Micro-injection Molded Droplet Generation System for Digital PCR Application.

Sensitive, effective, and quantitative analysis of infectious pathogens is an important task for the prevention of human health threats. Herein, we present an advanced approach to producing gene-encapsulated microdroplets for quantitative analysis using a micropatterned metal mold and injection molding technique with an automatically operated system. An injection molded microdroplet generation device was successfully fabricated with a minimum channel width of 30 µm and optimized to produce 100 µm diameter droplets. The optimized microchannel design and flow rate also enable the production of stable numbers of microdroplets (~ 16,000 droplets). To verify the applicability of our device and system to droplet-based digital PCR analysis, Escherichia coli (E. coli) O157:H7 was selected as a model bacterial pathogen, and the stx2 gene was amplified in the microdroplets. The generated microdroplets exhibit both chemical and mechanical stability, and our results are similar to those obtained by a commercially available method. Accordingly, the usefulness of the microdroplet generative device and system is confirmed as a simple, fast, and reliable tool for the quantitative molecular analysis of infectious diseases.

Touchable 3D hierarchically structured polyaniline nanoweb for capture and detection of pathogenic bacteria.

A bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling.

3D Hierarchical Polyaniline-Metal Hybrid Nanopillars: Morphological Control and Its Antibacterial Application.

Effective and reliable antibacterial surfaces are in high demand in modern society. Although recent works have shown excellent antibacterial performance by combining unique hierarchical nanotopological structures with functional polymer coating, determining the antibacterial performance arising from morphological changes is necessary. In this work, three-dimensional (3D) hierarchical polyaniline-gold (PANI/Au) hybrid nanopillars were successfully fabricated via chemical polymerization (i.e., dilute method). The morphology and structures of the PANI/Au nanopillars were controlled by the reaction time (10 min to 60 h) and the molar concentrations of the monomer (0.01, 0.1, and 1 M aniline), oxidant (0.002, 0.0067, 0.01, and 0.02 M ammonium persulfate), and acid (0.01, 0.1, 1, and 2 M perchloric acid). These complex combinations allow controlling the hierarchical micro- to nanostructure of PANI on a nanopillar array (NPA). Furthermore, the surface of the 3D PANI/Au hierarchical nanostructure can be chemically treated while maintaining the structure using initiated chemical vapor deposition. Moreover, the excellent antibacterial performance of the 3D PANI/Au hierarchical nanostructure (HNS) exceeds 99% after functional polymer coating. The excellent antibacterial performance of the obtained 3D PANI/Au HNS is mainly because of the complex topological and physicochemical surface modification. Thus, these 3D PANI/Au hierarchical nanostructures are promising high-performance antibacterial materials.

3D Hierarchical Nanotopography for On-Site Rapid Capture and Sensitive Detection of Infectious Microbial Pathogens.

Effective capture and rapid detection of pathogenic bacteria causing pandemic/epidemic diseases is an important task for global surveillance and prevention of human health threats. Here, we present an advanced approach for the on-site capture and detection of pathogenic bacteria through the combination of hierarchical nanostructures and a nuclease-responsive DNA probe. The specially designed hierarchical nanocilia and network structures on the pillar arrays, termed 3D bacterial capturing nanotopographical trap, exhibit excellent mechanical reliability and rapid (<30 s) and irreversible bacterial capturability. Moreover, the nuclease-responsive DNA probe enables the highly sensitive and extremely fast (<1 min) detection of bacteria. The bacterial capturing nanotopographical trap (b-CNT) facilitates the on-site capture and detection of notorious infectious pathogens (Escherichia coli O157:H7, Salmonella enteritidis, Staphylococcus aureus, and Bacillus cereus) from kitchen tools and food samples. Accordingly, the usefulness of the b-CNT is confirmed as a simple, fast, sensitive, portable, and robust on-site capture and detection tool for point-of-care testing.

Flexible and highly ordered nanopillar electrochemical sensor for sensitive insulin evaluation.

In line with growing interest in obesity management, there has been an increase in the amount of research focused on highly sensitive analysis systems for a small number of biomarers. In this paper, we introduce the highly ordered nanopillar electrode, featuring a high aspect ratio surface area that enables enhanced electron transfer. For fabrication of the flexible electrode, gold was evaporated by electronic beam lithography on polyurethane (PU), which has high flexibility. The fabricated nanopillar is 500 nm in diameter and 1500 nm in height. Based on the highly ordered nanostructure electrode, insulin was selected as a biomarker to monitor the insulin resistance associated with obesity. To effectively analyze the insulin, the self-assembled monolayer chemical was used to introduce the enzyme catalysis-based electrochemical immunoassay, leading to the analysis of the insulin concentration range from 0.1 to 1.0 ng/mL in the real sample. The square wave voltammetry principle was used to measure HRP-based electrochemical signal both electrochemically and quantitatively. Based on the nanostructural properties of significant electrochemical behavior, we successfully analyzed insulin in the plasma sample with high sensitivity (LOD of 0.1 ng/mL) and with high reproducibility (<10%). The obtained sensitivity of nanopillar electrode is approximately 10 times (1020%) greater than that of commercial electrode. The results demonstrated that the nanopillar electrode is suitable for precise and sensitive analysis of low-level biomolecules in medical and commercial fields.

A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen.

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL-1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

A microfluidic electrochemical aptasensor for enrichment and detection of bisphenol A.

Bisphenol A (BPA) is an organic monomer used to make common consumer goods such as plastic containers, sports equipment, and cosmetics which are heavily produced worldwide. A growing interest has been drawn to general public as BPA is one of the major endocrine disrupting chemicals threating human health. To date, numerous BPA sensors have been attempted to be developed but important challenges still remained such as limited linearity range, easy to use, and long term response time. To address the present issues, a microfluidic channel should be integrated into an electrochemical aptasensor and it is called Geometrically Activated Surface Interaction (GASI) chip. The vigorous generation of the micro-vortex in the GASI fluidic chamber provides the high collision chances between BPA and anti-BPA aptamer (BPAPT) and consequently more BPA molecules can be captured on the aptasensor surface, which finally results in high sensitivity of the aptasensor. To construct the integrated aptasensor, a miniaturized gold electrode is fabricated using shadow mask and e-beam evaporation process. Afterward, BPAPT is immobilized on a nanostructured gold electrode via thiol chemistry, and other terminus of the aptamer is labeled with a ferrocene (Fc) redox probe. Then, the microfluidic channel is mounted over the miniaturized gold electrode to introduce and enrich BPA to the aptasensor. Upon the specific interaction between BPA and its aptamer, configuration of aptamer is changed so that Fc tag approaches to the electrode surface and direct oxidation signal of Fc and BPA are followed as analytical signals. The unique microfluidic integrated electrochemical aptasensor delivers a wide linear dynamic range over 5 × 10-12 to 1 × 10-9 M, with a limit of detection 2 × 10-13 M. This aptasensor provides a precise platform for simple, selective and more importantly rapid detection of BPA. Such kind of sensing platforms can serve as a fertile ground for designing miniaturized portable sensors.

Flexible nanopillar-based electrochemical sensors for genetic detection of foodborne pathogens.

Flexible and highly ordered nanopillar arrayed electrodes have brought great interest for many electrochemical applications, especially to the biosensors, because of its unique mechanical and topological properties. Herein, we report an advanced method to fabricate highly ordered nanopillar electrodes produced by soft-/photo-lithography and metal evaporation. The highly ordered nanopillar array exhibited the superior electrochemical and mechanical properties in regard with the wide space to response with electrolytes, enabling the sensitive analysis. As-prepared gold and silver electrodes on nanopillar arrays exhibit great and stable electrochemical performance to detect the amplified gene from foodborne pathogen of Escherichia coli O157:H7. Additionally, lightweight, flexible, and USB-connectable nanopillar-based electrochemical sensor platform improves the connectivity, portability, and sensitivity. Moreover, we successfully confirm the performance of genetic analysis using real food, specially designed intercalator, and amplified gene from foodborne pathogens with high reproducibility (6% standard deviation) and sensitivity (10 × 1.01 CFU) within 25 s based on the square wave voltammetry principle. This study confirmed excellent mechanical and chemical characteristics of nanopillar electrodes have a great and considerable electrochemical activity to apply as genetic biosensor platform in the fields of point-of-care testing (POCT).

Ultrasonic fabrication of flexible antibacterial ZnO nanopillar array film.

Antibacterial activity is essential and highly demanded in worldwide to prevent potential bacterial infection. Here in this work, we report a new approch for the fabrication of flexible zinc oxide nanopillar arrays (ZG-NPA) film with an efficient antibacterial activity. A flexible NPA film served as a substrate for the rapid formation of ZnO by using ultrasound-assisted method. The enhancement of antibacterial activity were induced by cellular damages because of nano topological effects and electrostatic interaction between bacteria and ZG-NPA. Owing to the benefits of combination with flexibility, high surface areas from nano-features and excellent antibacterial efficiency (>80%) of ZG-NPA, the film can show great potential for use as novel biomaterials for preventing bacterial infections.

A film-based integrated chip for gene amplification and electrochemical detection of pathogens causing foodborne illnesses.

Given the increased interest in public hygiene due to outbreaks of food poisoning, increased emphasis has been placed on developing novel monitoring systems for point-of-care testing (POCT) to evaluate pathogens causing foodborne illnesses. Here, we demonstrate a pathogen evaluation system utilizing simple film-based microfluidics, featuring simultaneous gene amplification, solution mixing, and electrochemical detection. To minimize and integrate the various functionalities into a single chip, patterned polyimide and polyester films were mainly used on a polycarbonate housing chip, allowing simple fabrication and alignment, in contrast to conventional polymerase chain reaction, which requires a complex biosensing system at a bench-top scale. The individual integrated sensing chip could be manually fabricated in 10 min. Using the developed film-based integrated biosensing chip, the genes from the pathogens causing foodborne illnesses were simultaneously amplified based on multiple designed microfluidic chambers and Hoechst 33258, which intercalates into double-stranded DNA, to generate the electrochemical signal. The target pathogen gene was accurately analyzed by square wave voltammetry (SWV) within the 25 s, while the gel electrophoresis required about 30 min. Based on the developed integrated biosensing chip, the 1.0 × 101 and 1.0 × 102 colony-forming unit (CFU) of Staphylococcus aureus and Escherichia coli were sensitively detected with high reproducibility in the 25 s. On the basis of the significant features of the film-based molecular analysis platform, we expect that the developed sensor could be applied to the screening of various pathogens as a POCT device.

Development of bufferless gel electrophoresis chip for easy preparation and rapid DNA separation.

This work presents a handy, fast, and compact bufferless gel electrophoresis chip (BGEC), which consists of precast agarose gel confined in a disposable plastic body with electrodes. It does not require large volumes of buffer to fill reservoirs, or the process of immersing the gel in the buffer. It withstands voltages up to 28.4 V/cm, thereby allowing DNA separation within 10 min with a similar separation capability to the standard gel electrophoresis. The results suggest that our BGEC is highly suitable for in situ gel electrophoresis in forensic, epidemiological settings and crime scenes where standard gel electrophoresis equipment cannot be brought in while quick results are needed.

Fabrication of newspaper-based potentiometric platforms for flexible and disposable ion sensors.

Paper-based materials have attracted a great deal of attention in sensor applications because they are readily available, biodegradable, inexpensive, and mechanically flexible. Although paper-based sensors have been developed, but important obstacles remian, which include the retention of chemical and mechanical stabilities when paper is wetted. Herein, we develop a simple and scalable process for fabrication of newspaper-based platforms by coating of parylene C and patterning of metal layers. As-prepared parylene C-coated newspaper (PC-paper) provides low-cost, disposable, and mechanically and chemically stable electrochemical platforms for the development of potentiometric ion sensors for the detection of electrolyte cations, such as, H+ and K+. The pH and K+ sensors produced show near ideal Nernstian sensitivity, good repeatability, good ion selectivity, and low potential drift. These disposable, flexible ion sensors based on PC-paper platforms could provide new opportunities for the development of point-of-care testing sensors, for diagnostics, healthcare, and environment testing.

Flexible and Disposable Sensing Platforms Based on Newspaper.

The flexible sensing platform is a key component for the development of smart portable devices targeting healthcare, environmental monitoring, point-of-care diagnostics, and personal electronics. Herein, we demonstrate a simple, scalable, and cost-effective strategy for fabrication of a sensing electrode based on a waste newspaper with conformal coating of parylene C (P-paper). Thin polymeric layers over cellulose fibers allow the P-paper to possess improved mechanical and chemical stability, which results in high-performance flexible sensing platforms for the detection of pathogenic E. coli O157:H7 based on DNA hybridization. Moreover, P-paper electrodes have the potential to serve as disposable, flexible sensing platforms for point-of-care testing biosensors.

Contribution of actin filaments and microtubules to cell elongation and alignment depends on the grating depth of microgratings.

BACKGROUND: It has been reported that both chemical and physical surface patterns influence cellular behaviors, such as cell alignment and elongation. However, it still remains unclear how actin filament and microtubules (MTs) differentially respond to these patterns. RESULTS: We examined the effects of chemical and physical patterns on cell elongation and alignment by observing actin filament and MTs of retinal pigment epithelium-1(RPE-1) cells, which were cultured on either fibronectin (FN)-line pattern (line width and spacing: 1 µm) or FN-coated 1 µm gratings with two different depths (0.35 or 1 µm). On the surface with either FN-line pattern or micrograting structure, the cell aspect ratios were at least two times higher than those on the surface with no pattern. Cell elongation on the gratings depended on the depth of the gratings. Cell elongation and alignment on both FN-line pattern and 1 µm gratings with 0.35 µm depth were perturbed either by inhibition of actin polymerization or MT depletion, while cell elongation and alignment on 1 µm gratings with 1 µm depth were perturbed only by MT depletion. CONCLUSIONS: Our results suggest that the contribution of actin filaments and MTs to the elongation and alignment of epithelial cells on microgratings depends on the groove depth of these gratings.

Ultrasonic bonding method for heterogeneous microstructures using self-balancing jig.

Perfect sealing of heterogeneous microstructures in plastic-based microfluidic devices is a significant and urgent challenge to be able to apply them in various microfluidic-based applications, including biosensing, biofiltering, chemical reactors and lab-on-a-chip. In this study we report a simple but practical and effective method to bond a microstructure-incorporated microfluidic device using an ultrasonic bonding method. The specially designed hemisphere-shaped jig, which is called a self-balancing jig, provides a free motion in all x, y, and z directions. These unique properties of the jig allow us to precisely adjust and bond the heterogeneous microstructures in the device. A conventional jig shows in solution leakages around the heterogeneous microstructures while the self-balancing jig did not show any leakages in devices. Furthermore, the bonding performance was also confirmed by using a black ink and fluorescent dye solution. The micro-pillar arrays in the device also demonstrated its capability for selective filtering of microbeads. We believe that this technique would be a useful tool for producing microfluidic devices with heterogeneous microstructures.