Novel Microfluidic Biosensor for Rapid and Specific ... Novel Microfluidic Biosensor for Rapid and...

1

Novel Microfluidic Biosensor for Rapid and Specific Methicillin-Resistant

Staphylococcus aureus Detection

Amanda Bukhtia1, Kaitlyn Tingley1, Mansoor Nasir2, Yawen Li2, Therese Bou-Akl3, Gregory Auner3

Lawrence Technological University

Abstract

Methicillin-Resistant Staphylococcus aureus (MRSA) is an antibiotic resistant staph

infection that impacts many individuals in the United States. At the moment, there is no

revolutionary diagnostic tool for MRSA; either turnaround time is large or the procedure is

expensive and lacks specificity. Therefore, the goal of this project was to design and develop a

quicker cost effective diagnostic tool for MRSA screenings. To develop a quick and cost

effective method to detect if a patient has (MRSA) is the need statement for this project. When

researching different diagnostic tools, it was determined that micro-electromechanical systems

(MEMS) would be the best method for MRSA diagnosis due to the quick turnaround times and

inexpensive test costs. The MEMS device we developed uses changes in impedance to determine

if MRSA is present. This process works by immobilizing antibodies on the surfaces of gold

electrodes which have a high specificity towards MRSA. Everything that is not MRSA will not

bind to the antibodies and will flow out of the MEMS device. When MRSA does bind to the

antibodies a change in impedance occurs which we can measure with an impedance analyzer.

The device was designed and fabricated through two different prototypes. The impedance

mechanism of the device has been validated. Last, the specificity mechanism of the device has

been verified.

2

Background

MRSA is a bacterial staph infection that is passed through skin contact. This bacteria has

built up a resistance to antibiotics which makes it difficult to kill. If the bacteria is left untreated

it can enter the bloodstream from a cut. At that point, it can infect the heart and liver which will

ultimately lead to death (6). This is the main concern with MRSA in hospitals because many

patients have open wounds and are more susceptible to catching infections. The most common

way to test for MRSA takes 48-72 hours and in that time a patient can spread the disease to other

patients or even health care professionals (6). Over the summer, a team met with Maureen Raab,

Susan Allen, MD and Rocco Ottolino at the elderly care facility in Beaumont Hospital. One of

the ideas that was discussed was a dip stick device for MRSA testing. They suggested that the

development of an easier and quicker test would greatly improve their facility.

The unmet need that we pursued was to develop a quick and cost effective method to

detect if a patient has MRSA. There are many stakeholders in our project. Hospitals and nursing

homes already test for MRSA and would buy our device even if it costs more; as long as it

functioned better. It could be used in school systems after an outbreak occurs like the recent

outbreak at Novi high school. It could determine which students are infected in order to stop the

spread of the bacteria. Some hospitals are even looking into screening all of their incoming

patients and VA hospitals already screen all of their patients. In 2008 the VA treated 773,000

patients and if our device was set at the price on the standard test then there would be a market

value of 30.94 million; just in VA hospitals alone (9).

Current Diagnostics

There are currently only two diagnostic methods that are used to detect MRSA clinically;

Chart 1 compares these methods. One method is plate based culture screenings where the

3

samples are placed in a customized dish of nutrients that encourage bacteria growth in order for

the sample to grow large enough for analysis. This test is easy to conduct and does not cost much

but the detection time takes around 48 hours; at times it goes up to 72 hours (7). A newer

method utilizing a polymerase chain reaction (PCR) that only takes a few hours for detection

results is now being used in some clinics (7). The downside to this technique is the high cost of

the equipment required to perform the PCR as well as its tendency for false-positive results.

Recent advances in micro-electromechanical systems (MEMS) have allowed the fabrication of

what is referred to as lab-on-chip devices to be used in the detection of infections. Currently all

these devices are in the research development stage and can be characterized into four different

groups; magnetic, mechanical, electrochemical and optical. In general, when compared to the

clinically used methods, MEMS devices are cost efficient, have high sensitivity, and have a one

hour or less detection time (2).

Chart 1: Current MRSA Diagnostic Methods

Specificity

In order to understand specificity to MRSA, it is crucial to first understand the distinctive

biology that MRSA cells possess. The presence of MRSA can be isolated using a highly specific

binding interaction. Research shows that a protein known as PBP-2a controls the mecA gene and

cell wall of MRSA; this is precisely what differentiates MRSA from all other staph bacteria (3).

Because of this specificity, anti PBP-2a proteins could be used to bind to the cell wall of MRSA

4

cells, shown in Image 1. Antibodies work well in this application because they only accept the

antigen or protein that fits into the antigen-binding site. When an antigen reaches an antibody

that does not have a matching antigen-binding site no polypeptide bond will form. This is the

principle that was applied to our project (5).

Image 1: Anti-PBP2a binding interactions

Methods

There are specific parameters and stages that are needed to consider when diagnosing

MRSA. Before one can detect if a patient has the infection they need to separate the MRSA

bacteria from every other type of cell. The reason concentration is a factor is because we need to

consider how it will impact sensitivity. When you pull a swab from either the nares or blood

there is only a set amount of MRSA bacteria in that sample. This is why amplification is needed

in clinically used methods. The last consideration needed is how to detect the actual MRSA cells.

5

This can be related to concentration because with detection you either need a large amount of

MRSA cells or you need to have a device that is very specific in order to detect a smaller amount

of cells (4). Our goal for the project was to focus on the detection mechanism of this diagnostic

process.

Device Design

This device from the bottom-up contains a glass base with two electrodes on its top

surface and is shown in Image 2. The electrodes will have antibodies present on their surface.

PDMS is also used and comprises of a flow channel that is bonded directly to the glass surface

and will comprise of the electrodes in its channel. The device will contain an inlet followed by a

flow channel that flows over highly sensitive electrodes in which PBP-2a antibodies will be

contained on its surface. In order to get our sample into the device we will inject a sample into

the inlet with a needle and syringe. When the solution flows over the antibody covered

electrodes the MRSA bacteria will bind to them. Since this binding isn’t visible to the naked eye

we will then use an impedance measurement to detect the impedance change resulting from the

bound antibodies to the antigen. The gold electrodes are precisely what allow us to measure

impedance. As MRSA bacteria binds to the antibodies, an insulation layer will form onto the

electrodes. This will decrease the conductivity of the electrode resulting in an increased

impedance. The specific impedance values and differences will be determined during the future

work stages of our device. Our concept is novel in that no impedance measurement method

exists to detect MRSA. This concept is extremely cheap and detection time is almost immediate

since no sample amplification is needed.

6

Image 2: 3D image of the concept

Two different prototypes where designed and can be seen in Image 3 and Image 4.

Device A is a simple two lead design. The thinner region of the leads is where the antibodies will

be immobilized. Device B involves an interdigitated lead design. This design covers a larger

surface area for higher sensitivity impedance measurements. In other words, minimal changes in

conductivity will yield more obvious impedance changes. It is important to note that the left and

right side of design B are not connected. Wires where soldered to the end of each lead on both

designs. These wires were used to ultimately connect to the impedance analyzer. The blue

rectangles are the channel regions where the bacteria sample would flow across the electrodes.

The pink regions are areas that underwent passivation during fabrication. Passivation was

conducted in order to limit the amount of noise and external artifacts that would otherwise affect

the impedance signals. The full AutoCAD wafer design can be found in Appendix A.

7

Image 3: Device A design Image 4: Device B design

Fabrication

In order to create our device, we had to undergo three different protocols. First, the glass

wafer was fabricated to add gold (electrodes) and silicon oxide (passivation) in desired regions.

Next, the PDMS substrate was fabricated. Last, the PDMS substrate was bonded to the glass

devices. We fabricated our devices beginning with the electrode containing glass wafer, the

process can be found in Diagram 1. To begin this fabrication process, a glass wafer was

obtained. The wafer will underwent gold evaporation through thin-film deposition. The next step

involved a lithography process in order to define specific areas for the gold electrodes. The last

step utilized a wet etching technique to clear out the unwanted gold sections. The mask that is

used during the lithography process is a transparency that was printed using the orange layer in

our AutoCAD designs, Image 3 and Image 4. The detailed protocol can be found in Appendix B.

The detailed cross-sectional view process flow can be found in Appendix F.

After we fabricated the electrode containing glass wafer, the wafer underwent passivation

layer fabrication shown in Diagram 2. To begin this process, the wafer from protocol one was

8

obtained. The wafer underwent silicon oxide deposition. The next step involved a lithography

process in order to define specific areas for passivation. The last step utilized a wet etching

technique to clear out the unwanted passivated sections. The mask that is used during the

lithography process is a transparency that was printed using the pink layer in our AutoCAD

designs, Image 3 and Image 4. The regions that where passivated are gold regions that did not

have a flow across them or wires soldered onto them. The detailed cross-sectional view process

flow can be found in the appendix F. The actual resultant devices after electrode and passivation

fabrication are shown in Image 5 and Image 6.

Image 5: Actual device A fabricated glass Image 6: Actual device B fabricated glass

In part three, we created the PDMS substrate that would ultimately be bonded to the glass

slide; the process is shown in Diagram 3. We used a soft lithography protocol to create the

channel containing PDMS. The first step was to create a master mold which is resembled in the

first three steps on Diagram 3. This was accomplished by applying a thin layer of negative

photoresist and applying a lithography process in which unwanted photoresist is removed. After

this process was complete, we had a master mold which is when the PDMS solution was poured.

9

Finally, we cured the PDMS and peeled it off the master mold. This was when we were left with

just the blue layer. All our fabrication protocols were executed over at Wayne State’s class-1,000

nFab clean room with the exception of pouring, curing, and pealing our PDMS.

The final step involved bonding the PDMS substrate onto the glass devices. The two

pieces where bonded by oxygen plasma treating the PDMS and glass devices and then

compressing the two pieces together to make them adhere. The devices were then heated for 10

minutes onto a hot plate at 100 degrees Celsius to solidify bonding. Device A proved to have

better adhesion during this process. This may be due to the fact that more of the PDMS substrate

could be left on the device; this helped to facilitate bonding. The reason more of the substrate

had to be removed from device B is we needed more room in which to solder the wires onto the

electrode pads.

Diagram 1: Electrode fabrication cross-sectional process flow

10

Diagram 2: Passivation layer fabrication cross-sectional process flow

Diagram 3: PDMS channel fabrication process

Impedance Testing

Impedance testing was conducted onto our devices by using a PBS solution. The solution

was flowed through the device via the inlet at a constant .5 ml/min flow rate. A syringe pump

was used to accomplish the flow rate consistency, Image 6. Separate syringes, syringe needles,

and tubing were used for each PBS concentration. An impedance analyzer was connected to the

11

device. In order to do this, two wires were soldered onto each device, one on each lead, Image 7.

These wires were then connected to the impedance analyzer. A frequency sweep from 0 to

100,000 Hz was used to collect the data.

Image 6: Impedance testing set-up and equipment

Image 7: Device A final

12

Our impedance testing consisted of two parts. For the first part, we flowed four different

concentrations of PBS through the device (1X, 2.5X. 5X, and 10X); one concentration at a time.

Each concentration experiment was repeated three times. The averages between the trials where

calculated and a frequency vs. impedance graph was plotted for each concentration using excel.

Since PBS is a salt water solution, as we flow more ions across the two electrdes we would

essentialy be shorting them. Also, when there is no flow and the device is static, we have an open

circuit, hence an infinite impedance. Due to this, we hypothesiszed that as higher concentrations

of PBS are intrduced (increasing the number of ions), a decrease in impedance will result.

In part two, we used a solution of 1X PBS solution comprised of steel particles. A

magnetic platform was added while the device was placed directly on top of it. As the solution

was flowed through the device via the inlet, the steel particles would be captivated onto the

electrodes due to magnetism. As more and more particles are captivated we expected to see a

decrease in impedance due to the similar concept explained in part one. Our data was ploted

using frequency vs. impedance and the 1X solution was used as our control. The detailed

protocol can be found in Appendix B. The Impedance data can be found in Appendix C.

Antibody Binding Testing

For this project, we tested with MRSA which is a bio level 2 hazard. Due to its infectious

nature we received training from Wayne State University and worked with a trained professional

at all times. We also tested with methicillin-susceptible Staphylococcus aureus (MSSA) as a

control. Our goal was to show that our device can detect MRSA even if other strains of staph are

present. MRSA will react to the Anti PBP-2a because of the Penicillin-Binding Protein 2 (PBP2)

in the cell walls of the MRSA (4). The detailed protocol can be found in Appendix B. For this

experiment we needed pipets, centrifuge tubes, glass slides, a fluorescence microscope, MRSA,

13

MSSA, PBS, PBS BSA 3% and an alexa fluor. The Alexa fluor was important because it was

used to detect binding. The alexa flour binds to our antibody. When the antibody binds with the

MRSA the fluor with fluoresce under a fluorescent microscope. This is what we were able to

detect.

To begin the experiment created three antibody solutions with concentrations at 1:1000,

1:2000 and 1:5000. This was done to show what concentration of antibodies we would need in

order to achieve maximum binding. We pipeted a solution of bacteria, of antibody, PBS and

alexa flours into centrifuge tubes one tube for each individual concentration. We had one tube of

just bacteria and alexa flours to serve as a control to show that the alexa was not binding to the

bacteria creating a false positive.

Antibody Immobilization

In order to get our antibodies onto our device, we need to immobilize them onto the

electrodes. In order to achieve this, we used EDC ((1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride)) which is a chemical that binds to a

carboxylate on NHS (N-hydroxysulfosuccinimide) and an amine on our antibodies. To test our

immobiliztion process, we treated our devices with EDC and NHS, then applied the antibodies,

refer to Diagram 4. We wanted to look at our devices under an x-ray photoelectron microscope

but could not get acces to one. Instead, we were able to image one device using an atomic force

microscope. The detailed protocol can be found in Appendix B.

14

Diagram 4: Surface chemistry for antibody immobilization

Results

Impedance Testing

Part one of the impedance testing yeilded the expected results. Graph 1 proves that as

PBS concentration is increased, the impedance is decreased. This data was then used to create a

second plot. Three random frequencies greater than 20 Hz were plotted onto a concentration vs.

impedance graph shown in Graph 2. This was graphed to analyze the correlation between

concentration and impedance of our device. It is shown that our impedance is inversally

hyperbolic to ion concentration.

15

Graph 1: Frequency vs. Impedance graph for different concentrations

Graph 2: Concentration vs. Impedance graph for three different frequencies

16

Part 2 was used to compare our control (1X PBS solution) to the same solution comprised

of steel particles. Graph 3 displays our results. Similar to part one, the impedance is decreased

when the steel particals are present due to the increased ions that are introduced. In future work,

our device will be tested with MRSA and the antibodies. We will expect to see an opposite

correlation. For example, as more MRSA binds to the antibodies, an insulation layer will form on

the electrodes. This will increase impedance rather than decrease it; the hyperbolic correlation is

still expected.

Graph 3: Effects on frequency vs. impedance of a steel particle PBS solution

Antibody Binding Testing

Our results showed that MRSA did bind to the PBP2-a antibody which are the pictures

shown below in Image 8. MSSA is not shown in the images because nothing lit up when viewing

the solutions under the microscope. We also saw we had the most binding at a 1:2000 dilution

17

but we saw binding at all concentrations. We were able to show MSSA would not bind to the

MRSA this proved the specificity of our antibody. Also there are different PBP-2a antibodies

sold from a verity of suppliers. We were able to prove that MRSA binds to this specific PBP-2a

antibody we bought from myboisource.com.

Image 8: Antibody binding to MRSA under flourescent micrscopy

Antibody Immobilization

We wanted to look at our devices under an atomic force microscope in order to see if we

were able to bind our antibodies to our gold. The picture shown below, in Image 9, is the only

result we were able to get from the AFM. We can see there is something on the gold’s surface

but we cannot be sure if it is a drop of EDC/NHS or a group of antibodies clumped

together. We would need to complete more testing to confirm immobilization. Once we have

verified it, we could optimize our immobilization protocols.

18

Image 9: AFM immobilization results

Project Management

Timeline

Our executed project is shown in Timeline 1. The team received OSHA training in

January. We then started the design phase in February. It was decided to design and fabricate two

different prototypes in order to avoid future device modifications. Wafer fabrication was

completed in March as well as the antibody binding testing. We fabricated our PDMS substrate

early in April and were able to complete some device testing after.

Timeline 1: Project execution timeline

19

Task Division

Our team, MRSA Diagnostics, consists of two team members. Due to this small quantity,

we worked on most of the tasks together, Diagram 5. We both completed the OSHA training and

we collaborated on our device design. Even though we worked together on most aspects of the

project, each one of us is an expert on a certain section. Ms. Bukhtia oversaw fabrication and

impedance testing while Ms. Tingley oversaw antibody immobilization and antibody binding

testing. Our team communicated via text, emails and our shared calendar.

Diagram 5: Team task division

Expenses

Our project budget can be found in Budget 1. All of our expenses were paid through a

grant provided by the LEGENDS Entrepreneurial Student Awards (LESA) foundation. Our total

was just over 1,300 dollars.

20

Budget 1: Project budget

Discussion

Overall this project will greatly improve the health care industry. MRSA is a growing

problem in many hospitals. Quicker diagnostic times will help health care providers determine

who has MRSA and treat them appropriately in order to stop them from spreading the disease.

The low cost of a MRSA device would allow many people access to our device. This is the

impact our device will have once it is complete. The impedance mechanism of the device has

been validated, the antibody specificity has been proven, and antibodies have been immobilized

onto the device.

Future Work

The next steps for this project include validation of the antibody immobilization onto the

device. Also, flow through testing conditions can be determined using Comsol modeling. Last,

impedance testing using a MRSA containing solution can be conducted. Future outlook consists

of integrating an analysis mechanism onto the device itself and creating packaging for the

device.

22

Appendix

A. Design

AutoCAD Design

B. Protocols

Electrode Fabrication

1. Rinse and Dry (Cleans the wafer)

-Place wafers on rack and then into the VERTEQ Superclean 1600 for one cycle

23

2. Polymer Coat (Promotes better adhesion for step 3)

- Place rack into the MicroMaster (150 C for 25 minutes)

3. Photo Resist (Applies a coating of photo resist onto the wafers top surface)

-Place a dummy wafer onto the center platform of the spinner for a test run

-Place an actual wafer onto the center platform of the spinner (Gold side up)

-Pour 1811 positive photo resist onto the center of the wafer two cover its surface by 2/3

-Immediately activate the spinner (4,000 rpm for 30 seconds)

4. Post Baking (Heats up the wafer for preparation of step 5)

-Place the wafer onto a hot plate (95 C for two minutes)

5. Lithography (Patterns the wafer with the desired design)

-Obtain a 7” X 7” glass piece and firmly tape the transparency onto it

-Place the glass piece onto the platform and activate

24

-Place wafer onto the platform while using the alignment marks and activate

6. Wet Develop (Removes the unwanted photo resist)

-Place wafer in a glass pan full of (for 40 seconds)

-Rinse and Dry

7. Wet Etch (Removes the unwanted gold)

-Place wafer in a glass pan full of (solution name and quantity for 15 minutes)

-Rinse and dry

-Place wafer in bath full of (for 10 seconds)

-Rinse and dry

25

Impedance Testing

1. Clean device well with isopropyl and acetone.

2. Cut the PDMS channels

3. Punch a hole in the inlet and the outlet in the PDMS.

4. Oxygen plasma treat both the slides and the PDMS channels. Do not bond them together

yet.

5. After the treatment is done, carefully bond the PDMS channels (feature side down) to the

device.

6. Put devices on a hot plate set at 100 °C for ten minutes.

7. While that is happening, cut the tubing for the device.

8. After devices are cool, put on tube into the inlet and one into the outlet.

9. Obtain two wires for each device. Solder one wire to each electrode. Use glue to help

adhesion if necessary.

10. Obtain 1x, 10x PBS, syringe, two needles, impedance analyzer and a syringe pump.

11. Sandpaper steel wool into a cup to obtain particles.

12. Suspend steel particles in 1x PBS

13. Dilute 10x PBS with water to obtain 5x PBS and 2.5x PBS. Add 100 ml of water and 100

ml of 10x PBS to obtain 5x PBS. Add 50 ml of water and 50 ml of 5x PBS into a separate

container to make 2.5x PBS.

14. Hook up your device to the syringe pump and the impedance analyzer.

15. Take a set of data without flowing anything through the device.

16. Start the pump at 5ml/min for 1x PBS. Take another set of data once you see flow

through the device.

26

17. Repeat steps 15-17 for 2.5x, 5x and10x PBS.

18. For testing the steel place a magnet under the device before flowing the solution through.

Then follow steps 15-17.

Drop Test

1. Culture MRSA bacteria

2. Pipet 200 microliters of BHA and PBS 3% into four 1.5ml centrifuge tube

3. Add 100 microliters of MRSA into BHA/PBS tubes

4. Spin tubes for 5 minutes in centrifuge. Remove BHA and PBS after. Add PBS back into

one tube.

5. Create 1:200 work stock of PBP2-A (1:2000 and 1:5000) from 1:1000 stock solution.

Add 100 microliters of 1:1000 and 100 microliters of PBS into one 1.5 ml tube (1:200).

Add 100 microliters of stock solution into a tube with 500 microliters of PBS (1:500).

Add 200 microliters of stock solution into a tube.

6. Incubate tubes for 45 minutes.

7. Centrifuge and wash tubes with PBS three times.

8. Make alexa flour dilution. Add 100 microliters with 100 microliters of PBS.

9. Add 100 microliters of alexa flour solution into tubes.

10. Incubate for 45 minutes

11. Wash tubes.

12. View/qualitatively the result using a fluorescent microscope

27

1 2 3

A No Antibody No Antibody No Antibody

B Low 1:5000 Low 1:5000 Low 1:5000

C Medium 1:2000 Medium 1:2000 Medium 1:2000

D High 1:1000 High 1:1000 High 1:1000

Immobilization of PBP2a on gold coated surfaces

Proposed protocols

Option 1

1. prepare four gold coated cover slips (arrange cover slips on a glass slide and sputter coat

with gold for 4 min)

1. Stabilize the coated surfaces by immersion in PBS for 5 min

2. Prepare blocking buffer 3% BSA in PBS for blocking of nonspecific binding ( 0.3g of

BSA into 10ml of PBS)

3. have your antibody dilutions ready (1:1000, 1:2000 and 1:5000)

4. Prepare your EDC/NHS solution ( dissolve 0.4mg of EDC and 0.6mg of NHS in 1 ml of

PBS with pH between 6 and 7.1)

5. Cover the coated surfaces with 100ul of antibody solution ( all conditions without

the control)

6. Incubate the surfaces for ~ one hour at room temperature

7. wash the surfaces with PBS 3 times

8. cover the surfaces including the control with 100ul of blocking buffer (3%BSA/

PBS)

9. incubate for 15-30min

10. wash surfaces with PBS 3 times

11. Apply 100ul of EDC/NHS solution

28

12. incubate for 30 min


14. Save the cover slips in PBS at 4º C till the time for MRSA application

15. Now surfaces are ready to apply the MRSA

16. Apply 100ul of MRSA on the coated surfaces

17. Incubate for 45 min at room temperature


19. Apply 100ul of Alexafluor (1:200)

20. incubate for 45min

21. wash surfaces with PBS once

22. Keep surfaces covered with PBS

23. View under fluorescent microscope

Option 2









7. Combine 100ul of antibody with 100ul of EDC/NHS mixture ( do it for each

concentration of the antibody)

8. Cover the coated surfaces with 100ul of antibody/EDC/NHS solution

29

9. cover the control with 100ul of EDC/NHS only




PBS)













Option 3






30




6. Apply 100ul of EDC/NHS mixture to the coated surfaces ( do it for each


7. Incubate for 15 min

8. Remove EDC/NHS mixture

9. Cover with 100ul of antibody solution

10. cover the control with 100ul of PBS only




PBS)











31



Option four AFM

1. Prepare six devices




4. have your antibody dilution ready (1:1000)



6. Apply 100ul of EDC/NHS mixture to the coated surfaces ( do it for each


7. Incubate for 15 min

8. Remove EDC/NHS mixture

9. Cover with 100ul of antibody solution

10. cover the control with 100ul of PBS only




PBS)



16. Save the cover slips in PBS at 4º C

32

17. Look at device services using AFM

C. Impedance Data

Part 1 (Averages)

FREQUENCY(Hz)

Z[Ω] FREQUENCY(Hz)(1x)

Z[Ω] FREQUENCY(Hz)(2.5X) Z[Ω]

` 1.00E+01 1.01E+10 1.00E+01 2.57E+04 10 2391.408

5.10E+02 2.68E+08 5.10E+02 2.30E+04 509.95 1517.906

1.01E+03 1.40E+08 1.01E+03 2.13E+04 1009.9 1375.292

1.51E+03 9.32E+07 1.51E+03 2.03E+04 1509.9 1319.725

2.01E+03 6.96E+07 2.01E+03 1.97E+04 2009.8 1290.438

2.51E+03 5.58E+07 2.51E+03 1.92E+04 2509.8 1273.862

3.01E+03 4.65E+07 3.01E+03 1.87E+04 3009.7 1261.898

3.51E+03 3.98E+07 3.51E+03 1.82E+04 3509.7 1252.501

4.01E+03 3.48E+07 4.01E+03 1.78E+04 4009.6 1245.41

4.51E+03 3.08E+07 4.51E+03 1.73E+04 4509.6 1239.451

5.01E+03 2.77E+07 5.01E+03 1.70E+04 5009.5 1234.542

5.51E+03 2.52E+07 5.51E+03 1.68E+04 5509.5 1230.366

6.01E+03 2.30E+07 6.01E+03 1.66E+04 6009.4 1226.909

6.51E+03 2.12E+07 6.51E+03 1.65E+04 6509.4 1223.693

7.01E+03 1.97E+07 7.01E+03 1.61E+04 7009.3 1220.862

7.51E+03 1.84E+07 7.51E+03 1.61E+04 7509.3 1218.225

8.01E+03 1.72E+07 8.01E+03 1.60E+04 8009.2 1215.723

8.51E+03 1.62E+07 8.51E+03 1.58E+04 8509.2 1213.429

9.01E+03 1.52E+07 9.01E+03 1.57E+04 9009.1 1211.52

9.51E+03 1.44E+07 9.51E+03 1.56E+04 9509.1 1209.562

1.00E+04 1.37E+07 1.00E+04 1.56E+04 10009 1206.388

1.05E+04 1.30E+07 1.05E+04 1.54E+04 10509 1203.356

1.10E+04 1.24E+07 1.10E+04 1.54E+04 11009 1200.497

1.15E+04 1.19E+07 1.15E+04 1.53E+04 11509 1197.884

1.20E+04 1.14E+07 1.20E+04 1.51E+04 12009 1195.54

1.25E+04 1.09E+07 1.25E+04 1.50E+04 12509 1193.334

1.30E+04 1.05E+07 1.30E+04 1.48E+04 13009 1191.277

1.35E+04 1.01E+07 1.35E+04 1.49E+04 13509 1189.064

1.40E+04 9.72E+06 1.40E+04 1.47E+04 14009 1187.135

1.45E+04 9.38E+06 1.45E+04 1.44E+04 14509 1185.361

1.50E+04 9.05E+06 1.50E+04 1.42E+04 15009 1183.774

1.55E+04 8.75E+06 1.55E+04 1.40E+04 15508 1182.252

1.60E+04 8.47E+06 1.60E+04 1.37E+04 16008 1180.823

1.65E+04 8.21E+06 1.65E+04 1.37E+04 16508 1179.495

1.70E+04 7.97E+06 1.70E+04 1.37E+04 17008 1178.056

1.75E+04 7.74E+06 1.75E+04 1.37E+04 17508 1176.175

33

1.80E+04 7.53E+06 1.80E+04 1.37E+04 18008 1174.691

1.85E+04 7.32E+06 1.85E+04 1.37E+04 18508 1173.326

1.90E+04 7.12E+06 1.90E+04 1.37E+04 19008 1172.054

1.95E+04 6.94E+06 1.95E+04 1.37E+04 19508 1170.874

2.00E+04 6.76E+06 2.00E+04 1.36E+04 20008 1169.763

2.05E+04 6.59E+06 2.05E+04 1.36E+04 20508 1168.697

2.10E+04 6.43E+06 2.10E+04 1.36E+04 21008 1167.652

2.15E+04 6.28E+06 2.15E+04 1.36E+04 21508 1166.695

2.20E+04 6.14E+06 2.20E+04 1.37E+04 22008 1165.797

2.25E+04 6.00E+06 2.25E+04 1.36E+04 22508 1164.934

2.30E+04 5.87E+06 2.30E+04 1.36E+04 23008 1164.115

2.35E+04 5.74E+06 2.35E+04 1.36E+04 23508 1163.294

2.40E+04 5.62E+06 2.40E+04 1.36E+04 24008 1162.487

2.45E+04 5.50E+06 2.45E+04 1.37E+04 24508 1161.724

2.50E+04 5.39E+06 2.50E+04 1.36E+04 25008 1161.013

2.55E+04 5.29E+06 2.55E+04 1.36E+04 25507 1160.315

2.60E+04 5.18E+06 2.60E+04 1.36E+04 26007 1159.608

2.65E+04 5.08E+06 2.65E+04 1.36E+04 26507 1158.911

2.70E+04 4.98E+06 2.70E+04 1.36E+04 27007 1158.245

2.75E+04 4.89E+06 2.75E+04 1.36E+04 27507 1157.584

2.80E+04 4.80E+06 2.80E+04 1.36E+04 28007 1156.944

2.85E+04 4.72E+06 2.85E+04 1.36E+04 28507 1156.338

2.90E+04 4.64E+06 2.90E+04 1.36E+04 29007 1155.698

2.95E+04 4.56E+06 2.95E+04 1.36E+04 29507 1155.077

3.00E+04 4.48E+06 3.00E+04 1.37E+04 30007 1154.32

3.05E+04 4.40E+06 3.05E+04 1.37E+04 30507 1153.534

3.10E+04 4.33E+06 3.10E+04 1.36E+04 31007 1152.775

3.15E+04 4.26E+06 3.15E+04 1.37E+04 31507 1151.994

3.20E+04 4.20E+06 3.20E+04 1.37E+04 32007 1151.213

3.25E+04 4.13E+06 3.25E+04 1.37E+04 32507 1150.483

3.30E+04 4.07E+06 3.30E+04 1.37E+04 33007 1149.771

3.35E+04 4.01E+06 3.35E+04 1.36E+04 33507 1149.095

3.40E+04 3.95E+06 3.40E+04 1.36E+04 34007 1148.43

3.45E+04 3.89E+06 3.45E+04 1.35E+04 34507 1147.765

3.50E+04 3.83E+06 3.50E+04 1.36E+04 35007 1147.127

3.55E+04 3.77E+06 3.55E+04 1.36E+04 35506 1146.457

3.60E+04 3.72E+06 3.60E+04 1.36E+04 36006 1145.507

3.65E+04 3.67E+06 3.65E+04 1.36E+04 36506 1144.706

3.70E+04 3.62E+06 3.70E+04 1.35E+04 37006 1143.935

3.75E+04 3.57E+06 3.75E+04 1.36E+04 37506 1143.425

3.80E+04 3.52E+06 3.80E+04 1.36E+04 38006 1142.775

3.85E+04 3.48E+06 3.85E+04 1.36E+04 38506 1142.159

3.90E+04 3.43E+06 3.90E+04 1.35E+04 39006 1141.578

34

3.95E+04 3.39E+06 3.95E+04 1.35E+04 39506 1141.015

4.00E+04 3.35E+06 4.00E+04 1.35E+04 40006 1140.48

4.05E+04 3.30E+06 4.05E+04 1.34E+04 40506 1139.964

4.10E+04 3.26E+06 4.10E+04 1.34E+04 41006 1139.467

4.15E+04 3.22E+06 4.15E+04 1.35E+04 41506 1138.978

4.20E+04 3.18E+06 4.20E+04 1.34E+04 42006 1138.527

4.25E+04 3.14E+06 4.25E+04 1.34E+04 42506 1138.102

4.30E+04 3.11E+06 4.30E+04 1.33E+04 43006 1137.651

4.35E+04 3.07E+06 4.35E+04 1.34E+04 43506 1137.201

4.40E+04 3.04E+06 4.40E+04 1.34E+04 44006 1135.711

4.45E+04 3.00E+06 4.45E+04 1.34E+04 44506 1135.035

4.50E+04 2.97E+06 4.50E+04 1.34E+04 45006 1134.483

4.55E+04 2.94E+06 4.55E+04 1.34E+04 45505 1134.178

4.60E+04 2.90E+06 4.60E+04 1.33E+04 46005 1133.658

4.65E+04 2.87E+06 4.65E+04 1.32E+04 46505 1133.179

4.70E+04 2.84E+06 4.70E+04 1.32E+04 47005 1132.477

4.75E+04 2.81E+06 4.75E+04 1.33E+04 47505 1132.011

4.80E+04 2.78E+06 4.80E+04 1.33E+04 48005 1131.534

4.85E+04 2.75E+06 4.85E+04 1.33E+04 48505 1130.976

4.90E+04 2.72E+06 4.90E+04 1.33E+04 49005 1130.564

4.95E+04 2.70E+06 4.95E+04 1.33E+04 49505 1130.195

5.00E+04 2.67E+06 5.00E+04 1.33E+04 50005 1129.828

5.05E+04 2.64E+06 5.05E+04 1.33E+04 50505 1129.474

5.10E+04 2.62E+06 5.10E+04 1.32E+04 51005 1129.122

5.15E+04 2.59E+06 5.15E+04 1.32E+04 51505 1128.697

5.20E+04 2.57E+06 5.20E+04 1.32E+04 52005 1126.957

5.25E+04 2.54E+06 5.25E+04 1.32E+04 52505 1117.221

5.30E+04 2.52E+06 5.30E+04 1.32E+04 53005 1116.902

5.35E+04 2.49E+06 5.35E+04 1.32E+04 53505 1116.603

5.40E+04 2.47E+06 5.40E+04 1.31E+04 54005 1116.325

5.45E+04 2.45E+06 5.45E+04 1.31E+04 54505 1116.079

5.50E+04 2.42E+06 5.50E+04 1.31E+04 55005 1115.832

5.55E+04 2.40E+06 5.55E+04 1.31E+04 55504 1115.593

5.60E+04 2.38E+06 5.60E+04 1.31E+04 56004 1115.346

5.65E+04 2.36E+06 5.65E+04 1.31E+04 56504 1115.162

5.70E+04 2.34E+06 5.70E+04 1.30E+04 57004 1114.917

5.75E+04 2.32E+06 5.75E+04 1.30E+04 57504 1114.712

5.80E+04 2.30E+06 5.80E+04 1.30E+04 58004 1114.505

5.85E+04 2.28E+06 5.85E+04 1.30E+04 58504 1114.292

5.90E+04 2.26E+06 5.90E+04 1.29E+04 59004 1114.082

5.95E+04 2.24E+06 5.95E+04 1.29E+04 59504 1113.877

6.00E+04 2.22E+06 6.00E+04 1.28E+04 60004 1113.71

6.05E+04 2.20E+06 6.05E+04 1.27E+04 60504 1113.526

35

6.10E+04 2.18E+06 6.10E+04 1.28E+04 61004 1111.62

6.15E+04 2.16E+06 6.15E+04 1.28E+04 61504 1107.316

6.20E+04 2.15E+06 6.20E+04 1.28E+04 62004 1106.524

6.25E+04 2.13E+06 6.25E+04 1.27E+04 62504 1106.053

6.30E+04 2.11E+06 6.30E+04 1.27E+04 63004 1105.547

6.35E+04 2.09E+06 6.35E+04 1.27E+04 63504 1105.055

6.40E+04 2.08E+06 6.40E+04 1.27E+04 64004 1104.633

6.45E+04 2.06E+06 6.45E+04 1.26E+04 64504 1104.234

6.50E+04 2.05E+06 6.50E+04 1.26E+04 65004 1103.914

6.55E+04 2.03E+06 6.55E+04 1.26E+04 65503 1103.627

6.60E+04 2.01E+06 6.60E+04 1.26E+04 66003 1103.312

6.65E+04 2.00E+06 6.65E+04 1.25E+04 66503 1103.014

6.70E+04 1.98E+06 6.70E+04 1.25E+04 67003 1102.732

6.75E+04 1.97E+06 6.75E+04 1.24E+04 67503 1102.447

6.80E+04 1.95E+06 6.80E+04 1.24E+04 68003 1102.243

6.85E+04 1.94E+06 6.85E+04 1.24E+04 68503 1101.991

6.90E+04 1.93E+06 6.90E+04 1.23E+04 69003 1101.572

6.95E+04 1.91E+06 6.95E+04 1.21E+04 69503 1100.798

7.00E+04 1.90E+06 7.00E+04 1.21E+04 70003 1100.496

7.05E+04 1.88E+06 7.05E+04 1.20E+04 70503 1100.287

7.10E+04 1.87E+06 7.10E+04 1.20E+04 71003 1100.08

7.15E+04 1.86E+06 7.15E+04 1.20E+04 71503 1099.843

7.20E+04 1.84E+06 7.20E+04 1.19E+04 72003 1099.595

7.25E+04 1.83E+06 7.25E+04 1.18E+04 72503 1099.334

7.30E+04 1.82E+06 7.30E+04 1.18E+04 73003 1099.103

7.35E+04 1.80E+06 7.35E+04 1.19E+04 73503 1098.938

7.40E+04 1.79E+06 7.40E+04 1.18E+04 74003 1098.742

7.45E+04 1.78E+06 7.45E+04 1.18E+04 74503 1098.538

7.50E+04 1.77E+06 7.50E+04 1.18E+04 75003 1098.33

7.55E+04 1.76E+06 7.55E+04 1.17E+04 75502 1098.137

7.60E+04 1.75E+06 7.60E+04 1.17E+04 76002 1097.983

7.65E+04 1.74E+06 7.65E+04 1.17E+04 76502 1097.828

7.70E+04 1.72E+06 7.70E+04 1.17E+04 77002 1097.649

7.75E+04 1.71E+06 7.75E+04 1.16E+04 77502 1097.461

7.80E+04 1.70E+06 7.80E+04 1.16E+04 78002 1097.273

7.85E+04 1.69E+06 7.85E+04 1.16E+04 78502 1097.098

7.90E+04 1.68E+06 7.90E+04 1.15E+04 79002 1096.972

7.95E+04 1.67E+06 7.95E+04 1.26E+04 79502 1096.827

8.00E+04 1.66E+06 8.00E+04 1.25E+04 80002 1096.666

8.05E+04 1.65E+06 8.05E+04 1.25E+04 80502 1096.521

8.10E+04 1.64E+06 8.10E+04 1.25E+04 81002 1096.372

8.15E+04 1.63E+06 8.15E+04 1.25E+04 81502 1096.24

8.20E+04 1.62E+06 8.20E+04 1.25E+04 82002 1096.127

36

8.25E+04 1.61E+06 8.25E+04 1.25E+04 82502 1096.004

8.30E+04 1.60E+06 8.30E+04 1.25E+04 83002 1095.879

8.35E+04 1.59E+06 8.35E+04 1.25E+04 83502 1095.747

8.40E+04 1.58E+06 8.40E+04 1.25E+04 84002 1095.613

8.45E+04 1.57E+06 8.45E+04 1.24E+04 84502 1095.512

8.50E+04 1.56E+06 8.50E+04 1.24E+04 85002 1095.412

8.55E+04 1.55E+06 8.55E+04 1.24E+04 85501 1095.287

8.60E+04 1.54E+06 8.60E+04 1.24E+04 86001 1095.152

8.65E+04 1.53E+06 8.65E+04 1.24E+04 86501 1095.02

8.70E+04 1.52E+06 8.70E+04 1.23E+04 87001 1096.271

8.75E+04 1.51E+06 8.75E+04 1.23E+04 87501 1089.171

8.80E+04 1.51E+06 8.80E+04 1.23E+04 88001 1089.101

8.85E+04 1.50E+06 8.85E+04 1.23E+04 88501 1089.033

8.90E+04 1.49E+06 8.90E+04 1.23E+04 89001 1088.957

8.95E+04 1.48E+06 8.95E+04 1.23E+04 89501 1088.882

9.00E+04 1.47E+06 9.00E+04 1.22E+04 90001 1088.81

9.05E+04 1.46E+06 9.05E+04 1.22E+04 90501 1088.744

9.10E+04 1.45E+06 9.10E+04 1.22E+04 91001 1088.677

9.15E+04 1.45E+06 9.15E+04 1.22E+04 91501 1088.615

9.20E+04 1.44E+06 9.20E+04 1.22E+04 92001 1088.541

9.25E+04 1.43E+06 9.25E+04 1.21E+04 92501 1088.472

9.30E+04 1.42E+06 9.30E+04 1.21E+04 93001 1088.398

9.35E+04 1.42E+06 9.35E+04 1.21E+04 93501 1088.202

9.40E+04 1.41E+06 9.40E+04 1.21E+04 94001 1087.943

9.45E+04 1.40E+06 9.45E+04 1.21E+04 94501 1087.753

9.50E+04 1.39E+06 9.50E+04 1.21E+04 95001 1087.583

9.55E+04 1.39E+06 9.55E+04 1.21E+04 95500 1087.433

9.60E+04 1.38E+06 9.60E+04 1.21E+04 96000 1087.325

9.65E+04 1.37E+06 9.65E+04 1.20E+04 96500 1087.224

9.70E+04 1.36E+06 9.70E+04 1.20E+04 97000 1087.105

9.75E+04 1.36E+06 9.75E+04 1.20E+04 97500 1086.993

9.80E+04 1.35E+06 9.80E+04 1.20E+04 98000 1086.886

9.85E+04 1.34E+06 9.85E+04 1.20E+04 98500 1086.789

9.90E+04 1.34E+06 9.90E+04 1.20E+04 99000 1086.72

9.95E+04 1.33E+06 9.95E+04 1.19E+04 99500 1086.63

1.00E+05 1.32E+06 1.00E+05 1.14E+04 100000 1090.341

FREQUENCY(Hz)(5X) Z[Ω] FREQUENCY(Hz)(10X) Z[Ω]

10 185.7601 1.00E+01 1.43E+02

509.95 152.9999 5.10E+02 1.18E+02

1009.9 149.8559 1.01E+03 1.15E+02

37

1509.9 147.9439 1.51E+03 1.14E+02

2009.8 146.2508 2.01E+03 1.13E+02

2509.8 144.8413 2.51E+03 1.11E+02

3009.7 143.6436 3.01E+03 1.10E+02

3509.7 142.6084 3.51E+03 1.10E+02

4009.6 141.6672 4.01E+03 1.09E+02

4509.6 140.8241 4.51E+03 1.08E+02

5009.5 140.0743 5.01E+03 1.08E+02

5509.5 139.3118 5.51E+03 1.07E+02

6009.4 138.7037 6.01E+03 1.07E+02

6509.4 138.1615 6.51E+03 1.06E+02

7009.3 137.6697 7.01E+03 1.06E+02

7509.3 137.2187 7.51E+03 1.06E+02

8009.2 136.8035 8.01E+03 1.05E+02

8509.2 136.4286 8.51E+03 1.05E+02

9009.1 136.0832 9.01E+03 1.05E+02

9509.1 135.7634 9.51E+03 1.04E+02

10009 135.4862 1.00E+04 1.04E+02

10509 135.1879 1.05E+04 1.04E+02

11009 134.9112 1.10E+04 1.04E+02

11509 134.6441 1.15E+04 1.04E+02

12009 134.3953 1.20E+04 1.03E+02

12509 134.1696 1.25E+04 1.12E+02

13009 133.9529 1.30E+04 1.12E+02

13509 133.7372 1.35E+04 1.11E+02

14009 133.5272 1.40E+04 1.11E+02

14509 133.3231 1.45E+04 1.11E+02

15009 133.154 1.50E+04 1.11E+02

15508 132.9914 1.55E+04 1.11E+02

16008 132.824 1.60E+04 1.11E+02

16508 132.672 1.65E+04 1.11E+02

17008 132.531 1.70E+04 1.10E+02

17508 132.3986 1.75E+04 1.10E+02

18008 132.2684 1.80E+04 1.10E+02

18508 132.1455 1.85E+04 1.10E+02

19008 132.0278 1.90E+04 1.10E+02

19508 131.9154 1.95E+04 1.10E+02

20008 131.8109 2.00E+04 1.10E+02

20508 131.7055 2.05E+04 1.10E+02

21008 131.602 2.10E+04 1.10E+02

21508 131.5061 2.15E+04 1.10E+02

22008 131.4112 2.20E+04 1.10E+02

22508 131.3233 2.25E+04 1.09E+02

38

23008 131.2404 2.30E+04 1.09E+02

23508 131.1619 2.35E+04 1.09E+02

24008 131.0825 2.40E+04 1.09E+02

24508 130.9851 2.45E+04 1.09E+02

25008 130.8953 2.50E+04 1.09E+02

25507 130.8216 2.55E+04 1.09E+02

26007 130.7509 2.60E+04 1.09E+02

26507 130.6841 2.65E+04 1.09E+02

27007 130.6194 2.70E+04 1.09E+02

27507 130.5556 2.75E+04 1.09E+02

28007 130.4934 2.80E+04 1.09E+02

28507 130.4372 2.85E+04 1.09E+02

29007 130.3683 2.90E+04 1.09E+02

29507 130.3051 2.95E+04 1.09E+02

30007 130.3309 3.00E+04 1.09E+02

30507 129.5221 3.05E+04 1.08E+02

31007 129.4647 3.10E+04 1.08E+02

31507 129.4073 3.15E+04 1.08E+02

32007 129.3478 3.20E+04 1.08E+02

32507 129.2944 3.25E+04 1.08E+02

33007 129.2477 3.30E+04 1.08E+02

33507 129.2026 3.35E+04 1.08E+02

34007 129.1488 3.40E+04 1.08E+02

34507 129.0949 3.45E+04 1.08E+02

35007 129.0438 3.50E+04 1.08E+02

35506 129.0017 3.55E+04 1.08E+02

36006 127.8603 3.60E+04 1.07E+02

36506 127.8196 3.65E+04 1.07E+02

37006 127.781 3.70E+04 1.06E+02

37506 127.7426 3.75E+04 1.06E+02

38006 126.7572 3.80E+04 1.06E+02

38506 126.725 3.85E+04 1.06E+02

39006 126.6958 3.90E+04 1.06E+02

39506 126.665 3.95E+04 1.06E+02

40006 126.6353 4.00E+04 1.06E+02

40506 126.5894 4.05E+04 1.05E+02

41006 126.5579 4.10E+04 1.05E+02

41506 126.5292 4.15E+04 1.05E+02

42006 126.5026 4.20E+04 1.05E+02

42506 126.4697 4.25E+04 1.05E+02

43006 126.4384 4.30E+04 1.05E+02

43506 126.4143 4.35E+04 1.05E+02

44006 126.3893 4.40E+04 1.05E+02

39

44506 126.3643 4.45E+04 1.05E+02

45006 126.3372 4.50E+04 1.05E+02

45505 126.3117 4.55E+04 1.05E+02

46005 126.2858 4.60E+04 1.05E+02

46505 126.2563 4.65E+04 1.05E+02

47005 126.1643 4.70E+04 1.05E+02

47505 125.945 4.75E+04 1.05E+02

48005 125.923 4.80E+04 1.05E+02

48505 125.9017 4.85E+04 1.05E+02

49005 125.881 4.90E+04 1.05E+02

49505 125.8459 4.95E+04 1.05E+02

50005 125.797 5.00E+04 1.05E+02

50505 125.7978 5.05E+04 1.05E+02

51005 125.7869 5.10E+04 1.05E+02

51505 125.7779 5.15E+04 1.05E+02

52005 125.7664 5.20E+04 1.05E+02

52505 125.7528 5.25E+04 1.05E+02

53005 125.7382 5.30E+04 1.05E+02

53505 125.7251 5.35E+04 1.05E+02

54005 125.6853 5.40E+04 1.05E+02

54505 125.7304 5.45E+04 1.05E+02

55005 125.6655 5.50E+04 1.05E+02

55504 125.6497 5.55E+04 1.05E+02

56004 125.6337 5.60E+04 1.05E+02

56504 125.6168 5.65E+04 1.05E+02

57004 125.5995 5.70E+04 1.05E+02

57504 125.5823 5.75E+04 1.05E+02

58004 125.5657 5.80E+04 1.05E+02

58504 125.549 5.85E+04 1.05E+02

59004 125.531 5.90E+04 1.05E+02

59504 125.5143 5.95E+04 1.05E+02

60004 125.4975 6.00E+04 1.05E+02

60504 125.4813 6.05E+04 1.05E+02

61004 125.467 6.10E+04 1.05E+02

61504 125.4516 6.15E+04 1.05E+02

62004 125.4391 6.20E+04 1.05E+02

62504 125.4262 6.25E+04 1.05E+02

63004 125.413 6.30E+04 1.05E+02

63504 125.4027 6.35E+04 1.05E+02

64004 125.3887 6.40E+04 1.04E+02

64504 125.3733 6.45E+04 1.04E+02

65004 125.3593 6.50E+04 1.04E+02

65503 125.3454 6.55E+04 1.04E+02

40

66003 125.3301 6.60E+04 1.04E+02

66503 125.314 6.65E+04 1.04E+02

67003 125.2994 6.70E+04 1.04E+02

67503 125.2847 6.75E+04 1.04E+02

68003 125.272 6.80E+04 1.04E+02

68503 125.2599 6.85E+04 1.14E+02

69003 125.25 6.90E+04 1.14E+02

69503 125.241 6.95E+04 1.14E+02

70003 125.2311 7.00E+04 1.14E+02

70503 125.2169 7.05E+04 1.14E+02

71003 125.206 7.10E+04 1.14E+02

71503 125.1991 7.15E+04 1.14E+02

72003 125.1912 7.20E+04 1.14E+02

72503 125.1835 7.25E+04 1.14E+02

73003 125.1699 7.30E+04 1.14E+02

73503 125.16 7.35E+04 1.14E+02

74003 125.1528 7.40E+04 1.14E+02

74503 125.1449 7.45E+04 1.14E+02

75003 125.1354 7.50E+04 1.14E+02

75502 125.1272 7.55E+04 1.14E+02

76002 125.1193 7.60E+04 1.14E+02

76502 125.1115 7.65E+04 1.14E+02

77002 125.1047 7.70E+04 1.14E+02

77502 125.0986 7.75E+04 1.14E+02

78002 125.0921 7.80E+04 1.14E+02

78502 125.0863 7.85E+04 1.14E+02

79002 125.0796 7.90E+04 1.14E+02

79502 125.0733 7.95E+04 1.14E+02

80002 125.0665 8.00E+04 1.14E+02

80502 125.0607 8.05E+04 1.14E+02

81002 125.0544 8.10E+04 1.14E+02

81502 125.0471 8.15E+04 1.14E+02

82002 125.0096 8.20E+04 1.14E+02

82502 124.9473 8.25E+04 1.14E+02

83002 124.945 8.30E+04 1.14E+02

83502 124.9311 8.35E+04 1.14E+02

84002 124.915 8.40E+04 1.14E+02

84502 124.9014 8.45E+04 1.14E+02

85002 124.8925 8.50E+04 1.14E+02

85501 124.8848 8.55E+04 1.14E+02

86001 124.8779 8.60E+04 1.14E+02

86501 124.8727 8.65E+04 1.14E+02

87001 124.8622 8.70E+04 1.14E+02

41

87501 124.8564 8.75E+04 1.14E+02

88001 124.8543 8.80E+04 1.14E+02

88501 124.8506 8.85E+04 1.14E+02

89001 124.8462 8.90E+04 1.13E+02

89501 124.8453 8.95E+04 1.13E+02

90001 124.8417 9.00E+04 1.13E+02

90501 124.8363 9.05E+04 1.13E+02

91001 124.8301 9.10E+04 1.13E+02

91501 124.8246 9.15E+04 1.13E+02

92001 124.8184 9.20E+04 1.13E+02

92501 124.8141 9.25E+04 1.13E+02

93001 124.8093 9.30E+04 1.13E+02

93501 124.8039 9.35E+04 1.13E+02

94001 124.7991 9.40E+04 8.91E+01

94501 124.7948 9.45E+04 9.60E+01

95001 124.7904 9.50E+04 9.60E+01

95500 124.7852 9.55E+04 9.60E+01

96000 124.7802 9.60E+04 9.60E+01

96500 124.7764 9.65E+04 9.60E+01

97000 124.7703 9.70E+04 9.60E+01

97500 124.7679 9.75E+04 9.60E+01

98000 124.765 9.80E+04 9.60E+01

98500 124.7622 9.85E+04 9.60E+01

99000 124.759 9.90E+04 9.60E+01

99500 124.7558 9.95E+04 9.60E+01

100000 124.751 1.00E+05 9.60E+01

Part 2 (Averages)

FREQUENCY(Hz)(Steel) Z[Ω] FREQUENCY(Hz)(1x) Z[Ω]

1.00E+01 2.39E+03 1.00E+01 2.57E+04

5.10E+02 1.51E+03 5.10E+02 2.30E+04

1.01E+03 1.41E+03 1.01E+03 2.13E+04

1.51E+03 1.37E+03 1.51E+03 2.03E+04

2.01E+03 1.35E+03 2.01E+03 1.97E+04

2.51E+03 1.33E+03 2.51E+03 1.92E+04

3.01E+03 1.32E+03 3.01E+03 1.87E+04

3.51E+03 1.30E+03 3.51E+03 1.82E+04

4.01E+03 1.29E+03 4.01E+03 1.78E+04

4.51E+03 1.28E+03 4.51E+03 1.73E+04

5.01E+03 1.28E+03 5.01E+03 1.70E+04

5.51E+03 1.27E+03 5.51E+03 1.68E+04

6.01E+03 1.27E+03 6.01E+03 1.66E+04

42

6.51E+03 1.26E+03 6.51E+03 1.65E+04

7.01E+03 1.26E+03 7.01E+03 1.61E+04

7.51E+03 1.25E+03 7.51E+03 1.61E+04

8.01E+03 1.25E+03 8.01E+03 1.60E+04

8.51E+03 1.25E+03 8.51E+03 1.58E+04

9.01E+03 1.25E+03 9.01E+03 1.57E+04

9.51E+03 1.24E+03 9.51E+03 1.56E+04

1.00E+04 1.24E+03 1.00E+04 1.56E+04

1.05E+04 1.24E+03 1.05E+04 1.54E+04

1.10E+04 1.23E+03 1.10E+04 1.54E+04

1.15E+04 1.23E+03 1.15E+04 1.53E+04

1.20E+04 1.23E+03 1.20E+04 1.51E+04

1.25E+04 1.23E+03 1.25E+04 1.50E+04

1.30E+04 1.22E+03 1.30E+04 1.48E+04

1.35E+04 1.22E+03 1.35E+04 1.49E+04

1.40E+04 1.22E+03 1.40E+04 1.47E+04

1.45E+04 1.22E+03 1.45E+04 1.44E+04

1.50E+04 1.22E+03 1.50E+04 1.42E+04

1.55E+04 1.22E+03 1.55E+04 1.40E+04

1.60E+04 1.21E+03 1.60E+04 1.37E+04

1.65E+04 1.21E+03 1.65E+04 1.37E+04

1.70E+04 1.21E+03 1.70E+04 1.37E+04

1.75E+04 1.21E+03 1.75E+04 1.37E+04

1.80E+04 1.21E+03 1.80E+04 1.37E+04

1.85E+04 1.21E+03 1.85E+04 1.37E+04

1.90E+04 1.21E+03 1.90E+04 1.37E+04

1.95E+04 1.20E+03 1.95E+04 1.37E+04

2.00E+04 1.20E+03 2.00E+04 1.36E+04

2.05E+04 1.20E+03 2.05E+04 1.36E+04

2.10E+04 1.20E+03 2.10E+04 1.36E+04

2.15E+04 1.20E+03 2.15E+04 1.36E+04

2.20E+04 1.20E+03 2.20E+04 1.37E+04

2.25E+04 1.20E+03 2.25E+04 1.36E+04

2.30E+04 1.20E+03 2.30E+04 1.36E+04

2.35E+04 1.20E+03 2.35E+04 1.36E+04

2.40E+04 1.19E+03 2.40E+04 1.36E+04

2.45E+04 1.19E+03 2.45E+04 1.37E+04

2.50E+04 1.19E+03 2.50E+04 1.36E+04

2.55E+04 1.19E+03 2.55E+04 1.36E+04

2.60E+04 1.19E+03 2.60E+04 1.36E+04

2.65E+04 1.19E+03 2.65E+04 1.36E+04

2.70E+04 1.19E+03 2.70E+04 1.36E+04

2.75E+04 1.19E+03 2.75E+04 1.36E+04

43

2.80E+04 1.18E+03 2.80E+04 1.36E+04

2.85E+04 1.18E+03 2.85E+04 1.36E+04

2.90E+04 1.18E+03 2.90E+04 1.36E+04

2.95E+04 1.18E+03 2.95E+04 1.36E+04

3.00E+04 1.18E+03 3.00E+04 1.37E+04

3.05E+04 1.18E+03 3.05E+04 1.37E+04

3.10E+04 1.18E+03 3.10E+04 1.36E+04

3.15E+04 1.18E+03 3.15E+04 1.37E+04

3.20E+04 1.18E+03 3.20E+04 1.37E+04

3.25E+04 1.18E+03 3.25E+04 1.37E+04

3.30E+04 1.18E+03 3.30E+04 1.37E+04

3.35E+04 1.17E+03 3.35E+04 1.36E+04

3.40E+04 1.17E+03 3.40E+04 1.36E+04

3.45E+04 1.17E+03 3.45E+04 1.35E+04

3.50E+04 1.17E+03 3.50E+04 1.36E+04

3.55E+04 1.17E+03 3.55E+04 1.36E+04

3.60E+04 1.17E+03 3.60E+04 1.36E+04

3.65E+04 1.17E+03 3.65E+04 1.36E+04

3.70E+04 1.17E+03 3.70E+04 1.35E+04

3.75E+04 1.17E+03 3.75E+04 1.36E+04

3.80E+04 1.17E+03 3.80E+04 1.36E+04

3.85E+04 1.17E+03 3.85E+04 1.36E+04

3.90E+04 1.17E+03 3.90E+04 1.35E+04

3.95E+04 1.17E+03 3.95E+04 1.35E+04

4.00E+04 1.17E+03 4.00E+04 1.35E+04

4.05E+04 1.17E+03 4.05E+04 1.34E+04

4.10E+04 1.17E+03 4.10E+04 1.34E+04

4.15E+04 1.17E+03 4.15E+04 1.35E+04

4.20E+04 1.17E+03 4.20E+04 1.34E+04

4.25E+04 1.17E+03 4.25E+04 1.34E+04

4.30E+04 1.16E+03 4.30E+04 1.33E+04

4.35E+04 1.16E+03 4.35E+04 1.34E+04

4.40E+04 1.16E+03 4.40E+04 1.34E+04

4.45E+04 1.16E+03 4.45E+04 1.34E+04

4.50E+04 1.16E+03 4.50E+04 1.34E+04

4.55E+04 1.16E+03 4.55E+04 1.34E+04

4.60E+04 1.16E+03 4.60E+04 1.33E+04

4.65E+04 1.16E+03 4.65E+04 1.32E+04

4.70E+04 1.16E+03 4.70E+04 1.32E+04

4.75E+04 1.16E+03 4.75E+04 1.33E+04

4.80E+04 1.16E+03 4.80E+04 1.33E+04

4.85E+04 1.16E+03 4.85E+04 1.33E+04

4.90E+04 1.16E+03 4.90E+04 1.33E+04

44

4.95E+04 1.16E+03 4.95E+04 1.33E+04

5.00E+04 1.16E+03 5.00E+04 1.33E+04

5.05E+04 1.16E+03 5.05E+04 1.33E+04

5.10E+04 1.16E+03 5.10E+04 1.32E+04

5.15E+04 1.16E+03 5.15E+04 1.32E+04

5.20E+04 1.16E+03 5.20E+04 1.32E+04

5.25E+04 1.16E+03 5.25E+04 1.32E+04

5.30E+04 1.16E+03 5.30E+04 1.32E+04

5.35E+04 1.16E+03 5.35E+04 1.32E+04

5.40E+04 1.16E+03 5.40E+04 1.31E+04

5.45E+04 1.16E+03 5.45E+04 1.31E+04

5.50E+04 1.16E+03 5.50E+04 1.31E+04

5.55E+04 1.16E+03 5.55E+04 1.31E+04

5.60E+04 1.16E+03 5.60E+04 1.31E+04

5.65E+04 1.16E+03 5.65E+04 1.31E+04

5.70E+04 1.16E+03 5.70E+04 1.30E+04

5.75E+04 1.16E+03 5.75E+04 1.30E+04

5.80E+04 1.16E+03 5.80E+04 1.30E+04

5.85E+04 1.15E+03 5.85E+04 1.30E+04

5.90E+04 1.15E+03 5.90E+04 1.29E+04

5.95E+04 1.15E+03 5.95E+04 1.29E+04

6.00E+04 1.15E+03 6.00E+04 1.28E+04

6.05E+04 1.15E+03 6.05E+04 1.27E+04

6.10E+04 1.15E+03 6.10E+04 1.28E+04

6.15E+04 1.15E+03 6.15E+04 1.28E+04

6.20E+04 1.15E+03 6.20E+04 1.28E+04

6.25E+04 1.15E+03 6.25E+04 1.27E+04

6.30E+04 1.15E+03 6.30E+04 1.27E+04

6.35E+04 1.15E+03 6.35E+04 1.27E+04

6.40E+04 1.15E+03 6.40E+04 1.27E+04

6.45E+04 1.15E+03 6.45E+04 1.26E+04

6.50E+04 1.15E+03 6.50E+04 1.26E+04

6.55E+04 1.15E+03 6.55E+04 1.26E+04

6.60E+04 1.15E+03 6.60E+04 1.26E+04

6.65E+04 1.15E+03 6.65E+04 1.25E+04

6.70E+04 1.15E+03 6.70E+04 1.25E+04

6.75E+04 1.15E+03 6.75E+04 1.24E+04

6.80E+04 1.15E+03 6.80E+04 1.24E+04

6.85E+04 1.15E+03 6.85E+04 1.24E+04

6.90E+04 1.15E+03 6.90E+04 1.23E+04

6.95E+04 1.15E+03 6.95E+04 1.21E+04

7.00E+04 1.15E+03 7.00E+04 1.21E+04

7.05E+04 1.15E+03 7.05E+04 1.20E+04

45

7.10E+04 1.15E+03 7.10E+04 1.20E+04

7.15E+04 1.15E+03 7.15E+04 1.20E+04

7.20E+04 1.15E+03 7.20E+04 1.19E+04

7.25E+04 1.15E+03 7.25E+04 1.18E+04

7.30E+04 1.15E+03 7.30E+04 1.18E+04

7.35E+04 1.15E+03 7.35E+04 1.19E+04

7.40E+04 1.15E+03 7.40E+04 1.18E+04

7.45E+04 1.15E+03 7.45E+04 1.18E+04

7.50E+04 1.15E+03 7.50E+04 1.18E+04

7.55E+04 1.14E+03 7.55E+04 1.17E+04

7.60E+04 1.14E+03 7.60E+04 1.17E+04

7.65E+04 1.14E+03 7.65E+04 1.17E+04

7.70E+04 1.14E+03 7.70E+04 1.17E+04

7.75E+04 1.14E+03 7.75E+04 1.16E+04

7.80E+04 1.14E+03 7.80E+04 1.16E+04

7.85E+04 1.14E+03 7.85E+04 1.16E+04

7.90E+04 1.14E+03 7.90E+04 1.15E+04

7.95E+04 1.14E+03 7.95E+04 1.26E+04

8.00E+04 1.14E+03 8.00E+04 1.25E+04

8.05E+04 1.14E+03 8.05E+04 1.25E+04

8.10E+04 1.17E+03 8.10E+04 1.25E+04

8.15E+04 1.17E+03 8.15E+04 1.25E+04

8.20E+04 1.17E+03 8.20E+04 1.25E+04

8.25E+04 1.17E+03 8.25E+04 1.25E+04

8.30E+04 1.17E+03 8.30E+04 1.25E+04

8.35E+04 1.17E+03 8.35E+04 1.25E+04

8.40E+04 1.17E+03 8.40E+04 1.25E+04

8.45E+04 1.17E+03 8.45E+04 1.24E+04

8.50E+04 1.17E+03 8.50E+04 1.24E+04

8.55E+04 1.17E+03 8.55E+04 1.24E+04

8.60E+04 1.17E+03 8.60E+04 1.24E+04

8.65E+04 1.17E+03 8.65E+04 1.24E+04

8.70E+04 1.17E+03 8.70E+04 1.23E+04

8.75E+04 1.17E+03 8.75E+04 1.23E+04

8.80E+04 1.17E+03 8.80E+04 1.23E+04

8.85E+04 1.17E+03 8.85E+04 1.23E+04

8.90E+04 1.17E+03 8.90E+04 1.23E+04

8.95E+04 1.17E+03 8.95E+04 1.23E+04

9.00E+04 1.17E+03 9.00E+04 1.22E+04

9.05E+04 1.17E+03 9.05E+04 1.22E+04

9.10E+04 1.17E+03 9.10E+04 1.22E+04

9.15E+04 1.17E+03 9.15E+04 1.22E+04

9.20E+04 1.17E+03 9.20E+04 1.22E+04

46

9.25E+04 1.17E+03 9.25E+04 1.21E+04

9.30E+04 1.17E+03 9.30E+04 1.21E+04

9.35E+04 1.17E+03 9.35E+04 1.21E+04

9.40E+04 1.17E+03 9.40E+04 1.21E+04

9.45E+04 1.17E+03 9.45E+04 1.21E+04

9.50E+04 1.17E+03 9.50E+04 1.21E+04

9.55E+04 1.17E+03 9.55E+04 1.21E+04

9.60E+04 1.17E+03 9.60E+04 1.21E+04

9.65E+04 1.16E+03 9.65E+04 1.20E+04

9.70E+04 1.16E+03 9.70E+04 1.20E+04

9.75E+04 1.16E+03 9.75E+04 1.20E+04

9.80E+04 1.16E+03 9.80E+04 1.20E+04

9.85E+04 1.17E+03 9.85E+04 1.20E+04

9.90E+04 1.17E+03 9.90E+04 1.20E+04

9.95E+04 1.17E+03 9.95E+04 1.19E+04

1.00E+05 1.27E+03 1.00E+05 1.14E+04

D. Acknowledgements

We acknowledge the individuals who have helped us in pursuing our project goals. That

includes Wayne State’s Smart Sensors and Integrated microsystems lab for providing us with

training, machinery and raw materials in order to work on our project. Also, our advisors who

have been guiding us throughout this project. Lastly, the LESA foundation for providing us with

the grant necessary to complete our project. Our team will continue this development process

using an impedance MEMS Biosensor for the detection of MRSA.

E. Clinical application

Even though we are only working on the detection mechanism of MRSA, there are still

unique steps that need to be taken in a clinical setting to obtain a testable sample for our device.

These steps will be outside the scope of our project but we envision the steps to be included in

our devices future revisions. In order to complete our first separation a blood sample can be

taken from the patient as shown in in part one of Diagram 4. In step 2, filter paper could be used

to only allow cells that are smaller than 1.5 microns to flow through leaving a solution of

47

bacteria because human cells are the order of 2-120 microns (8). Step 3 is when Antibiotic

coated magnetic beads at the nano-scale size would be used to capture and control all the bacteria

with the exception of staph bacteria. The magnetic beads containing the bacteria will then be

disposed of leaving us with the staph bacteria solution. These three steps are steps that would be

taken in an actual clinical laboratory setting to have a testable sample of strictly staph bacteria to

insert into our detection device.

F. Detailed Cross-Sectional Process Flow

Electrode

1. The masks where printed using high resolution transparency by an external company

(Fineline Imaging)

2. Glass wafers will be spun with the gold by an external company (Wayne State nFab Lab)

4. Heat surface (900o C) of glass wafer to achieve oxygen formation

5. Apply positive (Microposit S1818) photoresist to top surface

-At a spin rate of 4500 RPM (30 seconds)

6. Soft bake the device (80o C) for 10 minutes

7. Apply mask to top surface and expose to UV light (400 nm)

8. Wet develop through a solvent bath (S2SO4)

9. Post baking (120o C) for 20 minutes

10. Wet etching

48

Passivation Layer

11. A SiO2 layer will be deposited by an external company (Wayne State nFab Lab)

12. Heat surface (900o C) of glass wafer to achieve oxygen formation

13. Apply positive (Microposit S1818) photoresist to top surface

-At a spin rate of 4500 RPM

14. Soft bake the device (80o C) for 10 minutes

15. Apply mask to top surface and expose to UV light (400 nm)

16. Wet develop through a solvent bath (S2SO4)

17. Post baking (120o C) for 20 minutes

18. Dry etching

50

Works Cited

1."InPhotonics: What Is Raman Spectroscopy?" 3 June 2011. Web. 16 Dec. 2014.

<http://www.inphotonics.com/raman.htm>.

2.Koydemir, Hatice Ceylan, Haluk Külah, Canan Özgen, Alpaslan Alp, and Gülşen Hasçelik.

"MEMS Biosensors for Detection of Methicillin Resistant Staphylococcus Aureus."

Biosensors and Bioelectronics (2009): 1-12. Print.

3.Leski, T. A., and A. Tomasz. "Role of Penicillin-Binding Protein 2 (PBP2) in the Antibiotic

Susceptibility and Cell Wall Cross-Linking of Staphylococcus Aureus: Evidence for the

Cooperative Functioning of PBP2, PBP4, and PBP2A." Journal of Bacteriology (2005):

1815-824. Print.

4.Lowy, Franklin. "STAPHYLOCOCCUS AUREUS INFECTIONS." New England Journal of

Medicine (1998): 520-32. Print.

5.Mansoor Nasir “Microfluidic Impedance-Based Biosensor” Print.

6."MRSA Infection." Definition. 4 Apr. 2007. Web. 16 Dec. 2014.

<http://www.mayoclinic.org/diseases-conditions/mrsa/basics/definition/con-20024479>.

7.Osiecki, John. "MRSA Screening: Culture or Molecular? Finding the Best Solution for Your

Facility Requires a Balanced Assessment of Performance, Speed and Cost." Advance

Healthcare Networks 19.20 (2010): 420. Print.

8."Staph Infection (Staphylococcus Aureus) Symptoms, Causes, Treatment - What Is Antibiotic-

resistant Staph Aureus? - MedicineNet." MedicineNet. 11 Nov. 2009. Web. 16 Dec.

2014. <http://www.medicinenet.com/staph_infection/page5.htm#what_is_antibiotic-

resistant_staph_aureus>.

9.U.S Department of Veterans Affairs 12 May 2008. Web. 16 Dec. 2014

<http://www.va.gov/opa/publications/factsheets/fs_department_of_veterans_affairs.pdf>.

Novel Microfluidic Biosensor for Rapid and Specific ... Novel Microfluidic Biosensor for Rapid and...

Documents

Transcript of Novel Microfluidic Biosensor for Rapid and Specific ... Novel Microfluidic Biosensor for Rapid and...