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.

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.

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.

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).

Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine.

Application of microchip phosphate-affinity electrophoresis to measurement of protease activity in complex samples.

We previously demonstrated microchip phosphate-affinity electrophoresis (µPAE) for activity measurement of kinases and phosphatases. In the current work, we extend the µPAE application to a protease: caspase-3 (CASP3). We designed a substrate peptide modified with both a fluorescent tag for label and a phosphate tag for separation. The CASP3 cleavage reaction split the two tags and made the fluorescent fragment migrate electrophoretically. The CASP3 activity was correlated with the amount of the migrating fragment. This assay was proven to be compatible with a crude cell lysate.

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.

Activity measurement of protein kinase and protein phosphatase by microchip phosphate-affinity electrophoresis.

We previously proposed microchip-based phosphate-affinity electrophoresis (µPAE) and demonstrated its application to activity measurement of a tyrosine kinase, c-Src. In this study, we extended the µPAE application to a serine/threonine kinase, protein kinase A (PKA), and to a tyrosine phosphatase, leukocyte antigen-related protein tyrosine phosphatase (LAR PTPase). For standard peptide samples, we obtained linear calibration plots, and the limits of detection were 1.2% (PKA) and 1.5% (LAR PTPase) product peptides in the total peptides. The µPAE was also proven to be effective for unpurified enzyme reaction products.

Rapid microRNA detection using power-free microfluidic chip: coaxial stacking effect enhances the sandwich hybridization.

We present a new method for rapid microRNA detection with a small volume of sample using the power-free microfluidic device driven by degassed PDMS. Target microRNA was detected by sandwich hybridization taking advantage of the coaxial stacking effect. This method allows us to detect miR-21 in 20 min with a 0.5 µL sample volume at a limit of detection of 0.62 nM. Since microRNAs can act as cancer markers, this method might substantially contribute to future point-of-care cancer diagnosis.

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.

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.

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.

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.

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.

Spatial distribution of laminar flow-assisted dendritic amplification.

In this paper, we report spatial distribution of laminar flow-assisted dendritic amplification (LFDA) product. LFDA is a recently invented signal amplification method dedicated to biomolecular binding events on microchannel walls. Onto the bound biomolecule, a dendritic structure is constructed by supplying two building blocks from laminar streams produced by a Y-shaped microchannel. In view of the extension of LFDA to simultaneous amplification of multiple binding spots, we have investigated the distribution of the LFDA product across and along the microchannel with the course of time. We fabricated a Y-shaped microchannel with a cross section of 110 microm x 22 microm using poly(dimethylsiloxane). As the LFDA building blocks, FITC-labeled streptavidin and biotinylated anti-streptavidin were injected from the two inlets of the microchannel at a mean flow velocity of 6.2 mm s(-1) (after the confluence). Nonspecific adsorption of the building blocks formed the seed layer of LFDA. The progress of LFDA was monitored with a fluorescence microscope up to 10.1 mm of microchannel length. After 5 min or later, the fluorescence intensity profile across the microchannel showed a peak at the center of the channel. With the course of time, the peak height grew exponentially except for slight saturation, but the peak width was almost constant. Along the microchannel, the peak height decreased almost linearly with the increasing logarithm of the distance, and the peak width was broadened in accordance with the 1/3 power law.

Alternating current cloud point extraction on a microchip for preconcentration of membrane-associated biomolecules.

Joule heating and negative dielectrophoresis, caused by AC electric fields in a microchannel, have been employed for cloud point extraction of phospholipid as a model of membrane-associated biomolecules.

SNP genotyping of unpurified PCR products by sandwich-type affinity electrophoresis on a microchip with programmed autonomous solution filling.

We demonstrate rapid single-nucleotide polymorphism (SNP) genotyping on a poly(dimethylsiloxane)-glass microchip. Sandwich-type affinity electrophoresis was employed to achieve sufficient specificity for reliable genotyping of unpurified PCR products. We tested three SNPs in different genes: CYP2D6 of artificial templates, and ALDH3A1 and CYP1A1 of human genomic samples. The target sequences were amplified by asymmetric PCR. For each SNP, we prepared a capture probe-poly(dimethylacrylamide) (CP-PDMA) conjugate and allele-specific, fluorescently-labeled detection probes (DPs). Prior to the electrophoresis, necessary solutions--the amplified sample, the CP-PDMA conjugate, the DPs, and a washer--were autonomously filled into their own regions of the microchannel in contact with each other. For precise control of this filling process, we have extended our published technique to a "programmed" version, in which additional passive stop valves synchronized the solution contacting events. Then we electrophoretically carried out a target DNA hybridization step, a DP hybridization step, and a washing step at the CP-PDMA conjugate region. This 3-step electrophoresis was completed in 2 min. The formation of the sandwich hybridization complex (CP-target-DP) was evaluated by fluorescence. Normalized fluorescence values of the different genotypes were clearly and reproducibly discriminated. The assay format presented here will be suitable for SNP genotyping at the point of care.

Controlling the number and positions of oligonucleotides on gold nanoparticle surfaces.

We describe a new method to immobilize a given number of oligonucleotides (ODNs) on gold nanoparticles (AuNPs) in a specific arrangement directed by a geometrical template made of DNA. The basic strategy is as follows. First, a set of thiolated ODNs for immobilization and a nonthiolated template are hybridized to make a DNA nanostructure. Next, the DNA nanostructure is reacted with AuNPs via the thiol groups to form a complex of the AuNP and the DNA nanostructure. Finally, the intended AuNP/ODN conjugate is obtained by removing the template from the complex. The above strategy enables us to make various formats of AuNP/ODN conjugates simply by changing the design of the DNA nanostructure. We demonstrate proof-of-concept experiments using a linear design of the DNA nanostructure.