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Programmable Microfluidic Paper Based Diagnostics

ABSTRACT
Paper is used in chromatography since the 19th century to detect the presence of certain molecules like proteins, amino acids etc. Point of Care Diagnostics using paper has seen an exponential growth amongst the research community since the last decade. This explosion in the interest was seen after the Whitesides group demonstrated the detection of glucose and protein in 5 µL of urine sample using a device containing hydrophobic and hydrophilic paper[1]. They find applications in a variety of field like the detection of infectious diseases, hormones and metabolites in biological fluids, food borne pathogen detection and even in environmental monitoring. In this study, we have used paper for designing devices capable of performing complex biological assays. We have designed a completely mechanical switch made of paper and mylar which is analogous to an electrical diode and mosfet. A microfluidic device capable of sequencing two is designed and biological assays are conducted to prove its capability. Further, Complex devices capable of sequencing three reagents are designed and tested.
ACKNOWLEDGEMENTS
I am very grateful to my thesis director and academic advisor Dr. V Korampally for his persistent guidance and support throughout the research period. I learnt many valuable research techniques and approaches to tackle the problem which are life changing lessons. Secondly, I am grateful to Dr. J Horn of the biochemistry department at NIU. This research would not have been possible without his expertise and help in performing biological assays. I would like to thank   Dr. D Zinger and Dr. M Tahernezhadi for being the committee members and giving a helping hand throughout the research process. Lastly I would like to acknowledge my research partner Mr. T Shapiro, Mr. D Pushparajan and Dr. J Horn’s research associates D Boland and H Eom for their contribution to the project.
 
LIST OF FIGURES
Figure 1. Schematic of paper-based microfluidic channel
Figure 2  Top & cross sectional view of fully assembled device 
Figure 3 a. Symbol and schematic of the diode  b. Microscopic schematic  c. Timed sequential photographs  d. Photograph at the end of the test
Figure 4. The cricut machine side view
Figure 5. The cricut machine top view
Figure 6. The wax printer side view
Figure 7. The wax printer top view
Figure 8. The original design
Figure 9. The second design
Figure 10 The third design
Figure 11. Modification fourth
Figure 12. Use of self-adhesive laminates
Figure 13.. Modification 6
Figure 14. Final and successful design
Figure 15. Actual AutoCAD design of the final design
Figure 16 A picture of a bare PCB
Figure 17. Picture of the device after printing and heating
Figure 18. Device after assembling the switches
Figure 19. Sequential flow of liquid……………………………………………………………..27
Figure 20. Sequential flow of liquid……………………………………………………………..

Figure 21 Activation of switch 1………………………………………………………………………………………27
Figure 22 Flow of liquid through the timing channel

Figure 23 Flow of liquid through the gate for switch 2…………………………………………28 
Figure 24. Activation of switch 2………………………………………………………………..
Figure 25. Liquid dye enters the drain region……………………………………………………28
Figure 26. End of the test (Dried wells)………………………………………………………….29
Figure 27. Location of various reagents for the ALP test
Figure 28. Black precipitation at the Spot
Figure 29. Switch mechanism
Figure 30. Activation of the switch
Figure 31. Connected channels through the flaps
Figure 32. Elastic behavior of the switches
Figure 33. Operation of the Gate
Figure 34. Operation of timed ON and timed OFF switch
Figure 35. AutoCAD design for self-aligned switches
Figure 36. An array of many switches
Figure 37. Photo of a physical array of the switches
Figure 38. Side view of the physical switch array
Figure 39. Getting one switch from the array
Figure 40. A switch ready for use in the device…………………………………………………40      
Figure 41. Picture of a switch after activation
Figure 42. The ON-OFF switch
Figure 43. The ON stage of the ON-OFF switch
Figure 44. The OFF stage of the ON-OFF switch
Figure 45. Planned design for multi-reagent device
Figure 46. AutoCAD design of device  Figure 47. Picture of device after the heating stage
Figure 48. Top view of the fully assembled device
Figure 49. Side view of the fully assembled device
Figure 50. AutoCAD design of the simplified prototype
Figure 51. Fully assembled simplified multi-reagent device
Figure 52. Photo of a test after the first two switches are activated
Figure 53. Photo of the device after the test (Dried up wells)
Figure 54. Experimental plan for the Caffeine test
Figure 55. Image of switch for measuring the angle
Figure 56. Image of the switch for measuring angle in the ImageJ software
Figure 57. Graph of the activation angle measurement
 
Table of Contents
1. INTRODUCTION
1.1 Thesis overview
2. MIROFLUIDICS
2.1 What is Microfluidics ?
2.2 Biological assay
2.3 Why is paper used to perform biological assay?
2.4 The ELISA
3. LITERATURE REVIEW
4. GOAL AND OBJECTIVES OF THE RESEARCH
5. THE MICROFLUIDIC DEVICE WITH TWO REAGENTS
5.1 Construction
5.2 The original design
5.3 The second modification
5.4 The third design of the device
5.5 The fourth design of device
5.6 The fifth design of device
5.7 The sixth design of the device
5.8 The final design
5.9 Fabrication details of the device
5.10 Proof of concept
5.11 The biological assay
6. THE SWITCH DESIGN
6.1 Basic paper switch design
6.2 Hybrid switch design (For ON, OFF and ON-OFF switching capability)
6.3 Self alligned switches fabrication
7. THE ON-OFF HYBRID SWITCH DESIGN
7.1 The ON-OFF switch design
8. THE MULTI-REAGENT DESIGN
8.1  The initial planned design for multi reagent assay
8.2 Problems associated with this device
8.3 The simpified prototype
8.4 The biological assay with the simplified multi-reagent device
9. THE SWITCH ACTIVATION ANGLE CALCULATION
10. CONCLUSION
11. REFERENCES

  1. INTRODUCTION

Paper chromatography was developed in the 19th century and has been used to separate and identify mixtures, small molecules, amino acids, proteins and antibodies[2]. Since the last decade the scientific community has seen an exponential growth in the research based on Point of Care Diagnostics. This explosion in the interest was seen after the Whitesides group demonstrated the detection of glucose and protein in 5 µL of urine sample using a device containing hydrophobic and hydrophilic paper [1].  Paper based diagnostics is a subset of microfluidics in which paper like porous materials are used to create analytical devices [3].  The results of the Whitesides group was applauded by the scientific community after  their patent in 2012 [4]. The Lateral Flow Immunoassays (LFIAs) have become very important tool for a wide range of applications [5]. Other than finding applications in the home pregnancy test kits, they are finding applications in a variety of field like the detection of infectious diseases, hormones and metabolites in biological fluids, food borne pathogen detection and even in environmental monitoring. These credit for these devices to be popular goes to their simplistic construction as they rely primarily on paper as an essential substrate through which the fluid flow occurs and detection takes place. As the entire device is built from paper, these devices were extremely inexpensive, and could be easily disbursed to resource limited remote locations. These early designs, while attractive, had their shortcomings. The relatively simplistic construction of these early devices implied that the fluid flow through the devices could not be effectively regulated thereby severely limiting the scope of these devices to simple biological assays. In the current state of art, the Lateral Flow Immunoassays (LFIAs) are not able to perform complex multi-step immunodetection tests because of their inability to introduce multiple reagents in a controlled manner to the detection area autonomously. Often, biological assays, for example, ELISA require complex multi-step fluid processing steps that these devices simply could not accommodate. Further, the early lateral flow assay based diagnostic strips were primarily meant to be qualitative assays and suffered from relatively low sensitivities [5]. We at NIU, have attempted to solve this problem by inventing a novel mechanical switch which has far better efficiency compared to the current state of art. Along with, we have designed a device capable of sequencing two and three reagents allowing us to perform complex biological assays as well.

1.1 Thesis overview

This thesis is divided into 10 chapters. Chapter 1 and chapter 2 gives you a background about microfluidics and introduces you to specific terms such as an assay etc. It discusses why paper is used in diagnostics and what are its advantages compared to other techniques. Chapter 3 gives you a literature overview of the current state of art. It discusses about the various methods using which we can pattern paper and justifies why we used wax printing for patterning paper. It also talks about the need of valves or similar structure on the devices. Chapter 4 states the goal and objectives for the research. Chapter 5 discusses the various design changes we had to go through so that we could reach the final design which gave us successful test results. It gives detail knowledge about how the device is designed, what software is used, the process of fabrication & final assembly. It also discusses the biological assay that we conducted as a proof on concept. The chapter 6 is the most important chapter shedding light on the switch design. This chapter discusses the fabrication and other technical details of the switch design. It shows images of how the layers of paper, tape and mylar together form a switch. It also talks about the self-alignment of the switches so that mass production is possible. Similarly, chapter 7, gives you details about the On-Off switch & its fabrication details. Chapter 8 & 9 discusses the need of multi-reagent design and what benefits do we have with such a design. We have made calculations for finding out the activation angle of the switch and they are discussed in chapter 9. Chapter 10 concludes the thesis giving a tabular comparison for prior work done and advancements occurred through my research.

  1. MIROFLUIDICS

Let us get started with studying the basic principle and the various commonly used terms in this field.

  1. What is Microfluidics ?

Microfluidics is the science which studies the behavior of fluids through microchannels including its design and fabrication of devices that are geometrically constrained to small typically millimeter and sub-millimeter levels[6], [7]. Microfluidics is often used and described in “lab on a chip” and “organ on a chip” technology, but microfluidics, can be applied to a wide range of applications. ‘Lab on a chip’ essentially is a miniaturized version of an actual lab. It refers to devices and technologies that allow us to perform experiments and assays, requiring a lab setting, to be performed on a portable handheld device. It is gaining interest in research community because of its advantages such as Precision of the experiment, Lower Limits of detection and the ability to run multiple assays at the same time. The applications of microfluidics are in a wide range of industries such as cosmetics, pharmaceuticals, health diagnosis, flow synthesis & stoichiometry and energy.

  1. Biological assay

An assay is an investigation for assessing qualitatively and measuring quantitatively the presence, amount or functional activity of a target entity[8]. A biological assay is an analytical in vitro procedure used to detect, quantify and/or study the binding or activity of a biological molecule, such as an enzyme[9]. It involves use of external reagents which will react with the target antibody or target reagent and give us a visible signal in form of color change or precipitation or similar results. At times, the target protein is bound to some other reagent which will then react with the incoming reagent and thus indirectly prove the presence of target reagent. Certain assays show very little or negligible amount of color change or such results. Hence, signal amplifiers are used to amplify the result signal within a detectable range. As an example, in a mixture of DNA sequences, only the specific target is amplified millions of times by using DNA polymerase enzyme [9]. The detection of the result signal can be through normal eyes, other chemical methods or by using sophisticated electronic and digital equipment.

  1. Why is paper used to perform biological assay?

The use of paper in diagnosis dated long back to the early nineteenth century. After the detection of the chemical substance which we call as ‘hormone’ today during the 1890s, there was a huge growth in the research based on detection of pregnancy [10]. Scientists were able to recognize a specific hormone called ‘hcg’ which was found only in pregnant women in the 1920s [10]. Initial testing methods took about three to four days for the results to show up and as technology advanced, the time came down to about 4 hours. The first paper based pregnancy test kit was invented in the 1960s and was marketed in Europe and then in North America in the mid-1970s [11].  Since then the significance and advantages of use of paper in diagnosis has led to a growth in the research in this field. This growth saw a breakthrough advancement in critical healthcare when Martinez et.al published a paper in analytical chemistry in 2007[2]. It was proven that paper can be used for detection of various types of substances and compounds. In fact, they can also be used for forensic applications. There is a wide variety of paper that can be employed to build microfluidic devices, with compositions ranging from cellulose to glass or polymer, and each type of paper can bring different functionality depending on the applications [12]. One of the first paper diagnostic devices created was for urine analysis [13]. They can also be used for detection of toxins and pathogens. These devices work on the principle of capillary action and hence eliminated the need of external power supply. Other than just that, these devices are made of paper and are extremely cheap. A simple μPAD typically can be fabricated for < $0.01 (for the cost of the paper and patterning)[2]. Patterned paper-based devices have been developed and demonstrated for diagnostic applications. Patterning involves creating distinct well defined regions of hydrophobicity and hydrophilicity thereby confining the fluid flow through specific hydrophilic channel patterns[14] . Several techniques have been reported to enable the patterning of such channels. The prominent ones include wax printing and plasma based approaches [15]. While a significant step towards the development of next generation point of care diagnostic devices, these approaches are still limited to simple single or few steps biological assays and often are qualitative or a semi-quantitative.

2.4  The ELISA

Diagram and comparison of common ELISA formats
Figure 1 [16]. The ELISA test
The ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones [16], [17].  ELISA is a very useful tool since it can detect the presence of antigen or the presence of antibody in a sample. The procedure of ELISA results to show color change whose intensity depends on the concentration of the target.
An antigen from a specific sample is attached to the surface. A specific antibody which can bind to that antigen is applied over the surface. Generally, this antigen-antibody linking doesn’t show any visible signs of the bonding. Hence it becomes difficult to check if the binding is really done or not. As a solution, the antibody is linked to an enzyme before applying on the surface. In the final step, a substance containing the enzyme’s substrate is added. The subsequent reaction produces a signal which most commonly is a detectable color change in the substrate.

2.5  Types of ELISA [18]

  • Direct ELISA – The antigen is detected by an antibody directly conjugated to an enzyme.
  • Indirect ELISA – The detection is a two-step process. In the first stage, an unlabeled primary antibody binds to specific antigen. And in the second stage, an enzyme conjugated secondary antibody that is directed against the host of the primary is applied.
  • Sandwich ELISA – This assay requires a matched antibody pairs. Each antibody is specific for a different non-overlapping region of the antigen. In this assay, the first step is to coat the plate with a capture antibody. The analyte is then added followed by a detection antibody. The detection antibody can be either labelled or unlabeled.
  1. LITERATURE REVIEW

The use of paper in diagnostics was first shown by Martinez et.al in his research work published in 2010 [2]. They called these device as Micropads (µ-pads).
Figure 2.[2] a. Schematic of paper-based microfluidic channel
b. Photolithography on paper
c. An example of paper fabricated by photolithography
d. Wax printing on the paper
e. An exmaple of the device fabricated by wax printing
In this paper they have researched on various methods of patterning paper. The various methods reported were –

  1. Photolithography
  2. Plotting
  3. Inkjet etching
  4. Plasma etching
  5. Cutting
  6. Wax printing

The researchers have found that the photolithography wax expensive and even after researching on lowering the cost, it was still significantly larger. The best approach they reported was use of inkjet wax printer in which the ink is supplied as solid low-melting wax which is melted right before being ejected from the print head but solidifies immediately on paper. This paper was then heated so that the wax seeps through and a hydrophobic layer is formed throughout its thickness.
We will be utilizing the same technique in our research for making these devices.
Another research paper published by the same research group is about 3 dimensional micro-pads [19]. This was the first research paper in which the concept on button was introduced. The devices were programmed by a single-use ON button with the help if a stylus or a ball point pen as shown in Figure 2. Pressing the button closed the small gap between two vertically arranged microfluidic channels and thereby made a wicking connection between them. They used mylar, paper and double sided adhesive tape for fabrication of these devices. They compared these devices to the Field-programmable gate arrays (FPGA).
Figure 3  [19]a. Top & cross sectional view of fully assembled device  b. View of the device after adding 10 µl of die  c. View of the device after compressing the paper with ball point pen  d. View of the device after addding blue dye in the left channel
Later they also developed programmable micro-pads for urinalysis with which the user can run any combination of calorimetric assays. The device was tested with sample urine containing known amounts of glucose, bovine serum albumin, acetoacetate and sodium nitrite.
Another research group uses strategy of manipulating the wettability to induce a slow down of fluid flow in the channel. Noh and Phillips used different concentrations of wax applied to porous channels to increase the contact angle in the paper system and demonstrated fine control of delay times of seconds to over one hour [3], [20]–[22].  Another research group Shin et.al worked on changing the pore size to observe the change in transport of fluid flow. They actually pressed the porous material in certain regions to slow down the fluid flow. The disadvantage was that it needed very accurate and precise positioning for the region to be pressed [23]. Toley et al. demonstrated a flow tool that is based on diverting fluid into an absorbent shunt parallel to the  porous channel in order to slow the progression of the fluid front. Specifically, the capillary force and fluidic resistance of the shunt material relative to the main channel determine the fluid time delay produced by the shunt[3], [24]. By changing the shunt length, the delay could be adjusted.   Another research group created what they called as a fluidic diode. This valve allowed the flow of liquid only in direction and prevented its flow in other direction[25]. This research paper proved the capability of their device by performing the Alkaline Phosphthase (ALP) test. We simulated the same test for our two reagent device to prove its capability of the performing biological assay. Figure 3 below shows the schematic of the diode valve and times sequence photographs of their test.
Figure 4[25]  a. Symbol and schematic of the diode  b. Microscopic schematic  c. Timed sequential photographs  d. Photograph at the end of the test
Other than just these, there are a lot of research paper published in ‘Lab-on-a-chip’ and ‘Analytical Chemistry’ journal based on paper based microfluidics.

  1. GOAL AND OBJECTIVES OF THE RESEARCH

The goal of the project is to design an inexpensive device capable of performing biological assays for point of care testing. The design enables fabrication of inexpensive diagnostic devices that have the capability of performing complex biological assays under resource poor settings with minimal external user intervention. Applications of this inventions would be for bed-side diagnostics (commercial applications), diagnostics for resource poor locations (Ex, third world countries, soldiers on the field, etc), food and water safety etc.
While there has been a significant interest in the development of paper based diagnostic devices, till date, there doesn’t exist techniques that could enable accurate control of the flow of fluids through paper networks. For the performance of multi-step complex biological assays, it becomes critical to control the amount of reagent delivered to the reaction zone, incubation time and the multiple washing steps, all in a pre-programmed manner. Since these devices are targeted towards resource poor settings, it is desirable for these devices to be able to perform the assays with minimal external user intervention such that users without any formal education/training could use these devices effectively.
Current state of the art devices are incapable of achieving such a control due their passive nature. Although there has been instances on the use of active switches in paper based diagnostic devices, these switches required external power sources (example, electromagnetic switches etc) that add additional complexity as well as cost to the device manufacture/operation. Our invention, which essentially teaches the integration  of active switches into traditional paper based microfluidic networks pushes the state of the art in making paper diagnostics devices much more amenable towards a host of biological assays while keeping the cost low. These switches operate entirely based off of power derived from gravity and capillarity and/or elastic energies.

  1. THE MICROFLUIDIC DEVICE WITH TWO REAGENTS

5.1 Construction

We started with making the design of the device on AutoCAD. We used AutoCAD 2014 © in the Biomed lab for designing the device. Once the design is done, the design is then converted to pdf and printed using the wax printer. The wax printer we used was ColorQube 8570  (Figure 6,7). We used Whatman grade filter paper as the base paper for printing. The printed design had wax printed design on one side and the other side had plain filter paper. The device then heated uniformly so that the wax seeps through and makes a hydrophobic channel even on the other side of the paper. Thus we now have hydrophobic channels on both side of the paper.
In order to prevent evaporation, the device is coated with transparent laminating sheets from the top. This will also protect the device from accidental contamination. We used Cricut Explore Air (Figure 4,5) for cutting the laminating in appropriate shapes.

5.2 The original design

The first design was made entirely of paper cut from the cricut machine as shown in Figure 8. This design had issues with shorting and leakage in the circuit. The two channels got shorted and the reagents would possibly mix with each other before the required time or before passing through the timing channel. This was seen during the portions of the timing channel when the paper was only a few millimeters apart. Also the timing channels were very thick so the test would take several hours to perform. This draft required a heat based laminate to insert the cut pieces of paper (later designed as switches) into as the device design. This design had various flaws and the process was time consuming.

Figure 8. The original design

5.3 The second modification

In order to reduce the time required for the process, the thickness of the channels was reduced. Also increasing the distance between two channels would prevent shorting between two channels. After reducing the thickness of the timing channel and increasing the distance between the channels, the final triangular section was increased. The thickness reduction of the timing channel reduced the amount of time it took for the experiment to be completed; the increase of the triangular section also complimented the reduction of time necessary. The spacing of the timing channels also helped reduce the potential for leakage.
Figure 9. The second design

5.4 The third design of the device

This design was meant to miniaturize the device so the time it took to complete the experiment would be significantly reduced, it was quickly discarded because of serious issues with leakage.
Figure 10 The third design

5.5 The fourth design of device

This was the first draft in which instead of cutting the pieces of paper out into three different components, a printed version is made so the only thing that needs to be done is print the device using a wax printer and then heating up the device to create hydrophobic surfaces replacing the need to cut out the paper individually and speeding up production time. This design also reduced the amount of leakage the device would cause from the previous method. The switches were also redesigned so they are easily installed and self aligning.
Figure 11. Modification fourth

5.6 The fifth design of device

After some testing, the device showed that it was a reliable design, yet speed was still a factoring issue. This revision was very similar to the previous draft, but the heat-based laminate (heat seals) was switched to an adhesive based laminate. The new laminate increased the time again and also resolved more issues with the leakage through the middle of the device.
Figure 12. Use of self-adhesive laminates

5.7 The sixth design of the device

To increase time again as well as minimizing leakage, the timing channels were decreased in thickness, but unfortunately the result actually made the liquid travel slower. Also the triangular portion of the device was increased to a larger size to absorb as much waste as possible at the end of the experiment. Also the switches began development to look appealing as well and not just placed on top.
Figure 13. Modification 6

5.8 The final design

The latest design consists a fully optimized thickness for the timing channel, the switches are neatly tucked away to look appealing. Also there is minimal leakage as long as it is not oversaturated with liquid and the switches were handled properly. This device has been optimized so there will be no shorting, leakage, a quick result, fast and easy to manufacture as well. This device doesn’t use the heat seals and thus the thickness of the overall u-pad is also reduced (since the heat seals were twice as thick as the self adhesive laminating sheets). Also, this design features fully pre-aligned switches which are easy to manufacture and assemble. This also gives a commercial up step to the design.
Figure 14. Final and successful design
Technical Specifications of current design-
The dimensional specifications of the u-pad are as follows-

  • Total length of the u-pad is  2.5 inch
  • Total width is 1 inch
  • Radius of each reagent well is 0.1 inch
  • Thickness of all timing channels is 0.06 inch

Figure 15. Actual AutoCAD design of the final design

5.9 Fabrication details of the device

The design of the device is essentially very simple to serve the goal. It is made completely of filter paper and laminating sheets and is modelled on the way modern PCBs works. A printed circuit board (PCB) mechanically supports and electrically connects electronic components using conductive tracks, pads and other features etched from copper sheets laminated onto a non-conductive substrate. A bare PCB is a one, onto which holes are drilled in a patterned manner & commercialized. The end user then attaches components such as resistor, capacitor, diodes etc. depending on the circuit design and the requirements in some of these holes. This design gives multi-functionality & flexibility to the circuit design. Our design approach reflects this approach. At this stage, we have holes punched onto the device on which the end user can install 2 switches which are analogous to the passive devices like R, L, C.
Figure 16 A picture of a bare PCB
The first step to fabricate the device is to draft its design in AutoCAD. AutoCAD is a drafting tool which can be used to design various industrial machine & equipment models with precise accuracy and repeatability. Our device is designed in AutoCAD with the scale set at 1:1. This makes the fabrication very easier since the exact dimensions of the real device are replicated on the software. Along with that, being the device smaller, we were able to make multiple copies of the same device in a single sheet of paper. This also makes the device commercially more viable since mass production becomes easier. Once the design is completed, the device patterns are printed using a wax printed on a filter paper(Whatman, grade).  To form the fluidic channels, the printed devices are subjected to a heating step that melts the wax in the regions corresponding to the printed regions. Regions not containing the wax remain hydrophilic thereby providing fluidic paths to the aqueous solutions. At this stage, the device looks as shown in Figure 17Once the fluidic paths have been formed on the paper, holes are then punched into the paper where the pre-fabricated switches are to be installed.
Figure 17. Picture of the device after printing and heating
The holes are made at the spots where the switches touch the device and from where they are actuated (the incoming channel). The incoming channel is connected to the actuation spot of the device using the gate as mentioned earlier. Hence, a total of 8 holes are made in the entire device, 2 for each of the two switches and 2 for each of the two gates.
At this stage, the device looks as shown in Figure 18.
Figure 18. Device after assembling the switches
Once we have the device ready, it can be used for testing and for conducting biological assays.

5.10 Proof of concept

To prove that the device works, we conducted a several tests with food dyes. The flow of food dyes simulate how the device will work.
Figures 19-26shows the flow of liquid through the channels at various times. The top part of every image is the top view while the part below is the side view of the device.
Figure 19. Sequential flow of liquid    Figure 20. Sequential flow of liquid
Figure 21 Activation of switch 1  Figure 22 Flow of liquid through the timing channel
Figure 23 Flow of liquid through the gate for switch 2 Figure 24. Activation of switch 2
Figure 25. Liquid dye enters the drain region  Figure 26. End of the test (Dried wells)
As shown in Figure 19blue and red colored dyes are pipetted in the two wells. The dyes start flowing through the channels as depicted in Figure 20. After a specific time, the blue dye flows through the gate and activates switch 1. Thus there is a connection between switch 1 and the channel to the right as visible in the side view image of Figure 21. The liquid then starts flowing through the timing channel and travels all the way through to activate the second switch. As seen in Figure 24, the liquid activates the second switch and thus a connection with the drain is established. Since the pressure in the drain at this point of time is minimum, all the fluid start flowing to the drain. This is visible from Figure 25.The process continues till all the liquid in both the wells move into the drain region. This can be confirmed from Figure 26.

5.11 The biological assay

Proven that the device is capable of sequentially loading two fluids with the use of completely mechanical switches, we can now conduct a simple biological assay to detect alkaline phosphathase (ALP) [8]. 2 µL solution of ALP is pipetted on the device at the spot as shown in Figure 27.The ALP was allowed to dry at room temperature for about one hour. A 40 µL enzyme substrate solution of NBT/BCIP (20 mg l-1)was pipetted in the second well. NBT/BCIP substrate was used because it gives a black precipitate on reacting with ALP. A 40 µL solution of 2 molar NaOH was used as a stop buffer which would stop the further precipitation of the ALP. This solution of NaOH was deposited in the first well. The solvent used for all the solutions for the experiment was DI water.
As soon as the enzyme came in contact with the ALP, the black precipitate started to appear near the spot as shown in Figure 28, indicating that the reaction has started. The stop buffer in the left activated the first switch and thus made a short with the ongoing reaction between the ALP and the enzyme. The stop buffer stopped the reaction and thus prevented further precipitation and thereby blackening at the spot.
Figure 27. Location of various reagents for the ALP test
Figure 28. Black precipitation at the Spot

  1. THE SWITCH DESIGN

The novelty of our design comes from the simplistic design of using a single layered paper & the fabrication and integration of active switches into paper based diagnostic networks that function without the requirement of any additional external power sources. These switches are capillary powered and derive power from elastic energy sources and could be configured to be both On/Off switches (valves) such that fluid flow within a paper based microfluidic network could be accurately controlled.
To the best of our knowledge, such simple microlfuidic paper based switches that could be configured to be under multiple states (for instance OFF  ON  OFF) at set time intervals has not been realized before. Such switches offer unique capabilities to fine tune control of fluid flow through microfluidic networks in paper. Further, these switches could also be used to serve as  time delays  in delaying the fluid flow through a given region. All of these features of the switches makes them very attractive from the point of view of fabricating inexpensive paper based diagnostic devices.
Another important aspect of the design is that the entire microfluidic device structure is completely modular in design. Each component of the device, including the microfluidic switches, may be separately realized and later integrated into the final structure making this approach highly modular, and facilitating easily scaled up production of these devices.

6.1 Basic paper switch design

The basis of this switch design relies on the intrinsic properties of paper. Paper is made of cellulose fibers that are hydrophilic and have a natural tendency to absorb moisture and as a consequence, undergo volume expansion. Paper strips when mechanically bent about an axis essentially strains the cellulose fibers at the bent region. When this paper strip featuring the bent section is exposed to water, water infiltration (through capillary action) through the fibrous network and the accompanying volume expansion of each individual fiber, particularly at the bent section, tend to actuate the bent paper flap back towards it natural (unbent) configuration. The flap rotates about the bent line towards the normal, unstrained position; the extent of actuation depends on the initial angle of the switch, the force applied for bending the paper as well as the thickness and density of the paper material. By choosing the appropriate thickness, the actuation angle may be controlled such that the folded paper flap actuates to near horizontal configuration. Figure 29 illustrates this mechanism.
Timed ON-switches may thus be realized through the incorporation of two of these switches along the path of a microfluidic channel defined in paper as illustrated in Figure 30. As shown below, when a sample is deposited on each side of the switch, the flap starts to attain its original position and the this rate is directly proportional to the fluid front on the flap.
Figure 31 shows the schematic of a switch mechanism that mimics the electrical analog of transistor operation. In this illustration fluid control is desired such that sequential delivery of fluid may occur to the sink region (region C). Here the fluid A should reach the drain, denoted by C; followed by fluid B. Incorporation of time delays here is simply accomplished either by changing the path length of the fluid that each individual channel has to tranverse before reaching the sink region or by varying the dimensions of each individual channel guided by the classic Washburn’s equation to accurately control the timing of fluidic delivery.
Figure 29. Switch mechanism
Figure 30. Activation of the switch
Figure 31. Connected channels through the flaps

6.2 Hybrid switch design (For ON, OFF and ON-OFF switching capability)

More functional forms of switches that may be seamlessly intergrated into the final paper based microfluidic device is that incorporating both the basic paper switches described earlier as well as switches made of plastic (mylar) strips. Here, we rely on the intrinsic properties of the mylar transparency strip for the realization of these switches. Akin to paper, mylar strips when bent about an axis undergoes a plastic deformation, slightly expanding it at the bent region. The bent mylar strip consequently assumes the new bent configuration. When the flap is deformed to its original position through the application of an external force, the removal of this force quickly restores the flap to its bent position. The strip follows elastic deformation in this case as the applied external force on the flap does not lead to any new permanent elongation or contraction of the mylar material at the bent region. This behavior, will be exploited to realize a wide variety of switching configurations in the paper networks. Figure 32 shows these configurations.  This behavior, will be exploited to realize a wide variety of switching configurations in the paper networks.
Figure 32. Elastic behavior of the switches
In order to activate the switch, a small piece of filter paper connecting the channel to the switch is used. This piece of paper resembles the entry point of the liquid to the switch mechanism and hence is called as ‘Gate’. The thickness of the gate is equal to that of the channel and is made of the same filter paper. This is shown in Figure. 33.
Figure 33. Operation of the Gate
As the fluid front flows through the gate, the adhesive strength of the double sided tape reduces due to the presence of water. This leads to the springing back of the flap to its bent position. Figure 34 – a, illustrates the operation of a Type 1 – “timed OFF” switches incorporated within the microfluidic network based on this working principle. Here, the paper switch (paper strip) is initially bridging the channels A and B. Fluidic disconnection between the channels occur when the mylar switch activates, disconnecting the bridging paper strip from contacting the channel B thereby disconnecting the flow between the two channels.
Figure 34 – b illustrates the operation of a Type 2 “timed ON” switches. Here, in the initial configuration, channel A and Channel B are disconnected from each other due to the lack of a connecting fluidic path. The switching configuration uses one paper switch (on the channel A side which is OPEN initially) and a paper strip on the channel B side overlying on the mylar switch. As the fluidic front reaches the end of the channel A, paper switch (1) activates, actuating the flap towards the horizontal position, stopping short of fully contacting the channel B; the final resting position being a function of the thickness of the paper, making up the switch. The time ‘ON’ operation occurs with the activation of the mylar switch, which would essentially raise the horizontally lying paper strip (2) to contact the switch (1) thereby establishing a continuous fluidic path from channel A to channel B.
Figure 34 – c illustrates the operation of a Type 3 “timed ON – Timed OFF” switches. In this configuration, initially channel A and channel B are disconnected from each other. The ON operation of this structure mimics the ON switch detailed above. With the incorporation of an additional Mylar switch underneath the paper switch (1), the switching behavior may be configured to also achieve the timed OFF operation, the OFF timing provided by the second mylar switch. Thus, one can obtain both a timed-ON operation followed by a timed OFF operation to control the fluid flow from one channel to the other. This flexibility is especially important from the view point of automation of the assays wherein often times, an accurate timing control is needed to control the amount of reagent delivered to the detection zone from individual channels of the device; each channel in-turn holding a particular reagent in its dried form.
Figure 34. Operation of timed ON and timed OFF switch

6.3 Self alligned switches fabrication

To facilitate modularity in the design, switches are fabricated separately and installed into the pre-printed paper fluidic network, akin to installing integrated circuit chips onto a printed circuit board.  These individual switch modules, because of the way these are fabricated, come pre-aligned to facilitate reliable operation during switching.
The switches and the supports are fabricated as usual as per the process described below. A new sheet of mylar is taken and these switches and supports are pasted over it. This mylar sheet is printed with the exact dimensions showing where to stick the switch and the flap or support. Also, a small gap of about 4 mm for the gate is also present on the mylar sheet thereby showing the user exactly where the switch will fit on the device. The left side is where the supports are pasted and the switches are pasted. The gap in between the switch and support shown in yellow ink is used for the gate. Thus with a single printed mylar sheet, many switches can be made easily making mass production easier.
Figure 35. AutoCAD design for self-aligned switches
Figure 36. An array of many switches
The use of this mylar sheet makes the job tremendously easier and makes it commercially more viable. The image below shows the actual realisation of how a printed strip of mylar sheet for the self alligned switch looks like.
Figure 37. Photo of a physical array of the switches
Figure 38. Side view of the physical switch array
This strip is then cut into small switches of proper thickness and gates are installed in the designated area.
Figure 39. Getting one switch from the array
Figure 40. A switch ready for use in the device       Figure 41. Picture of a switch after activation

  1. THE ON-OFF HYBRID SWITCH DESIGN

As the research proceeded, we were very acquinted with the ON switches. They were easy to assemble and could be fabricated quickly and in bulk quantities. But the problem associated with these ON switches was their inabilty to turn OFF after delivering the liquid to its destination. Hence a mechanism was needed to turn the switch OFF within a certain time of it being turned ON. This resulted in the design of the ON-OFF switch. These switches had the capability to turn off the flow of liquid after a certain time. This time again can be controlled by changing the length and thickness of the channel.

7.1 The ON-OFF switch design

The design for the ON-OFF switch is based on similar grounds as the regular ON switch. We used a sheet of mylar as a base to the switch and coated it with double sided tape. Instead of using the support flaps, two strpis of switch mylar design were utilised. A new design for base mylar on which the switches are assembled was made. Essentially the ON-OFF switch is the realization of two switch flaps stuck opposite to each other. The difference is that one of the switch flap will function as the support flap in the initial stage which will then work as the OFF switch in later stage. The Figure 42. below shows the ON-OFF switch.
The Figure 43. shows the first stage in which the switch is turned ON. The Fig shows the second stage in which the switch is turned OFF.
Thus by changing the length of the channel carrying the liquid for ON stage and OFF stage, we can change the time at which both the stages are activated.
Figure 42. The ON-OFF switch
Figure 43. The ON stage of the ON-OFF switch
Figure 44. The OFF stage of the ON-OFF switch

  1. THE MULTI-REAGENT DESIGN

After the successful test of the device capable of sequencing two reagents, we moved a step forward towards researching on a multiple reagent holding device.  The need for multi-reagent device arises from the necessity to perform complex assays that require more steps to be performed. Such assays generally feature a wash step in which a buffer washes away excess of the reagent so that the incoming enzyme can react properly with the susbtrate. Although the 2 reagent device can perform various biological assays, the number of reagents it can sequence allowed hinders its use for complex assays.

8.1  The initial planned design for multi reagent assay

We had rough drafts for sequencing 3 and 5 reagents simultaneously. Since we already had a design for ON-OFF switch, we incorporated the ON-OFF switch into the design for multi-reagent device.
Hence we started with the AutoCAD design for these devices. The AutoCAD design of the device as is shown in Figure 46.
Once the device is printed using the wax paper, we heat it up uniformly so that the wax seeps through and forms a hydrophobic layer even on the other side.
The Figure 47 below shows how the device looks after the heating stage is done. After this stage the device is now ready for final assembly of switches and then the testing.
A complete device with the switches assembled is as shown below in Figure 48.
Figure 45. AutoCAD design of device
Figure 46. Picture of device after the heating stage
Figure 47. Top view of the fully assembled device
Figure 48. Side view of the fully assembled device

8.2 Problems associated with this device

After the initial few tests with this device, we found certain problems in the design of this device. The problems are as listed below –

  • 8 switches were used in a single device and hence the device took long time for fabrication.
  • Because of presence of more switches in a very compact area, the probability of leakage was high.
  • There were some issues with the OFF stage of the ON-OFF switch which needed to be addressed before incorporation into an actual device.

Hence we simplified the device to work with 3 reagents and designed it in a modular way such that with little or no change, the device can be used for 4 or more reagents.

8.3 The simpified prototype

We designed a simple device capable of sequencing 3 reagents without any complexity. The new design featured the use of 3 reagents and was thus easier to fabricate. Alongwith, it used only 3 switches and thus the possibility of leakage was low. Nevertheless with the use of double sided tape, the leakage issue was very well addressed and was not observed in later devices.
The Figure 49 below shows the simplified prototype. The Figure 50 shows a fully assembled device ready to be tested.
Figure 49. AutoCAD design of the simplified prototype
Figure 50. Fully assembled simplified multi-reagent device
After building the prototype, we tested the device for proper sequencing. The Figure 51, 52 below shows a screenshot of the actual test.
Figure 51. Photo of a test after the first two switches are activated
Figure 52. Photo of the device after the test (Dried up wells)

8.4 The biological assay with the simplified multi-reagent device

We have collaborated with the department of biochemistry at NIU for conducting the caffeine test on our device. This test will prove that the device is capable of performing biological assays.
Caffeine test is a classic example of use of paper based device for detecting the presence of caffeine in any sample. We will be using nitroccellulose paper sotted with caffeine molecules. This site will be our actual detection area where we will observe signal change. We will be using Rhizavidin AP as the enzyme and PNPP as the substrate. The reaction between these two give a signal change from colorless to yellow color.
Rhizavidin AP has a property to attach with caffeine molecules. Hence the first well will have Rhizavidin AP which will travel and get attached with caffeine. The excess of Rhizavidin AP molecules will have to be washed away and hence a wash step in necessary. We will be utilising PBS- tween for washing the excess Rhizavidin AP. This will ensure that the excess molecules are washed away or are rendered inactive and only the molecules combined with caffeine will be stay on the surface.
In the last well, we will put our substrate PNPP which will combine with the Rhizavidin AP stuck to caffeine molecules and show a signal of rendering colorless to yellow. The experimental plan for the Caffeine test is as shown in Figure 53.
Figure 53. Experimental plan for the Caffeine test

  1. THE SWITCH ACTIVATION ANGLE CALCULATION

We also worked on the finding out the exact activation angle of the switch. This data is rather useful for packaging purposes and to make sure that the supports are at a reachable angle for the switches.
We used ImageJ © software to measure the angle of activation for the switch. We took data from 14 switches from successful tests.
Pictures of each of the switches were taken so that parallax can be avoided. These pictures were then processed in the software and the activation angle was found out. Figure 54 and 55 are the screesnhots of the actual software use for measuring the activation angle.
Figure 54. Image of switch for measuring the angle
Figure 55. Image of the switch for measuring angle in the ImageJ software
The data is tabulated in the table 1 –
Table 1
Data showing the activation angle for the switches

Switch No. Activation Angle (in degrees)
1 23.66
2 28.33
3 24.73
4 25.1
5 28.81
7 25.38
8 25.46
9 26.57
10 22.11
12 22.7
13 26.57
14 26.57
Average Activation angle 25.499


Figure 56. Graph of the activation angle measurement
Thus from the graph it is visible that average activation angle is about 25.50 degrees. Also, from this we can calculate the optimum angle that is required by the supports to maintain.
25.5 + 90 + (x) = 180 degrees
X = 64.5 degrees
Thus if measured in anticlockwise direction, the optimum angle that a support flap should maintain is (180 – 64.5) = 115.5 degrees.
Maintaining this angle will ensure that the switch will touch the support flap after activation.

  1. CONCLUSION

In this study, we explored how paper has found a way in the point of care diagnosis and in other biological assays. We learnt how paper can be  patterned to make hydrophobic and hydrophilic channels and thus sequence the flow of liquids. We have studied diffferent ways in which paper can be patterned and chosen the simplest and most inexpensive one. The benefits of using paper over the other conventional methods of diagnosis are low cost, ability to operate without any external power supply, high throughput & easily accessibility to resource poor settings.
We have designed a device capable of sequencing two different liquids in a controlled manner. The device employes mechanical switches which are capable of allowing or stopping the flow of liquid. Mass production of switches is possible with the help of self-aligned switches.These switches act like a passive electrical components and can be used as per requirement. In order to prove its capabilities, we performed the ALP test and thereby showed that it can perform biological assays. The device was designed keeping a multi-functional approach similar to a PCB so that the user will employ specific switches based on the assay.
We also designed a larger device capable of sequencing three reagents at a time. This device still needs some optimization but the main blue print is ready. The On-Off switches are optimized and the self aligned On-Off switches are designed for mass production. The caffeine test would be performed to prove the biological capabilities of this device and we have an experimental plan setup.
We have our design specifications backed by evidences from the capillary test. We have also perfomed the switch angle measurement test to find out the average value of activation angle. The advancements in the field due to this study is summarized in the following table –
Table 2.   Critical Evaluation of Prior work in relation to the proposed work

Ref. No Prior work Pros and Cons of Prior Work Significance and advantages of proposed work
[25] Use of chemical valves and surfactant based diodes to allow or stop flow of liquids. Use of completely mechanical switches. Mechanical switches do not fail as often as their previous counterparts did. 
Uses only the elastic energy of the mylar & no external entity.
[26] Design had a large envelope. Design is completely planar & is made up of single layer of paper. Easy to use and package. Has no moving parts and hence can be transported easily.
[27] Required user’s interference after the test has begun. Our device has automated switches and flow of liquids through the channels. Requires no attention from the user end until the test in complete & hence makes it feasible for use even for unskilled professionals.
[27] The chemical switches might interact with the reagent themselves. We use mylar based mechanical switches. Due to absence of any chemical or biological molecule, they do not interfere with the biological assay unlike the chemical based ones.
[28] Use of selective wetting to raise or lower the actuator’s tip for allowing the flow of liquid. Use of an activation channel through which the switches are activated automatically. Selective activation might face difficulties such as improper location for pippetting. With the use of our design, this problem is mitigated. Use of our switch design will guarantee the flow of fluid to be engaged or broken.
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