Multiplex MicroRNA Detection on a Power-free Microfluidic Chip with Laminar Flow-assisted Dendritic Amplification

Abstract

MicroRNA (miRNA) profile-based point-of-care (POC) diagnostic methods have attracted considerable attention. In our laboratory, singleplex miRNA detection on a power-free poly(dimethylsiloxane) (PDMS) microfluidic chip with laminar flow-assisted dendritic amplification (LFDA) has been developed. In this study, to obtain the miRNA profile and to improve the reliability of the diagnosis, multiplex miRNA detection on the same system is demonstrated without compromising any advantages of the singleplex miRNA detection. The limit of detection (LOD) was at the femto-to picomolar level and the assay time was 20 min. The sensitivity, rapidity, and portability of the microfluidic chip are adequate for POC diagnosis.

Detection of Methylated DNA on a Power-Free Microfluidic Chip with Laminar Flow-Assisted Dendritic Amplification

“…[17][18][19][20][21] As a preliminary experiment, we had confirmed that B-anti-IgG is as effective as B-anti-SA for induction of LFDA using the well-established miRNA detection system (Fig. S1 in Supporting Information).…”

“…CP-1 or CP-2 was immobilized onto the glass surface as described elsewhere. 20,21 Briefly, 1% glutaraldehyde in carbonate buffer was reacted with an aminated glass slide (SD00011, Matsunami Glass) at 37 C for 2 h. A PDMS part having a straight microchannel was attached onto the glass surface. CP-1 or CP-2 (100 μM) in ultrapure water was injected into the microchannel and incubated at 37 C for 1 h. The PDMS part was detached from the glass surface in a stopping buffer (25 mM Tris, 0.05% Tween 20), and the glass substrate was washed by ultrasonication in the stopping buffer, followed by deionized water.…”

“…We have also invented a signalamplification technology specialized for microfluidic chips: laminar flow-assisted dendritic amplification (LFDA). 17 The combination of these two technologies, the power-free microfluidic chip and LFDA, has been proven to be effective for the detection of proteins, 17,18 DNA, 19 and microRNA (miRNA) 20,21 with a typical assay time of 20 min. In this paper, we report on the detection of 5mC in target single-stranded DNA on a power-free microfluidic chip for the first time.…”

Specificity of MicroRNA Detection on a Power-free Microfluidic Chip with Laminar Flow-assisted Dendritic Amplification

“…[19][20][21] Our method has two distinct features. First, the microfluidic chip requires no external power source for fluid pumping, because the pumping is achieved by degassed poly(dimethylsiloxane) (PDMS), which is widely employed as a microfluidic chip material.…”

“…We have also reported that our method can easily be expanded to the simultaneous triplex detection of miR-16, miR-21, and miR-500. 21 These previous studies were carried out with artificial samples containing only purified target miRNAs. Although the latter paper 21 revealed that our method produced no cross-reaction between those totally different sequences, the detection specificity of our method has been largely unexplored.…”