Abstract
Colorimetric loop-mediated isothermal amplification (LAMP) on microfluidic paper-based analytical devices (μPADs) offers a low-cost, disposable, and equipment-free alternative to liquid LAMP assays. However, amplification on μPADs is consistently slower, by 5–46%, than reactions in tubes. To identify the origin of this delay, we evaluated heat transfer, diffusion in porous cellulose, and nonspecific adsorption of LAMP components across both high- and low-copy input regimes. Our results show that once thermal equilibrium is reached, reduced effective diffusion is the dominant contributor to the kinetic lag at low copy numbers, whereas nonspecific adsorption becomes the primary barrier at higher template concentrations. Pre-coating the paper with bovine serum albumin (BSA) mitigates adsorption. It narrows the tube-to-paper gap, thereby accelerating amplification of the SARS–CoV-2 ORF7ab synthetic gene by an average of 6 minutes, from 1E3 to 1E5 copies per reaction. These findings provide a mechanistic basis for the copy-number-dependent behavior of μPAD LAMP and offer simple, low-cost strategies to improve the speed and reliability of μPAD nucleic acid assays.
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Abstract
Colorimetric loop-mediated isothermal amplification (LAMP) on microfluidic paper-based analytical devices (μPADs) offers a low-cost, disposable, and equipment-free alternative to liquid LAMP assays. However, amplification on μPADs is consistently slower, by 5–46%, than reactions in tubes. To identify the origin of this delay, we evaluated heat transfer, diffusion in porous cellulose, and nonspecific adsorption of LAMP components across both high- and low-copy input regimes. Our results show that once thermal equilibrium is reached, reduced effective diffusion is the dominant contributor to the kinetic lag at low copy numbers, whereas nonspecific adsorption becomes the primary barrier at higher template concentrations. Pre-coating the paper with bovine serum albumin (BSA) mitigates adsorption. It narrows the tube-to-paper gap, thereby accelerating amplification of the SARS–CoV-2 ORF7ab synthetic gene by an average of 6 minutes, from 1E3 to 1E5 copies per reaction. These findings provide a mechanistic basis for the copy-number-dependent behavior of μPAD LAMP and offer simple, low-cost strategies to improve the speed and reliability of μPAD nucleic acid assays.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The language has been refined, and the data analysis has been enhanced by categorizing low copy concentration and high copy concentration. Additionally, comprehensive information on CFD is provided.
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