Team:Queens Canada/Device-Design
Device Design
Description
When testing ticks for Lyme disease, a challenge that the team encountered was to safely remove the innards for the ticks and apply them to a sample pad of the Lateral Flow Immunoassay (LFIA). Ticks have notoriously tough outer skin due to the presence of chitin within their exoskeletons which is responsible for protecting the ticks from punctures or squishes. The Lyme disease carrier ticks that we are targeting, the black-legged ticks of North America, are incredibly small. When unfed, they are approximately 3mm in length as can be observed in Figure 1 below.
Due to their composition and small size, an array of solid, stainless-steel spikes the size of microneedles (150m in height and a tip diameter of 90m), can puncture the tick when arranged on the bottom of a mortar. A lysis buffer would then be applied to the crushed tick to dissolve the released midguts and lyse the bacteria Borrelia Burgdorferi. This lysed aqueous solution is then applied to the LFIA. The LFIA employs a competitive design meaning that positive results will be indicated by the absence of the test line, whereas a negative test will be indicated by a line. The process and design of the LFIA are detailed here Assay Design.
Functionality
To employ our synthetic protein (developed in-situ), the dry lab has developed a low-cost, easy, and reusable kit to determine the presence of Lyme disease within a captured tick. The kit is comprised of a lateral flow assay in conjugation with an extraction and lysis buffer system. The various components are modelled using Solid Edge below.
Top Left: Complete device. Top Center: Exploded complete device. Top Right: Exploded bottom part of the device that houses the assay. Bottom Left: Stainless steel microspikes embedded onto the mortar. Bottom Right: Lysis application syringe, with and without the needle component.
To use the device, the following steps must be followed:
- Remove the pestle from its chamber and place the tick into its chamber. Puncture the tick' hard exoskeleton with the pestle, embedded with solid spikes the size of microneedles to release the midguts into the chamber.
- Once the tick is sufficiently crushed and its innards released, inject the provided syringe that is pre-loaded with the lysis buffer solution into the well containing the tick innards.
- Once sufficient time has passed for the proteins to be lysed, screw the provided needle onto the top of the syringe to ease the removal of the lysed tick mid-gut solution from the tick chamber.
- Attach the needle to the syringe to extract the solution from the chamber.
- Apply the solution to the remaining chamber, where it will be applied to the sample pad of the lateral flow assay.
- The solution will move through the assay and determine the presence of the target bacterium. The details of the assay are shown in Assay Design and Chemical Composition
- A positive test will be indicated by a singular strip on the control line, whereas a negative test will be indicated by two lines; one at the control strip and the other at the test strip.
The chemical composition and design details for the LFIA can be observed on the Assay Design pages.
Required Applied Force
To ensure the feasibility of the design, it must be confirmed that the spikes can puncture the tough outer shell of the tick to remove the sample.
Before determining the required applied force, the resting force exerted by the pestle through the microspikes is calculated to determine if it can puncture the tick’s outer shell without the use of applied force.
When the pestle is at rest on top of the skin, the only two forces at are gravity and the normal force. Before the force exerted by the pestle can be calculated, it’s mass must first be determined. The microneedles and the syringe will be comprised of stainless steel which has a density of 7,200 kg/m3 [1]. Due to the size of the microneedles in comparison to that of the pestle, their weight can be assumed to be negligible. Negating the mass of the microneedles, the total mass is determined to be 0.0168 kg. The force exerted can be calculated using the following formula:
∑F = ma (1)
Since the object is at rest, the sum of force is equal to 0. Therefore, the normal force can be calculated using the following equations:
∑F = 0
FN = Fg
FN = mg
FN = (0.01685kg)(9.81m/s2)
This force can now be used to calculate the applied pressure which can be compared to the tensile strength of chitin. Tensile strength is defined as the ‘‘maximum load that a material can support without fracture when being stretched, divided by the original cross-sectional area of the material.’’ [2]. Therefore, if the applied pressure were to exceed that, it would mean the spikes would be able to break through the surface. The values related to the tensile strength of chitin are used for this estimation since it is a primary component of the tick’s exoskeleton and provides the hard and tough characteristics of the exoskeleton [3].
Before the pressure can be calculated, first the surface area the force is applied over must be calculated. The surface area of the tip of one spike with a tip diameter of 90μm can be calculated to be 6.362x10-9 m2. [4]. With multiple tips, the needles in contact with the surface can be estimated using the following equations:
The surface area (SA) of the tick can be calculated utilizing the Knud Thomsen formula [5].
By using the approximate dimensions of an unfed black-legged tick, the surface area is calculated as followed:
SA = 7.7mm2 = 0.077cm2
The spikes can be manufactured in densities of 400-700 needles/cm2 [4]. Taking the average density of 550 spikes/cm2, the average number of spikes (S) in contact with the surface of a tick can be calculated using the following equation:
SA = 43S x (6.362 x 10-2m2) / S
SA = 2.7 x 10-7m2
The pressure that the resting pestle exerts is calculated as followed:
P = F/A
P = 0.165N / 2.7 x 10-7m2
P = 603,145Pa = 0.603MPa
Since the pressure due to gravity is less than that of the tensile strength of chitin (250 MPa), the spikes are unable to puncture the tick without any applied pressure [6]. Therefore, the required applied pressure to overcome the tensile strength can be back-calculated as followed:
F > P x A
F > 2.50 x 108Pa x 2.7 x 10-7m2
F > 67.5N
Subtracting the force due to gravity gives an applied force of 67.3 N. Therefore, to puncture the tick, a force greater than 67.5 N must be applied. This is feasible as a study proved on average that a human can apply 107.7 N ± 30.68 with their thumb and 56.7 N ± 12.62 with their index finger individually [6]. Since the calculated value falls within the standard deviations, it can be assumed to be an accurate and easily applied force for an average user.
Physical Casing
Once the iterations for the casing prototype were completed as detailed on the CAD Modelling page, the majority of the components for the fifth and final iteration were printed using a 3D printer. Components comprised of non-printable materials (such as stainless steel) or that were too small (the spikes) were not feasible to print. For the purpose of the prototype, the pestle was printed but when manufactured industrially, it will be comprised of stainless steel.
The process of 3D printing was an iterative experience, similar to the design process. It was a continuous learning experience with small adjustments having to be made to the design file and final pieces. For example, the team learned that when printing a design, it was important to take into account tolerance to ensure that the pieces would interact with one another as intended. When printing, we often made one component slightly bigger or smaller to ensure that the piece would fit. The difference in dimensions is dependent if a snug fit or a smooth glide is desired. Another lesson we learned is that it is best to print rudimentary designs of your components in order to optimize tolerances and ensure proper fit/design before printing the final detailed components. This is an effective way to save time, materials and ensure components will print properly. Additionally, if there was an issue with the printed pieces, due to the soft nature of the plastic material used, they were also easily sanded down until the proper fit was attained.
Pictures of the completed casing are shown below:
References
1. Materials Services Materials UL, "Density of Stainless Steel," thyssenkrupp, 2018. [Online]. Available: https://www.thyssenkrupp-materials.co.uk/density-of-stainless-steel. [Accessed 16 June 2021].
2. The Editors of Encyclopaedia Britannica, "Tensile Strength," Britannica, 11 March 2020. [Online]. Available: https://www.britannica.com/science/tensile-strength. [Accessed 16 June 2021].
3. P. Junquera, "The Biology of Ticks.," Parasitipedia.net, 22 May 2021. [Online]. Available: https://parasitipedia.net/index.php?option=com_content&view=article&id=2530&Itemid=2822. [Accessed 16 June 2021].
4. Micropoint, "Micropoint Patch," Micropoint, 2019. [Online]. Available: https://micropoint-tech.com/products/micropoint-patch/. [Accessed 15 June 2021].
5. P. C. Flynn and W. Reubem Kaufman, "Female ixodid ticks grow endocuticle during the rapid phase of engorgement," Exp Appl Acarol, vol. 53, pp. 167-178, 2011.
6. J. F. Vincent and U. G. Wegst, "Design and mechanical properties of insect cuticle," Arthropod Structure & Development, vol. 33, pp. 187-199, 2004.
7. K. János Bretz and Á. Jobbágy, "Force measurement of hand and fingers," Biomechanica Hungarica, vol. 111, pp. 61-66, 2010.