Grain boundary interface mechanics in strain gradient crystal plasticity

Interactions between dislocations and grain boundaries play an important role in the plastic deformation of polycrystalline metals. Capturing accurately the behaviour of these internal interfaces is particularly important for applications where the relative grain boundary fraction is significant, such as ultra fine-grained metals, thin films and microdevices. Incorporating these micro-scale interactions (which are sensitive to a number of dislocation, interface and crystallographic parameters) within a macro-scale crystal plasticity model poses a challenge. The innovative features in the present paper include (i) the formulation of a thermodynamically consistent grain boundary interface model within a microstructurally motivated strain gradient crystal plasticity framework, (ii) the presence of intra-grain slip system coupling through a microstructurally derived internal stress, (iii) the incorporation of inter-grain slip system coupling via an interface energy accounting for both the magnitude and direction of contributions to the residual defect from all slip systems in the two neighbouring grains, and (iv) the numerical implementation of the grain boundary model to directly investigate the influence of the interface constitutive parameters on plastic deformation. The model problem of a bicrystal deforming in plane strain is analysed. The influence of dissipative and energetic interface hardening, grain misorientation, asymmetry in the grain orientations and the grain size are systematically investigated. In each case, the crystal response is compared with reference calculations with grain boundaries that are either 'microhard' (impenetrable to dislocations) or 'microfree' (an infinite dislocation sink). © 2013 Elsevier Ltd. All rights reserved.

DNA diagnosis by capillary electrophoresis and microfabricated electrophoretic devices

DNA diagnosis is experiencing an impressive progression towards the development of novel technology to identity various clinically relevant categories of genetic changes and to meet the exponential growth of genomics. The introduction of capillary electrophoresis has dramatically accelerated the completion of the first draft of the human DNA sequence in the Human Genome Project, and thus, has become the method of choice for analysis of various genetic variants. The recent development of microfabricated electrophoretic devices has led to the possibility of integrating multiple sample handling with the actual measurement steps required for automation of molecular diagnostics. This review highlights the most recent progress in capillary electrophoresis and electrophoretic microdevices for DNA-based diagnostics, including the important areas of genotyping for point mutation, single nucleotide polymorphisms, short tandem repeats and organism identification. The application of these techniques for infectious and genetic disease diagnosis, as well as forensic identification purpose, are covered. The promising development and the challenges for techinical problems are also discussed.

Morphology of electrodeposited poly(3,4-ethylenedioxythiophene)/poly(4-styrene sulfonate) films

Poly(4-styrene sulfonate)-doped poly(3,4-ethylenedioxythiophene) (PEDOT/PSS) films with ring-, arrow-, and bubble-like microstructures have been electrochemically generated simply by a one-step cyclic voltammetry in an aqueous media. Influences of applied potentials and surfactant/dopant-PSS on morphology of the resulting film were investigated, and a gas bubble template mechanism has been proposed. The result confirmed a well-doping of PSS in the PEDOT film. Electrochemical property and conductivity of the micro-structured PEDOT/PSS film were investigated further. Similar preparation with potential applications in fabrication of microdevices and micro-sensors can be extended to other micro-structured conducting polymers.

In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Evaluating the process of borosilicate glass capillaries used for fabrication of in-vitro fertilization (IVF) micro-pipettes

In this paper we investigate a number of gas flames for fire polishing borosilicate glass capillaries used in the manufacturing of IVF micro-pipettes. Hydrofluoric acid (HF) was also used as an alternative to finish the pipette end. Glass micro tools in the IVF industry are drawn from hollow glass capillaries of diameter 1 mm. These capillaries are cut manually to a length of 100 mm from hollow glass rods resulting in sharp and chipped edges. These capillaries are held in a customised holder having padding of soft silicone or rubber. Sharp and uneven edges of these capillaries pick up particles of rubber or soft silicone shavings, rendering them ineffective for IVF treatments. The working range of borosilicate glass is 800-1,200 degrees C. The experiments involved analysis of fire polishing process for borosilicate glass capillaries using candle, butane, propane, 2350 butane propane, oxyacetylene gas flames, finding the optimum distance of the capillary relative to the flame, optimum time for which the capillary should be held in the flame and optimum region of the flame which gives the required temperature range. The results show that 2350 butane propane gas mix is optimum for fire polishing of borosilicate glass capillaries. The paper is concluded by comparing the results of fire polishing with the results of acid polishing, in which HF of 1.6% concentration is used to etch the ends of the borosilicate glass pipettes.

Gold Nanoparticles-Coated SU-8 for Sensitive Fluorescence-Based Detections of DNA

SU-8 epoxy-based negative photoresist has been extensively employed as a structural material for fabrication of numerous biological microelectro-mechanical systems (Bio-MEMS) or lab-on-a-chip (LOC) devices. However, SU-8 has a high autofluorescence level that limits sensitivity of microdevices that use fluorescence as the predominant detection workhorse. Here, we show that deposition of a thin gold nanoparticles layer onto the SU-8 surface significantly reduces the autofluorescence of the coated SU-8 surface by as much as 81% compared to bare SU-8. Furthermore, DNA probes can easily be immobilized on the Au surface with high thermal stability. These improvements enabled sensitive DNA detection by simple DNA hybridization down to 1 nM (a two orders of magnitude improvement) or by solid-phase PCR with sub-picomolar sensitivity. The approach is simple and easy to perform, making it suitable for various Bio-MEMs and LOC devices that use SU-8 as a structural material.

Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with similar to 270 mu m, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 mu L of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/mu L., (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of lambda-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Surface patterns induced by laser irradiation on thin polymer bilayer films

We report on the preparation of wavelike surface patterns with characteristic wavelengths on thin bilayers of poly(methyl methacrylate) on azobenzene liquid crystalline polymer films (LCP/PMMA) by irradiation of a single polarized pulsed laser beam. The formation of such patterns was influenced by the thickness of the upper layer and the laser fluence. We were also able to guide the wavelike pattern to have a specific orientation by placing an elastic polydimethylsiloxane (PDMS) mold on the surface of bilayer film prior to laser irradiation. Moreover, the property of the laser irradiation, that is, the selectivity through mask-projection systems, allowed us fabricating complicated micropattems for novel microdevices. (c) 2007 Elsevier B.V. All rights reserved.

Critical parameter determination of sonic flow controller diamond microtubes and micronozzles

In this article, the authors measure throughput of sonic diamond microtubes and micronozzles that can work as passive gas flow controllers and flow meters under choking conditions. The behavior of the outlet pressure through the microdevices using an experimental setup with constant volume and constant temperature was determined in order to obtain the critical throughput, the critical mass flow rate, and the discharge coefficients of the diamond sonic microdevices. © 2007 American Vacuum Society.

3D nanostructured microcarriers for cell therapy in regenerative medicine

Supercritical Emulsion Extraction technology (SEE-C) was proposed for the production of poly-lactic-co-glycolic acid microcarriers. SEE-C operating parameters as pressure, temperature and flow rate ratios were analyzed and the process performance was optimized in terms of size distribution and encapsulation efficiency. Microdevices loaded with bovine serum insulin were produced with different sizes (2 and 3 µm) or insulin charges (3 and 6 mg/g) and with an encapsulation efficiency of 60%. The microcarriers were characterized in terms of insulin release profile in two different media (PBS and DMEM) and the diffusion and degradation constants were also estimated by using a mathematical model. PLGA microdevices were also used in a cultivation of embryonic ventricular myoblasts (cell line H9c2 obtained from rat) in a FBS serum free medium to monitor cell viability and growth in dependence of insulin released. Good cell viability and growth were observed on 3 µm microdevices loaded with 3 mg/g of insulin. PLGA microspheres loaded with growth factors (GFs) were charged into alginate scaffold with human Mesenchimal Steam Cells (hMSC) for bone tissue engineering with the aim of monitoring the effect of the local release of these signals on cells differentiation. These “living” 3D scaffolds were incubated in a direct perfusion tubular bioreactor to enhance nutrient transport and exposing the cells to a given shear stress. Different GFs such as, h-VEGF, h-BMP2 and a mix of two (ratio 1:1) were loaded and alginate beads were recovered from dynamic (tubular perfusion system bioreactor) and static culture at different time points (1st, 7th, 21st days) for the analytical assays such as, live/dead; alkaline phosphatase; osteocalcin; osteopontin and Van Kossa Immunoassay. The immunoassay confirmed always a better cells differentiation in the bioreactor with respect to the static culture and revealed a great influence of the BMP-2 released in the scaffold on cell differentiation.

Development and in-vitro characterization of an implantable flow sensing transducer for hydrocephalus

An implantable transducer for monitoring the flow of Cerebrospinal fluid (CSF) for the treatment of hydrocephalus has been developed which is based on measuring the heat dissipation of a local thermal source. The transducer uses passive telemetry at 13.56 MHz for power supply and read out of the measured flow rate. The in vitro performance of the transducer has been characterized using artificial Cerebrospinal Fluid (CSF) with increased protein concentration and artificial CSF with 10\% fresh blood. After fresh blood was added to the artificial CSF a reduction of flow rate has been observed in case that the sensitive surface of the flow sensor is close to the sedimented erythrocytes. An increase of flow rate has been observed in case that the sensitive surface is in contact with the remaining plasma/artificial CSF mix above the sediment which can be explained by an asymmetric flow profile caused by the sedimentation of erythrocythes having increased viscosity compared to artificial CSF. After removal of blood from artificial CSF, no drift could be observed in the transducer measurement which could be associated to a deposition of proteins at the sensitive surface walls of the packaged flow transducer. The flow sensor specification requirement of +-10\% for a flow range between 2 ml/h and 40 ml/h. could be confirmed at test conditions of 37 degrees C.

ALTERNATING CURRENT DIELECTROPHORETIC MANIPULATION OF ERYTHROCYTES IN MEDICAL MICRODEVICE TECHNOLOGY

Medical microdevices have gained popularity in the past few decades because they allow the medical laboratory to be taken out into the field and for disease diagnostics to happen with a smaller sample volume, at a lower cost and much faster. Blood is the human body's most readily available and informative diagnostic fluid because of the wealth of information it provides about the body's general health including enzymatic, proteomic and immunological states. The purpose of this project is to optimize operating conditions and study ABO-Rh erythrocytes dielectrophoretic responses to alternating current electric signals. The end goal of this project is the creation of a relatively inexpensive microfluidic device, which can be used for the ABO-Rh typing of a blood sample. This dissertation presents results showing how blood samples of a known ABO- Rh blood type exhibit differing behavior to the same electrical stimulus based on their blood type. The first panel of donors and experiments, presented in Chapter 4 occurred when a sample of known blood type was injected into a microdevice with a T-shaped electrode configuration and the erythorcytes were found to rupture at a rate specific to their ABO-Rh blood type. The second set of experiments, presented in Chapter 5, were originally published in Electrophoresis in 20111. Novel in this work was the discovery that treatment of human erythrocytes with β-galactosidase successfully removed ABO surface antigens such that native A and B blood no longer agglutinated with the proper antibodies. This work was performed in a medium of conductivity 0.9S/m which is close to the measured conductivity of pooled plasma (~1.1S/m). The ability to perform dielectrophoresis experiments at physiological conductivities conditions is advantageous for future portable devices because the device/instrument would not need to store dilution buffers. The final results of this project, presented in Chapter 6, explore the entire dielectrophoretic spectra of the ABO-Rh erythrocytes including the cross-over frequency and the magnitudes of the positive or negative dielectrophoretic response. These were completed at lower medium conductivities of 0.1S/m and 0.01-0.04S/m. These results show that by using the sweep function built into the Agilent alternating current generator it is possible to explore how a single group of blood cells will react to rapid changes in frequency and will provide the user with curve that can be matched the theoretical dielectrophoretic response curves. As a whole this project shows that it is possible to distinguish human erythrocytes by their ABO-Rh blood type via three different dielectrophoretic methods. This work builds on the foundation of that it is possible to distinguish healthy from infected cells2-7, similar cell types1,7-14 and other work regarding the dielectrophoresis of human erythrocytes1,10,11. This work has implications in both medical diagnostics and future dielectrophoretic work because it has shown that ABO-Rh blood type is now a factor, which must be identified when working with a human blood sample. It also shows that the creation of a microfluidic device that subjects human erythrocytes to a dielectrophoretic impulse and then exports an ABO-Rh blood type is a near future possibility.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Cell force measurements in 3D microfabricated environments based on compliant cantilevers.

We report the fabrication, functionalization and testing of microdevices for cell culture and cell traction force measurements in three-dimensions (3D). The devices are composed of bent cantilevers patterned with cell-adhesive spots not lying on the same plane, and thus suspending cells in 3D. The cantilevers are soft enough to undergo micrometric deflections when cells pull on them, allowing cell forces to be measured by means of optical microscopy. Since individual cantilevers are mechanically independent of each other, cell traction forces are determined directly from cantilever deflections. This proves the potential of these new devices as a tool for the quantification of cell mechanics in a system with well-defined 3D geometry and mechanical properties.

Measuring localized viscoelasticity of the vitreous body using intraocular microprobes.

Vitrectomy is a standard ophthalmic procedure to remove the vitreous body from the eye. The biomechanics of the vitreous affects its duration (by changing the removal rate) and the mechanical forces transmitted via the vitreous on the surrounding tissues during the procedure. Biomechanical characterization of the vitreous is essential for optimizing the design and control of instruments that operate within the vitreous for improved precision, safety, and efficacy. The measurements are carried out using a magnetic microprobe inserted into the vitreous, a method known as magnetic microrheology. The location of the probe is tracked by a microscope/camera while magnetic forces are exerted wirelessly by applied magnetic fields. In this work, in vitro artificial vitreous, ex vivo human vitreous and ex vivo porcine vitreous were characterized. In addition, in vivo rabbit measurements were performed using a suturelessly injected probe. Measurements indicate that viscoelasticity parameters of the ex vivo human vitreous are an order of magnitude different from those of the ex vivo porcine vitreous. The in vivo intra-operative measurements show typical viscoelastic behavior of the vitreous with a lower compliance than the ex vivo measurements. The results of the magnetic microrheology measurements were validated with those obtained by a standard atomic force microscopy (AFM) method and in vitro artificial vitreous. This method allows minimally-invasive characterization of localized mechanical properties of the vitreous in vitro, ex vivo, and in vivo. A better understanding of the characteristics of the vitreous can lead to improvements in treatments concerning vitreal manipulation such as vitrectomy.