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Nano Report Draft 1 Engineering 1282.02H

Spring, 2016

Team Y6

Jack Canaday, Seat 24

Eric Glowacki, Seat 22

Justin Iovino, Seat 23

Shane Riddle, Seat 21

A. Theiss

Date of Submission: 04/11/2016

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Executive Summary

The development of new nanotechnology concepts has opened the door to the creation of new

lab on a chip (LOC) devices that would allow for cheap, simple point of care diagnosis of

diseases without the need for expensive laboratory equipment. The emerging Zika virus is a

prime candidate for the development of a LOC device as the World Health Organization (WHO)

has declared the disease a public health emergency with estimates of four million people being

infected before the end of 2016. This report presents an easy to use, disposable LOC device

capable of Zika virus diagnosis under eight hours.

The device uses plasmonic ELISA with gold nanoparticles as the primary detection

method for the virus. The main advantage of this technique is that it does not require any outside

reader in order to operate but rather can be read with the naked eye. This is accomplished

through a distinct color of blue and red for the positive and negative test results as a result of

differing structures of the gold nanoparticles.

In addition to not needing a reader, the other primary advantage of the device is that all

reagents are already preloaded into the device. Other Zika testing kits require the user to have

supplementary materials to perform the test. Also, the other testing kits require the test to sit

overnight before results can be read while this device produces results in less than eight hours.

The primary drawbacks of this device are that it has a one month shelf life and must be

refrigerated because of the natural decay of the gold and hydrogen peroxide solutions.

The cost of the device will be around $6.50. In conclusion, this device would provide a

major advantage in the treating and containing the emergent Zika virus.

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Acknowledgement

We would like to extend out thanks to everyone within the FEH program and our

wonderful TAs, GTAs, and Professors.

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Table of Contents

Executive Summary ................................................................................................... 1

Acknowledgement ..................................................................................................... 2

List of Tables and Figures .......................................................................................... 5

Team Introduction ...................................................................................................... 5

1. Introduction .......................................................................................................... 5

1.1. Outline........................................................................................................................................... 6

2. Problem Statement ............................................................................................... 6

2.1. Lab on a Chip Requirements ......................................................................................................... 6

2.2. Lab On A Chip Parameters ........................................................................................................... 7

3. Zika Virus ............................................................................................................ 7

4. Previous Work and Similar Devices .................................................................... 9

5. NANOLYSER Brainstorming ............................................................................. 9

5.1. Disease Selection .......................................................................................................................... 9

5.2. Viral Detection Methods ............................................................................................................. 10

5.3. Fluid Transport Methods ............................................................................................................. 11

5.4. Sample Loading Methods ........................................................................................................... 12

5.5. Device Reading Methods ............................................................................................................ 12

5.6. Initial Design ............................................................................................................................... 13

6. NANOLYSER ...................................................................................................14

6.1. Design Considerations ................................................................................................................ 14

6.2. Final Design ................................................................................................................................ 15

6.2.1. Final Processing Algorithm ................................................................................................ 16

6.2.2. Final Design of The Physical System ................................................................................. 16

6.2.3. Micro Components of Physical System .............................................................................. 18

6.2.4. Nano Components of Physical System ............................................................................... 19

6.3. Ideal Operating Conditions ......................................................................................................... 20

6.3.1. Safety and Ease of Disposal ................................................................................................ 21

6.3.2. Accuracy (Lack of False Positives)..................................................................................... 21

6.4. Fabrication .................................................................................................................................. 22

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6.5. Costs ............................................................................................................................................ 22

7. Summary and Conclusion ..................................................................................23

References ...............................................................................................................24

APPENDIX A: Sample Processing Algorithm ....................................................... A0

APPENDIX B: Not in Use Yet ............................................................................... B0

APPENDIX C: Not in Use Yet ............................................................................... C0

APPENDIX D: Not in Use Yet ............................................................................... D0

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List of Tables and Figures

Figure 1: Initial SolidWorks rendering of NANOLYSER Bottom ............................................................. 13 Figure 2: Initial SolidWorks rendering of NANOLYSER Top .................................................................. 14 Figure 3: NANOLYSER SolidWorks Assembly ........................................................................................ 17 Figure 4: SolidWorks Model Of Bubble Gate Mechanism ......................................................................... 19 Figure 5: Composition of Reagents in ELISA Positive Test [6] ................................................................. 20 Figure A0: Sample Processing Algorithm……………………………………………………………… A1

Team Introduction

Team Y6 is made up of Jack Canaday, Shane Riddle, Justin Iovino, and Eric Glowacki.

Jack Canaday is a Materials Science and Engineering major; his focus was on the project notebook

documentation and research. Shane Riddle is a Mechanical Engineering major; his focus was on

chip design. Eric Glowacki is a Chemical and Biomolecular Engineering major with a minor in

Leadership Studies; his focus was on research and formulation. Justin Iovino is a Biomedical

Engineering major; his focus was on documentation and research.

1. Introduction

In the medical field, few things are valued more than efficiency. In a product, efficiency

can include effectiveness, cost, ease of use, and speed. In diagnosis, especially, it is important to

attain accurate results in a timely manner, so appropriate action could be taken after the results of

the test are known. In less developed locations where current medical technology is not as

prominent, many cases of treatable diseases go undiagnosed and untreated. Most diagnostic

medical procedures require expensive machinery and large amounts of sample, but it is not

feasible to do this in remote places. Thus, it would save many lives if these procedures were

shrunk down to a smaller scale that would make the whole process easier, cheaper, quicker, and

more accessible.

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The present paper discusses the design of an acrylic NANOLYSER (Nanofunctionalized

Assay Nested in an Onboard Laboratory Yielding Specific Expeditious Results) for use in

diagnosis of Zika Virus. Zika Virus is a virus prominent in tropical areas that is difficult to

diagnose without proper medical equipment. This device would provide fast point of care

diagnosis of Zika Virus without the use of sample transportation, expensive machinery, and large

amounts of reagents. This device would need to acquire accurate results from one analyte in a

drop of blood, and the researchers chose to use the color-changing nature of certain antibody-

virus interactions.

1.1. Outline

The present chip, along with relevant research and documentation, was designed by Jack

Canaday, Eric Glowacki, Justin Iovino, and Shane Riddle, students at The Ohio State University.

The following section, Medical Applications and Background Information, will cover

background regarding Zika Virus and relevant research in the medical field. Section 3, will

explain design constraints and considerations, as well as the design strategies used by the

researchers. Sections 4 and 5 will discuss initial designs/redesigns and key design components,

respectively. Next, Section 6 will describe the final NANOLYSER design and process, and

Section 7 will provide summary and conclusions of the project.

2. Problem Statement

2.1. Lab on a Chip Requirements

Requirements of the NANOLYSER chip were: a fluid circuit to load blood sample, a

fluid circuit to load any reagents needed, a fluid circuit for separation/capture of analyte, a

nanoscale strategy for specific detection of analyte, an ability to interface appropriately with

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requisite technology for reading sample. In addition, all fluid movement will be either

electromotive, pressure driven, or capillary force and appropriate valves where needed to control

flow. If the chip will be reusable the cleaning and sterilizing approach must be discussed. The

chip must be able to plugged into a “reader” that is capable of gathering data from chip using

selected detection method(s) (e.g. must have light transparent section for collecting fluorescence

data or electrical connection for field effect sensing). These requirements were taken into

consideration when formulating the NANOLYSER.

2.2. Lab On A Chip Parameters

Additional parameters included in the problem statement state that: The NANOLYSER

should be able to process and diagnose a single drop of blood with a volume of around .05 mL

and the analyte to be detected in the bloodstream will be Zika virus antigens. Human interaction

with the device is limited to: (a) loading blood sample and reagents, and (b) inserting the chip

into a reader and/or pump. If you decide to use an external reader/pump (i.e., fluorescence

reader), you must explain how the external device works and include specific parameters (i.e.,

size, wavelengths used for a fluorescence reader, pressure applied for a pump). The parameter on

time restricts the operating time of the device to under 8 hours. Additionally, The method of

detection should be simple to operate without requiring extensive training and the equipment

used will be thoroughly described and the results should be reliable, no false positives should

appear within testing. If the virus is present, the chip will indicate the virus is present.

3. Zika Virus

Since the 1950’s the Zika Virus had only been known to exist near the equator in parts of

Africa and Asia. However, in recent years the virus has spread across the Pacific Ocean to tropical

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parts of Oceania and South America, which has caused it to gain more public attention. It is mostly

spread through the activity of the Aedes mosquito and sexual contact, with the former to a much

greater extent. There is currently no known cure or vaccine for the Zika Virus. The symptoms of

the Zika Virus are not especially severe or dangerous, with most cases ranging from no symptoms

at all to fever and joint pain. This also causes many people who have the disease to be

misdiagnosed as influenza or not diagnosed at all [1]. This poses a threat to pregnant women

because the virus is linked to an increased chance in the birth defect microcephaly. This condition

is characterized by the child develop a smaller than average head, which can cause many

developmental disorders, especially in motor function, cognitive development, and personality

disorders [2]. As a result, many pregnant women would prefer to know if they have the virus before

continuing with a pregnancy and risking potential birth defects. However, Zika Virus is mostly

prominent in poorer countries that do not have the medical technology of developed nations, so

the disease goes largely undiagnosed.

Most current Zika Virus diagnoses involves sending blood samples to large labs, which is

both time consuming and expensive [3]. However, recent studies have looked into more efficient

methods of detection, such as Gourinat et al. (2015) that found evidence that Zika Virus RNA can

be detected from a urine sample with the use of a real-time reverse transcription polymerase chain

reaction [4]. Other studies were integral to the researchers’ design process. Cheng et al. (2008)

provides an overview on recent advancements in viral detection methods and served as a basis for

the present researchers’ detection strategy [5]. Furthermore, Rica et al. (2012) investigates the use

of plasmoic ELISA for disease detection and provided a stepping stone procedure for the

NANOLYSER [6]. The NANOLYSER device proposed in this paper would provide and point-of-

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care diagnosis from a small sample of blood that is much cheaper and faster than the current lab-

based method mentioned previously.

4. Previous Work and Similar Devices

While not many diagnostic tests exist for the Zika virus the group found one preexisting

test for the disease not requiring a full laboratory to perform. This test comes in packs of 25 and

utilizes the Zika virus IgG/IgM antibody for a lateral flow immunoassay to detect the virus from

human serum, plasma, or whole blood [7]. The producers of the product do not provide the

method for how the test works but claim that results can be obtained within fifteen minutes.

Additionally, the test has not been approved for sale yet in North America by the Canadian

Department of National Health and Welfare so no price data could be obtained. However, it

would have to be commercially viable as the company is pushing to get it approved for sale.

5. NANOLYSER Brainstorming

5.1. Disease Selection

There were a multitude of initial ideas for what disease the NANOLYSER could detect.

The first idea was using the device to test for the Staphylococcus bacteria. This was due to one of

the group members having the disease and being frustrated with the difficulty in diagnosis of the

disease however this idea was discarded as all bacterial infections can be diagnosed fairly easily

through the growth of a culture from a patient sample.

Since bacterial infections were not desired, viral diseases were examined next. Since the

NANOLYSER device would be especially suited for use in third world countries due to their

lack of medical infrastructure, viral diseases affecting these countries were studied. Some of

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these viruses considered included, West Nile, dengue, and yellow fever. However, due to the

reasons discussed previously, Zika virus was seen as a much larger health threat to the world

than these other viruses. Additionally, these other viral diseases are fairly old and have a great

deal of pre-existing methods of effective diagnosis in third world countries. Zika virus is very

new emerging having its first outbreak in 2013 in French Polynesia and the more recent major

outbreak in South and Central America starting in April 2015 [8]. Therefore, Zika virus was

selected as the viral disease that the NANOLYSER would test.

5.2. Viral Detection Methods

Two primary methods of detecting the virus were considered for the NANOLYSER device.

First, the use of reverse transcriptase polymerase chain reaction (RT-PCR) coupled with

electrophoresis was looked at as a possible mechanism. This method was looked into because it

is currently the method used by the United States’ Center for Disease Control to accurately

diagnose the disease [9]. This method takes advantage of the presence of the RNA present within

the viral capsid to mass produce a large quantity of DNA that could then be used in gel

electrophoresis [10]. The first step of RT-PCR is attaching sequence specific primers to the RNA

nd allowing the enzyme reverse transcriptase to attach to the primer and create a complimentary

DNA (cDNA) strand to the RNA. The cDNA is separated from the RNA and a similar process

repeats attaching a primer to the cDNA and allowing DNA polymerase to create another cDNA

strand. This cycle repeats allowing for exponential increase of the amount of cDNA in solution

[11]. The cDNA is then loaded into an agarose gel to undergo electrophoresis. The cDNA is

mixed with a dye and a current is ran across the gel while it is submerged in a buffer solution.

The cDNA samples are loaded on the negative end of the cell because DNA is a negative

molecule and will move towards the positive terminal [11]. While this method provided very

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accurate results, it exceeded the time requirements of the device with most methods taking

around 10 -12 hours. Additionally, this method would require a thermocycler in order to adjust

the temperature to allow each enzyme to work in a fast manner. Therefore, the other method

considered to allow for detection was the use of the enzyme-linked immunosorbent assay

(ELISA) method. This method takes advantage of the lock and key aspect of antibodies and

antigens meaning that the human body produces a unique antibody for every antigen [12]. The

ELISA considered was the sandwich ELISA. This method starts with the complimentary

antibody being seeded onto the bottom of the well and then the virus represented by the blue oval

attaches to the antibody. The well is then washed and then a primary antibody which can adhere

to the virus is added. This is followed by a wash and a secondary antibody coupled with an

enzyme which can attach to the first antibody. A solution is then added which the enzyme can

react with in order to induce a color change or other reaction to indicate that the analyte is

present [13]. This method was deemed promising and was decided on as the main detection

strategy for the NANOLYSER device.

5.3. Fluid Transport Methods

Moving the blood and the other reagents posed a problem as conventional fluid dynamics

is altered at the microscopic level so conventional pumping mechanisms are ineffective. The

team decided that a vacuum pump would be the most effective way of transporting fluid within

the NANOLYSER. This pump works by creating a vacuum pressure differential at the end of the

chip which pulls the fluid towards the pump [14].

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5.4. Sample Loading Methods

Getting the blood sample into the NANOLYSER without contamination was another design

aspect that had multiple solutions. One solution that was thought of was having the blood be

directly sucked into the NANOLYSER from the patient’s skin. This idea would eliminate the

need for a transfer apparatus, however the problem of possible blockages was considered to be

too big of a potential problem to allow into the device. Instead, loading a capillary tube into the

device would prevent blockages and allow for blood samples to be transported easily before

using the NANOLYSER device if need be.

5.5. Device Reading Methods

Utilizing the ELISA method, a number of detection methods were made available. Three main

detection methods were considered. The first method was the use of a fluorescent detection

under a backlight. The other method was to utilize a color change and a spectrophotometer to

measure the intensity of the color [13]. However, both of these methods shared similar problems.

Both of them required some sort of outside equipment in order to be read. Additionally, the

amount of virus in the sample may have not been enough leading to a negative test when the

patient does have the virus. Therefore, the group looked at plasmonic ELISA detection to solve

these problems. Plasmonic ELISA utilizes gold nanoparticle formation in with regards to the

concentration of hydrogen peroxide in order to diagnose the disease [6]. Having a high

concentration of hydrogen peroxide results in spherical, separate nanoparticles that appear red in

solution while a lack of hydrogen peroxide yields gold nanoparticles that are not spherical and

clump together leading to a blue solution. This method of ELISA is a special type of ELISA with

catalase as the enzyme attached to the secondary antibody which catalyzes the decomposition of

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hydrogen peroxide. Therefore, if the virus is present, the concentration of hydrogen peroxide will

be low leading to a blue solution [6]. The distinct red blue color shift of the solution eliminates

the need for an outside reader device. Finally, this method of ELISA is ultra-sensitive allowing

for detection of concentrations down to 10-18 M [6].

5.6. Initial Design

The initial design of the chip took a layered approach and the bottom layer containing all the

processes to detect the virus, as shown below in Figure 1.

Figure 1: Initial SolidWorks rendering of NANOLYSER Bottom The bottom layer of the chip consisted of one main channel connected to a series of wells

containing the different reagents present in the NANOLYSER. Since the ELISA method does

not require the reagents to be mixed, no mixing strategies were employed in the chip such as

curves for passive mixing. The chip had an opening for a capillary tube to be inserted into the

chip as the first large well on the right. This well would be separated from the other wells using

an air bubble barrier which is how all the other wells were also separated. The next well was

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filled with a buffer solution followed by a well with the primary antibody. The next well would

have another wash solution and then the final secondary detection antibody. The blood from the

main well on the right would be pulled by an external vacuum pump to bull the blood to the

reaction well on the upper left corner. Moving south east from the detection well is the final

waste well where all excess reagents would be stored and the vacuum pump attached. The top

layer of the chip is shown below in Figure 2.

Figure 2: Initial SolidWorks rendering of NANOLYSER Top

The top layer of the chip was two openings cut into the top of the chip. The upper right

opening was to allow for a capillary tube to be inserted into the chip. The top left opening is a

clear glass opening to see the detection well. Both of the top and bottom would be made of solid

plastic or acrylic.

6. NANOLYSER

6.1. Design Considerations

The final design of the NANOLYSER needed to be relatively fast, efficient, cost effective,

and disposable. The procedure described in the blood processing algorithm was altered from the

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plasmonic ELISA method, in order to make it faster [6]. This involved reducing the number of

washes and minimizing all of the wait times for the reactions which was justified by the chip’s

reduced size which called for the use of less reagent which in turn meant the reactions would not

take as long. The efficiency was optimized by reducing the number of times the test was performed.

Due to the precision of the NANOLYSER, only one test is needed for an accurate diagnosis. This

also allowed for a significant cost reduction, as only one set of reagents is needed for each device

which also means the device only has to be big enough to house one complete fluid circuit. A

minimized size translates to a reduction in materials and a minimal number of fluid circuits reduces

the cost of labor involved with machining them. The chip was also made to be disposable since an

overly complex design would have made it extremely difficult to prepare and use without large

amounts of training due to the many reactants and precise volumes of each the device requires. It

would also be almost impossible to clean since that would involve removing the reagents from the

antibodies attached to the reaction well which would simply end up removing the antibody layer

itself. Coincidentally these decisions ended up reducing costs as well by making it necessary to

use a cheaper material for the chip body.

6.2. Final Design

The final design of the NANOLYSER was an amalgamation of what were deemed the best

components and processes considered during the brainstorming stage. These were melded together

in a rugged design that encompassed the goals of speed, efficiency, accuracy and cost

effectiveness.

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6.2.1. Final Processing Algorithm

The processing algorithm behind the NANOLYSER was based on a general procedure for

plasmonic ELISA [6]. The first step is to get the blood sample into the device. This is achieved by

way of a capillary tube which is inserted into the blood inlet well. At this point the vacuum pump

pulls the blood sample into the detection well where it is allowed to settle for one hour. Once the

hour has passed, the blood is pumped out and the standard wash procedure is performed. This

involves pumping a PBS wash buffer solution through the reaction chamber to clear out the

reagents used in the previous step. Next, 100 µL of primary antibody diluted in PBS-BSA buffer

is added to the detection well and allowed to sit for one hour. The chamber is then emptied and the

standard wash procedure performed. After the wash, 100 µL of biotinylated secondary antibody

diluted in PBS-BSA is pumped into the detection well and allowed to incubate for another hour.

Once again, the detection well is washed with the standard wash procedure. Then, 100 µL of

streptavidin-catalase conjugate in PBS-BSA is added and allowed to incubate for yet another hour.

This solution is also be pumped out after incubation and the reaction chamber washed according

to the standard wash procedure, this time followed by a wash with distilled water. Next, 100 µL

of 240 mM hydrogen peroxide buffered in 1mM 2-(n-morpholino)ethanesulfonic acid (MES) is

added to the detection well and allowed to incubate for thirty minutes. Without washing, 100 µL

of .2 mM gold (III) in 1 mM MES solution is added to the reaction chamber and allowed to sit for

an hour to see if a color change develops. Should the blood sample contain Zika Virus, the solution

will turn blue, if not it will remain red. This color change, or potential lack thereof depending on

the sample, can be observed with the naked eye through the reaction chamber which serves as a

detection well. This Algorithm can be found as Figure A1 in A1.

6.2.2. Final Design of The Physical System

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The physical NANOLYSER design incorporates multiple components, including some on

the micro and nanoscale, into a single fluid circuit. The general layout can be seen in the exploded

view shown below in Figure 3.

Figure 3: NANOLYSER SolidWorks Assembly

The fluid circuit is clearly outlined on the top layer which also shows the blood inlet and reagent

holding wells. The channels connect the wells to the reactant chamber/ detection well which is

shown on the right side and which is connected to a waste collection well which makes up the

majority of the base layer. On the far side of the middle layer there is an outlet hole which runs

through the layer to the waste well and connects it to the vacuum pump. This pump creates the

suction/ pulling force that moves the reagents through the fluid circuit by creating a pressure

gradient. The lower pressure end is at the vacuum pump meaning the reagents flow through the

channels to the reaction chamber and down into the waste well. To prevent the formation of

vacuums behind the reagents, which could stop flow completely in such small channels, the

holes used in manufacturing to insert the reagents are plugged in such a way that a tiny hole is

left, large enough to allow air in but not to let reagents out. The flow of each reagent is

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controlled by a series of bubble gates which stop all of them flowing to the reaction chamber and

waste well the second the pump is turned on. The reaction chamber itself is a well, created just

like the holding wells but with an added layer of antibodies on the bottom. These are what allow

the detection reactions to occur.

The NANOLYSER’s dimensions are approximately 1 inch by 1 inch by .3 inches. Each

of the wells is just large enough to hold the precise volume of reagent used in the processing

algorithm and the waste collection well is large enough to collect all the reagents. The channels

have a square cross section

6.2.3. Micro Components of Physical System

The only microscale components of this device were the bubble gates used to control the

flow of the reagents in the channels [15]. The bubble gates are operated by a tiny pneumatic system

attached to the device by way of the rectangular port on the front face as shown in Figure 3. The

port has nine openings, one for every bubble gate. Although there are ten wells that hold reagents,

one of these is the blood injection well which does not require a gate since the blood is vacuum

pumped into the reaction well as soon as it is inserted. Each of the nine openings is the connection

point for one of the nine pneumatic pumps in the pneumatic system, each one connected to a gate

by tiny channels not shown in Figure 3 due to their relatively miniscule size. The pump device is

programmed to open and close the gates for each reagent well as called for by the processing

algorithm meaning the only human interaction it requires is for setup and turning it on.

The integral component of a bubble gate is the bubble. A simple air bubble can be pushed

into and out of a region of the channel outlined by entrapment pillars which allow the reagent

fluids by, but not the bubble. By pushing the bubble into this entrapment chamber it blocks the

reagents from moving through the channel without moving itself. This position is deemed the

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closed position while the open position occurs when the bubble is retracted into the air pocket

attached to the channel as shown in blown up view of the gate mechanism, Figure 4, on the next

page.

Figure 4: SolidWorks Model Of Bubble Gate Mechanism

The pneumatic pump controls the bubble by contracting and expanding the fluid inside to move a

piston which forces the bubble into the channel or causes it recede back into the holding

chamber.

6.2.4. Nano Components of Physical System

The nanoscale components consisted of all the reagents and an antibody layer in the

reaction chamber. In the reaction chamber what essentially happens is the reagents build up from

the antibody layer as illustrated on the next page in Figure 5.

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Figure 5: Composition of Reagents in ELISA Positive Test [6]

Any virus in the blood sample will attach to the capture antibody layer, the primary

antibody will attach to the virus, the secondary antibody to the primary antibody, and the premade

streptavidin complex to the secondary antibody. Should the virus not be present, the rest of the

reagents have no place to attach to the capture antibody and therefore do not stick around in the

reaction chamber. The lack of these reagents means that the color change does not occur and

therefore the test would produce a negative result.

6.3. Ideal Operating Conditions

Since it was made for use in third world countries, the NANOLYSER is a rather versatile

device and can operate under a wide range of conditions. As with most microscale devices, it

ideally works best in controlled environments, such as in a lab setting. This being said, it works

perfectly well in moderately hot and cold environments, humid or dry air, and a slew of other

conditions due mainly to the fact that the fluid circuit is nearly completely sealed off from its

surroundings. The only significant external contaminant introduction point is the blood injection

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site and since the device is built to handle contaminants in the blood, it should be able to take care

of anything that enters with the blood with its many washing steps.

6.3.1. Safety and Ease of Disposal

None of the materials used within the NANOLYSER are harmful to humans. As blood is

involved in the process, the entire assembly should be classified as biohazardous waste

6.3.2. Accuracy (Lack of False Positives)

The device is very accurate, due mostly to its relation to an ELISA method commonly used

in standard diagnostic lab setups. While in a traditional setting the test would be run multiple times

to ensure that no false positives occur, a device that ran multiple tests could end up more costly

than most third world countries can afford. For this reason the multiple tests were replaced by

precision, the product of a completely automated pumping and gating system that runs itself with

minimal human interference. Since the pump and bubble gates, explained in the final design

section, can be programmed to move extremely precise volumes of reagent, the chance of a false

positive occurring due to human error, possible in a lab, are effectively eliminated.

To even further reduce potential misreadings of the results, this chip has once distinct

advantage over most ELISA methods. Rather than the solution turning a shade or two darker to

indicate the presence of a virus, as with most ELISA methods, this chip’s reaction well turns from

red to blue. The distinct color change allows the device to be read and understood perfectly by

visual inspection of the reaction site, which doubles as a detection site. This also means no external

device is needed to read/ interpret the results which further cuts product cost.

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6.4. Fabrication

Due to the nature of the chip’s design it must be created in at least 4 parts. The bottom layer

consists of one large well which is CNC machined from a piece of acrylic. The CNC machine is

essentially a computer guided drill bit which carves out a physical version of a 3D computer

modeled part. The CNC is also used to machine the channels and wells in a second layer of acrylic.

The channels in this layer consist of both the fluid circuit channels and the pneumatic pump

connector channels. This design was mirrored on the underside of the next layer, also machined

from acrylic, and the two were sealed together. The top consisted of another acrylic layer with

holes drilled into it to allow the insertion of all the reagents into the wells. The bottom 3 layers are

sealed together with a thin layer of epoxy leaving the wells open on the third layer. In the reaction

chamber 100 µL of .1M phosphate buffered saline (PBS) solution mixed with the capture

antibodies is allowed to seed in the chamber overnight at 4⁰ C. The well then undergoes the

standard washing procedure of filling the chamber with PBS buffer and removing it. 300 µL of

albumin from bovine serum (BSA) and BSA with a density of 1 mg/mL is then inserted into the

reaction chamber and allowed to sit for one hour. The standard wash procedure is repeated once

more and the top layer of the chip sealed.

6.5. Costs

The cost of an individual chip was determined by the cost of its materials and the cost of

labor involved in manufacturing and machining. As quantities of each material are not purchasable

in the amounts used, the values of the volumes used were estimated from the prices of purchasable

amounts. The acrylic used in one chip was estimated to be about $0.12. Most of the reagents cost

very little; the required amount of gold is about $0.24, the bovine serum less than a penny, the

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buffer about $0.75, and the hydrogen peroxide and ethanesulfonic acid costs were practically

negligible [16] [17]. Prices for the antibodies were searched for but not found. The estimated cost

of the amounts used is between three and five dollars, based on the price of other antibodies found

online, but without an actual cost this estimate is extremely rough [18]. When added up, the overall

materials cost is approximately $6.00- $7.00. This cost, however, is likely an overestimate as

buying materials in bulk for mass production should cost less, in the long run, than buying the

amounts used for these calculations. This also does not account for the price of labor since this

price could not be researched without having a machinist make it. That being said, we can assume

the cost of labor and machining would be significantly less than that of the materials used in

traditional ELISA methods since there are fewer parts and the device is much smaller than those

commonly used.

7. Summary and Conclusion

This paper presents a nano-scale chip design for use in quick point of care diagnosis of Zika

Virus. The chip will be cheap, easy to use, and will not require large amounts of sample. The chip

will use a plasmoic ELISA process to isolate and mark virus cells so a positive reading can be seen

by the naked eye. Future research that could be beneficial would be applying a similar process as

the present chip to the diagnosis of other diseases that are prominent in remote locations and hard

to detect traditionally.

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References

[1] Mcneil, Donald G., Catherine Saint Louis, and Nicholas St. "Short Answers to Hard Questions About Zika Virus." The New York Times. The New York Times, 15 Jan. 2016. Web. 29 Mar. 2016. Zika information [2] Ellis, Ralph. "Zika: Studies Strengthen Link between Virus, Birth Defects." CNN. Cable News Network, 4 Mar. 2016. Web. 29 Mar. 2016. [3] "Diagnostic Testing." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 05 Apr. 2016. Web. 07 Apr. 2016. [4] Gourinat, Ann-Claire et al. “Detection of Zika Virus in Urine.” Emerging Infectious Diseases 21.1 (2015): 84–86. PMC. Web. 11 Apr. 2016. [5] Cheng, Xuanhong, Grace Chen, and William R. Rodriguez. "Micro- and Nanotechnology for Viral Detection." Anal Bioanal Chem Analytical and Bioanalytical Chemistry 393.2 (2008): 487-501. Web. 29 Mar. 2016. [6] Rica, Roberto De La, and Molly M. Stevens. "Plasmonic ELISA for the Ultrasensitive Detection of Disease Biomarkers with the Naked Eye." Nature Nanotech Nature Nanotechnology 7.12 (2012): 821-24. Web. 29 Mar. 2016. [7] "Zika Virus Rapid Test." Biocan Diagnostics Inc. N.p., 24 Dec. 2015. Web. 11 Apr. 2016.

[8] "Zika Virus." Microbe Wiki. Kenyon College, 15 Feb. 2016. Web. 8 Apr. 2016

[9] "Diagnostic Testing." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 05 Apr. 2016. Web. 07 Apr. 2016.

[10] "Basic Principles of RT-qPCR." ThermoFisher Scientific. ThermoFisher Scientific Inc., n.d. Web. 29 Mar. 2016.

[11] "What Is Gel Electrophoresis?" What Is Gel Electrophoresis? Your Genome, n.d. Web. 08 Apr. 2016.

[12] SrushtiK. "What Is Antigen-Antibody Complex? - Interactive Biology, with Leslie Samuel." Interactive Biology with Leslie Samuel. N.p., 15 May 2012. Web. 07 Apr. 2016.

[13] "Sandwich ELISA, Highly Sensitive." ELISA Encyclopedia. Sino Biological Inc., n.d. Web. 29 Mar. 2016.

[14] Stiles, T., R. Fallon, T. Vestad, J. Oakey, D. W. M. Marr, J. Squier, and R. Jimenez. "Hydrodynamic Focusing for Vacuum-pumped Microfluidics."Microfluidics and Nanofluidics Microfluid Nanofluid 1.3 (2005): 280-83. Web. 29 Mar. 2016.

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[15] Oskooei, Ali, Milad Abolhasani, and Axel Günther. "Bubble Gate for In-plane Flow Control." Lab on a Chip Lab Chip 13.13 (2013): 2519. Web. 07 Apr. 2016.

[16] "Sigma-Aldrich: Analytical, Biology, Chemistry & Materials Science Products and Services." Sigma-Aldrich. N.p., n.d. Web. 07 Apr. 2016. [17] "Lee Biosolutions." Biologics Manufacturing and More. N.p., n.d. Web. 07 Apr. 2016. [18] "Chicken Anti-Human IgG H&L (Biotin) (ab6863)." Chicken Anti-Human Biotin (IgG H&L) (ab6863). N.p., n.d. Web. 07 Apr. 2016.

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APPENDIX A

Sample Processing Algorithm

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1. The user will insert the capillary tube filled with blood into the device 2. Activate the vacuum pump to pull the blood into the detection well 3. Wait one hour to let the virus adhere to the capture antibody 4. Pump out the blood into the waste well 5. Pump 100 µL of .1 M phosphate buffered saline (PBS) solution into the detection well

to wash away any remnants from the previous solution. 6. Pump in 100 µL of primary antibody buffered in PBS and albumin from bovine serum

(BSA) to the detection well 7. Wait one hour to let primary antibody adhere to the virus. 8. Pump out solution to the waste well 9. Repeat step 5 10. Pump in 100 µL of biotynlated secondary antibody diluted in PBS-BSA to the detection

well 11. Wait one hour to let secondary antibody adhere to the primary antibody 12. Repeat step 8 13. Repeat step 5 14. Add 100 µL streptavidin catalase conjugate in PBS-BSA to the detection well. 15. Wait one hour 16. Repeat step 8 17. Repeat step 5 18. Pump in 100 µL of distilled water to the detection well. 19. Repeat step 8 20. Pump in 100 µL of 240 mM hydrogen peroxide buffered in 1 mM 2-(n-

morpholino)ethanesulfonic acid (MES) to the detection well 21. Wait thirty minutes 22. Add 100 µL of .2 mM gold (III) in 1 mM MES solution will be added to the detection

well. 23. Wait 1 hour 24. User observes the color of the final solution through the detection lens and diagnoses

patient Total waiting time is five and a half hours. All pumping steps would cumulatively take one hour or less so diagnosis would be received in 6 and a half hours.

Figure A1: Sample Processing Algorithm

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APPENDIX B

Not in Use Yet

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APPENDIX C

Not In Use Yet

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APPENDIX D

Not in Use Yet