Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

A Heart-Breast Cancer-on-a-Chip Platform for Disease Modeling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-ß 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

The impact of educational attainment on cardiorespiratory fitness and metabolic syndrome in Korean adults.

The aim of this study was to evaluate the relationship between educational attainment and cardiorespiratory fitness (CRF) as a predictor of metabolic syndrome in a Korean population.In this single-center, retrospective cross-sectional study, 988 healthy adults (601 men and 387 women) who underwent regular health check-up in Seoul St. Mary's Hospital were analyzed. Educational attainment was categorized into 3 groups according to their final grade of educational course: middle or high school (≤12 years of education), college or university (12-16 years of education), and postgraduate (≥16 years of education). CRF was assessed by cardiopulmonary exercise testing, biceps strength, hand grip strength, bioelectrical impedance analysis, and echocardiography. Metabolic syndrome was diagnosed according to the 3rd report of the National Cholesterol Education Program.Among the subjects, 357 (36.1%) had metabolic syndrome. The postgraduate group had significantly higher peak oxygen consumption (VO2), biceps strength, hand grip strength, and peak expiratory flow than other groups (all P < .001). This group showed better left ventricular diastolic function, in terms of deceleration time of mitral inflow, maximal tricuspid valve regurgitation velocity, and left atrial volume index than other groups. Peak VO2 (%) was significantly correlated with all the parameters of metabolic syndrome, including insulin resistance (r = -0.106, P = .002), waist circumference (r = -0.387, P < .001), triglyceride (r = -0.109, P = .001), high density lipoprotein-cholesterol (r = 0.219, P < .001), systolic blood pressure (r = -0.143, P < .001), and diastolic blood pressure (r = -0.177, P < .001). And Peak VO2 (%) was found to be a predictor of metabolic syndrome (adjusted ß = .988, P < .001). However, the level of education was not able to predict metabolic syndrome (postgraduate group; ß = .955, P = .801).Although the postgraduate group had better CRF than other groups, the educational attainment could not exclusively predict metabolic syndrome in this study. Further research is needed to reveal the socioeconomic mechanism of developing metabolic syndrome.

Measuring Bone Biomarker Alkaline Phosphatase with Wafer-Scale Nanowell Array Electrodes.

Biosensors that can analyze a single drop of biological fluid can overcome limitations such as extraction volume from humans or animals, ethical problems, time, and cost. In this work, we have developed a highly sensitive electrochemical (EC) biosensor based on a nanowell array (NWA) for the detection of alkaline phosphatase (ALP), a serum indicator of bone formation. The size of the electrode is 2 × 1 mm2 and has over 10 million nanowells (400 nm diameter) arranged uniformly on the electrode surface. For detecting ALP, anti-ALP was immobilized and oriented on the NWA surface using a self-assembled monolayer and protein G. EC impedance spectroscopy (EIS) was used to determine the amount of ALP in 10 µL of sample. The impedance was calibrated with ALP concentration. The NWA has a linear dynamic range from 1 pg/mL to 100 ng/mL with a limit of detection (LOD) at 12 pg/mL. We used the sensor to measure the ALP in real mouse serum from 4, 10, and 20 weeks old mice and compared the results to the standard photometric assay. This work demonstrates the potential of EC NWA sensors to analyze a single drop of a real body fluid sample and to be developed for broad applications.

Innovations in biomedical nanoengineering: nanowell array biosensor.

Nanostructured biosensors have pioneered biomedical engineering by providing highly sensitive analyses of biomolecules. The nanowell array (NWA)-based biosensing platform is particularly innovative, where the small size of NWs within the array permits extremely profound sensing of a small quantity of biomolecules. Undoubtedly, the NWA geometry of a gently-sloped vertical wall is critical for selective docking of specific proteins without capillary resistances, and nanoprocessing has contributed to the fabrication of NWA electrodes on gold substrate such as molding process, e-beam lithography, and krypton-fluoride (KrF) stepper semiconductor method. The Lee group at the Mara Nanotech has established this NW-based biosensing technology during the past two decades by engineering highly sensitive electrochemical sensors and providing a broad range of detection methods from large molecules (e.g., cells or proteins) to small molecules (e.g., DNA and RNA). Nanosized gold dots in the NWA enhance the detection of electrochemical biosensing to the range of zeptomoles in precision against the complementary target DNA molecules. In this review, we discuss recent innovations in biomedical nanoengineering with a specific focus on novel NWA-based biosensors. We also describe our continuous efforts in achieving a label-free detection without non-specific binding while maintaining the activity and stability of immobilized biomolecules. This research can lay the foundation of a new platform for biomedical nanoengineering systems.

Polypyrrole Films with Micro/Nanosphere Shapes for Electrodes of High-Performance Supercapacitors.

We demonstrate a simple and efficient one-step procedure for synthesizing a solid state polypyrrole (PPy) thin film for supercapacitor applications using alternating current impedance spectroscopy. By controlling the frequency and amplitude we were able to create unique PPy nano/microstructures with a particular morphology of the loop. Our PPy micro/nanosphere shows extremely high capacitance of 568 F/g, which is close to the theoretical value of 620 F/g and 20-100% higher than that of other reported PPy electrodes. Most of all, this material presents high capacitance and significantly improved electrochemical stability without pulverization of its structure, demonstrating 77% retention of the capacitance value even after 10 000 charge/discharge cycles. These results are a consequence of the larger surface area and adequate porosity generated due to the balance between the nano/micro PPy loops. This created porous structure also allows the favored penetration of electrolyte and high ion mobility within the polymer and prevents the mechanical failure of the physical structure during volume variation associated with the insertion/deinsertion of ions upon cycling.

Single Microfluidic Electrochemical Sensor System for Simultaneous Multi-Pulmonary Hypertension Biomarker Analyses.

Miniaturized microfluidic biosensors have recently been advanced for portable point-of-care diagnostics by integrating lab-on-a-chip technology and electrochemical analysis. However, the design of a small, integrated, and reliable biosensor for multiple and simultaneous electrochemical analyses in a single device remains a challenge. Here, we present a simultaneous microfluidic electrochemical biosensing system to detect multiple biomarkers of pulmonary hypertension diseases in a single device. The miniaturized biosensor, which is composed of five chambers, is precisely and individually controlled using in-house-built pneumatic microvalves to manipulate the flow pathway. Each chamber is connected to an electrochemical sensor designed to detect four different biomarkers plus a reference control. Our design allows for loading of multiple reagents for simultaneous analyses. On the basis of the developed microfluidic electrochemical sensor system, we successfully detected four well-defined pulmonary hypertension-associated biomarkers, namely, fibrinogen, adiponectin, low-density lipoprotein, and 8-isoprostane. This novel approach offers a new platform for a rapid, miniaturized, and sensitive diagnostic sensor in a single device for various human diseases.

Nonmediated, Label-Free Based Detection of Cardiovascular Biomarker in a Biological Sample.

Direct electrochemical (EC) monitoring in a cell culture medium without electron transporter as called mediator is attractive topic in vitro organoid based on chip with frequently and long-time monitoring since it can avoid to its disadvantage as stability, toxicity. Here, direct monitoring with nonmediator is demonstrated based on impedance spectroscopy under the culture medium in order to overcome the limitation of mediator. The applicability of EC monitoring is shown by detecting alpha-1-anti trypsin (A1AT) which is known as biomarkers for cardiac damage and is widely chosen in organoid cardiac cell-based chip. The validity of presented EC monitoring is proved by observing signal processing and transduction in medium, mediator, medium-mediator complex. After the observation of electron behavior, A1AT as target analyte is immobilized on the electrode and detected using antibody-antigen interaction. As a result, the result indicates limit of detection is 10 ng mL-1 and linearity for the 10-1000 ng mL-1 range, with a sensitivity of 3980 nF (log [g mL])-1 retaining specificity. This EC monitoring is based on label-free and reagentless detection, will pave the way to use for continuous and simple monitoring of in vitro organoid platform.

Label-Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes.

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label-free microfluidic electrochemical (EC) biosensor with a unique built-in on-chip regeneration capability for continual measurement of cell-secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver-on-a-chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme-linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long-term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

Role of fatty acid composites in the toxicity of titanium dioxide nanoparticles used in cosmetic products.

It has been recognized that the use of nanoparticles (NPs) in the cosmetic industry results in products with better efficacy and functionality. However, recent advances in molecular toxicology have revealed that NP exposure can promote cytotoxicity and oxidative damage, which has raised health concerns in the use of NPs in personal care products. Nevertheless, the mechanistic basis for the toxicity and safety of cosmetic NPs is poorly understood. The goal of the study was to determine the cytotoxicity and intracellular distribution of titanium dioxide (TiO2) NPs containing fatty acid composites (palmitoleic acid, palmitic acid, stearic acid and oleic acid) commonly used in cosmetic products. Two types of cells, human fibroblast skin cells and adenocarcinoma lung cells, were exposed to either bare TiO2 NPs or TiO2 NPs mixed with fatty acids for up to 48 hr. NMR analysis confirmed that the fatty acid composites remained in the NPs after wash. The cytotoxicity of TiO2 NPs was determined by cell viability measurement using quantitative confocal microscopy, and the localization of two different forms of TiO2 NPs were assessed using electron spectroscopic imaging with transmission electron microscopy. TiO2 NPs containing fatty acids posed significantly reduced cytotoxicity (80-88% decreases) than bare NPs in both cell types. Furthermore, there was less intracellular penetration of the NPs containing fatty acid composites compared with bare NPs. These results provide important insights into the role of fatty acids in protecting the cells from possible toxicity caused by NPs used in the production of cosmetic products.

Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor.

We have reported that nanowell array (NWA) can enhance electrochemical detection of molecular binding events by controlling the binding sites of the captured molecules. Using NWA biosensor based amperometric analysis, we have detected biological macromolecules such as DNA, protein or aptamers at low concentrations. In this research, we developed an impedimetric immunosensor based on wafer-scale NWA for electrochemical detection of stress-induced-phosphoprotein-1 (STIP-1). In order to develop NWA sensor through the cost-effective combination of high-throughput nanopattern, the NWA electrode was fabricated on Si wafer by krypton-fluoride (KrF) stepper semiconductor process. Finally, 12,500,000 ea nanowell with a 500 nm diameter was fabricated on 4 mm × 2 mm substrate. Next, by using these electrodes, we measured impedance to quantify antigen binding to the immunoaffinity layer. The limit of detection (LOD) of the NWA was improved about 100-fold compared to milli-sized electrodes (4 mm × 2 mm) without an NWA. These results suggest that wafer-scale NWA immunosensor will be useful for biosensing applications because their interface response is appropriate for detecting molecular binding events.

Phospholipase-catalyzed hydrolysis in an artificial cell membrane in the presence of melittin.

Biomimicry involves the use of the structure and function of biological systems as models for the design and engineering of materials and machines. An artificial cell membrane was developed using biomembrane components, and the membrane, formed by a lipid bilayer, was analyzed using surface plasmon resonance (SPR) to monitor hydrolysis by phospholipase (PL). The simultaneous atomic force microscope (AFM) images show that PL catalyzed the nanometer-scale hydrolysis of the artificial lipid biomembranes through enzymatic hydrolysis. In addition, it was confirmed that the combination of PL and melittin allowed the control of enzyme hydrolysis for the degradation of the lipid bilayer. Regarding the expected activating effect of melittin on hydrolysis, no difference with respect to the non-treated lipid membrane was observed in the AFM images. It is assumed that the partitioning of melittin into the membrane might prevent the binding or hydrolysis of Phospholipase A2 (PLA2). This study provides basic knowledge on a new approach for patterning biomimicking lipid membranes on a nano-scale.

Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH.

In an effort toward determining the feasibility of single molecule analysis, we describe a case whereby the binding of one biotinylated DNA to one streptavidin molecule via electrostatic interactions was controlled by altering in pH 4.0-9.0 and 0.16 of the ion strength. The quantitative analysis of immobilized probe ssDNA was realized in real-time via a quartz crystal microbalance (QCM) and electrochemical (EC) measurement in the range 100 pM to 50 microM of probe oligonucleotide concentration. The variation amount of biotinylated ssDNA immobilized on the streptavidin-modified surface at pH 7.5 was about 0.16 pmol, giving a ratio of streptavidin to biotinylated ssDNA of about 1:1.1. On the other hand, at pH 4.9, it was immobilized about 0.29 pmol. From the shape of the Langmuir plot and QCM, the immobilization efficiency of biotinylated DNA via streptavidin at pH 4.9 was approximately twofold that at pH 7.5. In view points of the reaction velocity, it was increased with decreasing buffer solution pH, indicating a strong interaction of negatively charged probe DNA with the positively charged streptavidin. And also the EC response value of deltaI/I(streptavidin) for the immobilized biotinylated ssDNA in pH 4.9 was about 49%, while the corresponding value for the pH 7.5 was approximately 34%. As DNA molecules possess negative charges, electrostatic repulsion occurred between streptavidin and biotinylated ssDNA at pH 7.5. At pH 4.9, the attraction between the biotinylated ssDNA and streptavidin resulted in increased adsorption which has an isoelectric point of about 5.9. It was deduced that the binding of biotinylated ssDNA to one or two of the four binding sites of streptavidin can be controlled by adjusting the pH-controlled electrostatic interaction.

Atomic force microscopy observation of highly arrayed phospholipid bilayer vesicle on a gold surface.

Tapping mode atomic force microscopy (TM-AFM) imaging of a phospholipid bilayer vesicle (liposome) immobilized on a gold surface was performed to investigate morphologies of the electrode surfaces produced through application of three different sample preparation methods. We compared both methods from a morphological viewpoint using TM-AFM images. Liposomes, composed of zwitterionic and anionic phospholipids, were prepared by extrusion. Results indicate that the surface with immobilized L1-liposome, which was fabricated by the amino coupling method, seemed to form large amounts of aggregated or fused liposomes. In contrast, L2-liposome-containing 1-octadecanthiol that was directly attached on the gold surface using thiol-gold binding force was immobilized as a uniform surface topology without liposome aggregation. Finally, we attempted to arrange individual L3-liposome, prepared by mixing zwitterionic and anionic phospholipids, onto the gold layer by electron-beam (e-beam) lithography technique. A third method, L3-liposome formation on the sensor surface, is greatly anticipated for biosensor applications.

A novel route for immobilization of oligonucleotides onto modified silica nanoparticles.

A novel approach for immobilization of probe oligonucleotides that uses zirconium phosphate modified silica nanoparticles is proposed. The surface modification of nanoparticles was carried out in two stages. Initially binding of Zr4+ to the surface of silica nanoparticles and later treated with phosphoric acid for terminal phosphate groups. Oligonucleotide probes modified with amine group at 5'-end were strongly binds to the phosphate terminated silica nanoparticles with imidazole in presence of 0.1 mol L(-1) EDC [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide], as phosphate groups are more reactive towards amine group. Various studies, i.e., synthesis of silica nanoparticles, their surface modification, probe immobilization, measurement of hybridization and effect of bovine serum albumin (BSA) were carried out during optimization of reaction conditions. The significant reduction in the background signal was observed by treating the probe modified silica nanoparticles with bovine serum albumin prior to hybridization. The probe modified silica nanoparticles were retained their properties and the hybridization was induced by exposure of single-stranded DNA (ssDNA) containing silica nanoparticles to the complementary DNA in solution. The decrease in the fluorescence signal for one mismatch and three mismatch was observed upon hybridization of probe with target DNAs, while there was no response for the random target ssDNA under the same experimental conditions. The intensity of fluorescence signal was linear to the concentration of target DNA ranging from 3.9 x 10(-9) to 3.0 x 10(-6)mol L(-1). A detection limit of 1.22 x 10(-9) mol L(-1) of oligonucleotides can be estimated. The proposed hybridization assay is simple and possesses good analytical characteristics and it can provide an effective and efficient route in the development of DNA biosensors and biochips.

Electrochemical assay of nonlabeled DNA chip and SNOM imaging by using streptavidin-biotin interaction.

The assay of DNA biosensor-based nucleic acid recognition using microfabrication technology provides for high sensitivity, good surface coverage and reproducibility. We have achieved efficient immobilization and hybridization of nonlabeled DNA using cyclic voltammetry (CV), square wave voltammetry (SWV) and scanning near-field optical microscopy (SNOM) techniques. The increased electrochemical response observed following the immobilization of biotinlyated ssDNA probe suggests that nucleic acid is a somewhat better medium for electronic transfer. We demonstrated the high coverage of immobilized FITC-labeled biotinylated DNA probe on a streptavidin-modified surface using SNOM imaging. SNOM imaging of FITC-labeled complementary DNA also exhibited fluorescent light spots of hybridization distributed throughout. No fluorescent light was observed with the hybridization of non-complementary DNA.

Electrochemical detection of nonlabeled oligonucleotide DNA using biotin-modified DNA(ss) on a streptavidin-modified gold electrode.

In the electrochemical detection of nonlabeled DNA, it is important to control the bonding at the interface between the DNA and the electrode. Atomic force microscope (AFM) was taken for the commonly used thiol-modified DNA on a gold surface. It was found that the coverage of the DNA was very low. On the other hand, a streptavidin-modified gold electrode provided a much better alternative where DNA hybridization resulted in large changes in the electrochemical reaction responses. This work demonstrates that streptavidin-modified gold electrodes could be used in the development of a new electrochemical protocol for the detection of nonlabeled DNA.

Highly dense protein layers confirmed by atomic force microscopy and quartz crystal microbalance.

Protein adsorption on a gold surface is investigated by comparing the results of quartz crystal microbalance method and atomic force microscopy. The adsorption of streptavidin on functional gold surfaces is directly monitored by a quartz crystal microbalance, and confirmed by atomic force microscopy. For this investigation, a modified gold substrate is fabricated to obtain a topographic image of streptavidin molecules. Both methods show a correlation in terms of the highly dense protein single-layer formation, and the modified gold electrode shows a slightly denser protein layer formation because of the difference in substrate geometry as compared with that of a mica surface.

Near-field optical imaging of abasic sites on a single DNA molecule.

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.