Development of a multifunctional bioreactor to evaluate the promotion effects of cyclic stretching and electrical stimulation on muscle differentiation.

A multifunctional bioreactor was fabricated in this study to investigate the facilitation efficiency of electrical and mechanical stimulations on myogenic differentiation. This bioreactor consisted of a highly stretchable conductive membrane prepared by depositing polypyrrole (PPy) on a flexible polydimethylsiloxane (PDMS) film. The tensile deformation of the PPy/PDMS membrane can be tuned by adjusting the channel depth. In addition, PPy/PDMS maintained its electrical conductivity under continuous cyclic stretching in the strain range of 6.5%-13% for 24 h. This device can be used to individually or simultaneously perform cyclic stretching and electrical stimulation. The results of single stimulation showed that either cyclic stretching or electrical stimulation upregulated myogenic gene expression and promoted myotube formation, where electrical stimulation improved better than cyclic stretching. However, only cyclic stretching can align C2C12 cells perpendicular to the stretching direction, and electrical stimulation did not affect cell morphology. Myosin heavy chain (MHC) immunostaining demonstrated that oriented cells under cyclic stretching resulted in parallel myotubes. The combination of these two stimuli exhibited synergetic effects on both myogenic gene regulation and myotube formation, and the incorporated electrical field did not affect the orientation effect of the cyclic stretching. These results suggested that these two treatments likely influenced cells through different pathways. Overall, the simultaneous application of cyclic stretching and electrical stimulation preserved both stimuli's advantages, so myo-differentiation can be highly improved to obtain abundant parallel myotubes, suggesting that our developed multifunctional bioreactor should benefit muscle tissue engineering applications.

Paper/PMMA hybrid device with a microvalve-controlled design for exosome isolation and analysis.

This study proposes a paper/PMMA hybrid device designed to isolate exosomes and extract exosomal miRNA, followed by quantitative analysis. It aims to provide simplified and convenient sample preparation for potential point-of-care testing (POCT) processes. In contrast to previous work conducted by our research team, which focused on isolating exosomes and exosomal nucleic acids, this study introduces a novel approach by integrating paper and a PMMA mold with a microvalve controlled design. This innovative method enables the entire process to be performed on paper. The pressure on the paper could be adjusted by turning the screw upon the valve to change the pore size and permeability of the paper, which achieved the effect of controlling the flow rate of fluids. The paper was designed to have an immunoaffinity area for capturing exosomes and a sol-gel silica coating area for extracting miRNA. The paper-based ELISA (p-ELISA) exhibited a limit of detection and a limit of quantitation of 6 × 107 and 5.4 × 108 particles/mL, respectively, for exosome measurement. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that the Ct (threshold cycle) value for quantifying the miR-21 in the miRNAs extracted by the proposed paper/PMMA hybrid device was comparable to the Ct value of the commercial extraction kit. The developed paper/PMMA hybrid device with a microvalve-controlled design should be incorporated into the POCT system to extract exosomal miRNAs.

Microwave-Assisted Solvent Bonding for Polymethyl Methacrylate Microfluidic Device.

This paper demonstrated a microwave-assisted solvent bonding method that uses organic solvent to seal the thermoplastic substrates with microwave assistance. This direct bonding is a simple and straightforward process that starts with solvent application followed by microwave irradiation without the need for expensive facilities or complex procedures. The organic solvent applied at the bonding interface is used in dissolving and dielectric heating of the thermoplastic surfaces to seal the thermoplastic substrates under microwave assistance. We evaluated acetone and ethanol to seal the polymethyl methacrylate (PMMA) microfluidic device. The bonding performance, such as bonding coverage, geometry stability, and bonding strength (tensile) were observed and compared with the oven-heating and non-heating control experiments under the same force applications. Results showed that the microwave-assisted solvent bonding method presents a high bonding yield (maximum > 99%) and bonding strength (maximum ~2.77 MPa) without microchannel distortion, which can be used for various microfluidic applications.

Recent Advances in Thermoplastic Microfluidic Bonding.

Microfluidics is a multidisciplinary technology with applications in various fields, such as biomedical, energy, chemicals and environment. Thermoplastic is one of the most prominent materials for polymer microfluidics. Properties such as good mechanical rigidity, organic solvent resistivity, acid/base resistivity, and low water absorbance make thermoplastics suitable for various microfluidic applications. However, bonding of thermoplastics has always been challenging because of a wide range of bonding methods and requirements. This review paper summarizes the current bonding processes being practiced for the fabrication of thermoplastic microfluidic devices, and provides a comparison between the different bonding strategies to assist researchers in finding appropriate bonding methods for microfluidic device assembly.

Surface-enhanced Raman scattering (SERS) spectroscopy on localized silver nanoparticle-decorated porous silicon substrate.

Surface-enhanced Raman scattering (SERS) spectroscopy is a rapid and non-destructive optical detection method that has been applied in various applications. Recently, three-dimensional (3D) substrate-based silicon nanostructures have been widely used as SERS substrates due to their high detection sensitivity, repeatability, and reusability. This paper uses a simple and low-cost electroless etching deposition process to generate silver nanoparticle-decorated porous silicon (Ag-PS) substrates. We propose a contact deposition process to generate localized Ag-PS (LocAg-PS) for SERS analysis. Due to the hydrophilic LocAg-PS pad on the hydrophobic PS background, the sample droplets self-aligned to the predefined LocAg-PS pads and condensed into a higher local concentration for high sensitivity SERS detection without extensive search for the hot spot. The effects of critical fabrication parameters and SERS analysis on the LocAg-PS surface were evaluated.

The effect of channel aspect ratio on air entrapment during imbibition in soil-on-a-chip micromodels with 2D and 2.5D pore structures.

We developed a low-cost method for fabricating "soil-on-a-chip" micromodels with 2D and 2.5D pore structures by stacking layers made with a conventional low-cost tabletop CNC router followed by tape bonding. The pore structure was extracted from an X-ray micro-computed tomography scanning image of a medium-grain sandstone sample. The imbibition experiments performed in the 2D and 2.5D micromodels showed the trends of the residual saturation versus capillary number (Ca). The channels showed opposing trends for low-aspect-ratio 2D and high-aspect-ratio 2.5D micromodels. As the channel aspect ratio increased, the location of air entrapment changed from dead-end pores to transport pores. The sizes of trapped air bubbles in the transport pores decreased as the injection flow rates increased. To show the relationship between the air trapped size and Ca, we derived equations that described the competition between the bulk menisci and the corner flow in the channels for different Ca based on the "supply principle." The relative contributions of the piston displacement and corner film flow, which were dependent on the cross-sectional shapes of the pores and Ca, determined the size and location of the air bubbles trapped in the 2.5D micromodel.

Polymer Microchannel and Micromold Surface Polishing for Rapid, Low-Quantity Polydimethylsiloxane and Thermoplastic Microfluidic Device Fabrication.

Polymer-based micromolding has been proposed as an alternative to SU-8 micromolding for microfluidic chip fabrication. However, surface defects such as milling marks may result in rough microchannels and micromolds, limiting microfluidic device performance. Therefore, we use chemical and mechanical methods for polishing polymer microchannels and micromolds. In addition, we evaluated their performance in terms of removing the machining (milling) marks on polymer microchannel and micromold surfaces. For chemical polishing, we use solvent evaporation to polish the sample surfaces. For mechanical polishing, wool felt polishing bits with an abrasive agent were employed to polish the sample surfaces. Chemical polishing reduced surface roughness from 0.38 µm (0 min, after milling) to 0.13 µm after 6 min of evaporation time. Mechanical polishing reduced surface roughness from 0.38 to 0.165 µm (optimal pressing length: 0.3 mm). As polishing causes abrasion, we evaluated sample geometry loss after polishing. Mechanically and chemically polished micromolds had optimal micromold distortion percentages of 1.01% ± 0.76% and 1.10% ± 0.80%, respectively. Compared to chemical polishing, mechanical polishing could better maintain the geometric integrity since it is locally polished by computer numerical control (CNC) miller. Using these surface polishing methods with optimized parameters, polymer micromolds and microchannels can be rapidly produced for polydimethylsiloxane (PDMS) casting and thermoplastic hot embossing. In addition, low-quantity (15 times) polymer microchannel replication is demonstrated in this paper.

Bonding of thermoplastic microfluidics by using dry adhesive tape.

In this study, we investigated the effects of adhesive tape structure, adhesive tape thickness (30, 60, and 80 µm), and bonding time (5 and 15 seconds) on the bonding of inflexible and flexible substrates. We performed microchannel bonding by using a manual scraper press or a hot press machine. Rapid prototyping and mass production capabilities were achieved in the dry adhesive tape bonding of polymer microfluidic systems with both the aforementioned approaches. With process control, 95.16% and 99.53% bonding coverage could be achieved for the inflexible and flexible substrates, respectively, by using a manual scraper press. When using a press machine, the bonding coverage could be further enhanced to 99.24% for the inflexible substrates and 99.81% for the flexible substrates. Due to the viscoelastic nature of the adhesive layer in the adhesive tapes, we observed Saffman-Taylor finger and air bubble formation around the microchannel under high pumping pressure. The results indicated that the probability of Saffman-Taylor finger formation was lower and the bonding pressure was higher when using the thinner adhesive tape than when using thicker tape. Moreover, due to their rigidity, the inflexible substrates exhibited a higher bonding strength than the flexible substrates did. Bonding stability tests indicated that the bonded substrates had high bonding quality and bonding strength under long-term storage of up to 60 days.

The effects of substrate-mediated electrical stimulation on the promotion of osteogenic differentiation and its optimization.

To explore the effect of electrical stimulation (ES) on osteogenesis, a polypyrrole (PPy)-made electrical culture system was developed to provide a direct-current electric field (DCEF). This DCEF device was applied to treat differentiated rat bone marrow stromal cells (rBMSCs) once in different stages of osteo-differentation to investigate its temporal effects. The mineralization results showed that the DCEF treatment not only accelerated cell differentiation but also promoted the saturation levels, and the ES on day 8 was the group demonstrated the optimal result. The gene regulation analysis indicated that the DCEF treatment immediately increased the levels of genes related to osteo-differentiation, especially Runx2. Because Runx2 is a crucial transcriptional factor of osteogenesis, the ES-caused improvement of mineralization was likely contributed by the extension of its expression. Further, different ES modes were investigated of their efficacy on bone matrix deposition. Square waves with different parameters including frequency, offset, amplitude, and duty cycle were systematically examined. In contrast to constant voltage, square waves demonstrated periodical changes of current through substrate to significantly improve mineralization, and the efficiencies highly depended on both frequency and intensity. Through this comprehensive study, DCEF treating condition was optimized, which should be beneficial to its application on osteogenesis promotion. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1607-1619, 2019.

Exploration of Fungal Metabolic Interactions Using Imaging Mass Spectrometry on Nanostructured Silicon.

Application of matrix-assisted laser desorption/ionization imaging mass spectrometry to microbiology and natural product research has opened the door to the exploration of microbial interactions and the consequent discovery of new natural products and their functions in the interactions. However, several drawbacks of matrix-assisted laser desorption/ionization imaging mass spectrometry have limited its application especially to complicated and uneven microbial samples. Here, we applied nanostructured silicon as a substrate for surface-assisted laser desorption/ionization mass spectrometry for microbial imaging mass spectrometry to explore fungal metabolic interactions. We chose Phellinus noxius and Aspergillus strains to evaluate the potential of microbial imaging mass spectrometry on nanostructured silicon because both fungi produce a dense mass of aerial mycelia, which is known to complicate the collection of high-quality imaging mass spectrometry data. Our simple and straightforward sample imprinting method and low background interference resulted in an efficient analysis of small metabolites from the complex microbial interaction samples.

A stretchable conductive Polypyrrole Polydimethylsiloxane device fabricated by simple soft lithography and oxygen plasma treatment.

This paper reports a simple method used to fabricate a stretchable conductive polypyrrole (PPy) rough pore-shape polydimethylsiloxane (p-PDMS) device. An abrasive paper is first used to imprint rough micro-structures on the SU-8 micromold. The p-PDMS microchannel is then fabricated using a standard soft-lithography process. An oxygen plasma treatment is then applied to form an irreversible sealing between the microchannel and a blank cover PDMS. The conductive layer is formed by injecting the PPy mixture into the microchannel which polymerizes in the rough pore-shape micro-structures; The PPy/p-PDMS hybrid device shows good electrical property and stretchability. The electrical properties of different geometrical designs of the PPy/p-PDMS microchannel under stretching were investigated, including straight, curved, and serpentine. Mouse embryonic fibroblasts (NIH/3 T3) were also cultured inside the PPy/p-PDMS device to demonstrate good biocompatibility and feasibility using the conductive and stretchable microchannel in cell culture microfluidics applications. Finally, cyclic stretching and bending tests were performed to evaluate the reliability of PPy/p-PDMS microchannel.

A piezo-ring-on-chip microfluidic device for simple and low-cost mass spectrometry interfacing.

Mass spectrometry (MS) interfacing technology provides the means for incorporating microfluidic processing with post MS analysis. In this study, we propose a simple piezo-ring-on-chip microfluidic device for the controlled spraying of MALDI-MS targets. This device uses a low-cost, commercially-available ring-shaped piezoelectric acoustic atomizer (piezo-ring) directly integrated into a polydimethylsiloxane microfluidic device to spray the sample onto the MS target substrate. The piezo-ring-on-chip microfluidic device's design, fabrication, and actuation, and its pulsatile pumping effects were evaluated. The spraying performance was examined by depositing organic matrix samples onto the MS target substrate by using both an automatic linear motion motor, and manual deposition. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was performed to analyze the peptide samples on the MALDI target substrates. Using our technique, model peptides with 10-6 M concentration can be successfully detected. The results also indicate that the piezo-ring-on-chip approach forms finer matrix crystals and presents better MS signal uniformity with little sample consumption compared to the conventional pipetting method.

Negative Pressure Induced Droplet Generation in a Microfluidic Flow-Focusing Device.

We introduce an effective method to actively induce droplet generation using negative pressure. Droplets can be generated on demand using a series of periodic negative pressure pulses. Fluidic network models were developed using the analogy to electric networks to relate the pressure conditions for different flow regimes. Experimental results show that the droplet volume is correlated to the pressure ratio with a power law of 1.3. Using a pulsed negative pressure at the outlet, we are able to produce droplets in demand and with a volume proportional to the pulse width.

Active droplet sorting in microfluidics: a review.

The ability to manipulate and sort droplets is a fundamental issue in droplet-based microfluidics. Various lab-on-a-chip applications can only be realized if droplets are systematically categorized and sorted. These micron-sized droplets act as ideal reactors which compartmentalize different biological and chemical reagents. Array processing of these droplets hinges on the competence of the sorting and integration into the fluidic system. Recent technological advances only allow droplets to be actively sorted at the rate of kilohertz or less. In this review, we present state-of-the-art technologies which are implemented to efficiently sort droplets. We classify the concepts according to the type of energy implemented into the system. We also discuss various key issues and provide insights into various systems.

Magnetic microparticle-polydimethylsiloxane composite for reversible microchannel bonding.

In this study, an iron oxide magnetic microparticles and poly(dimethylsiloxane) (MMPs-PDMS) composite material was employed to demonstrate a simple high-strength reversible magnetic bonding method. This paper presents the casting of opaque-view (where optical inspection through the microchannels was impossible) and clear-view (where optical inspection through the microchannel was possible) MMPs-PDMS. The influence of the microchannel geometries on the casting of the opaque-view casting was limited, which is similar to standard PDMS casting. Clear-view casting performance was highly associated with the microchannel geometries. The effects of the microchannel layout and the gap between the PDMS cover layer and the micromold substrate were thoroughly investigated. Compared with the native PDMS bonding strength of 31 kPa, the MMPs-PDMS magnetic bonding experiments showed that the thin PDMS film with an MMPs-PDMS layer effectively reduced the surface roughness and enhanced MMPs-PDMS reversible magnetic bonding strength. A thin PDMS film-coated opaque-view MMPs-PDMS device exhibited the greatest bonding strength of 110 kPa, and a clear-view MMPs-PDMS device with a thin PDMS film attained a magnetic bonding strength of 81 kPa.

Nanoscale silicon surface-assisted laser desorption/ionization mass spectrometry: environment stability and activation by simple vacuum oven desiccation.

Nanoscale silicon surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is an emerging matrix-free, highly sensitive MS analysis method. An important challenge in using nanoscale silicon SALDI-MS analysis is the aging and stability of silicon after storage in various environments. No proper nanoscale silicon SALDI-MS activation procedure has been reported to solve this issue. This study investigated the sensitivity, wettability, and surface oxidation behavior of nanoscale silicon surface SALDI-MS in a room, an inert gas atmosphere, and a vacuum environment. A simple vacuum oven desiccation was proposed to activate the SALDI-MS surface, and the limit of detection was further enhanced 1000 times to a 500 attomole level using this approach. The long-term stability and desorption/ionization mechanism were also investigated.

Polymer Microfluidics: Simple, Low-Cost Fabrication Process Bridging Academic Lab Research to Commercialized Production.

Using polymer materials to fabricate microfluidic devices provides simple, cost effective, and disposal advantages for both lab-on-a-chip (LOC) devices and micro total analysis systems (µTAS). Polydimethylsiloxane (PDMS) elastomer and thermoplastics are the two major polymer materials used in microfluidics. The fabrication of PDMS and thermoplastic microfluidic device can be categorized as front-end polymer microchannel fabrication and post-end microfluidic bonding procedures, respectively. PDMS and thermoplastic materials each have unique advantages and their use is indispensable in polymer microfluidics. Therefore, the proper selection of polymer microfabrication is necessary for the successful application of microfluidics. In this paper, we give a short overview of polymer microfabrication methods for microfluidics and discuss current challenges and future opportunities for research in polymer microfluidics fabrication. We summarize standard approaches, as well as state-of-art polymer microfluidic fabrication methods. Currently, the polymer microfluidic device is at the stage of technology transition from research labs to commercial production. Thus, critical consideration is also required with respect to the commercialization aspects of fabricating polymer microfluidics. This article provides easy-to-understand illustrations and targets to assist the research community in selecting proper polymer microfabrication strategies in microfluidics.

High Sensitivity and High Detection Specificity of Gold-Nanoparticle-Grafted Nanostructured Silicon Mass Spectrometry for Glucose Analysis.

Desorption/ionization on silicon (DIOS) is a high-performance matrix-free mass spectrometry (MS) analysis method that involves using silicon nanostructures as a matrix for MS desorption/ionization. In this study, gold nanoparticles grafted onto a nanostructured silicon (AuNPs-nSi) surface were demonstrated as a DIOS-MS analysis approach with high sensitivity and high detection specificity for glucose detection. A glucose sample deposited on the AuNPs-nSi surface was directly catalyzed to negatively charged gluconic acid molecules on a single AuNPs-nSi chip for MS analysis. The AuNPs-nSi surface was fabricated using two electroless deposition steps and one electroless etching step. The effects of the electroless fabrication parameters on the glucose detection efficiency were evaluated. Practical application of AuNPs-nSi MS glucose analysis in urine samples was also demonstrated in this study.

Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

BACKGROUND: Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. RESULTS: After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. CONCLUSION: Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

Electrical stimulation to promote osteogenesis using conductive polypyrrole films.

In this study, we developed an electrical cell culture and monitoring device. Polypyrrole (PPy) films with different resistances were fabricated as conductive surfaces to investigate the effect of substrate-mediated electrical stimulation. The physical and chemical properties of the devices, as well as their biocompatibilities, were thoroughly evaluated. These PPy films had a dark but transparent appearance, on which the surface cells could be easily observed. After treating with the osteogenic medium, rat bone marrow stromal cells cultured on the PPy films differentiated into osteoblasts. The cells grown on the PPy films had up-regulated osteogenic markers, and an alkaline phosphatase activity assay showed that the PPy films accelerated cell differentiation. Alizarin red staining and calcium analysis suggested that the PPy films promoted osteogenesis. Finally, PPy films were subjected to a constant electric field to elucidate the effect of electrical stimulation on osteogenesis. Compared with the untreated group, electrical stimulation improved calcium deposition in the extracellular matrix. Furthermore, PPy films with lower resistances allowed larger currents to stimulate the surface cells, which resulted in higher levels of mineralization. Overall, these results indicated that this system exhibited superior electroactivity with controllable electrical resistance and that it can be coated directly to produce medical devices with a transparent appearance, which should be beneficial for research on electrical stimulation for tissue regeneration.