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Studies on Cantilever based Triglyceride Biosensor Renny Edwin Fernandez, Soma Sekhar B. V., Enakshi Bhattacharya, Anju Chadha Abstract— We report detection of micromolar levels of triglycerides using surface micromachined polysilicon cantilever beams. Enzymatic hydrolysis of triglycerides produces glycerol which alters the viscosity and density of the solution. This affects the dynamic properties of cantilever beams immersed in the solution. The change in the resonance frequency of the cantilever beams in the solution is measured using Doppler Vibrometry and the concentration of triglyceride is determined by comparing with a predetermined calibration plot. Index Terms— biosensor, microcantilever, resonance frequency, triglyceride, vibrometer
I. INTRODUCTION
M
icrocantilevers have gained much popularity in
investigating many biological reactions, mainly due to its ability to transduce a variety of chemical and physical phenomena into a mechanical movement on a micrometer scale [1, 2]. The deflection of microcantilevers, due to surface induced stresses, caused by the enzymatic hydrolysis of glucose has been reported earlier [3]. We report detection of micromolar levels of triglycerides using microcantilever beams by monitoring the resonance frequency changes induced by the tributyrin hydrolysis. Tributyrin, in the presence of the enzyme Lipase, hydrolyses to butyric acid and glycerol. Various chemical and electrical methods have been developed for triglyceride detection [4, 5]. An Electrolyte - Insulator - Semiconductor CAPacitor (EISCAP) sensor for tributyrin detection has been reported by us earlier [4], which detects the change in pH of the solution due to enzyme mediated hydrolysis of triglycerides. However the sensitivity of the EISCAP sensor was limited to concentrations above 100µM. This paper reports results on a microcantilever based sensor to detect triglyceride levels well below 100µM. Manuscript received September 15, 2007 Renny Edwin Fernandez and Enakshi Bhattacharya are with Microelectronics and MEMS Laboratory, Department of Electrical Engineering, Indian Institute of Technology Madras, Chennai – 600036, e-mail:
[email protected], ph: 91-44-22574419 SomaSekhar B.V is with Center for Non-Destructive Evaluation and Department of Mechanical Engineering, Indian Institute of Technology Madras. Anju Chadha is with Department of Biotechnology and aNational Center for Catalysis Research, Indian Institute of Technology Madras, Chennai-600036,
[email protected], ph: 91-44-2257 4106
978-1-4244-1728-5/07/$25.00 ©2007 IEEE
II.
MATERIALS AND METHODS
Crystalline silicon, p-type, oriented, wafers of resistivity 1-10 Ω cm were thermally oxidized followed by polysilicon deposition by Low-pressure chemical vapor deposition (LPCVD). Lipase (Pseudomonas cepacia) was bought from Amano, Japan. All the chemicals used were of analytic grade and were purchased locally. The oxide anchored polysilicon cantilever beams, of length 200 µm, width 20 µm and thickness 2 µm with 1.6 µm gap between the beam and the substrate were fabricated in house by surface micromachining[6]. Figure 1 shows the SEM picture of the cantilever beams. A Doppler vibrometer was used to measure the resonance frequency of the cantilever beams. POLYTEC OFV-5000 laser vibrometer with DD-300 displacement decoder was used for measurements. It is a non-contact, non-destructive technique and ensures high measurement accuracy with reduced testing time. Modifications were made to the vibrometer setup to suit the measurements by integrating a microfocus, to focus the laser beam at the tip of the microcantilever, and a probe station to actuate the cantilevers. The whole system is integrated with a computer via a software program, written in LabVIEW so as to minimize any measurement errors.
Figure 1: SEM picture of cantilever beams III. RESULTS AND DISCUSSION
The beams were excited with a sine wave of 10Vp-p coupled with a dc-offset voltage of 100mV. The frequency of the signal was varied from 5 to 100 kHz. The frequency at which the amplitude of vibration was at the maximum was taken as the resonance frequency of the beam. Resonance frequency of the cantilever beam in air was found out to be 76.1 kHz (Figure 2a)
2 whereas the resonance frequency of the beam when immersed in water, was found to be 51.2 kHz ( Figure 2b). It is worth noting that, apart from the decrease in resonance frequency, the amplitude of vibration has also come down considerably when vibrating in water. Equation 1 shows that the resonance frequency of a cantilever in air is [7].
f air =
1 2π
k m
(1)
Beam in air 76 .17 kHz
2.2 2.0
amplitude(volts)
1.8 1.6 1.4 1.2
Resonance frequency measurements were done with the same beam in methanol and ethanol which have the same density (ρ = 0.79 g/cm-3) but different viscosities (ηethanol = 1.074 × 10−3 Pa.s, ηmethanol = 0.544 × 10−3 Pa.s). It was seen that the resonance frequencies of the microcantilevers, vibrating in these liquids, were almost same (Table 1) suggesting that the density had more effect on the dynamic characteristics of the cantilever than the viscosity of the solution. However the changes in the viscosity, which is expected to reflect on the amplitude of vibration of the cantilever and the quality factor, could not be monitored accurately due to the shortcomings in the measurement set up. The change in resonance frequency is monitored for the detetcion of triglyceride concentration.
1.0 0.8 0.6
A. TRIGLYCERIDE DETECTION USING MICROCANTILEVER SENSORS
0.4 0.2 40.0k
60.0k
80.0k 100.0k 120.0k 140.0k 160.0k 180.0k 200.0k 220.0k
freq uency(H z)
Figure 2a: Resonance frequency curve of a cantilever beam vibrating in air
Laser vibrometer tip Laser beam
Beam in water
0.7
Liquid
51.52 kHz 0.6
Cantilever beam
amplitude(volts)
0.5
~
0.4
0.3
Figure 3: Experimental setup 0.2
0.1 10k
20k
30k
40k
50k
60k
70k
80k
frequency(Hz)
Figure 2b: Resonance frequency curve of a cantilever beam vibrating in water
The wafer, containing the cantilever beams, was fixed at the bottom of a small Petridish to which 3.5 ml of the solution was added to immerse the sample completely (Figure 3). Tributyrin solutions of varying concentrations were prepared in deionized water to which 1 mg lipase (1mg/ 10ml of the solution) was added and the resulting solution was incubated for 20
Where ‘k’ is the effective spring constant of the cantilever beam and ‘m’ is its mass.
6.8
Table 1: Resonance frequency Vs Density
Ethanol Methanol
Density (g/cm³)
8.9 × 10-4
1
Resonance Frequency (kHz) 51.2
1.074 × 10
0.79
57.2
0.544 × 10−3
0.79
58.7
−3
6.4
6.0
pH
Water
Viscosity (Pa·s)
0.2 mg of lipase 0.3 mg 0.5 mg 1 mg 2 mg
5.6
5.2
4.8 0
10
20
30
40
Time in minutes
A liquid like water being denser and more viscous than air contributes an extra mass (∆M) to the cantilever mass thus reducing its resonance frequency as shown in equation 2 [7, 8].
f liquid ∝
1 2π
k m + ∆M
(2)
978-1-4244-1728-5/07/$25.00 ©2007 IEEE
Figure 4: Change in pH, during the hydrolysis of 5mM of tributyrin, with various amounts of enzyme
minutes for the hydrolysis. These parameters were optimized by monitoring the pH changes during the hydrolysis of a 5mM triglyceride solution, for various amounts of lipase, over a period of time. It was found that the rate of the hydrolysis reaction was highest when the lipase concentration was 1mg/ml and further
3 increase in the enzyme concentration had no effect on the reaction rate (Figure 4). A molecule of tributyrin hydrolyses to one molecule of glycerol and three molecules of butyric acid.
IV. CONCLUSION
50
glycerol + butyric acid solution hydrolyzed triglyceride solution
10 µM 45 20 µM
resonance frequency(kHz)
compares the response of the cantilever with butyric acid + glycerol solutions and tributyrin hydrolyzed solutions. The resonance frequency of the sensor was seen to reduce from 47.2 kHz to 36.8 kHz for 10 µM to 100µM of the solution concentration as shown in figure 5b. The sensor system did not show a decipherable output signal, for concentrations higher than 100µM, due to the formation of air bubbles.
50 µM
40
75 µM 100 µM
35
150 µM 250 µM
30
25
20
Surface micromachined polysilicon cantilever beams were used to detect triglycerides in a solution. The sensor was calibrated with different concentrations of butyric acid + glycerol solutions, which were equivalent to the stoichiometry of the hydrolyzed tributyrin solutions and later were tested with lipase hydrolyzed tributyrin solutions. The sensor was found to be sensitive for concentrations as low as 10 µM. The higher concentration range was limited to 100 µM due to the formation of air bubbles which hindered the measurements.
15 0
100
200
300
400
ACKNOWLEDGMENT
500
concentration(µM)
Figure 5a: Comparison between enzymatically hydrolyzed tributyrin solution and Butyric acid + glycerol solution 54
We thank Prof. Krishnan Balasubramanian, Center for Non-Destructive Evaluation, Department of Mechanical Engineering, Indian Institute of Technology Madras for helpful discussions. REFERENCES
Resonance frequency(kHz)
52 50
[1]
10 µM
48
30 µM
46
20 µM
44
[2]
50 µM
42 40
60 µM
38
70 µM
36
[3]
80 µM 90 µM
34 0
20
40
60
80
[4]
100
concentration of tributyrin(mM)
Figure 5b: Response of the sensor with 10- 100 µM hydrolyzed triglyceride solution
[5] [6]
[7]
The cantilever sensor was first calibrated using different concentrations of butyric acid + glycerol solutions, which were equivalent to the stoichiometry of the hydrolyzed tributyrin solutions. The concentrations of glycerol varied from 24µM to 480µM and butyric acid concentrations were thrice that of glycerol. Glycerol, being denser (ρ = 1.26 g/cm-3), contributes to the density changes where as the presence of butyric acid (ρ = 0.96 g/cm-3) does not effect the density of the solution. The experiments were repeated using enzymatically hydrolyzed tributyrin solutions and the results were found to be similar to the calibrated readings. Figure 5a 978-1-4244-1728-5/07/$25.00 ©2007 IEEE
[8]
R. Raiteri, M. Grattarola, H. J. Butt, “Micromechanical cantilever based Biosensor, Sensors and Actuators B, vol.79, 2001, pp.115-126 J. Tamayo, A.D.L Humphris, A.M. Malloy, M.J. Miles, “Chemical sensors and biosensors in liquid environment based on microcantilevers with amplified quality factor”, Ultramicroscopy, vol. 86, 2001, 167–173 J. Pei, F. Tian, T. Thundat, “Glucose Biosensor Based on the Microcantilever”, Analytical Chemistry, vol.76, 2004, pp. 292297 I.Basu, R.V. Subramanian, A. Mathew, A. M. Kayastha, A. Chadha, E.Bhattacharya, “Solid State Potentiometric Sensor for the Estimation of Tributyrin and Urea”, Sensors and Actuators B vol. 107, 2005, pp.418-423. D. G. Pijanowska, A. Baraniecka, R. Wiater, G. Ginalska, J. obarzewski, W. Torbicz, “The pH detection of triglycerides”, Sensors and Actuators B, vol.78, 2001, pp. 263-266. S. Bhat, E. Bhattacharya, “Parameter extraction from simple electrical measurements on surface micromachined cantilevers”, Journal of Micro/Nanolithography, MEMS and MOEMS, 2007, to be published G.A.Campbell, R.Mutharasan, “Detection of pathogen Escherichia coli O157:H7 using self- excited PZT-glass microcantilevers”, Biosensors and Bioelectronics, vol. 21,2005, pp.462–473. C. A. Van Eysden and J. E. Sader, “Resonant frequencies of a rectangular cantilever beam immersed in a fluid”, Journal of Applied physics, vol.100, 2006, pp.114916
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