Discussion

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Microfluidics is based on the principles of biology, chemistry, physics, material science, and microelectronics. This technology represents an effective and simple alternative to carry out molecular separations based on size, as well as chemical reactions. Some of their advantages include enhanced sensitivity, faster reaction time, portability, temperature control, lower costs, and easier automation and parallelization (Elveflow, 2023; Niculescu, et al., 2021). In the construction of biology-focused microfluidic devices, the properties of liquids and gases are used at a microscale for the modeling or engraving of patterns of microchannels that enable the injection and evaluation of fluids containing molecules by detecting and separating specific compounds (Elveflow, 2023; Niculescu, et al., 2021).

Microfluidics permits the usage of less volume of samples and reagents, at the same time it enables the execution of many operations simultaneously, offering excellent data quality and a good parameters’ control (Elveflow, 2023).

In our project, these principles apply when bacterial cellular components such as proteins, organelles, and debris travel through the channel by capillary forces. In addition to transportation, these forces, along with the porosity of the paper and the strong negative charges, allow the DNA to be isolated and reach the reaction chamber (Kaarj, et al., 2018). Additionally, the materials from which the device is made, which consist of chromatographic cellulose paper and wax, allow a simple, cheap, and easy-to-replicate design, which also meets scientific standards for a correct operation.

LAMP-mediated amplification, which is the reaction by which multiple copies of a specific sequence can be generated at a constant temperature, is easier to perform in the field rather than a traditional PCR. That is why a reaction of this type is the most suitable to be carried out in a portable microluidic device (Wang, et al., 2015; Kaarj, et al., 2018). In addition, the primers, necessary to achieve a successful amplification, can be used to generate a visual indicator that confirms the presence of the amplicon of interest. Specifically, it was proposed that the LB and LF primers were labeled with 6-FAM on the 5' end, and a Black Hole Quencher on the 3' end. Furthermore, in order for fluorescence to be emitted in response to amplification, a hairpin primer structure was proposed in order to allow proximity between the flurophore and the quencher. Thus, if they did not anneal to the CTX-M-15 sequence, they would maintain proximity, preventing fluorescence. However, if they did bind to the sequence of interest, the primer is then displaced by the polymerase. As a consequence, the fluorophore is released, and with loss of proximity a green fluorescent signal is emitted (Varona and Anderson, 2019; Zhang, et al., 2023). By taking advantage of the already-included primers, specificity is guaranteed, and the possibility of false negatives is reduced since fluorescence is emitted in response to the presence of a target sequence, and not due to the action of the polymerase or the change in pH like other colorimetry-based detection methods.

As the project was intended to be carried out outside the laboratory, the need to develop a microheater powered by batteries that could reach a temperature of 65°C to carry out the LAMP reaction became more evident. After an extensive review of the scientific literature, it was found that a resistor made of Nanosilver ink with dimensions of 2 cm long and 0.5 cm wide, connected to a 0.8-1.2 volt AAA battery, could reach the required temperature in 2-3 minutes, and that could be used to carry out up to 4 tests. These advantages can be attributed to the physical properties of this material, such as its resistance, enhanced by the printed pattern design. In addition, the production cost is around 0.17 dollars, making it cost-effective, sustainable, and reusable (Byers, et al., 2020).

The proposal presented in this project represents a first phase of development, in which the fundamental points to carry out the design of the microfluidic device, the design of the microheater, the LAMP reaction, and the detection of the fluorescent indicator are described. We anticipate that the continuous development based on gradual empirical experimentation will allow continuous adaptation of the protocol, with the aim of perfecting it and finally achieving maximum efficiency and effectiveness in order to bring this proposal to tangible reality.