Digital LAMP in a sample self-digitization (SD) chip

Abstract

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource—limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we showed the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.

Progress of Microfluidics for Biology and Medicine

“…They demonstrated accurate quantification of relative and absolute DNA concentrations using digital loop-mediated DNA amplification (dLAMP) [94]. It is an inexpensive and easy-to-operate device for DNA amplification.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…Following amplification and counting of number of compartments showed positive amplification result reveals the initial number of the targeted molecules in the sample allowing quantitative analysis of ultimate precision and accuracy [85]. This compartmentalization can be realized in form of water in oil emulsion [86,87] where each water droplet is considered as unique and independent reaction volume; or by distribution of the reaction mixture across hundreds and thousands of individual microfluidic vessels [88][89][90]. The approach was successfully shown both for PCR [87,88,91,92] and isothermal amplification techniques (LAMP) [89].…”

“…This compartmentalization can be realized in form of water in oil emulsion [86,87] where each water droplet is considered as unique and independent reaction volume; or by distribution of the reaction mixture across hundreds and thousands of individual microfluidic vessels [88][89][90]. The approach was successfully shown both for PCR [87,88,91,92] and isothermal amplification techniques (LAMP) [89]. Zhang et al [90] showed implementation of the microfluidic arrays full cycle of nucleic acids analysis including DNA/RNA extraction and their real-time amplification (Fig.…”

Automatic sequential fluid handling with multilayer microfluidic sample isolated pumping

“…Additionally, such actuation methods are vulnerable to bubble formation and not suitable to load up dead-end structures that are attractive for quantitative digitized and multiplexed assays. [12][13][14] Most of the passive fluidic actuation methods lack effective positive control method or actuation reproducibility. [15][16][17][18][19] Take degas-driven flow as an example, it takes advantage of the Polydimethylsiloxane (PDMS) material to overcome the undesired bubble formation and enclosed chamber loading issue; however, it does not support a) Author to whom correspondence should be addressed.…”