Team:UPF Barcelona/Implementation

Team:UPF Barcelona - 2021.igem.org

Project

End-to-end pipeline

Review of the proposed implementation, end users, safety warnings and future steps.

Potential users

Since the main goal is to reduce the number of deaths from pathological conditions derived from antibiotic resistance (AR) infections (around 700,000 in the world, data from 2019) [1], potential users are workers in the healthcare system, especially medical doctors and nurses. In terms of patients, around 2,8 million people catch one of these diseases per year in the US only [2]. The current treatments require long tests and analysis that can take days to complete before administering a drug that may or may not affect the patient. In this context, ARIA would help increase not only the speed of characterization to the hour scale but also to help make the therapeutic decision more accurate.

We have envisioned a medical device that considers the causative complexity of AR. Now let’s review how the ARIA pipeline would be implemented in real-life situations. The final prototype would consist of three main parts: the paper-based array, the reagents to activate the detection process, and a set of one-use sterile pipettes to transfer the patient samples into the array.

Our desired final product is intended to act as a point of care, allowing the medical user to quickly analyze its patient samples at the bedside and receive useful information to proceed with the best possible therapy. We have decided to offer two different options depending on the resources and needs of the recipient.

The first option consists of providing a single-use and ready-to-use array with the engineered bacteria lyophilized. Portable for use anywhere. The only reagents provided with the kit will be the needed inducers, and the doctor will need to add the patient sample with possible antibiotic resistance.

Option 2 is intended for hospitals with laboratory facilities and high capacity of technical research staff.

Option 2: This second option is provided with bacteria stock in which the biosensors are growing. It will be contained in single tubes so that the user can design and prepare its own personalized paper-based arrays.

1. Customize and prepare your own paper-based array.
2. Induce the expression of the biosensors contained in two plasmids: one for Cas12a and one corresponding to gRNA.
3. Trigger bacteria autolysis to release the biosensors and allow them to carry out the detection.
4. Transfer the lysate to the wells in the array.

Main pipeline

Now proceed with the main pipeline:

1. Add the inducers to activate the biosensors into the wells that conform the array.
2. Once the sample has been extracted from the patient, pipette it into the array.

3. Wait until seeing a visual fluorescent signal. The results are to be produced within a period of time estimated around 30 minutes. A very early or late detection can be a signal of an error in previous steps.
4. Open Iris on your smartphone or any intelligent device with a camera.
5. Take a picture of the array by following the position guidelines shown on the screen.

a) The app may ask you to move closer or further from the array.
b) Wait until the whole array is detected, this process should last a few seconds.
c) Once the reading is complete, the information will be sent to our servers.
d) You may specify Iris your will to send multiple arrays. If so, the same process must be made to all your desired trials.
e) Our servers will compare the configuration of the sample with the information found by the Alpha artificial intelligence.

6. Soon you will receive a report with a complete analysis of the resistance configuration of the pathogen as well as some recommendations on the best predicted procedure.
7. Additionally, the system may be used locally to account with situations were internet connection is a problem.

Safety aspects to consider when employing our device

Since the ARIA is designed to work with liquid samples, we must consider the treatment of these to preserve patient safety. In the case of blood samples, it needs to be ensured that the material is sterilized before usage. The puncture site needs to be cleaned with antiseptics to avoid infection. In more complex cases, such as needle biopsy, the physiological state of the patient must also be taken into account [3][4].

Our biosensors have been designed with non-pathogenic E. Coli strains (NZY5alpha, DH5alpha and BL21 Gold (DE3)) [5]. Although these strains of E. Coli are harmless, others can make you sick. Some kinds of E. Coli can cause diarrhea, while others cause urinary tract infections, respiratory illness and pneumonia, and other illnesses [6]. In cases where an amplification step prior to detection is required, we would like to recall that general safety measures must be followed to avoid eye and mouth contact by using gloves, a lab coat and glasses. Bacteria must not be released to the environment.

At a computational level, we suggest a mechanism to ensure the correct treatment of the data once it is sent to the main server. An encryption process would take place to protect the input data. Since the recommendations and reports are sent via e-mail and it already has a protection mechanism no further encryption would be needed.

However, more details regarding safety are explained in the Safety section.

Pre-clinical studies

Before deploying the whole structure to the general public, several studies of efficiency and safety must be carried out. As suggested by Dr Alex Smithson, we should carry out a comparison study of the elapsed time taken to carry out the whole pipeline versus current methods of treatment. More information on the Education page.

References

[1] (2021). Retrieved 4 August 2021, from https://www.who.int/docs/default-source/documents/no-time-to-wait-securing-the-future-from-drug-resistant-infections-en.pdfsfvrsn=5b424d7_6

[2] Antibiotic Resistance Threatens Everyone. (2021). Retrieved 10 September 2021, from https://www.cdc.gov/drugresistance/index.html

[3] Diccionario de cáncer del NCI. (2021). Retrieved 13 September 2021, from https://www.cancer.gov/espanol/publicaciones/diccionarios/diccionario-cancer/def/biopsia

[4] The 'Liquid' Biopsy. (2021). Retrieved 13 August 2021, from https://www.cap.org/member-resources/articles/the-liquid-biopsy

[5] Nos mueve tu salud | Hospital Clínic Barcelona. (2021). Retrieved 13 October 2021, from http://clinicbarcelona.org

[6] E. coli - Symptoms and causes. (2021). Retrieved 13 July 2021, from https://www.mayoclinic.org/diseases-conditions/e-coli/symptoms-causes/syc-20372058