of time. From the time dependent kinetics, we determined association/dissociation constant (kon/koff) values from multiple sensors and recalculated KD=392fM, which is highly consistent with the equilibrium state measurement result.[4]
CONCLUSION
In summary, we fabricated multiple MoS2 transistor biosensors and demonstrated such biosensors can be utilized to obtain the consistent calibrated sensor response quantity, which is able to draw the standard curve. We also executed time dependent measurement for dynamic association/dissociation kinetics of antibody-(TNF-α) pair. This work advances a biosensing capability in analyzing the sensor responses at a fM-level detection limit with using atomically layered TMDC-based FETs. ACKNOWLEDGEMENTS
This research is supported by the NSF grant # ECCS-1452916.
REFERENCES [1] D. Sarkar, W. Liu, X. Xie, A.C. Anselmo, S. Mitragotri, and K. Banerjee, “MoS2 Field-Effect Tran-
sistor for Next- Generation Label-Free Biosensors”, ACS Nano, 8, 3992-4003, 2014. [2] L. Wang, Y. Wang, J.I. Wong, T. Palacios, J. Kong, and H.Y. Yang, “Functionalized MoS2
Nanosheet-Based Field-Effect Biosensor for Label-Free Sensitive Detection of Cancer Marker Pro-teins in Solution”, Small, 10, 1101-1105, 2014.
[3] H. Nam, S. Wi, H. Rokni, M. Chen, G. Priessnitz, W. Lu, and X. Liang, “MoS2 Transistors Fabricat-ed by Plasma-assisted Nanoprinting of Few-Layer-MoS2 Patterns into Large Arrays”, ACS Nano, 7, 7, 5870-5881, 2013.
[4] H. Nam, B. Oh, P. Chen, M. Chen, S. Wi, W. Wan, K. Kurabayashi, and X. Liang, “Multiple MoS2 Transistors for Sensing Molecule Interaction Kinetics”, Sci. Rep., 5, 10546, 2015.
CONTACT * Xiaogan Liang ; [email protected]
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