Paper-based ion concentration polarization device for selective preconcentration of muc1 and lamp-2 genes
© The Author(s) 2017
Received: 13 October 2016
Accepted: 4 January 2017
Published: 11 January 2017
Recently, novel biomolecules separation and detection methods based on ion concentration polarization (ICP) phenomena have been extensively researched due to its high amplification ratio and high-speed accumulation. Despite of these bright advances, the fabrication of conventional ICP devices still have complicated and times-consuming tasks. As an alternative platform, a paper have been recently used for the identical ICP operations. In this work, we demonstrated the selective preconcentration of a muc1 gene fragment as human breast cancer marker and a lamp-2 gene fragment as the cause of Danon disease in paper-based ICP devices. As a result, these two DNA fragments were successfully concentrated up to ~60 fold at different location in a single paper-channel. The device would be a promising platform for point-of-care device due to an economic fabrication, the easy extraction of concentrated sample and an easy disposability.
Over the past several decades, the separation and concentration of biomolecules including DNA and proteins have been developed for micro-scale assay such as genetics, disease diagnostics and point-of-care applications . Recently, novel biomolecules separation and detection methods based on ion concentration polarization (ICP) phenomena have been extensively developed [2–6]. The ICP phenomena that is generated near the interface of a microfluidic channel and perm-selective nanojunction causes the depletion zone at the anodic side of the membrane. Due to electro-neutrality requirement at low concentration inside the depletion zone, any charged species were rejected into the ion depletion zone so that the molecules were accumulated at the boundary of the zone with a proper application of tangential electric (or pressure) field. Unfortunately, the fabrication of conventional ICP devices still have demanded complex photolithography and high cost reactive ion etching. Moreover an interfacing to downstream analyzer would be restricted due to the minute volume of preconcentrated samples and fast dispersion. While the accuracy, performance and reproducibility of paper-based ICP device were lower than conventional ICP device, it was always suffered from the extraction of preconcentrated sample and the instability. Since a paper is easily cut by any methods, the endeavor for the sample extraction for further processes become extremely simple [7–10]. With the advantage of cost-effective fabrication and easy extraction, paper-based ICP platform would be one of the major field of this ICP research society. In terms of electrokinetics, the cellulose-matrix of paper was able to suppress undesirable vortical flow  so that overall ICP process become significantly stable.
In this work, we demonstrated an economic and fast-fabricable paper-based microfluidic devices, which would be an alternative platform to easily generate ICP phenomena and selective preconcentrate biomolecules. The first experimental results demonstrated that the fluorescent tracers (Alexa) was successfully concentrated and migrated in a wetted paper channel using the reversal of the electric field, leading to the preconcentration factor of ~10. Moreover, the mixture of the target genes (cell surface associated mucin 1 (muc1, 945 bp) as the human cancer marker  and lysosome-associated membrane protein 2 (lamp-2, 185 bp) encoding gene as Danon disease marker ) was injected and they were selectively preconcentrated at different locations having the amplification ratio of 20 and 60, respectively.
Preparation of DNA samples
Nested PCR amplification of muc1 gene (NG_029383.1, NCBI) was accomplished with a template of human genomic DNA. Amplification conditions of 3.5 kb fragment gene were 94 °C for 90 s, 65 °C for 45 s, and 72 °C for 90 s, by 35 cycles. The specific oligonucleotides were designed as following: forward primer; 5′-CCACACCCTTTGCATCAAGC-3′, reverse primer; 5′-TCTTGG CACCCGGTTGTTAC-3′. The second PCR conditions of 947 bp fragment gene were 94 °C for 1 min, 65 °C for 45 s, and 72 °C for 1 min, by 35 cycles. The forward primer of 947 bp muc1 fragments gene with tagging fluorescence dye FAM was specific for the 5′-FAM-TTTCCAGCCCGGGATACCTA-3′, whereas the reverse primer was designed as 5′-CTCCTCCCTCTGCTCTCCTT-3′. The primary and secondary PCR conditions of a lamp-2 fragment gene (NM_001122606.1, NCBI) fragment were 94 °C for 1 min, 60 °C for 30 s, and 72 °C for 1 min, by 32 cycles. The specific oligonucleotides were designed for primary amplicon with 510 bp as following: forward primer; 5′-TACATCACCACCCCTCTC-3, reverse primer; 5′-GCCCCTTCTTACTCTCCT-3′. The forward primer of 185 bp lamp-2 fragments gene with tagging CY5 for secondary PCR was specific for the 5′-CY5-TTGCAGCTGTTGTTGTACCG-3′, whereas the reverse primer was designed as 5′-GTAGCTTTGAACTGGTGCCC-3′. All PCR products were eluted from 1.0% TBE agarose gels and resuspended on autoclaved DI water.
Fabrication of the paper-based microfluidic device
To operate the device, reservoirs were pre-wetted with 10 mM KCl and loaded the mixture of KCl and the fluorescent tracer (Alexa488) or prepared DNAs. Ag/AgCl electrodes were inserted into both reservoirs and connected to a power supply (Keithley 238, Keithley Instruments, USA). All fluorescent images were captured by inverted microscope (IX53, Olympus, Japan) and analyzed by ImageJ program (https://imagej.nih.gov/ij/). The external voltage was swept from 0 to 15 V at 0.2 V/30 s only for the electrical measurement.
Results and discussion
Overlimiting current in the paper-based ICP device
Preconcentration in paper-based microfluidic device using the reversal of electric field
Figure 3b showed an effect of the electric field reversal. For first 10 s, the electric field was inversely applied to the device (snapshot from 0 to 8 s in Fig. 3b) so that the tracer initially flocked together near the left side of the membrane. The mechanism was the same ICP principle, but the left side of the membrane has the ion enrichment zone instead the ion depletion zone. After switching back the polarity of the electrode for preconcentration of samples at the ion depletion zone (snapshot from 10 to 20 s in Fig. 3b), the pre-accumulated tracer were preconcentrated further at the boundary of the depletion zone. The measured maximum concentration ratio of the tracer based on fluorescence intensity was ~10 fold. The reference fluorescent intensity of 2, 5, 10, 20 and 100 fold of samples were measured in advance and, then, the fluorescent from the preconcentrated signal was compared by ImageJ program.
Selective preconcentration of two target genes in paper-based ICP device
Recently ICP phenomenon had been drawn significant attentions in biomedical and environmental research field as well as fundamental electrokinetic society, since its versatile capability of controlling charged species. In this work, we have demonstrated an economic and fast-fabricable ICP device on a commercial paper for selective preconcentration. The generation of ICP phenomena was confirmed by I–V responses of the paper-based device by comparing with to without nanoporous membrane. The fluorescent tracer (Alexa488) and DNA fragments (muc1 and lamp-2) associated with human diseases were successfully preconcentrated and separated. Such paper-based ICP devices would be a promising tool due to an economic fabrication, the easy extraction of concentrated sample and an easy disposability.
SYS performed the experiments; HL analyzed the dynamics of analyte; SJK supervised the project, designed experiments and wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
This work was supported by National Research Foundation of Korea (CISS-2011-0031870, 2012-0009563, 2016R1A1A1A05005032, 2016R1A6A3A 11930759) and Korean Health Technology RND Project (HI13C1468 and HI14C0559). All authors acknowledge the support from BK21+ program of Creative Research Engineer Development IT, SNU.
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