(G-247) 3D printed sample to answer microfluidic system for colorimetric detection of pathogen

Introduction:: The use of colorimetric readout for detecting infectious diseases is becoming popular at the point of care because it is easy to analyze and interpret, requires minimal training, and can be integrated with highly specific nucleic acid detection techniques like Loop-mediated amplification (LAMP). However, the colorimetric readout coupled LAMP approach faces several challenges, such as rapidity, inter-user variability, difficulty in quantifying colorimetric signals, and involvement of trained personnel in sequential LAMP assay steps. These challenges hinder its potential application at the point of care/need. To address these challenges, we present a portable setup that employs a 3D-printed microfluidic cartridge coupled with a modular imaging setup for colorimetric signal capture, quantification, and transmission, fully operated remotely via a mobile phone.

Materials and Methods:: The workflow from sample collection to endpoint detection is automated by a portable setup housing three core components, (i) a 3D printed microfluidic cartridge, (ii) an imaging module, and (iii) a control and data analysis unit. In brief, the saliva is collected in the funnel of the microfluidic cartridge and placed inside the portable setup. The sequential steps of the LAMP assay are mediated by angle-dependent actuation of elastomeric chambers for precise volumetric control in the microfluidic cartridge. These novel tunable elastomeric chambers facilitate reliable sequential flow handling in the cartridge. The LAMP reaction is initiated by a feedback loop-controlled heater in the portable setup. Subsequently, the colorimetric change over the novel nanostructured substrate is captured by the imaging module. The imaging module is a modular epi-illumination setup that houses an illumination column and a Raspberry Pi coupled CMOS sensor for image capture. The illumination column is a novel optical setup that offers exceptional control over the spatial and intensity of the illumination beam over the light-sensitive nanostructured platform. The image captured by the CMOS is analyzed, and the results are transmitted to the user’s mobile phone. The whole setup is centrally concerted by microcontrollers operated via mobile phone by tap of a button.

Results, Conclusions, and Discussions:: The automated and portable setup demonstrated the detection of SARS-CoV-2 wildtype nucleic acid in 15 minutes at clinically relevant viral loads. The elastomeric chambers housed in the cartridge were empirically calibrated for accounting for residual volumes, enabling on-demand fabrication of the chambers via 3D printed molds. Further, these tunable elastomeric chambers demonstrated excellent control over fluid pumping. The setup could pump fluid volumes down to 0.1 µl/per 30-degree angular rotation in the microchannels. Further, the tunable elastomeric chambers demonstrated no fluid volume loss in a full cycle of suctioning and pumping, pointing to the system's reliability. Further, the imaging setup offered better illumination, with the maximum relative intensity varying only 17% while achieving a resolution of 0.155µm/pixel and a field of view of 298µm.
This work demonstrates a novel mobile-operated setup for automating colorimetric amplification assays with minimal user involvement and within minutes. The setup marks a significant improvement in integrating all the steps (sample to answer) of a LAMP assay onto a single platform at the point of care/need in a rapid, portable, and cost-effective fashion. Significant improvements are shown in deploying additive manufacturing (3D printing) in furthering on-demand testing at the point of need.

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