Rapid and efficient nucleic acid (NA) extraction and concentration are required for point-of-care analysis in order to prevent an epidemic/pandemic disease outbreak. Typical silica-based NA ...extraction methods have limitations such as being time-consuming, requiring human intervention, and resulting in a low recovery yield. In this study, we have developed a pathogenic DNA extraction device based on electrokinetic separation incorporated with a silicon nitride (SiN
x
) nanofilter, which expedites the DNA extraction procedure with advantages of being convenient, efficient, and inexpensive. This DNA extraction device consists of a computer numerical control (CNC) milled-Teflon gadget with a
cis
-chamber as a cell lysate reservoir and a
trans
-chamber as a elution solution reservoir, with the SiN
x
nanofilter being inserted between the two chambers. The SiN
x
nanofilter was fabricated using a photolithographic method in conjunction with nanoimprinting. Approximately 7.2 million nanopores of 220 nm diameter were located at the center of the nanofilter. When a DC electric field is applied through the nanopores, DNA is transferred from the
cis
-chamber to the
trans
-chamber to isolate the DNA from the cell debris. To demonstrate the DNA extraction performance, we measured the absorbances at 260 and 280 nm and performed a real-time polymerase chain reaction (real-time PCR) using the recovered DNA to verify its feasibility for downstream genetic analysis. Moreover, the DNA extraction device was successfully operated using a 1.5 V alkaline battery, which verifies the portability of the device for point-of-care testing. Such an advanced DNA extraction system can be utilized in various fields including clinical analysis, pathogen detection, forensic analysis, and on-site detection.
A simple electrophoretic DNA extraction device has developed using a nanofilter for rapid and efficient DNA aquisition required for point-of-care analysis in order to prevent an epidemic/pandemic disease outbreak.
The noninvasive early detection of biomarkers for Alzheimer’s disease (AD) is essential for the development of specific treatment strategies. This paper proposes an advanced method for fabricating ...highly ordered and flexible nanopillar-based electrochemical biosensors by the combination of soft/photolithography and metal evaporation. The nanopillar array (NPA) exhibits high surface area containing 1500 nm height and 500 nm diameter with 3:1 ratio. In regard with physical properties of polyurethane (PU) substrate, the developed NPA is sustainable and durable to external pressure such as bending and twisting. To manipulate the NPA surface to biocompatible, the gold was uniformly deposited on the PU substrate. The thiol chemistry which is stably modified on the gold surface as a form of self-assembled monolayer was employed for fabricating the NPA as a biocompatible chip by covalently immobilize the antibodies. The proposed nanopillar-based immunoelectrochemical biosensor exhibited good and stable electrochemical performance in β-amyloid (Aβ) detection. Moreover, we successfully confirmed the performance of the as-developed sensor using the artificial injection of Aβ in human tear, with sensitivity of 0.14 ng/mL and high reproducibility (as a standard deviation below 10%). Our findings show that the developed nanopillar-based sensor exhibits reliable electrochemical characteristics and prove its potential for application as a biosensor platform for testing at the point of care.
A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the ...nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 10
8
/cm
2
. The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.
We report a laser-pyrolyzed carbon (LPC) electrode prepared from a black photoresist for an on-chip microsupercapacitor (MSC). An interdigitated LPC electrode was fabricated by direct laser writing ...using a high-power carbon dioxide (CO2) laser to simultaneously carbonize and pattern a spin-coated black SU-8 film. Due to the high absorption of carbon blacks in black SU-8, the laser-irradiated SU-8 surface was directly exfoliated and carbonized by a fast photo-thermal reaction. Facile laser pyrolysis of black SU-8 provides a hierarchically macroporous, graphitic carbon structure with fewer defects (ID/IG = 0.19). The experimental conditions of CO2 direct laser writing were optimized to fabricate high-quality LPCs for MSC electrodes with low sheet resistance and good porosity. A typical MSC based on an LPC electrode showed a large areal capacitance of 1.26 mF cm−2 at a scan rate of 5 mV/s, outperforming most MSCs based on thermally pyrolyzed carbon. In addition, the results revealed that the high-resolution electrode pattern in the same footprint as that of the LPC-MSCs significantly affected the rate performance of the MSCs. Consequently, the proposed laser pyrolysis technique using black SU-8 provided simple and facile fabrication of porous, graphitic carbon electrodes for high-performance on-chip MSCs without high-temperature thermal pyrolysis.
We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap ...interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400
nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen–antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1
μm particles were formed from 10
nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1
pg
mL
−1 with a wide dynamic range (from 10
−3 to 100
ng
mL
−1 concentrations).
An enzymatic reaction was employed as a means to enhance the sensitivity of an immunosensor based on localized surface plasmon resonance (LSPR). The reaction occurs after intermolecular binding ...between an antigen and an antibody on gold nano-island (NI) surfaces. For LSPR sensing, the gold NI surface was fabricated on glass substrates using vacuum evaporation and heat treatment. The interferon-γ (IFN-γ) capture antibody was immobilized on the gold NIs, followed by binding of IFN-γ to the antibody. Subsequently, a biotinylated antibody and a horseradish peroxidase (HRP) conjugated with avidin were simultaneously introduced. A solution of 4-chloro-1-naphthol (4-CN) was then used for precipitation; precipitation was the result of the enzymatic reaction catalyzed the HRP on gold NIs. The LSPR spectra were obtained after each binding process. Using this method, the enzyme-catalyzed precipitation reaction on the gold NI surface was found to effectively amplify the change in the signal of the LSPR immunosensor after intermolecular binding.
We have demonstrated the use of thermal transfer printing for scalable, inkless fabrication of a graphene-based textile microsupercapacitor (MSC). An adhesive film of a heat transfer paper was ...employed as a flat, buffer adhesive layer on a rough textile substrate. Laser-induced graphene (LIG) directly laser-written on polyimide (PI) films was transferred onto the adhesive film area of the textile substrates at elevated temperature and pressure. A thermally transfer-printed LIG pattern preserved porous structure with 3D interconnected pores and high electrical conductivity of LIG formed on PI films. The developed textile LIG-MSCs exhibit electrical double layer capacitive characteristics with areal capacitance of 0.76mFcm−2 and excellent capacitance retention of 96% after 1000 cycles of large bending (180°) deformation. Furthermore, scalable transfer of a large-area LIG pattern provided various array configurations of MSCs in series and in parallel to adjust the voltage and current for practical applications. Moreover, LIG-MSCs with a LIG-metal composite exhibit fast ion transport at a high scan rates of up to 20Vs-1, suggesting outstanding rate capability among graphene-based textile MSCs. In this regard, the proposed inkless transfer strategy provided low cost and scalable fabrication of a LIG composite electrode on a textile substrate without preparing costly graphene-based ink materials.
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•An inkless fabrication of textile MSCs is demonstrated by thermal transfer printing.•Thermal transfer printing preserved porous structure with 3D interconnected pores.•A LIGT-MSC showed capacitance retention of 96% after 1000 cycles of large bending.•A LIGT-MSC exhibited fast ion transport at a high scan rates up to 20 V s−1.
The commonly used trypan blue dye exclusion method and other modified cell viability methods, such as fluorescein dye and tetrazolium dye exclusion, artificially introduce toxic chemicals to cells ...and, thus, alter cellular organelles when measuring cell viability. Therefore, cell viability could be affected by the processes currently used to observe viability. In this study, the cell viability of Chinese hamster ovary (CHO) cells was measured by simply counting attached cells after the cultured CHO cells were attached on a Concanavalin A (Con A) substrate. The efficiency of cell attachment to Con A surfaces was different for live and dead cells allowing the cell viability of CHO cells to be measured without any chemical modifications to the cells.
High sulfur content polymers (HSCPs) are regarded as promising materials for infrared (IR) optics, especially the long-wave IR range, due to intrinsic properties of sulfur, but the poor thermal ...stability of HSCPs limits their reliable utilization in wider IR applications. We herein present a new HSCP, poly(sulfur-co-hexavinyl disiloxane) (pSHVDS), prepared directly from elemental sulfur through sulfur vapor chemical deposition (sCVD). By employing hexavinyl disiloxane (HVDS) comonomer with high functionality (f = 12) and weak absorption in the IR range, despite the poor compatibility between sulfur and HVDS, the sCVD process enables the preparation of highly cross-linked HSCP with 68 wt % sulfur. Furthermore, the combination of high refractive index (RI) of 1.842, high glass transition temperature (T g ) of 151 °C, and high transparency in visible, mid- and long-wave IR range is achieved successfully, which is an unprecedentedly unique property compared to HSCPs reported to date. These favorable properties of pSHVDS render the material ideal for antireflection applications in IR optics, and conformal pSHVDS coatings on germanium IR lenses significantly improved the transmittance in mid- and long-wave IR regions. The developed polymer will lead the new possibility for polymer based optical devices, especially in the IR range.
Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging ...analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(
l-lysine) for sequence-specific DNA–protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein–protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.
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