PDMS Microdevices Research Articles

Determination of total protein content in biomedical products by the PDMS-assisted lab-in-a-syringe assay using 3D printed scaffolds removal

The aim of our work was to develop a low-cost, portable device for the fast and easy determination of total protein content by using PDMS-based lab-in-a-syringe technology with removal of 3D-printed channels. We proposed two designs with a one-step PDMS curing and a two-step PDMS-curing fabrication procedure. The one-step PDMS microdevices were found to be the best in the view of preparation, repeatability, and stability of the reagent. This design was then applied for the determination of total protein content in biomedical products using the Bradford assay.

Rapid Production of PDMS Microdevices for Electrodriven Separations and Microfluidics by 3D-Printed Scaffold Removal

In our work, we produced PDMS-based microfluidic devices by mechanical removal of 3D-printed scaffolds inserted in PDMS. Two setups leading to the fabrication of monolithic PDMS-based microdevices and bonded (or stamped) PDMS-based microdevices were designed. In the monolithic devices, the 3D-printed scaffolds were fully inserted in the PDMS and then carefully removed. The bonded devices were produced by forming imprints of the 3D-printed scaffolds in PDMS, followed by bonding the PDMS parts to glass slides. All these microfluidic devices were then successfully employed in three proof-of-concept applications: capture of magnetic microparticles, formation of droplets, and isotachophoresis separation of model organic dyes.

Functionalization of microfluidic devices by microstructures created with proton beam lithography

Microfluidic devices have become important part of technology nowadays in numerous fields of application, such as chemical reactions, biosensors, synthesis of nanomaterials, diagnostic device for detection of biological samples, etc.In this work, as a new fabrication approach, poly(dimethyl-siloxane) (PDMS) microstructures were integrated into PDMS microdevices by combining proton beam lithography and conventional UV lithography fabrication techniques. This way, the microstructures and the microdevices can be made from the same material, which is not only useful, but in case of certain applications, it is essential. This work deals with the design of various microfluidic devices with and without integrated microstructures, for passive mixing purposes. The investigation involves the computer simulation and optimization, and then the fabrication of the chips with the above mentioned methods. Finally, the analysis of the mixing efficiency of the micromixer devices was realized with UV/vis spectroscopy at different fluid flow rates. Compared to the small size of the whole mixing chip, high mixing efficiency could be achieved.

Abstract 5500: A 3D tumor tissue microphysiological system for realistic tumor microenvironment mimicry and therapeutic modeling

Abstract Introduction: Although cancer is a heterogeneous disease, current chemotherapy is based on the use of anti-cancer agents that broadly affect cell proliferation. As a result of this non-specific tumor therapy there is often high toxicity and therapeutic resistance, which increases the risk of disease progression. There is thus a critical need to find novel anti-cancer compounds, however, as a result of preliminary screens often performed in 2D systems, and the use of mouse models for secondary screens, many drugs fail during full clinical trials. Replicating a complex, 3D microenvironment incorporating human cells is therefore likely to improve drug screening efficiency. Methods: Here we have developed a system for high throughput screening of drug efficacy. Our system features 3D tumor tissues incorporating human cells co-cultured with stromal cells and connected by human microvessels in a naturally occurring 3D matrix in a PDMS microdevice. Transduced colorectal cancer cells (CRC) such as HCT116, SW480 and SW620s that express green fluorescent protein (GFP) constitutively are introduced into the device in co-culture with human endothelial colony-forming cell-derived EC (ECFC-EC) and fibroblasts. ECFC-EC or fibroblasts that express longer wavelength fluorescent proteins are also used to track both tumor cells and vasculature/fibroblast growth. The tri-culture is resuspended in fibrinogen (10 mg/ml) and mixed with thrombin before injecting into the microchambers. Cells are fed with endothelial growth media-2 and drugs are delivered through the microfluidic channels using a hydrostatic pressure gradient. Results: A complete vascular network was formed in the chambers by 7-10 days and this carried medium into the tissue. CRC showed continuous growth, often forming spheroids. After 7-10 days of culture, cells were exposed to various FDA-approved drugs at concentrations reported for plasma levels in patients. Tumor growing in the device responded differently to most drugs compared to cells growing in 2D. We identified cytotoxic and cytostatic drug actions in the device with IC50s close to the in vivo pharmacologic plasma concentration, but higher than those obtained in parallel 2D experiments. Importantly, we also identified drugs that were active in our 3D-device but not in 2D cultures. Conclusions: Preliminary results using our 3D microphysiological system, in which we have created a more complex microenvironment than used in 2D cultures, support an improved strategy for drug validation. In addition, future experiments will include the use of non-invasive optical imaging techniques to better understand the human 3D tumor microenvironment, including the study of different tumor behaviors, tumor metabolism and pathological angiogenesis. This proof-of-concept study validates the use of 3D microphysiological systems in place of more simplistic 2D systems. Citation Format: Agua Sobrino, Dúc Phan, Steven C George, Christopher C.W Hughes. A 3D tumor tissue microphysiological system for realistic tumor microenvironment mimicry and therapeutic modeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2015-5500

The development of a counterflow‐assisted preconcentration technique in capillary electrophoresis electrospray‐ionization mass spectrometry

A preconcentration approach for CE-MS using counterflow-assisted electrokinetic injection was proposed. The proposed preconcentration method was based on a counterflow-compatible sheathless interface. The interface was fabricated using a capillary-assembled PDMS microdevice that allowed the application of a counterflow and provided liquid-film electrical conduction. During electrokinetic injection, a hydrodynamic counterflow was introduced into the separation capillary to retard the movement of the stacking boundary to the CE outlet. Accordingly, a long injection time was achieved without a loss of CE separation. With the use of reverse polarity mode and a dynamic polybrene-coated separation capillary (filled with a 1% formic acid solution), a counterflow-assisted electrokinetic injection of peptide samples (in a aqueous solution containing 1% ammonia and 50% methanol) for 10 min provided a sensitivity enhancement of 750-1480 and a detection limit of 20 pM for the five peptide standards. The proposed technique was applied to the analysis of low nanomolar concentrations myoglobin tryptic peptides.

Investigation and improvement of reversible microfluidic devices based on glass–PDMS–glass sandwich configuration

Reversibly assembled microfluidic devices are dismountable and reusable, which is useful for a number of applications such as micro- and nano-device fabrication, surface functionalization, complex cell patterning, and other biological analysis by means of spatial–temporal pattern. However, reversible microfluidic devices fabricated with current standard procedures can only be used for low-pressure applications. Assembling technology based on glass–PDMS–glass sandwich configuration provides an alternative sealing method for reversible microfluidic devices, which can drastically increase the sealing strength of reversibly adhered devices. The improvement mechanism of sealing properties of microfluidic devices based on the sandwich technique has not been fully characterized, hindering further improvement and broad use of this technique. Here, we characterize, for the first time, the effect of various parameters on the sealing strength of reversible PDMS/glass hybrid microfluidic devices, including contact area, PDMS thickness, assembling mode, and external force. To further improve the reversible sealing of glass–PDMS–glass microfluidic devices, we propose a new scheme which exploits mechanical clamping elements to reinforce the sealing strength of glass–PDMS–glass sandwich structures. Using our scheme, the glass–PDMS–glass microchips can survive a pressure up to 400 kPa, which is comparable to the irreversibly bonded PDMS microdevices. We believe that this bonding method may find use in lab-on-a-chip devices, particularly in active high-pressure-driven microfluidic devices.

Microfluidic based immunosensor for detection and purification of carbonylated proteins

A microchip has been developed on the basis of immno-precipitation approach for fast and sensitive enrichment of low abundant carbonylated proteins. This microfluidic method could enrich molecular biomarkers, which could be further analyzed in the proteomic study of age-related diseases and therapeutic development. In this study, an immunoaffinity-based PDMS micro-device was designed, fabricated, and chemically modified to specifically trap DNP-labeled PTM proteins of low abundance from a complex protein mixture. Carbonylated protein is selected as a representative PTM protein to illustrate the wide application of this immuno-based microchip for other PTMs which could be readily labeled by different antibody groups. Surface characterization methods such as atomic force microscopy and fluorescence microscopy were used to evaluate the construction of glutaraldehyde- and antibody- terminated PDMS substrates in the device fabrication. Quantitative study was also applied to study the target protein capture and elution efficiency of the device. In a testing mixture consisting of smaller amount of test model-In Vitro oxidized cytochrome c and large blocking protein BSA, a high sensitivity and specificity for only carbonylated protein biomarkers was demonstrated using this on-chip immnuoaffinity based extraction/enrichment. For this highly dense 193-post arrays μ-chip, a low abundance of 159 ng of standard in vitro test model- cytochrome c was enriched at flow speed of 5 μL/min within 110 min. We demonstrated that this nascent micro-immunoprecipitation (μ-IP) method is capable for enrichment of biomarkers in protein post-translation modification related diseases and promise great advance in early disease detection.

Patterned Paper as a Low-Cost, Flexible Substrate for Rapid Prototyping of PDMS Microdevices via “Liquid Molding”

This report describes the use of patterned paper as a low-cost, flexible substrate for rapidly prototyping PDMS microdevices via "liquid molding". The entire fabrication process consists simply of three steps: (1) fabrication of patterned paper in NC membrane by direct wax printing (or modified wax printing that we call "transfer wax printing"); (2) formation of liquid mold on wax-patterned NC membrane; (3) PDMS molding and curing on wax-patterned NC membrane anchored with liquid micropatterns. All these procedures can be finished within only 1.5 h without the use of a photomask, photoresist, UV lamp, etc. Through the use of wax-patterned NC membrane coupled with a liquid mold as a template, different PDMS microdevices such as microwells and microchannels have been fabricated to demonstrate the usefulness of the method for PDMS microfabrication. The height of microwells and microchannels can also be tailored flexibly by adjusting the liquid filling volume. This method for prototyping PDMS microdevices has some favorable merits including simple operation procedures, fast concept-to-device time, and low cost, indicating its potential for simple PDMS microdevice fabrication and applications.

On-demand micro-encapsulation utilizing on-chip synthesis of semi-permeable alginate-PLL capsules

This article presents an on-demand, multi-step synthesis scheme that is capable of forming semi-permeable micro-capsules on an integrated microfluidic system. Emulsion droplets functioning as templates and reactors are employed to realize the synthesis process. Three-layered PDMS micro-devices with pneumatically actuated diaphragm valves constructed on top of specially designed fluidic-channels are utilized to control the droplets and, consequently, the encapsulation process. A PDMS molding and bonding process is used to fabricate the proposed microfluidic devices. In the prototype demonstration, relatively small Na-alginate droplets are metered, trapped, and then drawn into relatively large CaCl2 droplets, while they react and form solid Ca-alginate micro-capsules on the interfaces. In addition, entrapment and transfer of the resulting capsules can also be performed on the same microfluidic system to further process Ca-alginate into semi-permeable alginate-poly-l-lysine (PLL). It has been demonstrated that: (1) both emulsion droplets and solid capsules could be manipulated; (2) multi-step reactions could be performed on droplet-in-droplet interfaces to synthesize alginate-PLL capsules; and (3) on demand, controlled encapsulation could be achieved on an integrated microfluidic system. As such, the demonstrated multi-step synthesis scheme could potentially fulfill the real-time controllability on micro-encapsulation, which is desired for a variety of biological and medical applications.

On-demand double emulsification utilizing pneumatically actuated, selectively surface-modified PDMS micro-devices

This article presents an active, two-step emulsification scheme that is capable of producing double emulsions with desired geometries and compositions on demand. Three-layer PDMS micro-devices with pneumatically actuated membrane-valves constructed on top of specially designed fluidic-channels are utilized to meter and shape immiscible fluids into double emulsions. By intermittently squeezing a fluid into another one, controlled emulsification is realized, and successive emulsification steps result in the formation of multiple emulsions. In the prototype demonstration, a three-layer PDMS molding and bonding process was employed to fabricate the proposed microfluidic devices, whose channel surfaces were selectively modified into hydrophilic by a photo-grafting process. A governing computer program cooperating with a set of control hardware was employed to coordinate the actuation of the prototype system. It has been demonstrated that: (1) both water-in-oil-in-oil and water-in-oil-in-water double emulsions can be produced; (2) the sizes of inner aqueous droplets and outer oil drops can be controlled independently; and (3) adjacent oil drops with varying overall sizes, and both diameters and numbers of inner aqueous droplets can be produced on demand. As such, the demonstrated emulsification scheme could potentially fulfill the real-time controllability on emulsion formation, which is desired for a variety of chemical and biological applications.

Ultrasensitive nanoelectrospray ionization-mass spectrometry using poly(dimethylsiloxane) microchips with monolithically integrated emitters

Poly(dimethylsiloxane) (PDMS) is a widely used substrate for microfluidic devices, as it enables facile fabrication and has other distinctive properties. However, for applications requiring highly sensitive nanoelectrospray ionization mass spectrometry (nanoESI-MS) detection, the use of PDMS microdevices has been hindered by a large chemical background in the mass spectra that originates from the leaching of uncross-linked oligomers and other contaminants from the substrate. A more general challenge is that microfluidic devices containing monolithically integrated electrospray emitters are frequently unable to operate stably in the nanoflow regime where the best sensitivity is achieved. In this report, we extracted the contaminants from PDMS substrates using a series of solvents, eliminating the background observed when untreated PDMS microchips are used for nanoESI-MS, such that peptides at concentrations of 1 nM were readily detected. Optimization of the integrated emitter geometry enabled stable operation at flow rates as low as 10 nL min(-1).

A vacuum manifold for rapid world-to-chip connectivity of complex PDMS microdevices

The lack of simple interfaces for microfluidic devices with a large number of inlets significantly limits production and utilization of these devices. In this article, we describe the fabrication of a reusable manifold that provides rapid world-to-chip connectivity. A vacuum network milled into a rigid manifold holds microdevices and prevents leakage of fluids injected into the device from ports in the manifold. A number of different manifold designs were explored, and all performed similarly, yielding an average of 100 kPa (15 psi) fluid holding pressure. The wide applicability of this manifold concept is demonstrated by interfacing with a 51-inlet microfluidic chip containing 144 chambers and hundreds of embedded pneumatic valves. Due to the speed of connectivity, the manifolds are ideal for rapid prototyping and are well suited to serve as "universal" interfaces.

A facile “liquid-molding” method to fabricate PDMS microdevices with 3-dimensional channel topography

This work deals with a facile "liquid-molding" technology to fabricate PDMS microdevices with complex structured microchannels, the main procedures of which involves: (1) Photo-lithographically fabricating chemical micropatterns of hydrogels on silanized glass substrates to form heterogeneous hydrophilic/hydrophobic surfaces; (2) Fabricating stable 3D surface relief patterns of a liquid via dip-coating the hydrogel patterned substrate in a polar solution; (3) Fabricating PDMS microfluidic devices via a slightly modified replica molding procedure, using a liquid patterned substrate as the molding template. In addition to its simplicity compared to conventional microfabrication methods, this technology produces microchannels with 3D surface topography, which allows for more flexibility in device design and applications. We discuss the main features of the liquid-molding method as well as the structural characteristics of liquid-molded microchannels. We demonstrate the potential application of these "liquid-molded" PDMS (LM-PDMS) devices by designing a cell trapping microdevice that is capable of trapping multiple or individual cells through simple operation procedures.

Study of osteoblastic cells in a microfluidic environment

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2×10 6 cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 μl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 μl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.

Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis

We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF-alpha and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phosphatidyl-serine, membrane blebbing and permeabilization of the cell membrane have been observed in real time with different individual cells. From the results, an interesting heterogeneicity in the apoptotic phenotype has been observed. The CRISC method is easy to use and provides biologist with a powerful additional tool to study in real-time processes of several hours of duration such as apoptosis. We predict that the period of cell viability obtained after protein coating of the PDMS devices (>80 h) will also allow monitoring of other biological processes of longer duration or long onset time, such as mitosis, phagocytosis and differentiation.

Towards single molecule analysis in PDMS microdevices: from the detection of ultra low dye concentrations to single DNA molecule studies

In this paper, we report on the performance of electrophoretical separation and laser-induced fluorescence (LIF) detection of dyes and fluorescently labeled biomolecules in poly(dimethylsiloxane) (PDMS) microdevices. The dyes fluorescein and fluorescein isothiocyanate (FITC) have been separated effectively in nM concentrations. Fluorescein injections gave linear concentration response in the range from 4 to 100 pM. As ultimate detection sensitivity, 100 fM injected fluorescein was obtained. Further, 100 fM injected fluorescein could be detected. This is to our knowledge the smallest electrokinetically injected dye concentration detected on a microchip. Injection studies of fluorescently labeled avidin revealed a theoretical detection limit of 25 nM for laser-induced fluorescence detection in good agreement with separations in glass chips. Furthermore, the injection of several and even one single DNA molecule using a PDMS cross injector has been demonstrated as well as free solution separation of λ- and T2-DNA (60 pM each) in periodically structured channels.