Measurement and Simulation Techniques For Piezoresistive Microcantilever Biosensor Applications

Applications of microcantilevers as biosensors have been explored by many researchers for the applications in medicine, biological, chemistry, and environmental monitoring. This research discusses a design of measurement method and simuations for piezoresistive microcantilever as a biosensor, which consist of designing Wheatstone bridge circuit as object detector, simulation of resonance frequency shift based on Euler Bernoulli Beam equation, and microcantilever vibration simulation using COMSOL Multiphysics 3.5. The piezoresistive microcantilever used here is Seiko Instrument Technology (Japan) product with length of 110 μm, width of 50 μm, and thickness of 1 μm. Microcantilever mass is 12.815 ng, including the mass receptor. The sample object in this research is bacteria EColi. One bacteria mass is assumed to 0.3 pg. Simulation results show that the mass of one bacterium will cause the deflection of 0,03053 nm and resonance frequency value of 118,90 kHz. Moreover, four bacterium will cause the deflection of 0,03054 nm and resonance frequency value of 118,68 kHz. These datas indicate that the increasing of the bacteria mass increases the deflection value and reduces the value of resonance frequency DOI:  http://dx.doi.org/10.11591/telkomnika.v10i6.1180 Full Text: PDF


Introduction
A simple detection system with a high sensitivity is desired in the fields of biotechnology and medical science. The basic principle of biologically-based sensor (called as biosensor) is an interaction between bio-molecules, especially antigen-antibody and deoxyribonucleic acid (DNA)-DNA.Such bio-molecules interaction is generally measured by using gas chromatography mass spectrometry (GCMS), a quartz crystal oscillator and a surface Plasmon resonance. However, these methodshave some disadvantages as follows [1]. First, GCMS has a high mass resolution of about 1 pg, but it takes long time to measure a sample. Second, the ISSN: 1693-6930 TELKOMNIKA Vol. 10, No. 4, December 2012 : 725 -732 726 quartz crystal oscillator method has a low sensitivity of about 30 pg/Hz, and third, the Surface Plasmon Resonance method is complex and expensive Micro-machined cantilever is a good candidate for sensor.This was first used as force probes in Atomic Force Microscopy (AFM). The extreme sensitivity to several environmental factors, such as noise, temperature, humidity and pressure was immediately evident. In 1994, research teams in Oak Ridge National Laboratory and IBM have converted the mechanism causing interference into a platform for a novel family of biosensors [1]. Some of characteristics of microcantilever are as follows: a) small size (in range micrometer) b) high sensitivity (scale in attogram, 10 -18 gram) c) low cost relatively d) low power consumption e) detectable of various object in one platform f) simple fabrication g) integrated in microarray form.
The microcantilever based sensors areapplicable for various fields in medicine and biology field and in chemistry & environmental monitoring [2], such as detection of DNA and protein, detection of myoglobin, detection of temperature and heat changes, detection of Escherichia Coli bacteria, detection of humidity, etc.This paper discusses a design of measurement technique and simuations for piezoresistive-typedmicrocantilever as a biosensor, including the design of Wheatstone bridge circuit as object detector, simulation of resonance frequency shift based on Euler-Bernoulli Beam equation, and microcantilever vibration simulation using COMSOL.

Research Method
In this research, the measurement techniques and simulations of microcantilever-based biosensor are discussed, especially the changes in deflection (∆δ) and the change in resonance frequency (∆f) ofmicrocantileverdue to the changes of object mass (∆m) absorbed on the microcantileversurface. The measurement techniqueswere designed using a Wheatstone bridge circuit and operational amplifier of INA 128. The microcantilever used here is piezoresistivetype made by Seiko Instrument Technology (Japan). Microcantilever surface will be coated by a material receptor that serves as a catcher and selector that only react with the selected object. The microcantilever size is 110 µm in length, 50 µm in width, and 1 µm in thickness. Microcantilever mass is 12.815 ng, including the mass receptor.The object used here is Escherichia Coli bacteria, which has the mass of0.3 pg. Simulation of the resonance frequency is performed based on Euler-Bernoulli Beam Equation, and the microcantilever motion simulation is done by using COMSOL Multiphysics 3.5. Figure 1 describes a simple structure of the microcantilever before and after deflection. In order to use the micro cantilever as a biosensor, it must be placed bio-receptor as functionalization layer on the surface of microcantilever and placing the electronics devices to detect the deflection of the microcantilever. Figure 2 shows a biosensor microcantilever (before (a) and after (b) deflections), where functionalization layer is coated on the microcantilever surface.

Microcantilever Structure
In case of the bacteria object detection, the microcantilever needs to be coated by antibody layer on its surface which only can react with bacteria. The mass detected here is the reaction result between both antibody and bacteria mass which is represented by BSA (Bovine Serum Albumin). BSA and the antibody wereimmobilized on the microcantilever surface using physicaladsorption as follows. First, the microcantilevers were cleaned using piranha solution (H 2 O 2 :H 2 SO 4 =1:1) and then immersed in ethanolbefore critical point dried (CPD). Second, the microcantilevers werethen rinsed for around 30 s in deionized (DI) water and treated with BSA at a concentration of 2 mg/ ml for 15 min. The resonance frequency of the microcantileveris then measured after both the antibody and BSA are attached tothe surface [3]. Commercial microcantilever dimensions used here is 50-200 µm in length, 10-40 µm in width, and 0.3-3 µm in thickness. Such microcantilever size is possible to detect attogram scale (10 -18 gram) object mass [4].

Microcantilever Deflection Modes
Microcantileverbiosensors can operate in two modes (see Figure3), i.e., static and dynamic modes. In the static mode, themicrocantileverbiosensor operates based on the difference ofsurface stress generated between the top and bottom surfacesduring binding or adsorption of target molecules on afunctionalized surface. The change of surface stress causes the cantilever to bend. This bending is known as deflection (δ).
On the other hand, in the dynamic mode, the microcantilever is oscillated at a certain frequency andthe change in oscillationfrequency due to mass change (∆m) loading is measured on its surface. Deflection values can be calculated using the two methods, i.e., distributed load and pointed loadas shown in Figure 4.  The calculation of deflection values in static mode for uniformly distributed load method usesequation (1), while the calculation of deflection values for pointed load method usesequation (2) [6]: where E isYoung's Modulus of material, P is the force in microcantilever beam, l is length of microcantilever, a is the object distancefromfixed end,and I is InertiaMoment ofmicrocantilever, which is calculated by the equation (3) wherewandtarewidth and thickness of microcantilever, respectively. To determine the amount of deflection in the dynamic mode, we use the Euler-Bernoulli Beam Equationwith uniformly distributed load method [7] as follows, wheren ρ is the density of material, and A is cross section area ( = * ). To change the partial differential equations into differential equations on the value of x only, we assume a harmonic input force applied to the system with the radial frequency (߱), and thenܲ(‫,ݔ‬ ‫)ݐ‬ = ܲ݁ ఠ௧ andߜ(‫,ݔ‬ ‫)ݐ‬ = ߜ݁ ఠ௧ . Equation (4) can be then simplified by replacing the factor݁ ఠ௧ , becomes [8]: whereሺ + ࢽሻ is a constant value of structural damping factor at the time of the vibrations and strains that occur in the microcantilever.
The value of resonance frequency is influenced by the mass and strain value factor (spring constant) of microcantilever [9].
where M is object mass on microcantilever surface and m is effective mass of microcantilever (݉ = ‫݈ܣߩ‬ = ‫)݈ݐݓߩ‬ [10]. By adding the mass, the resonance frequency changes (∆f) will reduce the value of the frequency and then mass change (∆m) can be determined. The resonance frequency change can be seen in the following equation [11].

Results and Discussions 3.1. Measurement Results of ∆V and ∆R
Here, the measurement of microcantileverdeflection was done using a Wheatstone bridge circuits which consists of potentiometers and resistors components ( Figure 5). Potentiometer can be considered to replace the piezoresistor inmicrocantilever. From the data  R 1 and R 2 components are piezoresistors within the piezo resistive microcantilever. Components R 3 , R 4 and R 5 arean external resistors. The amplifier INA128 is used to enhance the value of ∆V. In the experimental procedure, value R 1 is set close to the value R = 630 Ω and Vout is set to zero. Once the value of V out = 0 is obtained, then the value of V out is measured for all values of R 1 as shown in Table 1. The measurementof ∆V values for all values of R1 in Table  1 are shown in Figure 6.  Figure 6. Graphs of experimental Resultsof ∆V and ∆R+R as function of R1 For comparison, we performed the calculation of ∆V and ∆R using equation (9) and equation (10) [12]. The calculation results are shown in Table 2. The graphs of ∆V and ∆R+R as function of R1 from Table 2 can be shown in Figure 7.

Simulation Results of The Dynamic Mode
In this simulation, mass of bacteria as object is assumed to be 0.3 pg. We use Euler-Bernoulli Beam equation approach to simulate the deflection and resonance frequency. The simulation is done using Matlab software and the result is shown in Figure 8. To see the deflection shift (∆δ) and the change of resonance frequency (∆f), we will simulate two bacteria masses, i.e., 0.3 pg and 0.6 pg. Figure 8 shows that the changes of objectmass (∆m) causes the changes of frequency (X axis) and the changes of deflection value (Y axis). For bacteria mass is 3 pg, the resonance frequency value (f) is 118.90 kHz and the deflection value (δ) is 30.536 pm. On the other hand, for bacteria mass of 6 pg, the resonance frequency value (f) is 118.85 kHz and the deflection value (δ) is 30.539 pm. It shows that the change of the bacterial mass from 3 pg to 6 pg results in a decrease in the resonance frequency value of 0.05 kHz and an increase in the deflection value of 0.003 pm.

Simulation of Microcantilever Motion
It is well known that the deflection of microcantilever is influenced by several factors related to physical geometries such as length, width and thickness. Therefore, in order to see the difference in the deflection value, the simulation is done by using COMSOL Multiphysics 3.5 software [14][15][16][17]. Here, the dimensions of the microcantilever are the length of 110 µm, a width of 50 µm and a thick of1 µm. The results of this simulation can be seen in Figure 9.The values of frequency and microcantilever deflection can be seen. When we increase the length of the microcantilever, the value of deflection increases and the value of resonance frequency decreases.This indicates that the increasing of microcantilever dimension will cause a change in microcantilever mass and also the changes in the value of deflection and resonance frequency.The detail of simulation results for several bacterial masses can be seen in Table 3. From the result in Table 3, it can be seen that the increasing of bacteria masses on the microcantilever surface will decrease the value of the resonance frequency and increase the deflectionvalue of microcantilever.

Conclusion
The main results of this research as follows: 1. In static mode deflection, the increasing of object mass causes an increase in the value of the Wheatstone bridgevoltage change (∆V). 2. In dynamic mode deflection, one bacteria will cause the deflection value of 0.30535 pm and resonance frequency value of 118.90 kHz. Four objects of bacterium will cause the deflection value of 0.30544 pm and resonance frequency value of 118.68 kHz. It means that increasing of the object mass causes an increase in the value of deflection and decrease in the resonance frequency value.