Team:Athens/Applied Design

Applied Design

Applied Design


GENOMERS: A Point of Care diagnostic kit for MERS-CoV

As of this August, the Center for Disease Control and Prevention of the US has declared MERS in the Arabian Peninsula as an Alert Level 2 virus. This means that travelers are advised to take enhanced precautions in order to avoid potentially spreading the virus. In our effort to strengthen the response provisions in case of an outbreak, we propose GENOMERS, a MERS-CoV Detection Kit design that could be used in Point-of-Care (POC) and emergency testing facilities. In order to successfully satisfy the needs that occur in such facilities our kit should enclose certain characteristics. We have identified and listed these characteristics into three categories according to their importance: the ones that are critical, the ones that are desirable and those that are useful. Following this prioritization, we managed to implement in our design all of the critical and several of the desirable and useful characteristics.

Image 1: Needs in POC and Field Diagnostics.

In conclusion, this diagnostic kit meets the key requirements for use in emergency situations where rapid mass testing is needed, from busy airports around the world to low-resource healthcare facilities in remote villages. Since its operation does not require specially educated personnel nor complex laboratory equipment, the test could be performed by any nurse, assisting staff or volunteer that has received a short training.

The proposed kit design is a product of an open dialogue with experts, as well as with the society. Our ultimate goal was to keep our project aligned to the real societal needs around MERS-CoV detection and rapid containment during a potential outbreak. For more details on how we implemented the feedback in our design, visit our Integrated Human Practices page .

Image 2: A 3D view of the proposed diagnostic kit.



A Top-Down Approach

Our design flow follows a top-down approach, where the overall system is depicted to consist of distinct, consecutive tasks. First of all, we describe some general characteristics of the overall system, as mentioned above, and then we continue on specifying the needs of the separate sub-systems. These needs formulate the main driving force for the design process to be followed.

Figure 1: The Biosensor System consists of several separate subsystems.

The choice of a molecular method, the toehold switch technology, as the core detection mechanism constraints the subsystem definition, in the sense that the subsystems to precede the detection shall include the following steps:

  1. Cell lysis to release viral RNA
  2. RNA isolation and purification
  3. RNA amplification

Taking into consideration our limitations the system becomes more defined as shown in Figure 2:

Figure 2: The defined subsystems of the Biosensor System.



Using the Kit

The sample is collected with a safe and non-invasive method, with which the patients are familiar: the personnel collects pharyngeal smear with an oropharyngeal swab and then inserts the swab in the kit.

Image 3: A vertical section of the kit.

The user moves the slider, that is connected to the middle layer, for each next step, at known time intervals.




Start:

  • Swab is inserted in a well containing cell lysis buffers (1).
  • 50μL of solution fall into Disc (A).


Step1:

  • 200μL RNA purification washing buffers (2) added on Disc (A).
  • Buffers are collected on wash pad (C). Cell debris are removed and RNA is bound on the paper. [1]

Step 2:

  • Amplification Reagents (3) are added on Disc (A).

Step 3:

  • Disc (A) is sealed, amplification occurs.
  • Disc (B), containing the TX-TL system with the regulatory DNA circuits, is hydrated (4). Transcription occurs.

Step 4:

  • The hydrophobic film (D) is removed, Discs A & B come in direct contact.
  • Amplified sample wicks towards cell-free translation system, trehalase is produced.

Step 5:

  • Citric acid (5) is added to lower pH levels, from the TX-TL system’s optimum pH 7 to trehalase’s optimum pH 5.5. Trehalase hydrolyzes trehalose to glucose.

End:

  • The glucose meter strip is now inserted (6).
  • The strip is in direct contact with the detection reaction product on Disc A&B and absorbs sufficient quantity of solution (0,4-0,6μL).
  • The strip is then placed in the glucose meter to quantify glucose levels.



Design Specifications

Paper Discs
In our diagnostic kit, the reactions take place on paper discs. The main benefit is that thanks to the hydrophilicity of the paper and the capillary action we eliminate the need for pumps and power for the movement of fluids. At each step, the paper discs slide into different fluidic paths and the reactions propagate. [2] Paper is also suitable for our device as it is affordable, compatible with clinical samples, captures RNA and provides a high surface area for the reactions to occur. Lastly, reagents and DNA circuits can be easily freeze-dried on paper, simplifying storage requirements. [3]

Reagent Encapsulation
The extraction buffers and the amplification reagents will be encapsulated in polymer bubbles that will break with a plastic needle that slides alongside the second layer. The content will come in contact and be absorbed by the paper disc. Given the small volumes of the reagents, this storage method is ideal as it prevents evaporation and replaces the need for pipetting or mechanical pumps and valves.

Detection Reaction
The detection reaction occurs on Paper Disc B. This disc carries the freeze-dried cell-free transcription-translation system with the DNA circuit, as well as excess trehalose. [4] Transcription of the circuit begins upon rehydration of the disc, producing toehold regulated trehalase genes. When the trigger RNA is present, the enzyme trehalase is produced. In the right pH conditions, trehalase hydrolyzes the existing trehalose to glucose, whose levels are then measured with a glucometer.

Hydrophobic Barrier
The hydrophobic barrier is a thin film that keeps the two paper discs separated so that reactions can run on both of them in parallel, without interference. [5] The one end of the film is stuck on the front side of the device so that the film falls behind after a certain step of layer two's sliding, allowing direct contact of the two paper discs. This happens at the end of the RNA amplification on Paper Disc A, where the amplified sample must wick to Paper Disc B for the detection reaction to occur.

Viral RNA Amplification
The kit is designed to detect the virus with an initial viral load down to 104 copies/mL in the sample. In order for the device to be sensitive, the viral RNA should be amplified, raising the trigger RNA sequence concentration levels. We chose to implement RPA (Recombinase Polymerase Amplification), an isothermal amplification method that operates between 37-42oC. This temperature range is favorable as the freeze-dried cell-free protein expression system works best at 37oC. Besides, this method delivers a number of copies comparable to PCR in less than 30 minutes. [6] Briefly, RPA reagents are freeze-dried on paper Disc A, to extend the shelf life of the kit in areas where refrigeration is inaccessible. The blister containing the RPA buffers breaks, the disc absorbs them and amplification occurs.

Heater
The isothermal amplification method, as well as the cell-free transcription-translation system, require a constant temperature. Both subsystems, as defined, function optimally in 37oC, therefore we propose the use of a self-regulating PTC thermistor as a heater. Positive Temperature Coefficient (PTC) thermistors are resistors whose resistance increases with temperature. Coupling this with an aluminum sheet to spread the heat and an insulating material to surround the kit, we expect a uniform temperature profile, that would need minimum power to maintain. The heating element and the power source, two small 3.7V batteries, are a cost-effective, portable and reusable solution. You can view here the models for temperature distribution and temperature dependance on heat transfer coefficient.




Control Test

Our proposed device includes a control test to further ensure its reliability.

More specifically, it is a positive control that gives the maximum value of glucose levels in the final solution. The control is similar to the detection assay, except that the riboswitches are triggered by human RNA found in the parapharyngeal sample. This way, the detected glucose levels indicate if anything during the process does not follow the designed path. If the output value is not within the expected range but significantly lower, a false negative signal is identified.

Given the nature and the aim of our test, that is a tool for mass testing for a first immediate diagnosis, we concluded that a negative control would add complexity to our device rather than provide useful information. Avoiding a false positive would, of course, be desirable, however, for safety reasons, all signals positive to MERS-CoV diagnosis should be further tested by the conventional laboratory assays. In addition, a binary test is widely considered more user-friendly than one demanding three measurements and the subsequent comparisons.

Image 4: A positive control test is performed in parallel with the detection assay.


MERS-CoV Diagnosis

The output signal is a glucose concentration in mg/dL or mmol/L, as the most common glucometers display. The test operator will give a yes or no answer to the patient according to the threshold value. If the positive control value is higher than expected, the measurement is considered invalid.

We have chosen to use the existing glucose monitoring technology as it is an already robust and optimised measurement method. In addition, glucose meters are very common and low-cost equipment, broadly available in Point-of-Care facilities. The glucometer should be calibrated when switching from blood sample to processed pharyngeal sample, so as to avoid inaccurate measurement. A simple glucose aquatic solution is used for the calibration before the MERS-CoV diagnostic assay. Before reusing the glucometer for blood testing, it should be calibrated with a commercial control solution. Also glucometers should be sanitized after each use.

The detection system coupled with the glucose monitoring device is a semi-quantitative method as it could give an approximate indication of the viral load in the sample. For this to be reliable, we should make a series of experiments with clinical samples and calculate the correction factors and establish the error ranges.



Materials

Image 5: The materials used for the kit: ABS, ferrite magnet, aluminum, nylon and paper.
  • Acrylonitrile butadiene styrene (ABS) is a thermoplastic polymer commonly used in 3D printing. It is the polymer of which Lego bricks are made. It is suitable not only for prototyping but also for end-use applications. ABS has a high thermal stability and exhibits impact resistance as well as toughness. It is a non-toxic, non-leaching material, safe for human use and for the environment, as it may not be biodegradable but it is recyclable. ABS is suitable for a diagnostic kit since it shows high chemical resistance and it can be found at a low cost.
  • Ferrites are ceramic magnets derived from iron oxides. They have high thermal conductivity but insulate electrical current. They are resistant to corrosion, most chemicals, and biological samples. They are particularly low-cost and are commonly used in household products, such as fridge magnets. In this kit they are necessary in order to ensure thermal conductivity and seal the reaction chambers to avoid evaporation. [2]
  • Aluminium foil (or aluminum foil), is aluminium prepared in thin metal leaves with a thickness less than 0.2 mm. It has various uses such as, packaging, cooking and geochemical sampling. Here it is used to spread the heat on the whole reaction area.
  • Nylon, is a generic designation for a family of synthetic polymers, based on aliphatic or semi-aromatic polyamides. Nylon is a thermoplastic silky material that can be melt- processed into fibers, films or shapes. Here it acts as a hydrophobic barrier between the two paper discs.
  • Paper discs, the main benefit is that thanks to the hydrophilicity of the paper and the capillary action we eliminate the need for pumps and power for the movement of fluids. It is eco-friendly, affordable, suitable as a base for biological processing and facilitates reagents storage.


Cost Analysis

Below is a cost analysis of the MERS-CoV Diagnostic Kit. Clearly, the total cost does not respond to that of a fully developed product, as we have not taken into account the upscaling factor, that would significantly reduce the cost. It indicates, however, that the cost of the chosen materials and mechanisms would not limit the affordability of the kit, making it accessible to a large portion of the population.

Table 1: A cost analysis of the diagnostic kit [2].
Materials Cost (US$) 6,44
Battery 2,7
Thermistor 3
Timer 0,4
ABS plastic 0,34
Cost of Assay (US$) 3,15
Toehold mechanism 0,2
Sample preparation 2,2
Magnets 0,25
Nylon and lubricant 0,06
Paper discs 0,04
Glucose strips 0,4
Total Cost per Assay for a
max of 50 Assays (US$)		 3,28


Safe use and disposal

Safety has been a key factor throughout the designing of our kit. We are confident that we propose a complete design that does not sacrifice health and environmental quality over usability and cost.

More specifically, the kit consists of a detachable, disposable part and a reusable and recyclable one.

The first two layers of the kit come in contact with the infected sample, consequently bearing a high risk of carryover contamination if improperly handled. For this reason, they are considered biohazardous and should be discarded accordingly to local regulations. Therefore, the two upper layers will be detached and discarded in a disposal bag with a “biohazard” sign that will be provided with the kit along with detailed instructions, as suggested by World Health Organization.

The lower layer, containing the heater and the power source, will be reusable. This would reduce the cost per assay in Point of Care facilities, where one heating base will be used for multiple runs. The kit will include instructions on proper disposal of electronic waste.



Competitive Advantages

As of now there are two categories of detection methods for MERS-CoV. The first category is molecular tests, that can detect active infections. The second category is serology tests that detect antibodies to MERS-CoV, therefore indicating that the patient has been infected in the past. For this reason they are only suitable for surveillance and research purposes, not for diagnostic ones.

The proposed toehold switch mechanism that we employ in our kit belongs to the molecular methods. The table below is is an attempt to compare the proposed diagnostic kit to rRT-PCR, the main alternative molecular method for MERS-CoV detection.

Table 2: A comparison between the two molecular diagnostic methods, rRT-PCR and the proposed kit “GENOMERS”.
Characteristics Diagnostic Methods for MERS-CoV
rRT-PCR GENOMERS
Expertise requirements Training and experience is absolutely necessary No expertise is required, a short training is sufficient
Space requirements Laboratory environment and expensive equipment is required No laboratory environment is required, suitable for POC use
Run time 2 hours 1 hour
Hands-on time 20 minutes 5 minutes
Cost per assay 77.5 – 372.5 $[7] 3.28 $
Quantification Viral load can be quantified by PCR-based quantification techniques Semi-quantificational method, correlation of viral load to glucose levels
Sensitivity High Unknown yet
Availability to the public No public availability Available to everyone
Monitoring false results Difficult and costly Easy and affordable


Concerns on Negative Implications

The proposed design has been developed incorporating feedback from a number of experts and always considering the general safety. However, we could not but underline certain aspects that could lead to negative implications.

Firstly, we should take into account the risk of the virus transmitting to those that operate the test, whether they are professionals or volunteers, and make sure to educate them properly on keeping safety measures. Since part of the kit is reusable, it should be sanitized after each use. The same applies to the glucometer that should be disinfected according to the ISO 15197:2013, as recommended by the FDA. This procedure involves cleaning the glucometer with germicidal wipes for one minute and allowing it to dry at room temperature. In addition, the disposable part of the kit should be discarded according to the local policies. However, we are concerned that in some areas the biohazard disposal policies might be more loose due to lack of education or resources.

On the other hand, the diagnostic test alone is not sufficient for containing a virus outbreak. We are worried that, perhaps, some patients will not proceed to visiting a doctor and follow the suggested procedures. For this reason, the emergency testing facilities should be in direct contact with hospitals to facilitate and encourage patients to comply with safety rules.

Finally, we are concerned over the psychological factor of delivering the MERS-CoV diagnosis. The personnel’s training should involve instructions on how to announce the diagnosis, informing the patients on the next steps without panicking them. This is important especially as the diagnostic method does not contain a negative control for simplicity and cost reasons, thus a false positive cannot be detected.



References

[1]Ye, X., et al. (2018). Equipment-free nucleic acid extraction and amplification on a simple paper disc for point-of-care diagnosis of rotavirus A. Analytica Chimica Acta, 1018 , 78-85.
[2]Connelly, J., et al. (2015). “Paper Machine” for Molecular Diagnostics. Analytical Chemistry, 87 (15), 7595-7601.
[3] Rodriguez, N., et al. (2015). Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens. Analytical Chemistry, 87(15), 7872-7879.
[4]Pardee, K. et al. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell,165, 1255-1266.
[5]Singh, A., et al. (2018). Paper-Based Sensors: Emerging Themes and Applications. Sensors, 18(9), 2838.
[6]Daher, R., et al. (2016). Recombinase Polymerase Amplification for Diagnostic Applications. Clinical Chemistry, 62(7), 947-958.
[7]ARQ Genetics - Gene Expression Services. (2018). Retrieved from https://www.arqgenetics.com/gene_expression_services.php