Team:Leiden/Hardware
<!DOCTYPE html>
Rapidemic
Hardware
Our project aims to create a point-of-care tool for the detection of specific nucleic acid sequences. We envisioned this tool to ideally be a functional biochemical reaction housed in a user-friendly interface. This project would thus include the development of the reactions as well as the hardware. While our lab experiments focus on the development of biochemical reactions, our hardware design attempt aims to conceptualize the hardware of our point-of-care detection tool.
Introduction
The hardware serves as a platform for the biochemical reactions to take place. In our case, the hardware should therefore provide compartments for recombinase polymerase amplification (RPA), linear strand-displacement amplification (LSDA), and TMB oxidation reactions to occur. Moreover, it should facilitate liquid transfer from each preceding compartments to the next. As a point-of-care device, it should also serve as an interface for the user to interact with the core biochemical reactions, and vice versa. In short, it should satisfy the technical needs of our biochemical reactions while being intuitive enough for users to easily interact and gain information from.
In this section, we will discuss the conceptualization of our hardware. The study was performed mainly through literature research to discover the different options to enrich our kit design toolbox – much like an analogy to the biological ‘parts’ metaphor that has been popular in iGEM. We also had interviews with professionals in the field to learn from their experience regarding the practicalities of different kit designs as well as additional requirements of a point-of-care tool that is rarely discussed in the literature. We then reflected the information on the unique requirements of our biochemical reaction based on our lab results and proposed a preliminary design for the hardware - a prototype that we will refer to as the "minimum viable product" (Fig. 1).
Fig. 1 Preliminary hardware design.
Literature Study
At the start of our hardware design process, we performed a preliminary literature study to learn more about different materials and methods to automate the transfer of liquid from one to the next reaction. Specifically, we focused on popular techniques that were found to be compatible with RPA. Below is a short summary of the two promising techniques that we identified.
Microfluidics-based designs have been gaining a lot of attention in the field of point-of-care diagnostics. For RPA-based methods, microfluidics kits can be grouped into two general categories based on the driving force of the fluids in the device. These are centrifugal micro-devices and microchips1-3. As the name implies, centrifugal devices use the centrifugal force to drive fluid movement from one chamber to another. The device is therefore often shaped like a disk. A highly advanced design also implemented laser-controlled valves and a built-in signal reader into the device, allowing tight control of on-demand fluid movement using the centrifugal micro-device1,2. Unlike centrifugal devices, a microchip is a rather static device. Microchips often implement a vacuum to prompt liquid movement through the device. Though highly effective in providing a means to control sample flow, microfluidic devices often require an external power source to provide its driving force3. Microfluidic designs are also quite complex. Their implementation would thus create an additional challenge in manufacturing.
Unlike microfluidic devices, paper-based designs are simpler and more affordable. They rarely require an external power source since fluid movement through the device can easily be facilitated by capillary forces. However, compartmentalization and sensor integration into a paper-based design can be a challenge. Folded-paper or paper origami designs achieve kit compartmentalization through folding and sliding of the paper4,5. Nevertheless, sensor integration remained to be a challenge.
Both microfluidic and paper-based designs have their own advantages and disadvantages. Microfluidic devices often allow high-fluid controllability and implementation of supporting electronic devices into the kit. However, they can be quite complex, posing a huge challenge to design and manufacture. Paper-based devices lie on the other end of this extreme, which trades the ability to perform tight fluid control with plain simplicity. In the following section, we will assess how the two designs may fare in supporting our envisioned point-of-care device, and what would be required to optimize it into an effective diagnostic kit.
Expert interviews
Following the literature study, we interviewed some professionals in diagnostics and hardware design in the hope to gain some insights into the design of viable hardware. These interviews provided us with insights not only into the design but also the production and distribution of diagnostic devices. Here we discuss three critical aspects that we learned from these interviews.
The importance and challenges of sample extraction
Our biochemical reaction highly depends on access to genetic material of a target pathogen. Extraction is therefore an essential step to include in our diagnostic kit. However, many currently existing extraction methods are prone to interfere with the reactions of our detection method that would follow this extraction step. The implementation of extraction in a diagnostic tool is further complicated by the absence of a universal extraction buffer that can be used to effectively extract genetic material from any types of sample (blood, saliva, nasal swab) and any types of pathogen (bacteria, fungi, parasites). Mondial Diagnostics, one of our sources, pointed us to a pathogen DNA isolation kit by Molzym, an extraction kit that is applicable to a wide range of pathogens and offers the additional benefit of dismantling patient-derived DNA from the sample to remove noise and possible cross-reactivity.
The Dutch research organization Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek (TNO) pointed out the importance of implementing pathogen inactivation during the extraction step as this helps to assure the safety for our device, both during use and after its disposal.
User interface and minimizing human error
The appearance of a diagnostic device is another aspect of kit. Besides making our kit more appealing for end-users, the design should also provide clear clues as to how the test should be performed, and how the results should be interpreted. Therefore, we intend to come up with a color-coded, colorblind-proof design that will assist the users in the operation of the device. Each color will correspond to a step that has to be completed to ensure correct performance of our test and prevent human error. Limited user understanding of inherent false-positive signals is also one of the largest caveats of point-of-care diagnostics. A number of professionals thus suggested the inclusion of negative controls as a way to inform users of the occurrence of false-positive tests and the need to repeat the test with a new kit.
Minimization of human error is a very important aspect of a point-of-care tool. We were kindly guided by the IDE Group in finding strategies to approach the topic. This will be discussed in a later section: Mentorship with IDE Group.
Manufacturing, distribution, and storage
Although manufacturing, distribution, and storage lies at the very end of the kit development process, its efficiency as a long-term process is highly determined by the kit design.
The choice of material is one key aspect that determines the ease and cost of manufacturing. As we were investigating the possibility of a microfluidic device, we enquired about options for easy biodegradable options for polymers which are strong and flexible. Our investigation lead us to an interview with Prof. Aldrik Velders & Dr. Vittorio Saggiomo from Wageningen University, in which we were introduced to polydimethylsiloxane (PDMS). PDMS is a polymer that can be 3D-printed and is compatible with water-dissolved solutions6. More importantly, PDMS is highly moldable at lower temperatures, allowing it to be used to encase freeze-dried biochemical agents required for our reactions (especially RPA). However, PDMS is permeable to gases and water vapor. We would therefore need to design an airtight casing to prevent reagents from deteriorating over time as a result of gas and water vapor exposure.
Prevention of reagent deterioration is important for efficient distribution and storage. This is exceptionally true knowing the location of end-users, which are often far away from the production site. Although the final measures would depend highly on the composition of our final reagent mix, form, as well as encasement, the use of airtight casing and freeze-drying is often favorable to increase shelf-life and minimize the need of low-temperature storage.
Mentorship with IDE Group
During the project, we received mentorship from IDE Group, a company specialized in the design and commercialization of rapid diagnostic tests (RDTs). IDE Group is a home to industrial designers whose experience stems from developing a self-test kit for HIV in collaboration with Atomo Diagnostics.
Their main message was to make the kit as simple as possible to use. They found that in many cases, users find RDTs difficult to use. This causes them to make mistakes, such as adding incorrect amounts of sample (e.g., too much or too little blood). Sometimes this results in patients receiving a false-negative test result. This can be dangerous as it let users think that they are healthy and lead them to a false sense of safety in overlooking the measures required to prevent the disease that they contracted from spreading.
To help users perform the test correctly, they advised to make it very clear to the user whether a step has been completed. One of the problems they identified with existing tests was that users tend to add an incorrect amount of the buffer fluid, add the fluids to the wrong location, or add the fluids at a different time. This led IDE Group to design a blister pack filled with buffer fluid. The blister pack is then placed under the button in their kit hardware. This allows the blister pack to break and release its contents when the button is pressed. By minimizing user tasks, IDE Group successfully designed a buffer administer tool that ensures the correct amount of fluid will be added to the correct location. In one of their later designs, they also implemented a locking mechanism that prevents the button to be pressed before the completion of a required preceding task. Sequential control of fluid flow and volume is also important in our biochemical reaction. Thus, similar mechanisms may also be applicable in our kit even though specific optimization may be needed.
In the end, our mentors from the IDE Group advised us to start by creating a “minimum viable product” (MVP) – a minimum design that allows our kit to function as intended. This is because the development of a 'perfect' product (streamlined for production, user friendly, compatible with long-term shipment and storage) can take a significant amount of time and resources. It is better to look for the easiest and least expensive way to design a well-working product. This is also the route that they followed during the development of their HIV test kit. The first product did not contain blisters, since these were expensive to manufacture, and each version grew with complexity. Having a product that is simple but works helps to convince stakeholders and obtain financing for further development. Prototypes should thus begin from a simple design, and complexity and supporting functionality should be added gradually.
Minimum Viable Product
The conceptualization of our kit was rather complex since the kit consisted of multiple elements, which could be made in different forms. For example, some of the components could be packaged in liquid form, but they could also be freeze-dried. IDE Group advised us to approach this by making a matrix, where the packaging options for each element are listed. Then, we would be able to look at all the combinations in the matrix, and determine the best option to design a “minimum viable product” (MVP).
To design our kit, it was important to first gain a profound understanding of the components of our reactions. We decided to make a matrix containing all possibilities and restrains that came along with each reaction. For instance, we defined the compatibility of the reaction components. Because of the limited compatibility of DTT in RPA and the oxidation reaction, it became clear that we will have to implement a volume control step in our kit to reach the dilution that allows for optimal oxidation. Furthermore, we identified in what form the components should be integrated into our kit; some can be freeze-dried but others should remain in solution.
Table 1. Considerations for minimum viable product
| Component | Subcomponent | Storage in fluid/blister | Storage as freezedried pellet | Reaction possible on paper | Reaction possible in fluid | Not compatible with | Volume | Reaction time | Comments |
|---|---|---|---|---|---|---|---|---|---|
| Primers | Primers | Yes | Yes | Yes | Yes | - | 1-5 µL | X | Preferably added later to the kit: maybe second dripping vial with septum to separate demi water and freeze-dried powder |
| Swap and extraction | Swap, extraction buffer, patient material | No | No | Yes | Yes | Extraction buffer might inhibit subsequent reactions, not tested yet | 1-5 mL | ~5 min | In dripping vial, maybe extra volume control |
| RPA + SDA | Enzymes (pellet), buffer, MgOAc | Enzymes: no Buffer and MgOAc: yes |
Enzymes: yes Buffer and MgOAc: no |
Yes | Yes | RPA pellet contains DTT which inhibits TMB oxidation if not diluted properly (see Results page) | 10-20 µl | 25 min (see Model page) | |
| Oxidation | TMB, H2O2, KCl, hemin, pH 3.8 phosphate citrate buffer | Yes | To be investigated | Yes (seeProof Of Concept page) | Yes | TMB cannot be in contact with hemin until moment of testing | 100-1000 µL | 22 min (see Model page) | TMB is light-sensitive |
Based on this information, we created a preliminary concept of what the hardware should be - an MVP. The preliminary design can be seen in Fig. 1. Although the MVP design may still need to be streamlined and optimized, the design can serve as a platform to house the Rapidemic reactions. In the current design, we assumed that RPA and LSDA can be combined into a one-pot reaction. This prototype is envisioned to have two main reaction chambers, one sample inlet, and two buffer storage chambers. The two buffer storages will also be connected to two additional reaction chambers to accommodate for positive control reaction.
The MVP is a combination of at least devices, 1) the basic platform design, and 2) a volume controller to connect inlets and chambers within the hardware. Our studies lead us to identify microfluidics and paper-based designs for the basic platform design. The volume can be a microfluidic valve, in which case the hardware would need to have buttons for users to control the fluid movements, or a paper-based origami design, in which case a slider would be required. The selection is co-dependent as origami devices can only be implemented with paper-based designs. Nevertheless, the current design would have to be further optimized into a viable design. We are currently in discussion with the IDE group to discuss the viability of each choice and select device parts that can best fit our requirements.
References
- T.H. Kim, J. Park, C.J. Kim, Y.K. Cho. "Fully integrated lab-on-a-disc for nucleic acid analysis of food-borne pathogens" Analytical Chemistry, (2014)
- G. Choi, J.H. Jung, B.H. Park, S.J. Oh, et al. "A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria" Lab on a Chip, (2016)
- E.C. Yeh, C.C. Fu, L. Hu, R. Thakur, et al. "Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip" Science Advances, (2017)
- M.S. Cordray, R.R. Richards-Kortum. "A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA" Malaria Journal, (2015)
- L. Magro, B. Jacquelin, C. Escadafal, P. Garneret, et al. "Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics" Scientific Reports, (2017)
- McDonald J, Duffy D, Anderson J, Chiu D, Wu H, Schueller O et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis. 2000;21(1):27-40.