Self-actuated microfluidic chiplet for two-stage multiplex nucleic acid amplification assay

• Published in: Lab on a chip 2024-11, Vol.24 (23), p.5175-5183
• Main Authors: Ansah, Felix, Hajialyani, Marziyeh, Ahmadi, Fatemeh, Gu, Yuming, Tar m, Ergün Alperay, Mauk, Michael G, Awandare, Gordon A, Bau, Haim H
• Format: Article
• Language: English
• Subjects: Assaying; Chambers; Chemistry; Equipment Design; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques - instrumentation; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques - instrumentation; Nucleic acids; Pathogens; Valves
• ISSN: 1473-0197; 1473-0189; 1473-0189
• DOI: 10.1039/d4lc00752b

Effective diagnosis of comorbidities and infectious diseases that present similar symptoms requires point-of-need assays capable of co-detecting and differentiating among multiple co-endemic pathogens to enable timely, precision medicine and effective control measures. We previously developed a two-stage isothermal amplification assay dubbed Penn-RAMP to address this need. Penn-RAMP's first stage comprises a recombinase polymerase amplification (RPA), which amplifies all targets of interest in a single reaction chamber for a short duration. The RPA amplicons are then aliquoted into multiple loop-mediated isothermal amplification (LAMP) reaction chambers, each customized with pre-dried primers to amplify a single target or a group of targets. To enable Penn-RAMP at the point of need, we describe here a self-actuated Penn-RAMP chiplet that accommodates the Penn-RAMP assay. Our chiplet employs temperature-controlled phase change valves and capillary valves to self-aliquot first-stage amplicons into multiple (five) second-stage reaction chambers and to seal these chambers. The functionality of our device is demonstrated by co-detecting plant pathogens. The analytical performance of our chiplet is comparable to that of the benchtop Penn-RAMP assay and surpasses that of standalone LAMP assays. Our self-actuated chiplet can be operated standalone with purified nucleic acids or as the downstream amplification module of a sample preparation cassette. Self-actuated chiplet for Penn-RAMP assay.