A Preliminary Investigation on MEMS Based Immunosensor for E. coli O157:H7 Detection

M. H. Abu Bakar1*, M. H. Ibrahim1, N. M. Kassim1 and A. B. Mohammad1 1 Photonics Technology Centre, Faculty of Electrical Engineering,

Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

*hilal@fke.utm.my

Abstract− Bio-recognition layers on sensors can be designed in various ways; however the most popular approach is to immobi-lise antibodies for specific capture of analytes also known as im-munosensor. This paper presents a preliminary study on MEMS based immunosensor for detecting Escherichia coli 0157:H7. In

this paper, the recent progress within biosensors for E. coli O157:H7 detection will be reviewed. We will discuss the

strengths and limitations of current sensor technologies for the detection of E. coli 0157:H7 .This paper explores the previous methods that have been employed to detect E. coli O157:H7

based on their detection scheme, detection limit, and response time. The reasons focusing on sensor utilizing specifically defined microcantilever using piezoresistive detection scheme is also dis-

cussed. We will also explain briefly on the theoretical aspects governing the relation between surface stress and the bending of

the microcantilever.

Keywords − MEMS, Microcantilever, Piezoresistive, Immunosen-sor, Escherichia coli, O157:H7, Surface stress, Biosensor, antibo-dies, bacteria, detection.

I. INTRODUCTION As our country gears to become an independent food pro-

ducer to cater to internal demand by increasing the production of lifestocks and grazing animals, it is most vital that health related issues due to mass farming is taken into consideration. Contamination of water supplies by fecal waste from lifes-tocks has always been a major problem where mass farming is practiced. One of the most problematic causes of contamina-tion is Coliform bacterias. This explains why Coliform bacte-rias are commonly-used as an indicator of sanitary quality of foods and water.

Unlike the general coliform group, Escherichia coli (E. coli) are almost exclusively of fecal origin and their presence is thus an effective confirmation of fecal contamination. E. coli O157:H7 has caused numerous outbreaks associated with recreational and municipal drinking water [1][2][3][4].The E. coli O157:H7 can easily contaminate ground beef, raw milk, poultry products, fresh apple cider, cold sandwich, vegetables and drinking water. It can be transmitted not only via conta-minated foods but also from person to person [5].

The conventional methods for detecting E. coli O157:H7 take 2–3 days for selective and confirmative enrichments and

up to 4 days for a final biochemical and serological characte-rization [6]. For these reason biosensors could be used to speedily identify and isolate the bacteria of interest. Immuno-sensors are a class of biosensors that exploit the antibody-antigen interaction. They are generally more specific and sen-sitive than other biosensors because of the highly specific mo-lecular recognition and affinity between an antibody and its antigen [7].

II. REVIEW ON ESCHERICHIA COLI 0157:H7 SENSORS

Previous research has been carried out on detecting E. co-li 0157:H7 with varying sensing schemes and detection limits. A surface plasmon resonance biosensor was reported to detect E. coli O157:H7 in meat, environmental, liquid and food sam-ples [8][9][10][11]. Using porous filter membranes, a flow-through conductometric immuno-filtration biosensor was de-veloped for the detection of E. coli O157:H7 in liquid media [12].

A portable evanescent wave fiber-optic biosensor was used to detect E. coli O157:H7 in samples of water at 102 CFU mL with processing time of 35 minutes [13]. This sensor however needed the extra adding Na2S2O3 to the sensor sys-tem to detect the bacteria presence and the incubation of the fiber optic waveguide with the capture antibody which re-quired more than an hour.

Each sensor characteristic is summarized briefly below in table 1.

Table 1: E. coli 0157:H7 Sensors from Previous Research

Researcher Sensor Type Detection Limit (CFU/ml) (time)

Sample

Pyle et al. (1995)

Fluorescent la-beled antibody

1 x 105 Water

Abdel-Hamid et. al (1998)

Porous Filter Membrane Im-munofiltration Amperometric Sensor

1 x 102 (30minutes)

Liquid

DeMarco et. al (2002)

Evanescent Wave Fiber Op-

1 x 102 (35minutes)

Water

Proceedings of the 2009 IEEE 9th Malaysia International Conference on Communications 15 -17 December 2009 Kuala Lumpur Malaysia

978-1-4244-5532-4/09/$26.00 ©2009 IEEE 51

tic Song & Vo-Dinh (2004)

ELISA Immuno capillary reactors

3 X 101 (60 minutes)

Liquid

Meusen et.al (2005)

SPR Immuno-sensor

8.7 x 106 (35minutes)

Environment

Waswa et al. (2006)

SPR Immuno-sensor

1 x 102 Food

Subramanian et. al (2006)

SPR Immuno-sensor

1 x 103 Liquid

Song & Vo-Dinh (2004)

ELISA based Capilarry reac-tors

0.3 x 101 Liquid

The infective dose of E. coli O157:H7 has been estimated

to be as low as 1 to 10 colony-forming units (100 - 101 CFU/mL) [15]. Because of this it can be said that more effi-cient and sensitive sensors need to be develop as past research have generally fall short of detecting these pathogens at the infective dose required for it to be declared clinically signifi-cant.

A miniature biochip system for detection of E. coli O157:H7 based on antibody-immobilized capillary reactors and enzyme-linked immunosorbent assay (ELISA) has also been documented [16]. The detection limits of E. coli O157:H7 using this particular method were 3 and 230 cells. This achieved a good detection limit, however as this detec-tion scheme uses the ELISA based labeling immunoassays, it requires fluorescent labeling and bacteria enrichment which is not cost effective and requires experience personnel handling. Owing to its small size scale and sensitive application, Micro-Electro Mechanical System (MEMS) based sensors will be able to provide an easy and quick detection scheme for bac-terial detection.

III. MEMS SENSOR TECHNOLOGY Among the MEMS platforms, microcantilevers have been

proven to be an outstanding platform for chemical and biolog-ical sensors, with detection limits reaching the femtomolar range [17] and capable of measuring as low as attogram (10–18 grams) mass change, aiding in single molecule or cell detec-tion, and are superior over other signal conversion methods [20]. This explained the great surge of research focusing on such this technology [18][19].

The typical dimensions of commercially available mi-cromachined, mass-produced microcantilevers are 50-200 μm long, 10-40 μm wide and 0.3-3 μm thick, with mass in the range of a few nanograms [21]. Depending on the dimensions, the cantilever spring constant can vary from 0.008 - 30 N/m. Deflections of these cantilevers can be detected with sub-angstrom precision using current techniques perfected for AFM technology such as optical, piezoresistive, piezoelectric, capacitive, and electron tunneling [22]. Unlike other proce-dures currently employed for diagnosing the bacteria, the mi-crocantilever based sensors will require less number of steps

involved, low cost, and ultra sensitive easy-to-use label-free microgravimetric procedure.

Microcantilevers deflection and surface stress measure-ment via piezoresistive layer has been proposed [23][24]. Pie-zoresistivity is the phenomenon of changes in the bulk resis-tivity with applied stress. When a silicon microcantilever with an appropriately shaped doped region is deformed, the change in the resistance of the doped region reflects the degree of deformation [25]. Piezoresistive cantilevers are usually used in force microscopy applications where difficulties in laser alignment make optical detection inconvenient.

This provides an excellent platform for environmental biosensing which have difficulty when applied with optical detection schemes. Modified microcantilevers can recognize target molecules through specified biological binding of anti-body-antigen complex which results in deflection of the canti-lever and the change of resistant due to the stress generated through the binding on the surface of the cantilever.

Fig. 1: Diagram of interactions between target and probe molecules on a microcantilever beam [26]

Previous research done on piezoresistant microcanti-lever immunosensor have been reported and proven to be suc-cessful. A piezoresistive immunosensor was developed for detecting Mycobacterium tuberculosis (M. tb) which ensures a rapid, sensitive, specific, and inexpensive detection of tuber-culosis in less than an hour [27]. Some researchers successful-ly explored the high sensitivity of cantilever transducers to interfacial stress changes in their work on an immunosensor for herbicide [28]. The cantilever with immobilized herbicide exhibited partially reversible bending in response to the anti herbicide antibodies concentrations of 5 and 25 μg/mL in a phosphate buffer saline.

IV. THEORETICAL BACKGROUND To explain the mechanism of the cantilever sensing properties, it is important to understand the cause of deflection on the cantilever surface. Uniform surface stress acting on an isotropic material increases (compressive stress) or decreases (tensile stress) the surface area. For an immunosensor the stress will act on one side of the beam, causing the whole structure to bend as shown in Fig. 2.

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Fig. 2: Side view of a thin beam. N the neutral plane with a curvature R due to force F. [29]

The bending is caused by change in the surface stress. This is due to the fact that between the areas of com-pressive stress and tensile stress, there is a neutral plane, N which is not deformed. Because of the bending, a force F is acting at a distance of x and in the neutral plane resulting in a bending moment, M=F·x. The radius of curvature R is thus given by [29]:

Where M is the bending moment, is the apparent Young’s modulus, and is the moment of inertia. For a rectangular beam, the moment of inertia , is given by [29]:

Where b is the cantilever’s width and t is the thickness respec-tively. In our design b>>t, so the apparent Young Modulus is [30]:

Change of surface stress at one side of the cantilever will cause a static bending with a bending moment given by [29]:

Where ∆σ= σ1 – σ2 with σ1 and σ2 as the surface stress on the upper and lower side of the cantilever. If external gravitation-al, magnetic and electrostatics force that exist on the cantilev-er surface are neglected due to the fact that the bending arises from surface stress generated on the functionalized surface, then the cantilever deformation can be related to the gradient of mechanical stress generated on the device. This phenome-non is denoted by Stoney’s Formula [31] which is given by substituting Eq. (4) into Eq. (1) which will give:

Where is the Poisson’s ratio, E is the substrate’s Young’s modulus, t is the cantilever thickness and is the differential surface stress. If we take into consideration the boundary con-

ditions of the cantilever (R>>L) [32] then this can be further solved and the cantilever displacement s given by;

This change in surface stress can be the result of ad-sorption process, or electrostatic interaction between the charged molecules on the surface of the microcantilever. This equation shows a linear relation between the cantilever bend-ing and the differential surface stress generated on the func-tionalized layer of the immunosensor. The reaction occurring on one side of the cantilever will maximize the relative stress between the two sides (∆σ).

V. PROPOSED WORK Based on the review of previous research showing promising prospects, we are planning a research to focus on designing and evaluating the performance of the MEMS based immunosensor which utilizes microcantilever detection schemes to detect the presence of E. coli 0157:H7. Our re-search will be focusing on optimizing the design of MEMS-based immunosensor for high sensitivity, fast response and accurate detection of E. coli 0157:H7. We will simulate the different dimensions of a microcantilever for optimization using the MemPZR module in CoventorWare which will in-crease the sensitivity of the immunosensor and maximize the output signals.

VI. CONCLUSION Detecting pathogens such as E. coli O157:H7 is im-

portant in the progress of efficient monitoring to avoid conta-mination and outbreak of bacteria related diseases. For this reason, the formulating of an efficient, accurate and viable sensor for detecting E. coli O157:H7 is of vital importance. MEMS based sensors have been proven to be an excellent platform for biosensing from previous observations thus mak-ing it one of the best options for biosensing applications. It is also shown from the discussion above that differential surface stress generated, results in the bending of the microcantilever which can then be measured. Hence, a research on a micro-cantilever MEMS based immunosensor for E. coli O157:H7 detection is proposed which is expected to create a more accu-rate and responsive sensing scheme.

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