Thermally multiplexed polymerase chain reaction

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.     

Disrupting the wall accumulation of human sperm cells by artificial corrugation

Many self-propelled microorganisms are attracted to surfaces. This makes their dynamics in restricted geometries very different from that observed in the bulk. Swimming along walls is beneficial for directing and sorting cells, but may be detrimental if homogeneous populations are desired, such as in counting microchambers. In this work, we characterize the motion of human sperm cells ∼60 μm long, strongly confined to ∼25 μm shallow chambers. We investigate the nature of the cell trajectories between the confining surfaces and their accumulation near the borders. Observed cell trajectories are composed of a succession of quasi-circular and quasi-linear segments. This suggests that the cells follow a path of intermittent trappings near the top and bottom surfaces separated by stretches of quasi-free motion in between the two surfaces, as confirmed by depth resolved confocal microscopy studies. We show that the introduction of artificial petal-shaped corrugation in the lateral boundaries removes the tendency of cells to accumulate near the borders, an effect which we hypothesize may be valuable for microfluidic applications in biomedicine.     

Microfluidic sampling system for tissue analytics

We have developed a microfluidics based sampling system for tissue analytics. The proof-of-concept of the sampling system was demonstrated by extracting lipid samples from tissue biopsies. The sample collection system consists of a disposable silicon based multiport microneedle integrated with polymer microfluidics. The polymethyl methacrylate polymer microfluidic chip has a 10 μl sample reservoir and actuation membranes for liquid pumping. A special automated robotic system was developed to control the positioning of the needle and the sampling procedure on preselected spots on the tissue. Real breast cancer tissue samples were used to test the feasibility of the sampling system. We successfully measured indicative cancer biomarkers from the tissue surface. Phosphatidylcholine and phosphoethanolamine were extracted from the tissue membrane with methyl tert-butyl ether solvent and detected by mass spectrometry. In the future, this tool could be used in characterization of preoperative biopsies and tumour tissues removed during surgery.     

Homogeneous agglutination assay based on micro-chip sheathless flow cytometry

Homogeneous assays possess important advantages that no washing or physical separation is required, contributing to robust protocols and easy implementation which ensures potential point-of-care applications. Optimizing the detection strategy to reduce the number of reagents used and simplify the detection device is desirable. A method of homogeneous bead-agglutination assay based on micro-chip sheathless flow cytometry has been developed. The detection processes include mixing the capture-probe conjugated beads with an analyte containing sample, followed by flowing the reaction mixtures through the micro-chip sheathless flow cytometric device. The analyte concentrations were detected by counting the proportion of monomers in the reaction mixtures. Streptavidin-coated magnetic beads and biotinylated bovine serum albumin (bBSA) were used as a model system to verify the method, and detection limits of 0.15 pM and 1.5 pM for bBSA were achieved, using commercial Calibur and the developed micro-chip sheathless flow cytometric device, respectively. The setup of the micro-chip sheathless flow cytometric device is significantly simple; meanwhile, the system maintains relatively high sensitivity, which mainly benefits from the application of forward scattering to distinguish aggregates from monomers. The micro-chip sheathless flow cytometric device for bead agglutination detection provides us with a promising method for versatile immunoassays on microfluidic platforms.     

Robust fluidic connections to freestanding microfluidic hydrogels

Biomimetic scaffolds approaching physiological scale, whose size and large cellular load far exceed the limits of diffusion, require incorporation of a fluidic means to achieve adequate nutrient/metabolite exchange. This need has driven the extension of microfluidic technologies into the area of biomaterials. While construction of perfusable scaffolds is essentially a problem of microfluidic device fabrication, functional implementation of free-standing, thick-tissue constructs depends upon successful integration of external pumping mechanisms through optimized connective assemblies. However, a critical analysis to identify optimal materials/assembly components for hydrogel substrates has received little focus to date. This investigation addresses this issue directly by evaluating the efficacy of a range of adhesive and mechanical fluidic connection methods to gelatin hydrogel constructs based upon both mechanical property analysis and cell compatibility. Results identify a novel bioadhesive, comprised of two enzymatically modified gelatin compounds, for connecting tubing to hydrogel constructs that is both structurally robust and non-cytotoxic. Furthermore, outcomes from this study provide clear evidence that fluidic interconnect success varies with substrate composition (specifically hydrogel versus polydimethylsiloxane), highlighting not only the importance of selecting the appropriately tailored components for fluidic hydrogel systems but also that of encouraging ongoing, targeted exploration of this issue. The optimization of such interconnect systems will ultimately promote exciting scientific and therapeutic developments provided by microfluidic, cell-laden scaffolds.     

Tracking of physical activity in pubertal boys with different BMI over two-year period

We examined the tracking of physical activity (PA) measured by accelerometers and subjective self- and parental reports in normal weight and overweight and obese pubertal boys over two-year period. In total, 156 boys with mean (±SD) age of 11.53 ± 0.76 at baseline and with mean age of 13.94 ± 0.74 at 2 year follow-up were studied. At baseline and approximately two years later, the boys completed self-report questionnaire and wore an accelerometer for seven consecutive days. On the basis of first year assessment’s body mass index (BMI), the children were grouped as normal weight and overweight and obese groups according to BMI cut-offs. Tracking correlations of objectively measured PA and subjectively measured PA were fairly similar across the 12–14 year-old-boys weight groups over two year period. Tracking correlations of objectively measured PA and subjectively measured PA were not significantly different over two-year period between both BMI groups. The results of the study show that pubertal boys objectively measured PA decreased over two-year period and so the boys started to be less active in their pubertal period.     

The use of spatial manipulation to examine goalkeepers’ anticipation

This study investigated whether anticipation and search strategies of goalkeepers are influenced by temporally and spatially manipulated video of a penalty. Participants were clustered into three groups depending on skill: goalkeepers (n = 17), field players (n = 20) and control group (n = 20). An eye tracker was worn whilst watching 40 videos of a striker kicking to four corners of a goal in random order. All 40 videos were temporally occluded at foot-to-ball contact, and the non-kicking leg of 20 videos was spatially manipulated. Results showed that goalkeepers had significantly better predictions than the two groups with no differences between the two testing conditions. According to effect size, the percentage of fixation location and viewing time of the kicking leg and ball were greater for the goalkeepers and field players group than the control group irrespective of testing conditions. The fixations on the kicking leg and ball in conjunction with comparable predictions between spatially manipulated and control conditions suggest that goalkeepers may not rely on the non-kicking leg. Furthermore, goalkeepers appear to use a global perceptual approach by anchoring on a distal fixation point/s of the penalty taker whilst using peripheral vision to obtain additional information.     

‘The Product of the Town Itself’: Community Representation and the Stanley Cup Hockey Challenges of the Kenora Thistles, 1903–1907

This case study of community representation in Canadian hockey analyzes media accounts of four Stanley Cup hockey series played by the Kenora Thistles between 1903 and 1907. It examines newspaper coverage of the Thistles hockey club as it moved from an amateur team represented by players with roots in their home community to a professional aggregation that included paid imports from outside the town. The essay explores the relationship between sport and civic identity during a key time of change in top-level hockey. The dominant narrative surrounding the Thistles portrayed players as true ‘members’ of the town of Kenora. Even after the turn to professionalism, narratives of small-town, amateur purity and close connections to the community characterized coverage of the Thistles as they pursued the Stanley Cup. At the same time, however, a growing emphasis on securing the personnel that could ensure victory led to praise for the club’s efforts to please its supporters, or ‘customers’. As a result, this study of notions of civic representation in early hockey provides insight into the process by which sports teams came to be viewed as symbolic representatives of their communities.     

Osmotically driven drug delivery through remote-controlled magnetic nanocomposite membranes

Implantable drug delivery systems can provide long-term reliability, controllability, and biocompatibility, and have been used in many applications, including cancer pain and non-malignant pain treatment. However, many of the available systems are limited to zero-order, inconsistent, or single burst event drug release. To address these limitations, we demonstrate prototypes of a remotely operated drug delivery device that offers controllability of drug release profiles, using osmotic pumping as a pressure source and magnetically triggered membranes as switchable on-demand valves. The membranes are made of either ethyl cellulose, or the proposed stronger cellulose acetate polymer, mixed with thermosensitive poly(N-isopropylacrylamide) hydrogel and superparamagnetic iron oxide particles. The prototype devices'' drug diffusion rates are on the order of 0.5–2 μg/h for higher release rate designs, and 12–40 ng/h for lower release rates, with maximum release ratios of 4.2 and 3.2, respectively. The devices exhibit increased drug delivery rates with higher osmotic pumping rates or with magnetically increased membrane porosity. Furthermore, by vapor deposition of a cyanoacrylate layer, a drastic reduction of the drug delivery rate from micrograms down to tens of nanograms per hour is achieved. By utilizing magnetic membranes as the valve-control mechanism, triggered remotely by means of induction heating, the demonstrated drug delivery devices benefit from having the power source external to the system, eliminating the need for a battery. These designs multiply the potential approaches towards increasing the on-demand controllability and customizability of drug delivery profiles in the expanding field of implantable drug delivery systems, with the future possibility of remotely controlling the pressure source.     

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.