1-1 Research Background and motivation
To secure health of human beings is the fundamental of pathogens detection.
Foodborne disease infected by pathogen has being a serious threat for decades in public health. Most of the diseases are caused commonly by the pathogenic bacteria, including Escherichia coli O157:H7, Salmonella Typhimurium, and Vibrio
parahaemolyticus. One pathogenic bacteria, Vibrio parahaemolyticus was
normally found in many species of fish, shellfish, and crustaceans in marine environments, shown in Figure 1-1. They survive in warm saline water with a temperature ranging from 5 to 43 degree Celsius. When infected, Vibrio parahaemolyticus causes watery diarrhea with abdominal cramping, nausea, vomiting, fever and chills. Usually these symptoms occur within 24 hours after ingestion. Illness is usually self-limited within 3 days. Due to the change of diet and the global warning, Vibrio parahaemolyticus is becoming a highest risk in food poisoning both in United State(Figure 1-2) and Taiwan(Table 1-1), Thus, the development of rapid, sensitive and specific detection as an assay is important to respond effectively.[1, 2]Biosensors have a wide range of applications for diseases detection and diagnosis. The biosensor is an analytical device, which converts a biological response into an electrical signal. It consists of two main components: the biorecepter element, which recognizes the target analytes, and the transducer element, for converting the recognition event into a measurable electrical signal. The bioreceptor can be a tissue, microorganism, cell, enzyme, antibody etc. The transduction may be optical, electrochemical, piezoelectric, and micromechanical combinations. All the biosensors were designed for fast detection, specificity, sensitivity, accuracy, and capacity to
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detect small amounts of target in samples.[3]The impedimetric detection is a potential technique for biosensors with advantages of low-cost, miniaturization, and flexibility. It can integrate with other analytic methods for higher sensitivity. The detection of bacterial cells using impedance analyzer is well-known.[4, 5] However, performance of the previous studies may be so far limited due to time and diffusion even the analytes are close to sensing surface. In the case of fluidic system applications for biomolecular detection, the targets displacement is dominated by diffusion of its nature. To overcome the constraints of biosensing, the sensing signal enhancement with AC electrokinetics is necessary and reduces the time of detection greatly.
Figure 1-1 SEM image of Vibrio parahaemolyticus.[1]
Table 1-1 Statistics of pathogen infection in Taiwan in 2010. [2]
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Figure 1-2 Relative rate of laboratory-confirmed infections with Campylobacter,
E. coli O157, Listeria, Salmonella, and Vibrio, compared with
1996-1998 rates by years. [1]4
1-2 Bacteria detection via Impedance analyzer
The recognition of pathogenic bacteria can help control the poisoning foods and prevent the foodborne diseases. Indeed, a wide range of biosensors in current on bacteria detection, such as surface plasmon resonance(SPR)[6], quartz crystal microbalance(QCM)[7], and electrochemical impedance spectroscopy(EIS), are mostly adapted to detect bacteria. The advantages of these methods are simple and rapid compared with conventional systems such as Enzyme-linked immunosorbent assay(ELISA)[8] and polymerase chain reaction(PCR) /DNA array[9], which are limited by length of sensing time and portability for biochemical testing procedures.[3]
The previous studies, Yang et al. in 2004 used indium tin-oxide(ITO) interdigitated electrode for the detection of viable Salmonella typhimurium in milk.[10] The impedance growth curves, against bacteria growth time, were recorded at four frequencies (10Hz, 100Hz, lkHz, and 10kHz) during the growth of
S.typhimurium. It was observed that impedance did not change until the cell number
reached 105-106 cfu/mL(determined by the plating method). The greatest change in impedance was observed at 10Hz. Figure 1-3 shows the results presented by Yang. He also fabricated a label-free electrochemical immunosensor for detection of E coli O157:H7 by immobilizing anti-E. coli antibodies on the surface of ITO interdigitated electrode and measuring impedance change in the presence of a redox probe label-free detection of Salmonella cells in DI water. Figure 1-4 shows the bacteria attachment on the ITO electrode and measured via redox probe.[11]Suehiro et al. in 2001, 2003, and 2006 studied the issue of capturing bacterial cells on the surface of electrodes using dielectrophoresis(DEP).[12, 13, 14, 15] The concentration of bacteria formed pear chain between electrodes followed by an
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impedance measurement, shown in Figure 1-5(a). The detection system was able to detect 105 cfu/mL in 10 min. The group has also presented specificity improvement by immobilization of antibody on DEP devices in a microfluidic environment, shown in Figure 1-5(b). This study was published as a proof of concept and only a high concentration (106 cfu/mL) of E.coli cells was demonstrated. (in Figure 1-6)
Figure 1-3 (a) Changes in impedance of the IME impedance sensor during the Bacterial growth in selective solution recorded at different frequencies.
Initial Salmonella cell number: 2.06 × 102 cfu/mL (b) a group of impedance growth curve during the growth of Salmonella, recorded at
10Hz. Initial cell number : a:control, b: 4.8 × 100, c: 2.84 × 101, d: 1.75
× 102, (e): 5.4 × 103, f: 5.3 × 104, g: 5.4 × 105 cfu/mL; AC amplitude:
5mV (c) SEM micrograph before test (d) after the test of bacteria growth (initial number:1.76*102cfu/mL).[10]
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Figure 1-4 The principle of the direct impedance immunosensor with interdigitated electrode.[11]
Figure 1-5 (a) Dielectrophoresis based impedance detection system (b) selective impedance analysis based on Dielectrophoresis using immobilized antibody.[13]
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Figure 1-6 Real-time Conductance inspected in 0.1M mannitol solution for (a) blank electrode (b) with antibody modification (c) E. coli suspension without antibody modification (d) E. coli suspension without antibody modification. (1V, 10kHz, 10min).[13]
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1-3 Electrodes configuration of microfluidic system for bioelectronics detection
There are plenty of studies about the concentration of bioparticles using AC electrokinetics on electrodes with various configurations, such as parallel integrated, castellated, potential-well, quadrupole, and concentric. AC electrokinetics as an approach in microfludic system for particles manipulation has also been reported by several research teams. AC electrokinetics can induce non-uniform AC electric field above electrodes. P. K. Wong had used the circular electrode (Figure 1-7) to concentrate the samples into the region at center of electrode, which has a advantage of smaller target preparation, such as E. coli Bacteria, quantum dot, and DNA, showed as Figure 1-8 and Figure 1-9.[16, 17, 18] Also, multiple interdigited electrode arrays in previous researches were widely used as impedance analyzer. The impedance spectroscopy in microfluidic system often uses the parallel interdigited electrode for incubation of the analytes. It has been present advantages of low ohmic drop, fast response of steady-state, and well performance in detecting small amounts of products.[19] Figure 1-10 shows the experimental set up of impedance measurement with interdigitated electrode.
In this work, concentric electrode combined with inside parallel integrated electrode enhances the capability of target concentration and bioassay. The bacteria were concentrated onto the inner circular electrodes, and impedance measurement was performed by using inner interdigitated electrode inside the inner circular.
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Figure 1-7 (a)Electrode design of the AC electroosmotic processor (topview).
(b) Schematic (side view) illustrating electrode polarization and formation of AC electroosmosis.[18]
Figure 1-8 Particles concentration (a-f) Video time series showing concentration of 200-nm fluorescence particles on the central electrode each picture is separated by 10s. (g) Concentration of E.coli bacteria at the center of the central electrode.[18]
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Figure 1-9 Fluorescence image demonstrating concentration of double-stranded λ phage DNA molecules (48.5kbp) on the central electrode.[18]
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Figure 1-10 (a) The experimental set up of impedance measurement with
interdigitated electrode for bacteria growth (b) impedance response with time for the growth of bacteria.[19]
(a) (b)
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1-4 AC electrokinetics in Microfluidics system
In microfluidic system, the phenomena under AC electric field that involve the interactions among applied electric fields , ions suspended in solution, and polarized particles result in ions and particles movement. There are three dominant mechanics during the AC signal applying, dielectrophoresis(DEP), AC Electroosmosis(ACEO), and electrothermal effect actuating motion of solid bodies( micro-/nano-particles or bio-particles ) in the process of applied electric field.[20, 21] The AC electrokinetics physically displaces particles and induces fluidic flow duo to the controllable parameters, such as voltage, frequency, electrode size, etc.[22] Electroosmosis is the motion of fluid induced by tangential electric field at the surface of electrode due to ionic movement in the electrical double layer (Figure 1-11(a)). Dielectrophoresis occurred during non-uniform signal applied in the solution as suspended particles is polarized. The polarized dipoles difference of dielectric particles between the interface inside and outside give rise to a net force, either toward the strong electric field, called Positive DEP(p-DEP), or away from electric field, called negative DEP(n-DEP), on the particles(Figure 1-11(b)). Electrothermal fluid is generated by electric field from the temperature heating of the fluid and the gradient in local conductivity and permittivity (Figure 1-11(c)). In this thesis, AC electrothermal effect is negligible due to the low conductivity of medium and undesirable mechanism while trapping the particles. These experiments reduce the response time by relying on forces integrated with electroosmosis and dielectriophoresis on analytes. The parameters are with low potential and low frequency that can induce merely dielectrophoresis on particles and electroosmosis in electrical double layer, but prevent the electrothermal fluid in the microfuidic cell.
The potential application of AC electrokinetics can facilitate methods for
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manipulating biological objects.[23, 24] Efficient bioparticles manipulation can shorten the detection time and improve the sensitivity.[25, 26]
In the article, bacteria were trapped onto the center of the inner electrode, experiencing a balance force from dielectrophoresis induced by polarized particles and drag force by AC electrosomsis under non-uniform electric fields without using bulk fluidic. It can largely reduce the time for immobilization of bacteria and enhance the sensitivity.
Figure 1-11 AC eletrokinetics behaviors (a)Dielectrophoresis (b)AC electroosmosis (c)Electrothermal effect.[22]
1-5 Structure of the thesis
This thesis reported enhancement of biosensor via AC electrokinetics on bacteria detection. We detected the Vibrio parahaemolyticus as pathogenic analytes via impedance biosensor and AC electrokinetics was adopted to enhance the capture of bacteria. First, a new configuration of electrodes for diagnosis was fabricated and simulated by COMSOL V. 4.2 for analysis of electric field and AC electroosmosis.
Second, the chip was cleaned and functionalized by chemical molecules ion gold electrode with confirm using characteristic methods. The specific biomolecules that recognize Vibrio parahaemolyticus uniquely was immobilized on the electrode before detection. Third, the AC electrokinetics was involved for bacteria trapping and the
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impedance was analyzed through electrodes. Modeling using equivalent circuit was also adopted to justify the impedance spectroscopy from the measurement. Finally, to confirm the selectivity and specificity in Vibrio parahaemolyticus detection, a non-pathogenic E.coli bacteria was mixed with Vibrio parahaemolyticus in the
analyte for detection. Figure 1-12 illustrates the experimental flowchart on achieving Vibrio parahaemolyticus detection.
Figure 1-12 Experimental flowchart of this thesis.