Automated Droplet-Based Microfluidic Platform for Multiplexed Analysis of Biochemical Markers in Small Volumes.

The ability to detect multiple analytes in a small sample volume has significance for numerous areas of research, including organs-on-chip, small animal experiments, and neonatology. The objective of this study was to develop an automated microfluidics platform for multiplexed detection of analytes in microliter sample volumes. This platform employed computer-controlled microvalves to create laminar co-flows of sample and assay reagent solutions. It also contained valve-regulated cross-junction for discretizing sample/reagent mixtures into water-in-oil droplets. Microfluidic automation allowed us to control parameters related to frequency of droplet generation and the number of droplets of the same composition, as well as the size of droplets. Each droplet represented an individual enzymatic assay carried out in a sub-nanoliter (0.8 nL) volume reactor. An enzymatic reaction involving target analyte and assay reagents produced colorimetric or fluorescent signals in droplets. Importantly, intensity of optical signal was proportional to the concentration of analyte in question. This microfluidic bioanalysis platform was used in conjunction with commercial "mix-detect" assays for glucose, total bile acids, and lactate dehydrogenase (LDH). After characterizing these assays individually, we demonstrated sensitive multiplexed detection of three analytes from as little as 3 µL. In fact, this volume was sufficient to generate multiple repeat droplets for each of the three biochemical assays as well as positive control droplets, confirming the quality of assay reagents and negative control droplets to help with background subtraction. One potential application for this microfluidic bioanalysis platform involves sampling cell-conditioned media in organ-on-chip devices. To highlight this application, hepatocyte spheroids were established in microfluidic devices, injured on-chip by exposure to lipotoxic agent (palmitate), and then connected to the bioanalysis module for daily monitoring of changes in cytotoxicity (LDH), energy metabolism (glucose), and liver function (total bile acids). Microfluidic in-droplet assays revealed increased levels of LDH as well as reduction in bile acid synthesis-results that were consistent with hepatic injury. Importantly, these experiments highlighted the fact that in-droplet assays were sufficiently sensitive to detect changes in functional output of a relatively small (∼100) number of hepatocyte spheroids cultured in a microfluidic device. Moving forward, we foresee increasing the multiplexing capability of this technology and applying this platform to other biological/medical scenarios where detection of multiple analytes from a small sample volume is desired.

Engineered cocultures of iPSC-derived atrial cardiomyocytes and atrial fibroblasts for modeling atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling. We developed a protein patterning process within multiwell plates to engineer patterned iPSC-aCM and atrial fibroblast coculture (PC) that significantly enhanced iPSC-aCM structural, electrical, contractile, and metabolic maturation for 6+ weeks compared to conventional mono-/coculture. PC displayed greater sensitivity for detecting drug efficacy than monoculture and enabled the modeling and pharmacological or gene editing treatment of an AF-like electrophysiological phenotype due to a mutated sodium channel. Overall, PC is useful for elucidating cell signaling in the atria, drug screening, and modeling AF.

Chip-based cartilage oligomeric matrix protein detection in serum and synovial fluid for osteoarthritis diagnosis.

We have developed a method to detect cartilage oligomeric matrix protein (COMP) as a specific biomarker of osteoarthritis (OA). In pathological conditions of the cartilage, COMP is released first into the synovial fluid (SF) and from there into the blood. Thus, measurement of COMP in the blood and SF facilitates OA diagnosis. To determine COMP, we developed a fluoro-microbead guiding chip (FMGC)-based immunoassay. The FMGC has four immunoreactive regions, each with five patterns, to allow multiple assays. A COMP-specific capture antibody was immobilized to the FMGC surface to create a self-assembled interfacial layer. SF or serum samples from patients with OA possessing the target COMP were applied to the COMP-sensing monolayer. To generate binding signal, COMP detection antibody-conjugated fluoro-microbeads were applied and the numbers of fluoro-microbeads bound specifically were counted to determine COMP concentrations. This FMGC-based immunoassay clearly distinguished immunospecific from nonspecific binding by comparing optical signals from inside and outside of the patterns. The optical signals showed linear correlations with serum and SF COMP concentrations. Optical detection and quantification of COMP using fluorescence microscopy correlated well with results from commercial enzyme-linked immunosorbent assay (ELISA). This FMGC-based immunoassay offers a new approach for detecting a clinically relevant biomarker for OA in human blood and SF.

Retroreflection-based optical biosensing: From concept to applications.

Optical biochemical assays that utilize traditional optical signaling labels, such as fluorophores and fluorescent nanoparticles, have been extensively applied in the development of optical biosensors. However, traditional optical-label-based analytical approaches require expensive and sophisticated optical instruments; thus, the application of traditional optical-label-based biochemical assays to optical biosensors in point-of-care testing (POCT) concepts that require cost-effectiveness and user-friendliness remains challenging. Retroreflection-based optical biosensing technology that utilizes micro-sized retroreflectors as an optical signaling label is being studied as a promising technological alternative to overcome the drawbacks of conventional optical-label-based biosensors. Retroreflection is an optical phenomenon whereby light rays strike a specific surface, a retroreflector, and are redirected to the light source along the inverse direction of the incident light. Biosensors that involve the retroreflection principle and retroreflector-type optical label offer distinctive advantages, such as the cost-effective simplification of optical instrument configuration, highly flexible applicability to various biochemical assays, and high analytical capability; therefore, their further applications toward the biosensing platform for POCT is highly promising. This review introduces the fundamentals of retroreflection and summarizes recent research achievements of retroreflection-based optical biosensor development from the perspective of how retroreflectors can be coupled and utilized with the optical biosensing principle as optical signal labels. The expected future applications of retroreflection-based optical biosensor technology is also discussed.

Mutant ANP induces mitochondrial and ion channel remodeling in a human iPSC-derived atrial fibrillation model.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can model heritable arrhythmias to personalize therapies for individual patients. Although atrial fibrillation (AF) is a leading cause of cardiovascular morbidity and mortality, current platforms to generate iPSC-atrial (a) CMs are inadequate for modeling AF. We applied a combinatorial engineering approach, which integrated multiple physiological cues, including metabolic conditioning and electrical stimulation, to generate mature iPSC-aCMs. Using the patient's own atrial tissue as a gold standard benchmark, we assessed the electrophysiological, structural, metabolic, and molecular maturation of iPSC-aCMs. Unbiased transcriptomic analysis and inference from gene regulatory networks identified key gene expression pathways and transcription factors mediating atrial development and maturation. Only mature iPSC-aCMs generated from patients with heritable AF carrying the non-ion channel gene (NPPA) mutation showed enhanced expression and function of a cardiac potassium channel and revealed mitochondrial electron transport chain dysfunction. Collectively, we propose that ion channel remodeling in conjunction with metabolic defects created an electrophysiological substrate for AF. Overall, our electro-metabolic approach generated mature human iPSC-aCMs that unmasked the underlying mechanism of the first non-ion channel gene, NPPA, that causes AF. Our maturation approach will allow for the investigation of the molecular underpinnings of heritable AF and the development of personalized therapies.

Multienzyme-modified biosensing surface for the electrochemical analysis of aspartate transaminase and alanine transaminase in human plasma.

We investigated the electrochemical detection of aspartate transaminase (AST) and alanine transaminase (ALT) by using a multienzyme-modified electrode surface. Determination of the activities of transaminases in human serum is clinically significant because their concentrations and ratios indicate the presence of hepatic diseases or myocardial dysfunction. For electrochemical detection of AST and ALT, enzymes that participate in the reaction mechanism of AST and ALT, such as pyruvate oxidase (POX) and oxaloacetate decarboxylase, were immobilized on an electrode surface by using an amine-reactive self-assembled monolayer and a homobifunctional cross-linker. In the presence of suitable substrates such as L-aspartate (L-alanine) and α-ketoglutarate, AST and ALT generate pyruvate as an enzymatic end product. To determine the activities of AST and ALT, electroanalyses of pyruvate were conducted using a POX and ferrocenemethanol electron shuttle. Anodically generated oxidative currents from multienzyme-mediated reactions were correlated to AST and ALT levels in human plasma. On the basis of the electrochemical analysis, we obtained calibration results for AST and ALT concentrations from 7.5 to 720 units/L in human plasma-based samples, covering the required clinical detection range.

Bioengineering approaches to mature induced pluripotent stem cell-derived atrial cardiomyocytes to model atrial fibrillation.

Induced pluripotent stem cells (iPSCs) serve as a robust platform to model several human arrhythmia syndromes including atrial fibrillation (AF). However, the structural, molecular, functional, and electrophysiological parameters of patient-specific iPSC-derived atrial cardiomyocytes (iPSC-aCMs) do not fully recapitulate the mature phenotype of their human adult counterparts. The use of physiologically inspired microenvironmental cues, such as postnatal factors, metabolic conditioning, extracellular matrix (ECM) modulation, electrical and mechanical stimulation, co-culture with non-parenchymal cells, and 3D culture techniques can help mimic natural atrial development and induce a more mature adult phenotype in iPSC-aCMs. Such advances will not only elucidate the underlying pathophysiological mechanisms of AF, but also identify and assess novel mechanism-based therapies towards supporting a more 'personalized' (i.e. patient-specific) approach to pharmacologic therapy of AF.

Instrumentation-Free Semiquantitative Immunoanalysis Using a Specially Patterned Lateral Flow Assay Device.

In traditional colorimetric lateral flow immunoassay (LFI) using gold nanoparticles (AuNPs) as a probe, additional optical transducers are required to quantify the signal intensity of the test line because it presents as a single red-colored line. In order to eliminate external equipment, the LFI signal should be quantifiable by the naked eye without the involvement of optical instruments. Given this objective, the single line test zone of conventional LFI was converted to several spots that formed herringbone patterns. When the sandwich immunoassay was performed on a newly developed semi-quantitative (SQ)-LFI system using AuNPs as an optical probe, the spots were colorized and the number of colored spots increased proportionally with the analyte concentration. By counting the number of colored spots, the analyte concentration can be easily estimated with the naked eye. To demonstrate the applicability of the SQ-LFI system in practical immunoanalysis, microalbumin, which is a diagnostic marker for renal failure, was analyzed using microalbumin-spiked artificial urine samples. Using the SQ-LFI system, the calibration results for artificial urine-based microalbumin were studied, ranging from 0 to 500 µg/mL, covering the required clinical detection range, and the limit of detection (LOD) value was calculated to be 15.5 µg/mL. Thus, the SQ-LFI system provides an avenue for the realization of an efficient quantification diagnostic device in resource-limited conditions.

Salmonella Typhimurium Sensing Strategy Based on the Loop-Mediated Isothermal Amplification Using Retroreflective Janus Particle as a Nonspectroscopic Signaling Probe.

Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.

An Optical Biosensing Strategy Based on Selective Light Absorption and Wavelength Filtering from Chromogenic Reaction.

To overcome the time and space constraints in disease diagnosis via the biosensing approach, we developed a new signal-transducing strategy that can be applied to colorimetric optical biosensors. Our study is focused on implementation of a signal transduction technology that can directly translate the color intensity signals-that require complicated optical equipment for the analysis-into signals that can be easily counted with the naked eye. Based on the selective light absorption and wavelength-filtering principles, our new optical signaling transducer was built from a common computer monitor and a smartphone. In this signal transducer, the liquid crystal display (LCD) panel of the computer monitor served as a light source and a signal guide generator. In addition, the smartphone was used as an optical receiver and signal display. As a biorecognition layer, a transparent and soft material-based biosensing channel was employed generating blue output via a target-specific bienzymatic chromogenic reaction. Using graphics editor software, we displayed the optical signal guide patterns containing multiple polygons (a triangle, circle, pentagon, heptagon, and 3/4 circle, each associated with a specified color ratio) on the LCD monitor panel. During observation of signal guide patterns displayed on the LCD monitor panel using a smartphone camera via the target analyte-loaded biosensing channel as a color-filtering layer, the number of observed polygons changed according to the concentration of the target analyte via the spectral correlation between absorbance changes in a solution of the biosensing channel and color emission properties of each type of polygon. By simple counting of the changes in the number of polygons registered by the smartphone camera, we could efficiently measure the concentration of a target analyte in a sample without complicated and expensive optical instruments. In a demonstration test on glucose as a model analyte, we could easily measure the concentration of glucose in the range from 0 to 10 mM.

Double-sided 3D printing on paper towards mass production of three-dimensional paper-based microfluidic analytical devices (3D-µPADs).

Recently, much effort has been focused on developing three-dimensional, paper-based microfluidic analytical devices (3D-µPADs) targeting in vitro diagnostics. However, 3D-µPAD fabrication typically requires tedious assembly that hinders mass production. Here, we report on a fabrication method for 3D-µPADs made of plastics without the need for additional assembly. Both sides of the paper were printed via liquid resin photopolymerization using a digital light processing (DLP) printer. The sample reservoir and detection zones are located on the top of the 3D-µPADs, and three microchannels are located on the bottom. The detection limits for glucose, cholesterol, and triglyceride (TG) in phosphate-buffered saline (PBS) were 0.3 mM, 0.2 mM, and 0.3 mM, respectively. The detectable ranges of glucose, cholesterol, and TG in human serum were 5-11 mM, 2.6-6.7 mM, and 1-2.3 mM. These results suggest that our fabrication method is suitable to mass produce 3D-µPADs with relative ease using simple fabrication processes.

Water-soluble mercury ion sensing based on the thymine-Hg2+-thymine base pair using retroreflective Janus particle as an optical signaling probe.

Herein, we report an optical sensing platform for mercury ions (Hg2+) in water based on the integration of Hg2+-mediated thymine-thymine (T-T) stabilization, a biotinylated stem-loop DNA probe, and a streptavidin-modified retroreflective Janus particle (SA-RJP). Two oligonucleotide probes, including a stem-loop DNA probe and an assistant DNA probe, were utilized. In the absence of Hg2+, the assistant DNA probe does not hybridize with the stem-loop probe due to their T-T mismatch, so the surface-immobilized stem-loop DNA probe remains a closed hairpin structure. In the presence of Hg2+, the DNA forms a double-stranded structure with the loop region via Hg2+-mediated T-T stabilization. This DNA hybridization induces stretching of the stem-loop DNA probe, exposing biotin. To translate these Hg2+-mediated structural changes in DNA probe into measurable signal, SA-RJP, an optical signaling label, is applied to recognize the exposed biotin. The number of biospecifically bound SA-RJPs is proportional to the concentration of Hg2+, so that the concentration of Hg2+ can be quantitatively analyzed by counting the number of RJPs. Using the system, a highly selective and sensitive measurement of Hg2+ was accomplished with a limit of detection of 0.027nM. Considering the simplified optical instrumentation required for retroreflection-based RJP counting, RJP-assisted Hg2+ measurement can be accomplished in a much easier and inexpensive manner. Moreover, the detection of Hg2+ in real drinking water samples including tap and commercial bottled water was successfully carried out.

Ambient light-based optical biosensing platform with smartphone-embedded illumination sensor.

We present a hand-held optical biosensing system utilizing a smartphone-embedded illumination sensor that is integrated with immunoblotting assay method. The smartphone-embedded illumination sensor is regarded as an alternative optical receiver that can replaces the conventional optical analysis apparatus because the illumination sensor can respond to the ambient light in a wide range of wavelengths, including visible and infrared. To demonstrate the biosensing applicability of our system employing the enzyme-mediated immunoblotting and accompanying light interference, various types of ambient light conditions including outdoor sunlight and indoor fluorescent were tested. For the immunoblotting assay, the biosensing channel generating insoluble precipitates as an end product of the enzymatic reaction is fabricated and mounted on the illumination sensor of the smartphone. The intensity of penetrating light arrives on the illumination sensor is inversely proportional to the amount of precipitates produced in the channel, and these changes are immediately analyzed and quantified via smartphone software. In this study, urinary C-terminal telopeptide fragment of type II collagen (uCTX-II), a biomarker of osteoarthritis diagnosis, was tested as a model analyte. The developed smartphone-based sensing system efficiently measured uCTX-II in the 0-5ng/mL concentration range with a high sensitivity and accuracy under various light conditions. These assay results show that the illumination sensor-based optical biosensor is suitable for point-of-care testing (POCT).

Encapsulation-Stabilized, Europium Containing Nanoparticle as a Probe for Time-Resolved luminescence Detection of Cardiac Troponin I.

The use of a robust optical signaling probe with a high signal-to-noise ratio is important in the development of immunoassays. Lanthanide chelates are a promising material for this purpose, which provide time-resolved luminescence (TRL) due to their large Stokes shift and long luminescence lifetime. From this, they have attracted considerable interest in the in vitro diagnostics field. However, the direct use of lanthanide chelates is limited because their luminescent signal can be easily affected by various quenchers. To overcome this drawback, strategies that rely on the entrapment of lanthanide chelates inside nanoparticles, thereby enabling the protection of the lanthanide chelate from water, have been reported. However, the poor stability of the lanthanide-entrapped nanoparticles results in a significant fluctuation in TRL signal intensity, and this still remains a challenging issue. To address this, we have developed a Lanthanide chelate-Encapsulated Silica Nano Particle (LESNP) as a new immunosensing probe. In this approach, the lanthanide chelate is covalently crosslinked within the silane monomer during the silica nanoparticle formation. The resulting LESNP is physically stable and retains TRL properties of the parent lanthanide chelate. Using the probe, a highly sensitive, sandwich-based TRL immunoassay for the cardiac troponin I was conducted, exhibiting a limit of detection of 48 pg/mL. On the basis of the features of the LESNP such as TRL signaling capability, stability, and the ease of biofunctionalization, we expect that the LESNP can be widely applied in the development of TRL-based immunosensing.

Boronate-functionalized hydrogel as a novel biosensing interface for the glycated hemoglobin A1c (HbA1c) based on the competitive binding with signaling glycoprotein.

According to recent increases in public healthcare costs associated with diabetes mellitus, the development of new glycemic monitoring techniques based on the biosensing of glycated hemoglobin A1c (HbA1c), a promising long-term glycemic biomarker, has become a major challenge. In the development of HbA1c biosensors for point-of-care applications, the selection of an effective biorecognition layer that provides a high reaction yield and specificity toward HbA1c is regarded as the most significant issue. To address this, we developed a novel HbA1c biosensing interfacial material by the integration of boronate hydrogel with glass fiber membrane. In the present study, a new boronate-functionalized hydrogel was designed and spatio-selectively photopolymerized on a hydrophilic glass fiber membrane by using N-hydroxyethyl acrylamide, 3-(acrylamido)phenylboronic acid, and bis(N,N'-methylene-bis-acrylamide). Using this approach, the boronic acid group, which specifically recognizes the cis-diol residue of glucose on the HbA1c molecule, can be three-dimensionally coated on the surface of the glass fiber network with a high density. Because this network structure of boronate hydrogel-grafted fibers enables capillary-driven fluid control, facile HbA1c biosensing in a lateral flow assay concept could be accomplished. On the proposed HbA1c biosensing interface, various concentrations of HbA1c (5-15%) in blood-originated samples were sensitively measured by a colorimetric assay using horseradish peroxidase, a glycoenzyme can generate chromogenic signal after the competitive binding against HbA1c to the boronic acid residues. Based on the demonstrated advantages of boronate hydrogel-modified membrane including high analytical performance, easy operation, and cost-effectiveness, we expect that the proposed biorecognition interfacial material can be applied not only to point-of-care HbA1c biosensors, but also to the quantitative analysis of other glycoprotein biomarkers.

Selective protein transport through ultra-thin suspended reduced graphene oxide nanopores.

The nanoporous free-standing graphene membrane is of great interest in high performance separation technology. In particular, the separation of biological molecules with similar sizes is one of the key challenges in the purification of biomaterials. Here, we report a reliable, cost-effective, and facile method for the fabrication of a graphene-based nanosieve and its application in the separation of similar-size proteins. A suspended reduced graphene oxide (rGO) nanosieve with ultra-thin, large-area, well-ordered, and dense 15 nm-sized pores was fabricated using block copolymer (BCP) lithography. The fabricated 5 nm-ultrathin nanosieve with an area of 200 µm × 200 µm (an ultra-high aspect ratio of ∼40 000) endured pressure up to 1 atm, and effectively separated hemoglobin (Hb) from a mixture of hemoglobin and immunoglobulin G (IgG), the common proteins in human blood, in a highly selective and rapid manner. The use of the suspended rGO nanosieve is expected to provide a simple and manufacturable platform for practical biomolecule separation offering high selectivity and a large throughput.

Retroreflective Janus Microparticle as a Nonspectroscopic Optical Immunosensing Probe.

We developed retroreflective Janus microparticles (RJPs) as a novel optical immunosensing probe for use in a nonspectroscopic retroreflection-based immunoassay. By coating the metals on the hemispherical surface of silica particles, highly reflective RJPs were fabricated. On the basis of the retroreflection principle, the RJPs responded to polychromatic white light sources, in contrast to conventional optical probes, which require specific monochromatic light. The retroreflection signals from RJPs were distinctively recognized as shining dots, which can be intuitively counted using a digital camera setup. Using the developed retroreflective immunosensing system, cardiac troponin I, a specific biomarker of acute myocardial infarction, was detected with high sensitivity. On the basis of the demonstrated features of the retroreflective immunosensing platform, we expect that our approach may be applied for various point-of-care-testing applications.

Cascadic multienzyme reaction-based electrochemical biosensors.

: Since the first glucose biosensor was developed by Clark and Lyons, there have been great efforts to develop effective enzyme biosensors for wide applications. Those efforts are closely related to the enhancement of biosensor performance, including sensitivity improvement, elevation of selectivity, and extension of the range of analytes that may be determined. Introduction of a cascadic multienzyme reaction to the electrochemical biosensor is one of those efforts. By employing more than two enzymes to the biosensor, its sensitivity and accuracy can be enhanced. Also, the narrow application range that is a typical limitation of single enzyme-based biosensor can be overcome. This chapter will discuss the fundamental principles for the development of cascadic multienzyme reaction-based electrochemical biosensors and their applications in clinical and environmental fields.

Bioelectrocatalytic sensor for triglycerides in human skin sebum based on enzymatic cascade reaction of lipase, glycerol kinase and glycerophosphate oxidase.

We report the development of an electrochemical biosensor for the quantification of triglycerides in human skin sebum, based on a multienzyme cascade reaction. The presence of excessive triglycerides in human sebum is one of the leading causes of various skin ailments. However, to the best of our knowledge, no bioelectrocatalytic approach for the quantification of sebum triglycerides has been made. In order to develop triglyceride biosensor, we fabricated a multienzyme-associated electrode incorporating lipase, glycerol kinase, and glycerophosphate oxidase. Enzymes were deposited by electrostatic force and further stabilized via crosslinking between enzymes and polymer matrices. The enzyme-modified biosensing electrode maintained its bioelectrocatalytic activity for five days. An additional constraint was the limited solubility of sebum triglycerides in aqueous electrolytes, impeding the analysis. To address this issue, triglyceride samples were prepared in the form of micelles, enabling efficient sample preparation for biosensor signaling. Calibration tests revealed that the designed assay had a detection range of 15-200mg/dL of micellar triglyceride, which covered the required determination range. The developed biosensing approach was successfully used to determine triglyceride concentrations in real sebum samples of unknown triglyceride content.

Use of a columnar metal thin film as a nanosieve with sub-10 nm pores.

A columnar-structured nanosieve is unique in the sense that it is a general thin film formed by physical vapor deposition (PVD). Instead of additional processes to make nanopores, the numerous voids naturally formed among columnar grains during PVD are used as nanopores. Since the thin film formed by PVD has vertically grown columnar grains, the fabricated nanosieve has numerous straight-opened nanopores, which is an ideal structure for a nanosieve.