Nanopillar Chips Research Articles

A smartphone-based electroporation system with highly robust and low-voltage silicon nanopillar chips

In this work, a novel smartphone-based electroporation (EP) system integrated with 3D scalable and robust gold-coated silicon-nanopillar Electroporation (Au-Si NP-EP) chip using projection photolithography is developed, for the first time, for both EP and electric cell lysing (ECL) at low voltages. Au-SiNP-EP chip consists of silicon nanopillars fabricated by using ASML stepper, Deep Reactive Ion Etching (DRIE) process and coated with a gold microelectrode. The silicon nanopillars were optimized based on theoretical analysis and numerical simulations to enhance the electrical field intensity and mechanical strength. The fabricated Au-SiNP-EP chips are tested with both permeable (Acridine Orange (AO) and impermeable (Propidium Iodide (PI)) molecules for HeLa cells at different volts (1–8 V) and pulse duration (1–9 μs). The fabricated chip achieved an optimized EP efficiency of 84.3% and cell viability of 81.4% at a much smaller voltage (4.5V) than reported planar electroporation (PEP) devices (8–100V). Compared with nanostructures-based devices (2–20 V), our devices show both higher mechanical strength and fabrication yield. Besides, a smartphone app integrated with a low-cost open-source portable Arduino-based system is developed to provide optimized electrical protocols for both EP and ECL. The electric cell lysing with ECL efficiency of 97.0% at 7 V and pulse duration of 9 ms has been successfully demonstrated. The experimental results show that the proposed smartphone-based EP system with Au-SiNP EP chips is promising for various applications, including intracellular delivery of various biomolecules, drugs, and release of DNA/RNA molecules from biological cells.

Au-Capped Nanopillar Immobilized with a Length-Controlled Glycopolymer for Immune-Related Protein Detection.

A Au-capped nanopillar chip was prepared using nanoimprint lithography (NIL) and Au sputtering onto a cyclo-olefin polymer film. The Au surface of the chip exerting localized surface plasmon resonance (LSPR) phenomena was immobilized with a glycopolymer for the detection of cytokines. The glycopolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization for controlled polymer chain length, and thiol-terminated glycopolymers with chain lengths of 20-, 100-, and 200-mers were designed. The thickness of the biomolecular layer on the Au surface was controlled by changing the polymer chain length of the immobilized glycopolymer, and the absorption of proteins onto the Au surface was detected by the shift of absorbance peak wavelength. The value of absorbance peak wavelength shift by protein adsorption increased as the glycopolymer layer thickness became thinner. This difference in LSPR signal response was remarkable for cytokine recognition compared to larger proteins. It was shown that controlling the biomolecular layer thickness was effective for the detection of small proteins, and our research suggested the usefulness of the controlled glycopolymer surface as a molecular recognition material for cytokine detection.

Micro- and Nanopillar Chips for Continuous Separation of Extracellular Vesicles.

Micro- and nanopillar chips are widely used to separate and enrich biomolecules, such as DNA, RNA, protein, and cells, as an analytical technique and to provide a confined nanospace for polymer science analyses. Herein, we demonstrated a continuous accurate and precise separation technique for extracellular vesicles (EVs), nanometer-sized vesicles (typically 50-200 nm) currently recognized as novel biomarkers present in biofluids, based on the principle of electroosmotic flow-driven deterministic lateral displacement in micro- and nanopillar array chips. Notably, the easy-to-operate flow control afforded by electroosmotic flow allowed nanoparticles 50-500 nm in size, including EVs, to be precisely separated and enriched in a continuous manner. By observation of the flow behavior of nanoparticles, we found that electroosmotic flow velocity in the nanopillar arrays did not solely depend on counterion mobility on the surface of nanopillar chips, but rather showed a parabolic flow profile. This hydrodynamic pressure-free and easy-to-use separation and enrichment technique, which requires only electrode insertion into the reservoirs and electric field application, may thus serve as a promising technique for future precise and accurate EV analysis, reflecting both size and composition for research and potential clinical diagnostic applications.

Pressure free nanoimprinting lithography using ladder-type HSQ material for LSPR biosensor chip

We present an ingenious pressure free NIL (Nanoimprint lithography) method by simply employing a very thin PDMS mold along with a Spin-on-Glass HSQ material. Fabricating nano-patterns (i.e. nano-gratings and 2 D nano-arrays) via NIL is advantageous for LSPR based biosensors for producing large working area with high-throughput. Unlike conventional NIL and other fabrication methods, no complicated equipment is required in this work; only a simple heating plate (at 100°C) was utilized for curing time reduction. Nano-pillar structure with definition down to 50–100nm has been successfully fabricated using this method. After Au-deposition, mushroom like Au-capped nano-pillar chip has been successfully validated as a LSPR bio-sensor chip for IgA detection. In addition, this method provides promising features for higher dimensional structures fabrication which is considered as a beneficial tool for LSPR based biosensor sensitivity enhancement.

ナノピラー・ナノワイヤチップによる核酸の高速分離法

ナノピラー・ナノワイヤチップによる核酸の高速分離法

Electroosmotic Flow in Microchannels with Nanostructures

Here we report that nanopillar array structures have an intrinsic ability to suppress electroosmotic flow (EOF). Currently using glass chips for electrophoresis requires laborious surface coating to control EOF, which works as a counterflow to the electrophoresis mobility of negatively charged samples such as DNA and sodium dodecyl sulfate (SDS) denatured proteins. Due to the intrinsic ability of the nanopillar array to suppress the EOF, we carried out electrophoresis of SDS-protein complexes in nanopillar chips without adding any reagent to suppress protein adsorption and the EOF. We also show that the EOF profile inside a nanopillar region was deformed to an inverse parabolic flow. We used a combination of EOF measurements and fluorescence observations to compare EOF in microchannel, nanochannel, and nanopillar array chips. Our results of EOF measurements in micro- and nanochannel chips were in complete agreement with the conventional equation of the EOF mobility (μ(EOF-channel) = αC(i)(-0.5), where C(i) is the bulk concentration of the i-ions and α differs in micro- and nanochannels), whereas EOF in the nanopillar chips did not follow this equation. Therefore we developed a new modified form of the conventional EOF equation, μ(EOF-nanopillar) ≈ β[C(i) - (C(i)(2)/N(i))], where N(i) is the number of sites available to i-ions and β differs for each nanopillar chip because of different spacings or patterns, etc. The modified equation of the EOF mobility that we proposed here was in good agreement with our experimental results. In this equation, we showed that the charge density of the nanopillar region, that is, the total number of nanopillars inside the microchannel, affected the suppression of EOF, and the arrangement of nanopillars into a tilted or square array had no effect on it.

DNA Separation in Nanowall Array Chips

A nanowall array structure was fabricated on a quartz chip as a separation matrix of DNA fragments, and a 30 s separation was realized for a mixture of DNA fragments (48.5 and 1 kbp fragments) by applying the electric voltage. A longer DNA fragment migrates faster than a shorter one in a nanowall array chip, and it is completely different from the separation of DNA based on gel electrophoresis, nanopillar chips, and nanoparticle array chips. Although the result is similar to DNA separation by entropic trapping, it could not be fully explained by entropic trapping phenomena. Direct observation of single-DNA molecular dynamics inside a nanowall array structure indicates that both confined elongation and relaxation recoiling of a DNA molecule occur, and an elongated DNA molecule migrates faster than a recoiled DNA molecule. Numerical fitting of DNA molecular dynamics reveals that the balance between times for the transverse of a DNA molecule in the nanowall array chip and the relaxation-recoiling of a DNA molecule governs the separation of DNA.

Construction of a more sensitive fluorescence sensing material for the detection of vascular endothelial growth factor, a biomarker for angiogenesis, prepared by combining a fluorescent peptide and a nanopillar substrate

We have established a highly sensitive and selective protein detection technology in combination with the nanofabrication technique. A silica nanopillar chip with a 200-nm pitch and 1000-nm height pillar substrate was fabricated by electron beam lithography and deep reactive ion etching method. Fluorescent peptides, with high affinity towards vascular endothelial growth factor (VEGF), were immobilized on nanopillar chip via a self-assembled monolayer made from 3-aminopropyltrimethoxysilane and glutaraldehyde under optimal conditions. The fluorescence intensity of the fluorescent peptide on the nanopillar substrate increased with increasing VEGF concentrations, as determined by a fluorescence spectrophotometer and fluorescent scanning image analysis. The dissociation constant ( K d value) calculated by the non-linear least square curve fitting method was 6.0 × 10 −9 M, which contributed to the highly sensitive detection of VEGF. The fluorescence intensity of the fluorescent reagent on the nanopillar substrate upon binding to VEGF was higher than that obtained using the flat substrate because the dense and tall nanopillar array increased the virtual protein binding area. The reproducibility tests and lifetime measurement indicate the fluorescent reagent to be a useful biosensor for the detection of VEGF in this system. These experimental results clearly showed that the combination of a fluorescent reagent and a nanopillar substrate may be widely applicable as a convenient method for the detection of VEGF.

Influences of electroosmotic flows in nanopillar chips on DNA separation: Experimental results and numerical simulations

AbstractThe various potential factors affecting the performance of nanopillar chips on DNA separation were investigated from the viewpoints of both numerical calculations and actual experiments. To yield higher performance and replace the conventional DNA separation techniques such as microchip electrophoresis, the phenomenon specific to the nanopillar chips should be deeply understood. In this paper, although various factors affecting the performance of the nanopillar chips are considered, we focused on the effect of electroosmotic flow, which is particularly noticeable in quartz‐made nanopillar chips. High‐resolution separation of DNA was realized when an electroosmotic flow was suppressed by simply using a higher concentration of buffer, but DNA separation failed in the presence of an electroosmotic flow. It was confirmed from the numerical simulations and the direct observations that the deformation of DNA band during the injection process was induced by electroosmotic flow and consequently led to a poor resolution.

Fabrication of nano-pillar chips by a plasma etching technique for fast DNA separation

The fabrication of quartz nano-pillars was investigated using dry etching with a Ni mask. The mask diameter increased during etching due to re-sputtering of the Pt/Cr seed layer. However, once the seed layer had been eroded the enlarged mask diameter did not increase any further. Hence, the use of the mask enabled the fabrication of nano-pillars with a high aspect ratio. In situ FTIR–ATR observation of HF quartz plate pressure bonding developed a new bonding technique involving the use of H 2SiF 6. The nano-pillar chips allowed then to size-separate DNA of 10 kbp and 38 kbp within 20 s.

Study of water properties in nanospace

Here we report an anomalous behavior of water, especially its viscosity and hydrodynamic flow, in a nanometer-confined space. As a typical model of a nanometer-confined space, the nanopillar chip, which was developed for DNA size-based separation was used, and single-particle tracking (SPT) technique was applied to investigate water viscosity and hydrodynamic flow in the nanopillar chip. The diffusion coefficients of nanospheres were almost one-third of the theoretical value derived from the Stokes-Einstein equation. This result gave indirect proof that water viscosity in a nanometer-confined space is higher than in a bulk solution. In order to improve resolution and throughput of the nanopillar chip for DNA separation, these potential factors affecting performance should be seriously considered.

Separation of long DNA molecules by quartz nanopillar chips under a direct current electric field.

We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1-38 kbp) and large DNA fragments (lambda DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas lambda DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.