International Workshop on Quantitative Biology 2017

[Background] In the field of clinical diagnostics, there is continuously growing demand for on-site analytical techniques. In this context, (microfluidic) paper-based analytical devices (µPADs) have evolved into popular tools for assays performed by untrained users. They find increasing use for point-of-care-testing in hospitals or in resource limited settings[1]. Relying on simple paper substrates makes them extremely low cost and allows for pump-free transportation of liquids by capillary wicking, overcoming a major disadvantage of conventional microfluidics. The high surface-to-volume ratio of paper substrates enables immobilization of reagents required for analytical assays, so that user interaction is limited to simple sample application.
[Methods] We focus on classical printing approaches for the fabrication of µPADs. Of particular interest is the inkjet printing technology, because it allows the contactless and highly precise deposition of picoliter volumes of liquids onto paper substrates[2]. Other pieces of office equipment, such as wax printers, paper cutters and hot laminators further contribute to the simple and highly reproducible fabrication of µPADs. A second important component for µPAD fabrication are (bio)chemically functional printing inks, which are used to inkjet deposit reagents and additives required for (bio)chemical assays on paper.
[Results] The first example shows the quantification of the glycoprotein lactoferrin in human tear fluid, which is of interest in clinical diagnostics of ocular diseases. The conventionally used immunoassay method has been replaced by a chemical assay no longer requiring costly and unstable antibodies. The assay relies on the fluorescence emission of lactoferrin bound terbium (Tb3+) cations[3]. To further simplify the assay method, a “distance-based” signal detection system like an analog thermometer has been developed, replacing computer-based data processing by naked eye direct signal readout[4]. The second example is a µPAD for the detection of Zn2+, Fe2+ and Cu2+. This device introduces a new method of sample transport on paper-based microfluidics, resulting in faster analysis times and lower detection limits[5]. The complete devices, including the immobilization of colorimetric reagents to the paper device surface, can be fabricated on conventional desktop inkjet printers.
[Conclusion] It has been demonstrated that simple, single-use microfluidic paper-based analytical devices can be highly reproducibly fabricated with the aid of ordinary office equipment. Some µPADs can compete with much more sophisticated conventional analytical techniques and offer identical results, but at significantly lower cost, within shorter time and without the need for highly skilled instrument operators.

[References]
1. K. Yamada, H. Shibata, K. Suzuki, D. Citterio*. “Toward practical application of paper-based microfluidics for medical diagnostics: State-of-the-art and challenges”. Lab on a Chip, in press, doi: 10.1039/c6lc01577h (2017)
2. K. Yamada, T. G. Henares, K. Suzuki, D. Citterio*. “Paper-based inkjet-printed microfluidic analytical devices”. Angewandte Chemie International Edition 54, 5294-5310 (2015)
3. K. Yamada, S. Takaki, N. Komuro, K. Suzuki, D. Citterio*. “An antibody-free microfluidic paper-based analytical device for the determination of tear fluid lactoferrin by fluorescence sensitization of Tb3+”. Analyst 139, 1637-1643 (2014)
4. K. Yamada, T. G. Henares, K. Suzuki, D. Citterio*. “Distance-based tear lactoferrin assay on microfluidic paper device using interfacial interactions on surface-modified cellulose”. ACS Applied Materials & Interfaces 7, 24864-24875 (2015)

[Background] The discrimination of normal and tumor cells is so attractive from a clinical viewpoint. However, the consistent different between normal and tumor cells are often not sufficient to distinguish them. Tumorous extracellular pH (average pH 6.8) is a slightly lower than that of normal tissue and the physiological pH of arterial blood (pH 7.4). Poly(N-isopropylacryl-amide) (PNIPAAm) exhibits a reversible temperature-responsive phase transition at a lower critical solution temperature (LCST) in aqueous solution. The LCST of PNIPAAm-based polymer can be precisely controlled to near body temperature for biomedical applications by copolymerization with hydrophilic monomers. Furthermore, PNIPAAm-based copolymer was functionalized with pH responsive characteristic by copolymerization with pH-responsive monomer. In this presentation, the design of temperature- and pH-responsive polymer probes, and its temperature- and pH-dependent intracellular uptake will be described [1-3].
[Methods] Temperature- and pH responsive polymers with carboxyl end-groups were synthesized by radical polymerization. LCST of polymers were determined as the temperature at 50% optical transmittance of polymer solutions (0.5w/v%) at 500 nm. Temperature- and pH-dependent hydrodynamic diameters were determined by the dynamic light scattering (DLS) method. Terminal carboxyl groups were labeled with amino fluorescein, and fluorescence polymer probes were obtained. Temperature- and pH-dependent intracellular uptakes were visualized by microscope. The quantitative analysis of fluorescence polymer probes internalized into cells was measured by flow cytometer.
[Results] Obtained temperature- and pH-responsive polymers exhibited temperature- and pH-dependent phase transition. The hydrodynamic diameters of these polymers drastically increased at temperatures greater than phase transition temperature, indicative of the change from hydrophilic to hydrophobic characteristic, followed by polymer aggregation caused by hydrophobic interactions. The temperature- and pH-dependent intracellular uptakes of amino fluorescein labeled polymers were evaluated with changing incubation conditions. Temperature- and pH-dependent intracellular uptakes were visualized with fluorescence microscopy images, and quantified with flow cytometry fluorescence histograms.
[Conclusion] Temperature- and pH-responsive fluorescence polymer probes were successfully designed and synthesized. Temperature- and pH-dependent intracellular uptakes opens up the visualization of tumors at the cellular level, which may offer the potential for development/use as a diagnosis tool for early tumor detection.
[References] 1. Y. Hiruta, M. Shimamura, M. Matsuura, Y. Maekawa, T. Funatsu, Y. Suzuki, E. Ayano, T. Okano, H. Kanazawa*. “Temperature-Responsive Fluorescence Polymer Probes with Accurate Thermally Controlled Cellular Uptakes”. ACS Macro Letters 3, 281-285 (2014) 2. Y. Hiruta*, T. Funatsu, M. Minami, J. Wang, E. Ayano, H. Kanazawa. “pH/temperature-responsive fluorescence polymer probe with pH-controlled cellular uptake”. Sensors and Actuators B: Chemical 207, 671-677 (2015) 3. Y. Hiruta*, R. Nemoto, H. Kanazawa. “Design and synthesis of temperature-responsive polymer/silica hybrid nanoparticles and application to thermally controlled cellular uptake”. Colloids and Surfaces B: Biointerfaces 153, 2-9 (2017)

Asymmetric cell division is considered to be one of the triggers of cell differentiation and tissue development. Wnt3a is a notable candidate for contributing to asymmetric cell division. However, the sensitivity of cells to Wnt3a signalling in deciding axis of cell division has not yet been analysed. We develop a microfluidic cell culture device, which can produce a gradient of signalling molecules to test the sensitivity of cells to Wnt3a signalling for controlling the axis of cell division. We confirm that 1 ~ 6 x 10^-3 nM/$\mu$m concentration gradient of Wnt3a is detected by cells and has an effect on the decision about the orientation of the asymmetric distribution of ODF2/cenexin between mother centrioles by late mitotic phases. Our results suggest that differences of only a few molecules in the receptor occupancy at both sides of an after-metaphase cell may be detected to control the axis of mitotic elements.

The majority of cancer cells show chromosomal instability (CIN), a condition in which chromosome missegregation occurs at a high rate. As defects in critical mitotic processes are lethal, CIN in cancer cells supposedly arise from defects in non-essential processes that allow cell survival. Merotelic attachment, an erroneous attachment of kinetochores to microtubules, plays a major role in the generation of CIN, by escaping the spindle assembly checkpoint and forming lagging chromosomes that result in chromosome missegregation. We found that efficient chromosome movement towards the spindle equator and subsequent chromosome oscillation around the spindle equator are important for high-fidelity chromosome transmission. We propose that these non-essential processes play a causal role in the generation of CIN through the formation of merotelic attachment.

Throughout life, the process of cell division ensures the proper replenishment of dead or injured cells. When a cell divides into two, microtubules capture and pull chromosomes apart into two equal sets. My research group studies how human cells ensure the accurate segregation of chromosomes using a combination of high-resolution microscopy, biochemistry and molecular biology techniques. Chromosome-microtubule attachment is facilitated by a macromolecular structure called the kinetochore. Our work and others’ have contributed to the identification of kinetochore proteins that are essential for establishing and monitoring chromosome-microtubule attachment, and in turn chromosome segregation fidelity. I will present our recent work which shows that chromosomes are first captured along the walls of microtubules and then brought to the ends of microtubules without detachment by a process called end-on conversion. Errors in the end-on conversion process can lead to chromosome mis-segregation. I will conclude with a brief overview of ongoing research efforts to discover the master regulators of the end-on conversion process that are essential to maintain a stable genome.