MB-ERC2 (miRNA Biomarker based Exponential RCA with CRISPR-Cas12a) for COPD

Overview

Traditionally, the diagnosis of COPD is complicated and time-consuming, with doctors examining family/medical history and exposure to lung irritants and ordering several tests (COPD - Diagnosis and Treatment - Mayo Clinic, 2020). Moreover, misdiagnosis is common, and oftentimes result in adverse physiological effects and add pressure to the healthcare system (Jeremias, 2021). To address, this issue, we created the MB-ERC2 (miRNA Biomarker based Exponential RCA with CRISPR-Cas12a) diagnosis system, using miR-223 as the biomarker for COPD, and employing the RCA/Cas12a-based EXTRA-CRISPR system (He et al., 2023). Optimization was performed in order to select the best reaction system and determine the biochemical mechanism (see more on the Experiments page). Furthermore, the MB-ERC2 system is potentially applicable to other diseases with miRNA biomarkers, such as tuberculosis (Lyu et al., 2019), malaria (Li et al., 2018), and HIV (Biswas et al., 2019).

Reagents List

Reagent Volume Price Volume per kit Price per reaction
dNTP 0.8 µl $66 800 µl $0.066
Cas12a 0.04 µl $70 70 µl $0.04
SplintR ligase 0.2 µl $452 250 µl $0.36
Reporter 0.2 µl $400 1290 µl $0.06
Phi29 polymerase 0.2 µl $239 125 µl $0.38

Schematics

The padlock (ssDNA) is designed to possess sequences complementary to the target miRNA biomarker on its two ends. After annealing, SplintR ligase circularises the padlock. Then, Phi29 polymerase drives the synthesis of DNA, using the padlock as template. The ribonucleoprotein, or RNP, is composed of Cas12a and crRNA (CRISPR RNA). crRNA, which is complementary to the amplicon, guides the RNP to bind with the newly synthesised DNA. Cas12a’s cis-cleavage is specific, and cleaves the amplification products of the miRNA-padlock circular duplex, resulting in more repeats of the process described above. The trans-cleavage, being non-specific, cleaves both the amplicon and the fluorophore-quencher reporting system described below, generating the fluorescence signal used for quantification.

Detection

A ‘reporter’ is typically composed of a quencher and a fluorophore. While the two are connected, the quencher absorbs energy from the fluorophore, thus preventing it from emitting fluorescence. But when cas12a trans-cleavage acts on the quencher-fluorophore and severs their link, the fluorophore would start giving out fluorescence. Thus, the fluorescence level is used to quantify the concentration of miRNA biomarkers in the sample tested.

Discussion

Based on the biochemical principles of the MB-ERC2 system, we believe that the assay is applicable in the detection of other diseases with miRNA biomarkers. The padlock, which contains miRNA-specific ends for annealing, would require to be re-designed and synthesised. But the rest of the reaction assay, including the middle-portion of the padlock sequence, is generalised and does not require further modifications. Furthermore, MB-ERCC’s various characteristics, such as wide detection range, high efficiency, and cost-effectiveness makes it a favourable alternative compared to many conventional diagnosis methods. Thus, we sincerely hope that MB-ERCC could help make a difference.

VBD3 (Visual Based Disease Detection Device)

Overview

During the process of detecting COPD diseases, our team performed a method named RCA (Rolling Circle Amplification). However, in the real world, detection often requires a PCR machine or a Thermal Cycler. Even in using RCA, up said two devices are used to detect the existence of the disease or virus. If we wanted to promote early detection, our objectives were simple, as a replacement for a device or machine that could detect disease at a low cost was vital. The major challenges encountered while developing this device were to effectively bring down the price and to guarantee the test accuracy at the same time. This required developing and designing the device by using entirely different approaches and methods. This leads to extensive amounts of testing and debugging over the process of development. The Device is named VBD3 (Visual Based Disease Detection Device), by utilising two ESP32 microcontrollers and sets of other electronic components, a cost-efficient design, this device is also capable of diagnosing diseases and quantifying the data for further analysis from doctors or lab members.

Parts List

Component Price
ESP32-Cam 25
ESP32-Wroom-U32 24
TEC Heater 54
Mosfet Module x2 8
MicroSD Adaptor 4.8
OLED Screen 19.5
MAX6675 Module 14

Design

The electric system of VBD3 is built under an Arduino environment using Arduino IDE. However, Arduino itself is not a very powerful development board, there are a lot of other alternatives including STM32, Raspberry Pi, Teensy boards ... etc. In our case, we chose to use ESP32 development boards with its benefits of wireless communication, stronger processing power, cost-efficiency…… etc. The electric system is mainly split into two parts: the heating and detecting mechanisms.

The heating mechanism is for heating the samples to a preferable temperature for them to react and perform RCA better, it uses a TEC1-12706 semi-conduct cooling pallet, a heating block, and a K-type thermocouple with MAX6675 module driving it for temperature sensing. These components are picked based on their reliability, their sensitivity, and their ability to heat or cool components. The detecting system uses an ESP32-Cam module to take photos and a 12V blue LED to excite the fluorescence. The device also has an OLED screen, encoder, SD card module buzzers, and fans for controlling and cooling purposes.

The detection mechanism of this device is very simple. The sample is put into an aluminium holder and a heat block. With the program started the heating system will start heating the sample to a preset temperature in our case 37 Celsius. The heating mechanism will sustain the temperature through PID control minimising the inconsistency of the temperature. After this, the ESP32-cam module will take a photo of the sample consistently, however right before it takes the photo it uses a Blue LED light to illuminate the sample. After the photo is taken, the ESP32-cam then sends the photo to the ESP32 development board through the ESP NOW wireless communication protocol. When the ESP32 board successfully receives the photo, it performs the MGV (Mean Grey Value) where a captured picture of the sample will be converted to its Mean Grey Value, and saves the data into an SD card. After the step of taking photos for 2 hours and converting them to data is over, the device can export the data via an SD card to a mobile device or desktop, and the data is converted to a 2-axis graph for better-examining purposes.

Schematics and Images

Below is a diagram showing the detection method VBDDD is using for estimating the fluorescence value of the sample. A blue LED light excites the sample and the emitted fluorescence light is captured by an ESP32-Cam camera through the filtration of lights through an optical filter.


As said the electric system for VBD3 is split into two parts detecting and heating, the detecting part is what the MPU (Main Processing Unit) is in charge of, and the heating part is what the SPU(Secondary Processing Unit) is in charge of. The Microcontroller in MPU is the ESP32-WROOM-32U and it is in charge of converting the images into values or MGV (Mean Grey Values). The other Microcontroller that is in SPU is the ESP32-Cam and it is in charge of capturing photos and for PID controls of the heating mechanism. A brief electric diagram is shown in the image below.




References