An integrated chip-based device for droplet-flow polymerase chain reaction

The polymerase chain reaction (PCR) is an important in-vitro technique in molecular biology for amplifying trace quantities of deoxyribonucleic acid (DNA). PCR is carried out by mixing the DNA molecules to be amplified with primers, polymerase enzymes and deoxynucleotide triphosphates (dNTPs) in a suitable buffer solution. A conventional thermal-cycler is then used to cycle the PCR mixture between multiple temperatures for denaturation, annealing and extension. Bench-top thermal cyclers have large thermal masses and use large sample volumes, leading to overly long cycling times, excessive energy and material consumption, inhomogeneity in the reaction environment, and an inability to handle large numbers of small volume aliquots. Microfluidic technologies overcome many of the limitations of bench-top thermal cyclers, providing a more controlled approach to PCR. Droplet-flow is one of the most promising microfluidic methods for carrying out PCR. The droplet-flow approach uses small water-in-oil droplets for compartmentalisation of the PCR reaction mixture, with each droplet behaving like an individual reaction chamber. By flowing the droplets over different temperatures for denaturation, annealing and extension, rapid thermal cycling can be achieved, greatly reducing the reaction time relative to bench-top thermal cyclers. The use of an oil phase to encapsulate the aqueous PCR mixture as droplets also prevents unwanted surface interactions and flow dispersion that can adversely affect the PCR yield. Here we describe an integrated microfluidic device for carrying out droplet-flow PCR. Instead of using multiple temperature zones to thermally-cycle the flowing droplets, the device used an on-chip radial temperature gradient. The droplets passed through microchannels arranged in a spoke-like geometry, causing them to pass backwards and forwards along the radial temperature gradient and so undergo the repeated thermal cycling required for PCR. The device reported here builds on an earlier plastic microfluidic PCR device (by Schaerli et al.) in which the radial temperature gradient was generated using a bulky external heater and a thermoelectric cooler, together with heat sinks and fans. In the silicon- and glass-based device reported here, integrated heaters, temperature sensors and air gaps (for passive cooling) were used to generate the temperature gradient, leading to significant miniaturisation of the device. The dimensions of the complete device assembly were 6.0 cm x 5.0 cm x 2.0 cm compared to 25.0 cm x 25.0 cm x 25.0 cm for the device by Schaerli et al.. Despite the small size of the device, the achievable temperature gradient on the chip was sizeable. For instance, when the central heater was set to 92.0 °C, the temperature at the periphery was ~60.0 °C, corresponding to a temperature difference of ~32.0 °C – easily sufficient for PCR applications. Using chemical modification, the hydrophilic walls of the microchannel were rendered hydrophobic. An on-chip T-junction or flow-focusing junction was subsequently used to merge the oil and aqueous streams to generate the PCR-containing water-in-oil droplets. A PCR recipe was optimised on a bench-top thermal cycle. With this recipe, droplet-flow PCR was conducted on the PCR device by flowing the generated droplets up and down the radial temperature gradient to induce thermal cycling. Gel electrophoresis analysis of the collected droplets from the device showed the presence of the PCR product, confirming the ability of the integrated device to conduct droplet-flow PCR. By varying the central temperature of the PCR device and the flow rate of the droplets, the yield of the PCR product could be tuned. By serially diluting the concentration of the DNA molecules, it was found that the PCR device was able to amplify concentrations as low as 0.01 pM to a level detectable by gel electrophoresis. When coupled to a laser-induced fluorescence detection system, the emission from the PCR mixture in the water-in-oil droplets could be successfully detected for each PCR cycle. The increase in the fluorescence over successive PCR cycles once again verified the feasibility of carrying out droplet-flow PCR on the integrated device.Open Acces

3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis

The emergence of microfluidic technologies has enabled the miniaturization of cell analysis processes, including nucleic acid analysis, single cell phenotypic analysis, single cell DNA and RNA sequencing, etc. Traditional chip fabrication via soft lithography cost thousands of dollars just in personnel training and capital cost. The design of these systems is also confined to two dimensions limited by their fabrication. To address the needs of smooth transition from technology to adoption by end-users, less complexity is urgently needed for microfluidics to be applied in pathogen detection under low-resource settings and more powerful integration of analyses to understand single cells. This dissertation presents my explorations in 3D microfluidics involving simulation-aided design of pretreatment devices for pathogen detection, fabrication through 3D printing, utilization of alternative commercial parts, and the combination with hydrogel material to link phenotypic analysis with in situ molecular detection for single cells. The main outputs of this dissertation are as follows: 1) COMSOL Multiphysics® was used to aid the design and understanding of microfluidic systems for environmental pathogen detection. In the development of an asymmetric membrane for concentration and digital detection of bacteria, the quantification requires Poisson distribution of cells into membrane pores; the flow field and particle trajectories were simulated to validate the cell distribution in capturing pores. In electrochemical bacterial DNA extraction, the hydroxide ion generation, species diffusion, and cation exchange were modeled to understand the pH gradient within the chamber. To address the overestimated risk by polymerase chain reactions (PCR) that detects all target nucleic acids regardless of cell viability, we developed a microfluidic device to carry out on-chip propidium monoazide (PMA) pretreatment. The design utilizes split-and-recombine (SAR) mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. Ten SAR mixers were employed based on fluid flow and diffusion simulation. High-resolution 3D printing was used for prototyping. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was experimentally demonstrated. 2) Water-in-oil droplet-based microfluidic platforms for digital nucleic acid analysis eliminates the need for calibration that is required for qPCR-based environmental pathogen detection. However, utilizing droplet microfluidics generally requires fabrication of sub-100 µm channels and complicated operation of multiple syringe pumps, thus hindering the wide adoption of this powerful tool. We designed a disposable centrifugal droplet generation device made simply from needles and microcentrifuge tubes. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil at the bottom of the tube. The average droplet size was tunable from 96 μm to 334 μm and the coefficient of variance (CV) was minimized to 5%. For droplets of a diameter of 175 μm, each standard 20 μL reaction could produce ~10⁴ droplets. Based on this calculated compartmentalization, the dynamic range is theoretically from 0.5 to 3×10³ target copies or cells per μL, and the detection limit is 0.1 copies or cells per μL. 3) Based on the disposable droplet generation device, we further developed a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol (PEG) hydrogel beads. The crosslinked hydrogel network in aqueous phase adds additional robustness to droplet microfluidics by allowing reagent exchange. The hydrogel beads demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital PCR, digital loop-mediated isothermal amplification (dLAMP), and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. This platform has the potential to advance the understanding of single cell genotype-to-phenotype correlations. 4) For effective sorting of the hydrogel beads after single cell phenotyping, a gravity-driven acoustic fluorescence-based hydrogel beads sorter was developed. The design involves a 3D-printed microfluidic tube, two sequential photodetectors, acoustic actuator, and a control system. Instead of bulky syringe pumps used in traditional cell or droplet sorting, this invention drives beads suspended in heavier fluorinated oil simply by buoyancy force to have the beads float through a vertical channel. Along the channel, sequential photodetectors quantify the bead acceleration and inform the action of downstream acoustic actuator. Hydrogel beads with different fluorescence intensity level were led into different collection chambers. The developed sorter promises cheap instrumentation, easy operation, and low contamination for beads sorting, and thus the full establishment of the single cell phenotype-genotype link. In summary, the work in this dissertation established a) the simulation-aided design and 3D printing to reduce the complexity of microfluidics, and thus lowered its barrier for environmental applications, b) a simple and disposable device using cheap commercial components to produce monodispersed water-in-oil droplets to enable easy adoption of droplet microfluidics by non-specialized labs, c) a hydrogel bead-based analysis platform that links single-cell phenotype and genotype to open new research avenues, and d) a gravity-driven portable bead sorting system that may extend to a broader application of hydrogel microfluidics to point of care and point of sample collection. These simple-for-end-user solutions are envisioned to open new research avenues to tackle problems in antibiotic heteroresistance, environmental microbial ecology, and other related fundamental problems.</p

マルチキャピラリーガラスプレートとポリジメチルシロキサンマイクロアレイを用いたマイクロイムノアッセイプラットフォームの開発

首都大学東京, 2014-09-30, 博士（工学）首都大学東

DEVELOPMENT OF MICROFLUIDIC PLATFORMS AS A TOOL FOR HIGH-THROUGHPUT BIOMARKER SCREENING

Droplet microfluidic platforms are in the early stages of revolutionizing high throughput and combinatorial sample screening for bioanalytical applications. However, many droplet platforms are incapable of addressing the needs of numerous applications, which require high degrees of multiplexing, as well as high-throughput analysis of multiple samples. Examples of applications include single nucleotide polymorphism (SNP) analysis for crop improvement and genotyping for the identification of genes associated with common diseases. My PhD thesis focused on developing microfluidic devices to extend their capabilities to meet the needs of a wide array of applications

Reactive inkjet of quantum dot-silicone composites

There is a need for high-resolution and high-sensitivity temperature sensing in fields such as micro/nanoelectronics, integrated photonics, and biomedicine; however, non-invasive integrated sensing is difficult and expensive to achieve in miniaturised devices, as fabrication is greatly complicated by multi-step processes, heat treatments, and material compatibility. Inkjet printing (IJP) is a direct writing technique in the material jetting AM category that is effective for maskless multi-material printing with <50 µm resolution, which enables production of end-use devices and could simplify sensor integration. Existing inkjet-printed temperature sensors comprise simple circuit devices, which use the change in the electrical resistance of a sensing area to measure temperature. While current examples are well-suited to wearable sensors, they do not achieve the spatial and thermal resolutions desired for printed devices such as microfluidics. Development of inks for luminescence nanothermometry would enable inkjet-printable sensing geometries for planar and 3D thermal imaging with submicron and subdegree resolutions. Silicones are polymers suitable for optical sensing due to their ultraviolet (UV) and thermal stability, optical transparency, and high refractive indices. Composite inks for luminescence nanothermometry can be formulated with quantum dots (QDs), fluorescent semiconductor nanocrystals with intrinsic, reversible temperature quenching. Printable optical sensing materials would enable in situ temperature monitoring for applications and geometries that are otherwise impossible to monitor by conventional means. This thesis describes the development of the first inkjet-printable QD-silicone composite, and the first ink for luminescence thermometry, for integrated optical sensing; these may also have use in lighting applications . 2-part addition cure silicone inks and 1-part UV cure silicone inks were explored and QD-silicone composites were synthesised; inkjet printing of an addition cure QD-composite was demonstrated. Printing of reactive addition cure inks, where Ink A contained crosslinker and Ink B contained catalyst, was demonstrated using drop-on-drop IJP with the smallest average drop diameters reported for silicone IJP to date (33 36 µm). To overcome poor contact pinning, a pinned grid strategy was used for single printhead IJP and a line-by-line strategy for dual printhead IJP. Curing was the greatest challenge in reactive inkjet of QD-silicone composites, as labile ligands on the QDs poisoned the platinum catalyst despite low QD loading (0.005 wt% QD-Ink A). PtCl2 catalyst was added at low loading to enable curing and to explore the interactions between QDs and the catalyst. However, quenching was observed, with 70% decrease in emission intensity as PtCl2 concentration doubled; it was theorised that the QDs and catalyst competed for ligands, leading to metal-induced aggregation. Printing of fluorescent QD-silicone composites was demonstrated on a single printhead system using a pinned grid strategy; inks with no PtCl2 had stronger fluorescence but did not cure, highlighting their greater vulnerability to delays or fluctuations in heating. Novel UV curable silicone inks were formulated for inkjet using a high throughput screening method. Two photoinitiators (PIs) were trialled: DMPA (2,2-dimethoxy-2-phenylacetophenone) and TPO (phenylbis(2,4,6-trimethylbenzoyl)-phosphine oxide). DMPA was associated with rapid loss of fluorescence in QD-silicones, whereas quenching was not observed with TPO. Detachment of passivating ligands followed by photo-oxidation was suggested as a mechanism: TPO radicals are more susceptible to recombination with oxygen radicals than DMPA derived radicals, which might result in better shielding of the QD surface. Printing of 1 wt% TPO silicone inks without quantum dots was carried out under nitrogen to prevent oxygen inhibition. Jetting was demonstrated with 34-42 µm average drop diameter on silanised glass slides, while printing of continuous films was demonstrated on glass slides coated in a release agent. The temperature sensing performance of novel QD-silicone composites was assessed via fluorescence spectroscopy and imaging. 100 nm diameter QD clusters were observed in transmission electron microscopy and micron-scale QD aggregates in optical microscopy. QD emission appeared to be largely unchanged by immobilisation in silicone, although QD aggregation was expected to reduce photostability of the composite. Intensity- and spectral shift-based optical thermometry was demonstrated using well-plate reading and confocal laser scanning microscopy. Emission sensitivity at 627 nm was found to be approximately -0.7 to -1.2 % °C-1 between 30 50 °C and spectral sensitivity 0.07 to 0.08 nm °C-1, in agreement with other values in QD-sensing literature. Intensity decreased between thermal cycles of the same sample, although values at 60 °C were unchanged, while spectral shift appeared repeatable without redshift. Overall, fluorescent QD-silicone composites were produced via IJP for the first time and were shown to have temperature-sensitive emission. These materials are suitable for inkjet-printable devices with embedded optical temperature sensors using luminescence nanothermometry

Biomimetic Based Applications

The interaction between cells, tissues and biomaterial surfaces are the highlights of the book "Biomimetic Based Applications". In this regard the effect of nanostructures and nanotopographies and their effect on the development of a new generation of biomaterials including advanced multifunctional scaffolds for tissue engineering are discussed. The 2 volumes contain articles that cover a wide spectrum of subject matter such as different aspects of the development of scaffolds and coatings with enhanced performance and bioactivity, including investigations of material surface-cell interactions