Digital PCR using a simple PDMS microfluidic chip and standard laboratory equipment.

Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments.

Design of a Sensitive Extracellular Vesicle Detection Method Utilizing a Surface-Functionalized Power-Free Microchip.

Extracellular vesicles (EVs), which are small membrane vesicles secreted from cells into bodily fluids, are promising candidates as biomarkers for various diseases. We propose a simple, highly sensitive method for detecting EVs using a microchip. The limit of detection (LOD) for EVs was improved 29-fold by changing the microchannel structure of the microchip and by optimizing the EV detection protocols. The height of the microchannel was changed from 25 to 8 µm only at the detection region, and the time for EV capture was extended from 5 to 10 min. The LOD was 6.3 × 1010 particles/mL, which is lower than the concentration of EVs in the blood. The detection time was 19 min, and the volume of EV solution used was 2.0 µL. These results indicate that an efficient supply of EVs to the detection region is effective in improving the sensitivity of EV detection. The proposed EV detection method is expected to contribute to the establishment of EV-based cancer point-of-care testing.

Generation of transmitochondrial cybrids using a microfluidic device.

Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ0) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ0143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2'-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ0143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ0 cells with puromycin-based selection in the microfluidic device.

Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Device.

Quantitative control of mitochondrial transfer is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because it enables precise modulation of heteroplasmy. Furthermore, single mitochondrion transfer from a mtDNA mutation-accumulated cell to a mtDNA-less (ρ0) cell potentially achieves homoplasmy of mutated mtDNA. Here we describe the method for quantitative control of mitochondrial transfer including achieving single mitochondrion transfer between live single cells using a microfluidic device.

Multiplex MicroRNA Detection on a Surface-Functionalized Power-Free Microfluidic Chip.

Circulating microRNAs (miRNAs) have emerged as promising cancer biomarkers because their concentration profiles in body fluids are associated with the type and clinical stage of cancer. For multiplex miRNA detection, a novel surface-functionalized power-free microfluidic chip (SF-PF microchip) has been developed. The inner surface of the SF-PF microchip microchannels was functionalized via electron beam-induced graft polymerization and immobilization of capture probe DNAs. Simultaneous and specific duplex miRNA detection was achieved on the line-type SF-PF microchip with detection limits of 19.1 and 47.6 nmol L-1 for hsa-miR-16 and hsa-miR-500a-3p, respectively. Moreover, simultaneous and specific triplex miRNA detection was achieved on the stripe-type SF-PF microchip. The sample volume required for this microchip was 0.5 µL, and the time required for detection was 17 min. These results indicate that up to six types of miRNAs could be detected without compromising the advantages of the previous SF-PF microchips for cancer point-of-care testing.

A Microfluidic Device for Modulation of Organellar Heterogeneity in Live Single Cells.

The quantitatively controlled organellar transfer between living single cells provides a unique experimental platform to analyze the contribution of organellar heterogeneity on cellular phenotypes. We previously developed a microfluidic device which can perform quantitatively controlled mitochondrial transfer between live single cells by promoting strictured cytoplasmic connections between live single cells, but its application to other organelles is unclear. In this study, we investigated the quantitative properties of peroxisome transfer in our microfluidic device. When cells were fused through a 10 or 4 µm long microtunnel by a Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel, and a subsequent recovery culture disconnected the fused cells. The peroxisome number being transferred through a 10 µm length of the microtunnel was smaller than that of 4 µm. These data suggest that our microfuidic device can perform a quantitative control of peroxisome transfer.

Biomarker Analysis on a Power-free Microfluidic Chip Driven by Degassed Poly(dimethylsiloxane).

Point-of-care testing (POCT) of biomarkers, such as proteins and nucleic acids, is a hot topic in modern medical engineering toward the early diagnosis of various diseases including cancer. Although microfluidic chips show great promise as a new platform for POCT, external pumps and valves for driving those chips have hindered the realization of POCT on the chips. To eliminate the need for pumps and valves, a power-free microfluidic pumping method utilizing degassed poly(dimethylsiloxane) (PDMS) was invented in 2004. In this article, the working principle of the degas-driven power-free microfluidic chip is first described, and then applications of those chips to biomarker analysis are reviewed. The biomarker analysis on the chip was typically achieved with a small sample volume of ∼1 µL and a short analysis time of ∼20 min. For protein analysis, the sandwich immunoassay format was adopted. The limit of detection (LOD) was improved by three orders of magnitude by using laminar flow-assisted dendritic amplification (LFDA), which was a newly devised amplification method specialized for microfluidic chips. For analysis of nucleic acids such as DNA and microRNA, the sandwich hybridization format was adopted, and the LFDA was also effective to reduce the LOD. With the LFDA, typical LOD values for proteins and nucleic acids were both around 1 pM.

Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device.

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.

Characterizing the non-crosslinked aggregation of DNA-modified gold nanoparticles: effects of DNA length and terminal base pair.

We previously reported that fully complementary DNA duplexes formed on gold nanoparticle (GNP) surfaces aggregate at high salt concentrations. We previously reported that DNA-functionalized gold nanoparticles (GNPs) aggregate by hybridization with fully complementary DNA at high salt concentrations. Although this behavior has been applied to some precise naked-eye colorimetric analyses of DNA-related molecules, the aggregation mechanism is still unclear and comprehensive studies are needed. In this paper, we reveal the key factors that influence GNP aggregation. The effects of temperature, electrolyte concentration, probe length, and particle size, which control the stabilities of double-stranded DNAs and GNPs, were investigated. Larger GNPs aggregated more easily, and GNP aggregates were easily formed with ∼15-mer-long probes, while longer probes prevented aggregation, perhaps by preventing the formation of rigid double-stranded DNA layers, compared to shorter probes. Furthermore, GNPs with purine bases at their 5' ends aggregated more easily than those with these bases at their 3' ends. This phenomenon is different from that based on the melting-temperature trend calculated using the nearest-neighbor method.

Sensitivity Enhancement of MicroRNA Detection Using a Power-free Microfluidic Chip.

We present a microRNA (miRNA) detection method that achieves enhanced sensitivity by means of a power-free microfluidic chip without the requirement of an external power source. The miRNA detection is completed by sandwich hybridization between probe DNAs and target miRNA with small sample volume (0.5 µL) within 20 min. Fluorescence signals after hybridization were amplified by laminar flow-assisted dendritic amplification (LFDA) using fluorescein isothiocyanate (FITC)-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) as amplification reagents. To enhance the sensitivity of on-chip miRNA detection, the hybridization buffer solution was newly optimized with three main components-sodium dodecyl sulfate (SDS), formamide and dextran sulfate-that are known to strongly influence hybridization. An on-chip miRNA detection test in the newly optimized hybridization buffer (0.2% SDS, 5% formamide and 1% dextran sulfate) revealed dramatic increases in both the LFDA signal in the sample channel and the signal-to-background ratio (S/B ratio). Moreover, the LFDA signals in a blank reference channel remained low due to the suppression of non-specific bindings and hybridizations. By changing the hybridization buffer, we obtained an improved limit of detection (LOD) that was 0.045 pM (miRNA-196a) and 0.45 pM (miRNA-331), which are around 30- and 10-fold better than that of when control hybridization buffer was used. The improved performance of our miRNA detection system with short running time and high sensitivity could contribute to future research, including point-of-care diagnostic systems.

Quantitative control of mitochondria transfer between live single cells using a microfluidic device.

Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because single mitochondrion transfer to a mtDNA-less (ρ0) cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 µm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 µm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

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

MicroRNAs (miRNAs) are attracting considerable attention as potential biomarkers for the early diagnosis of cancer. We have been developing a detection method for miRNAs on a microfluidic chip with external-power-free fluid pumping and enzyme-free amplification. The assay is completed within 20 min. Here, we describe the specificity of this miRNA detection method. First, the specificity against mismatched sequences was investigated. The nonspecific detection of a 2-nucleotide mismatched sequence was negligible, while that of a 1-nucleotide mismatched sequence was observed to a reasonable extent. Next, the disturbance in mature miRNA detection by existence of its precursor miRNA was evaluated. One precursor miRNA out of four tested showed significant nonspecific responses at 1 nM or higher concentrations. However, those responses were much lower than that of the target mature miRNA at 0.1 nM. Finally, we tried to detect three endogenous miRNAs, which are known to be potential cancer biomarkers, in human leucocyte total RNA. The measured concentraions of these miRNAs agreed well with those obtained by quantitative reverse transcription polymerase chain reaction. These results indicate that the on-chip miRNA detection method has good specificity, which is promising for applications to real biological samples.

Preparation of a Surface-functionalized Power-free PDMS Microchip for MicroRNA Detection Utilizing Electron Beam-induced Graft Polymerization.

We propose an easy microchannel surface functionalization method for a poly(dimethylsiloxane) (PDMS) microchip that utilizes electron beam-induced graft polymerization (EIGP) as a platform for microchip-based biomarker analysis. Unlike other grafting techniques, EIGP enables rapid surface modification of PDMS without initiator immobilization. The grafted microchip is preservable, and can be easily functionalized for versatile applications. In this study, the surface-functionalized power-free microchip (SF-PF microchip) was used for the detection of microRNA (miRNA), which is a biomarker for many serious diseases. The EIGP enables high-density three-dimensional probe DNA immobilization, resulting in rapid and sensitive miRNA detection on the portable SF-PF microchip. The limit of detection was 0.8 pM, the required sample volume was 0.5 µL, and the analysis time was 15 min. The SF-PF microchip will be a versatile platform for microchip-based point-of-care diagnosis.

Rapid and Easy Extracellular Vesicle Detection on a Surface-Functionalized Power-Free Microchip toward Point-of-Care Diagnostics.

Extracellular vesicles (EVs) are promising novel cancer biomarkers. However, rapid and easy analysis of EVs is challenging because conventional detection methods require large sample volumes and long detection times. Microchip-based analytical systems have particularly attracted attention for development of point-of-care (POC) diagnostics. Previously, various biomarker detection methods on a portable power-free poly(dimethylsiloxane) (PDMS) microchip using laminar flow-assisted dendritic amplification have been developed. Recently, for easy functionalization, we proposed a microchannel inner surface-functionalized power-free PDMS microchip (SF-PF microchip) utilizing electron beam-induced graft polymerization. In this study, we apply the technique and prepare a novel SF-PF microchip. On the microchip, EVs were successfully detected. The required sample volume was 1.0 µL, and the total analysis time was 20 min. The microchip can contribute to EV-based POC cancer diagnosis.

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

We report on a detection method for methylated DNA on a microfluidic chip, which needs no external power for fluid pumping. The methylated DNA was sandwiched by immobilized probe DNA and an anti-methylcytosine antibody. The fluorescence signal was amplified by our original amplification technology. The detection method was first optimized using a 22-mer DNA sequence, then further validated using a 60-mer DNA sequence adapted from the SEPT9 gene. We were able to detect the methylated 60-mer DNA at 0.4 nM within 18 min.

Cryopreservation of adhered mammalian cells on a microfluidic device: Toward ready-to-use cell-based experimental platforms.

In this paper, we describe cryopreservation of mammalian cells in the adhered state on a microfluidic device (microdevice) for the first time. HeLa, NIH3T3, MCF-7, and PC12 cells were cultured on a microdevice in which a commercial polystyrene dish surface was used as the cell adhesion surface. Without cell-detaching treatment, the microdevice was stored in a freezer at -80°C. After thawing, we observed a greater number of live cells on the microdevice than those on a control culture dish. Although the effectiveness of the microdevice varied depending on the cell type and surface coating, the trend was consistent. We confirmed that the phenotype of the PC12 cells to differentiate into neuron-like cells was kept after the on-chip cryopreservation, and that the results of cytotoxicity test of cisplatin against the HeLa cells were essentially unchanged by the on-chip cryopreservation. These findings will open up a new possibility of ready-to-use cell-based experimental platforms.

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

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.

Effects of ROCK inhibitor Y-27632 on cell fusion through a microslit.

We previously reported a direct cytoplasmic transfer method using a microfluidic device, in which cell fusion was induced through a microslit (slit-through-fusion) by the Sendai virus envelope (HVJ-E) to prevent nuclear mixing. However, the method was impractical due to low efficiency of slit-through-fusion formation and insufficient prevention of nuclear mixing. The purpose of this study was to establish an efficient method for inducing slit-through-fusion without nuclear mixing. We hypothesized that modulation of cytoskeletal component can decrease nuclear migration through the microslit considering its functions. Here we report that supplementation with Y-27632, a specific ROCK inhibitor, significantly enhances cell fusion induction and prevention of nuclear mixing. Supplementation with Y-27632 increased the formation of slit-through-fusion efficiency by more than twofold. Disruption of F-actin by Y-27632 prevented nuclear migration between fused cells through the microslit. These two effects of Y-27632 led to promotion of the slit-through-fusion without nuclear mixing with a 16.5-fold higher frequency compared to our previous method (i.e., cell fusion induction by HVJ-E without supplementation with Y-27632). We also confirmed that mitochondria were successfully transferred to the fusion partner under conditions of Y-27632 supplementation. These findings demonstrate the practicality of our cell fusion system in producing direct cytoplasmic transfer between live cells.

A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

Microfluidic perfusion cell culture system confined in 35 mm culture dish for standard biological laboratories.

An extremely simple, self-standing microfluidic cell culture system is reported. The whole system is confined in a 35 mm culture dish, and requires only a standard CO2 incubator. The culture medium is perfused by gravity. We successfully cultured NIH3T3-derived cells up to 10 days with a viability of ∼90%.