The following materials and chemicals are used:
Potassium hydroxide(KOH), Potassium ferrocyanide, bovine serum
albumin(BSA) , N-hydroxy-succinimide 97% (NHS) and
1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC) were purchased from Sigma-Aldrich . carboxytetramethyl-rhodamine succinimidyl ester (5(6)-TAMRA,SE) were obtained from AnaSpec, Inc. 11-mercaptoundecanoic acid(11-MUA) and 3-mercaptopropanol(3-MPOH) were provided by laboratory of Prof. Yaw-Kuen Li in Department of Applied Chemistry, NCTU. Vibrio parahaemolyticus and Polyclonal antibody of Vibrio parahaemolyticus purified by mouse Immunoglobulin G was prepared by laboratory of Prof. Tung-Kung Wu in Department of Biological Science and Technology, NCTU. Phosphate buffer was prepared as 4.4 mM Na2HPO4 and 1.4 mM KH2PO4.
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3-2 Design and Fabrication of electrodes for biosensing
3-2-1 Patterned electrodes Device
Geometry, area and gap between electrodes are important parameters that influence the function of AC electrokinetics. In the interest of the bioparticles and micro- or nano- particles manipulation and concentration, the concentric electrode was designed.[18] The interdigitated electrode for analytes detection in microfluidic system was beneficial for the impedance spectroscopy.[19] In this work, concentric electrode combined with inside interdigitated electrode was designed as shown in Figure 3-1. AC electrokinetics manipulation and impedance measurement was integrated together in this device. The size of the circular electrode part was defined for the effective trapping and for electrical impedance measurement. Due to the biomolecular modification onto the electrode, the material of external surface of electrode is limited to gold, which has also a great biocompatible affinity. Gap distance and interdigited arrays size influence the scale of impedance. Devices with 5-um gap and 5-um-electrode width were optimized for impedance measurement.
Figure 3-1 Innovative integration for electrodes. Right side as a device for detection.
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3-2-2 Electrode preparation
Each device used wet SiO2(wet oxide) as substrate. A thin layer of titanium(Ti) of 30 nm and gold(Au) of 300 nm in thickness were then deposited on SiO2 with the procedures of thermal coating by CHIPBOND Technology Corporation. Then, Au-Ti film electrode was printed by photolithography and etched by wet-etching processes:
I. Coating the positive photoresist(6400) by spinner II. Soft bake
III. Exposure
IV. Development and fixing
V. Gold etching with KI:I2:DI water = 68 mg: 17 mg: 24 mL (shake the etchant gently during etching the gold )
VI. Ti etching with DHF:H2O2:DI water = 1 mL:1 mL:80 mL
Finally, the photoresist on the electrodes was dissolved by acetone as shown in Figure 3-2
.
Figure 3-2 Electrode configuration.
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3-2-3 Definition of the sensing area for impedance analysis
For impedance analysis, the area of electrode dominates the measurement as well.
Though the microfluidic channel was used, various sizes of channel made artificially had effect the measurement. Hence, the SU-8 2005 (negative photoresist) as passivative layer created same area in all electrodes and reduce the effect on impedance measurement. A 5-μm SU-8 layer was patterned via photolithography. The defined SU-8 pattern was shown in Figure 3-3. The processes were depicted as followed:
I. Spin coating the SU-8: 500 rpm for 10 sec; 3000 rpm for 30 sec.
II. Soft bake: 65℃ for 60 sec; 95℃ for 120 sec; 65℃ for 60 sec.
III. Exposure (with light intensity 130 millijoule per square centimeter).
IV. Hard bake: 65℃, 60 sec; 95℃,600 sec; 65℃, 60 sec.
V. Development: 40 sec with SU-8 developer.
VI. Definition: 30 sec with IPA.
Figure 3-3 Sensing area definition of the chip after SU-8 passivation.
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3-3 Preparation of Self-assembly monolayer modification
3-3-1 Gold electrode cleaning methods
A clean gold surface provides a better interaction with thiol molecules during SAM process, which is also beneficial to electrochemical detection and impedance spectroscopy.[34, 35] Previous study had shown technique for cleaning gold electrodes. The gold electrodes were cleaned sequentially by following processes:
I. The bare gold chip was first cleaned by immersing chip in isopropanol and ethanol for 10 min each within 2 min sonication.
II. After rinsed with DI water, sample was immersed in a solution of 50mM KOH with 25% H2O2 for 10 min.
III. After the treatment described above, sample was in 50mM KOH
and connected to a potentiostat. The electrode potential was swept from -200 mV to -2000 mV (vs. Ag/AgCl) at 20 mV/s scan rate, and then rinsed in DI water again.
IV. Blow dry with N2.
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3-3-2 Preparation of mixed SAMs on gold electrode
The alkanethiol layer was modified onto the gold electrode by the self-assembly techniques. A SAM as linker molecule, 11-Mercaptoundecanoic acid(MUA), on the gold surface was modified to connect the proteins or other biomolecule via covalent bond.[36] In previous attempts, however, using a thiol mixture with a 4:1molar ratio of 3-mercaptopropionic acid(3-MOPHA) as a spacer to 11- mercaptoundecanoic acid(11-MUA) immobilized proteins for antibody detection exhibits a better performance than homogeneous SAM since the mixed SAM s reduces the steric hindrance caused by the carboxylic-terminated groups of SAMs.[37] Therefore, we designed a kind of mixed SAM, including hydroxyl- and carboxylic-terminated thiol for covalent bond with anti-Vibrio Parahaemolyticus antibody. The antibodies of bacteria were then exposed on the thiol-functionalized gold electrode. As a result, the biocompatible layer formed on gold surface for Vibrio Parahaemolyticus capturing.
Figure 3-4 shows a cartoon flowcart of the surface modification. The processes:
I. The cleaning gold electrode was immersed 8 mM 3-MPOH and 2 mM 11-MUA mixing thiol at absolute ethanol for 12 hours at room temperature II. Gold electrode were washed by sonication in ethanol, followed several times
by ethanol and DI water
III. The 50 mM EDAC and 30 mM NHS in 1xPBS for 30 min was attached the NHS group to –COOH terminated thiol on the gold electrode at room temperature and then the chip was washed by 1xPBS and DI water.
IV. Vibrio Parahaemolyticus antibody with a concentration of 100 μg/mL in 1xPBS injected into the channel through fluidic flow and incubated for 6 hours at 4℃
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V. The PBS and the DI water rinsed the chip
VI. After microfluidic channel made up, BSA with 1mg/mL for blocking was incubated in the chamber for 1hour and washed with 1xPBS and 0.01xPB.
Figure 3-4 Processes of modification: the clean gold electrode was modified by 11-MUA/3-MPOH mixed solution to assemble the carboxylate-thiol layer. EDC/NHS activated the carboxylate group for antibodies binding. After immobilizing the antibodies, the BSA was followed to block remain area of nonbinding.
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3-4 Surface molecular identification on Gold electrode
3-4-1 Electrochemical method for 11-MUA and 3-MPOH identification
Cyclic voltammetry is an electrochemical technique that probes the reduction and oxidation of the redox active species in the solution with the electrode. The stability of structural SAM on the gold electrode can be analyzed by CV method.[35]
Three electrode cells were comprised. Gold electrode with patterns on the SiO2 substrate was as a working electorde. A Pt wire and Ag/AgCl metal in 3 M KCL was also prepared as counter electrode and a reference electrode. During the CV scan, shape of I-E curve between the barely clean gold electrode and SAM–coveraged gold electrode would get a strong difference. The gold electrodes with SAM blocking the redox currents influences the electrons transfer in CV scan. The investigation gave the identification of the MUA/MH coating on the gold electrode. CV measurement was achieved by a CH Instruments Model 600B potentiostatic (CH Instruments, Austin,TX) with parameters:
1. Electrolyte and redox active species: 1xPBS(pH7.6) with 30 mM potassium ferrocyanide(K3Fe(CN)6)
2. Scan range: -0.4 V ~ 0.8 V 3. Scan rate: 100 mV/s
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3-4-2 Fluorescence labeling
Antibody of Vibrio Parahaemolyticus at concentration of 100 ug/mL was applied to NHS group on surface of electrode. Carboxytetramethyl-rhodamine succinimidyl ester (5(6)-TAMRA,SE) were diluted to 1 mg/mL in 1xPBS. The solution within rhodamine put on antibody-modified electrode to carry out the binding reaction.[38]
The reaction took 4 hr. PBS with 0.5% tween 20 and D.I water rinse heavily on the chip, followed by dry N2. Images of labeled antibody were obtained by fluorescent microscopy( Zeiss AX10). A filter of 515-560 nm was used for exciting the rhodamine. The binding method was show in Figure 3-5.
Figure 3-5 Process of sample preparation for fluorescence imaging. After binding antibodies on the electrode, the Rhodamine was labeled on antibodies for fluorescence detection.
3-4-3 TMB development
TMB solutions are chromogenic reagents for horseradish peroxidase(HRP) enzyme, wildly utilized in ELISA techniques normally. In the presence of HRP, the peroxide active the enzyme and thus convert TMB into a blue byproduct. The incubation for 6 hr with HRP conjugated second antibody on to the no-patterned gold electrode, which was immobilized antibody in previous. Figure 3-6 demonstrates the
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antibody modified on the thiol-modified electrode. After incubation of HRP-conjugated second antibody on the first-antibody modified electrode for 6 hr, the chip was washed with PBS/0.05% Tween-20 and D.I. water. Subsequently, the TMB substrate was dropped on the chip for a while and the measurable color-changed solution was then taken to read absorbance at 450nm wavelength. Here, the negative comparison was experimented by taking the absorbance change at 450nm for the
electrode without antibody modification.
Figure 3-6 Processes of TMP development: The antibody-modified gold electrode was followed by incubating the second antibody. Then the TMP substrate was dropped on the electrode and developed.
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3-5 Microfluidic channel and detection system set up
In recent years, the interest in the bacteria or cell detection using microfluidic system increase dramatically. Microfluidic channel was used for the stability and enhancement of biosensing. The integration of bacteria detection with microfluidic enables a faster detection. We use a simple microfluidic system to combine the chip and facilities for bacterial transport and detection.
3-5-1 Microfluidic settlement
Following the modification of biomolecular on the gold electrode of chip, the chip was located by a Teflon O-ring and acrylic broad to make a microfludic chamber.
The advantage of being removable and reusable Teflon ring on the top of the chip gives a compliance of experimental channel. Teflon at 1mm thickness was taken to a well with a volume of 25 uL.Figure 3-7(a) illustrates schematics of chamber settlement. Teflon tube was then inject into the chamber as inlet and outlet for sample delivery. The chip after settlement placed at the PCB, shown in Figure 3-7(b). Bovine serum albumin (BSA) was injected into chamber for blocking before measuring 0.01xPhosphate buffer as baseline.
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Figure 3-7 Processes of microfuidic system set-up (a) fabrication of microfluidic channel . (b) completion of microfluidic chip.
3-5-2 Processes of detection in microfluidic channel
Due to the design of electrode configuration, the electrical connection for bacteria trapping and bacteria detection is different. In order to manipulate the bacteria to the inner circular of gold electrode, inner electrode was connected with functional generator reverse to outer ring of electrode, shown in Figure 3-8(a).
However, measuring bacteria immobilizing on the electrode was completed by linking either side of pad in inner electrode to impedance analyzer, shown in Figure 3-8(b).
Phosphate buffer diluted 100 times is as solution for whole detections. The solution did not react electrochemically with electrodes during trapping and detection.
In this bioassay, Peristaltic pump for sample delivery was connected to inlet and outlet port and make the flow through the channel at rate of uL per minute, shown in
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Figure 3-9. The all assays of detection were investigated under microscopy. Figure 3-10 also states the process of detection. A modified- antibody electrode without AC electrokinetics was also inspected for comparison. Figure 3-10 shows the pictures of detection system.
Figure 3-8 Connection for (a) bacteria trapping (b) bacteria detection by electrode.
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Figure 3-9 Schematics illustration of assay.
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Figure 3-10 Pictures of the whole system.
3-6 Bacteria preparation
The fresh Vibrio Parahaemolyticus and E coli XL1-Blue strain were taken from frozen stock and then cultured in the solution of Tryptic Soy Broth (TSB) with 3%
sodium chloride, and Luria Broth (LB) containing 20μg/ml tetracycline at 37℃for 6 hour, respectively, until the value of OD600 reach about of 0.97 (109cfu bacteria). The bacteria were collected and diluted 100 fold in phosphate buffer that do not affect the electrode surface after applying via oscillate voltage. The bacteria were then trapped though AC electrokinetics and the impedance were sequentially detected.
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3-7 AC electrokinetics manipulation for analyte trapping
After antibodies were immobilized onto microelectrode surface, the AC electrokinetics processed so that only antibody specific bacteria can be left on the inner surface effectively even the electric field unapplied. The article demonstrates the enhancement of the binding of anayltes with its antibody via AC electrokinetics. The mechanism assist to reduce binding time. Several researches of AC electrokinetics forced on bioparticles trapping onto center of circular electrode. The designed electrode using in this detection, however, has not been applied for the bacteria or other bioparticles trapping. Here we show the approachable AC eletroosomosis and electric field by COMSOL V.4.2 simulation on the electrodes to make sure the probability of bacteria trapping.[22] The ideal result for bacteria trapping is manipulating it onto inner surface of electrode, depicted in Figure 3-11. There is one size of inner electrodes with diameters 500 μm, and each contains the interdigited electrode with 5 μm width and 5 μm gap. The diameter of outer electrode is 1050 with width 50 um.
Figure 3-11 Bacteria trapping via AC electrokinetics enhancement.
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3-8 Impedance measurement
Each detection method used Agilent 4294A impedance analyzer with two-end measurement. The data were collected via Labview programming. A set of inner circular electrodes was actuated with AC electrokinetics and another set of electrode was without AC electrokinetics as control part. The sensor detects the changes of dielectric properties of electrode surface up to analytes binding. Bactria were dragged in fluidic flow channel at various dilution (105~107cfu/mL) and mixed (Vibrio
Parahaemolyticus+XL1 Blue E coli) solution. The baseline was established by the
flow flushed thought the antibody-functionalized electrode.3-8-1 Parameters for impedance measurement
For frequency sweep measurement, the impedance was measured from 50 Hz to 500 kHz with oscillate voltage 100mV. Diluted Phosphate buffer with 100 times as a solution for measurement without and with bacteria. The fixed volume of solution was used to steady while measurement.
3-8-2 Normalized impedance change as impedance analysis
Using antibody-modified electrode to binding the bacteria without AC electrokinetics enhancement was used as control. To observe the effect of Bacteria binding, the magnitude of impedance was compared for bacteria binding in the presence of AC electrokinetics to antibody modified on electrode. Introducing a Normalized impedance change (NIC) as a function of frequency range from 50 Hz to 500 kHz and drawn based on the difference of magnitude of impedance with respect to the baseline. The value of NIC was given by following formula:
where is the magnitude of impedance for antibody-modified sample and
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is the magnitude of impedance for a sample containing bacteria, Vibrio
Parahaemolyticus and XL1 Blue E coli.
3-8-3 Equivalent circuit
In impedance analyzer as biosensor, total impedance of medium cannot directly be calculated from input voltage and current. Another alternative method, an equivalent circuit, can fit the experimental data. The information of equivalent circuit characterizes physical response of electrical parameters owing to the impedance change.
The electrical parameters used in the experiments were designed and fitted including bulk medium resistance (Rsol), double layer capacitance (CPEdl), and medium dielectric capacitance (CPEsol) between the surface of inner interdigitated electrodes , shown in Figure 3-12(a) and (b). When electrodes immerse in solution, it is consider that the bulk medium and electrical double layer in series. Bulk medium with ion species is as a resistance and the polarized interface of electrodes as capacitance. The dielectric capacitor explains dielectric properties of solution surrounding the electrodes. Instead of ideal capacitors, these capacitors mentioned above are described as constant phase element (CPE):[5]
T and P depend on the environment of medium and interface of electrodes. If p is 1, the CPE is an ideal capacitor. In reverse, p close to 0 means a simple resist emerging.
if the equivalent circuit is simulated, the detection plot as a function of frequency can be explained in three distinguishing range(Figure 3-12(c)).[39] The CPE double layer dominates at lower frequency since the higher impedance of interface capacitor. At intermediate frequency, the CPE interface and CPE solution obtain a balance that
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causes bulk medium resistance influences the total impedance changes. At higher frequency, the electrical double layer vanishes and impedance of the dielectric capacitor still diminishing, so the dielectric capacitor is become important.[40]
Figure 3-12 (a) Equivalent electrical circuit (b) Equivalent electrical circuit on the chip (c) Impedance spectra divided into dominant element.