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Introduction This presentation will focus on the lab-on-a-chip microsystems for different applications, ranging from cytotoxicity tests to the evaluation of the effectiveness of selected anticancer therapies. When in vitro tests are performed with the use of microsystems, manual activities that are required when seeding cells or administering solutions of tested compounds are eliminated. It allows for greater reproducibility and eliminates some limitations of studies conducted in the macroscale. Moreover, in microsystems it is possible to better reflect in vivo conditions and obtain a homogeneous (same size) population of multicellular spheroids (3D cell model) relatively easily [1]. This way, the results obtained in microfluidic systems can allow to better predict the response of an organism to the tested compounds/nanomaterials or applied anticancer treatment. Microsystems for in vitro studies The first stage in the development of a microsystems for in vitro studies is usually based on a selection of appropriate materials for their fabrication. Because non-toxic materials must be applied for this aim, glass and chosen polymers are among the most often used ones. In addition, the application of transparent materials enables observation of cell cultures during testing. The introduction of cells to microchambers, obtaining specific models of cell culture (monolayer or spheroids culture) as well as nutrient delivery to the cells is achieved thanks to the appropriate geometry of the microchannels and microchambers. In the framework of presented studies, microsystems u obtained by bonding several layers of PDMS or glass and PDMS will be discussed. Methods The cytotoxicity of selected nanomaterials (such as quantum dots or gold nanoparticles) was evaluated with the use of microsystems [2,3]. The research was carried out on two cell culture models (monolayer and spheroids) and the examination of cell viability was based on the fluorescence staining of live and dead cells or Alamar blue test. Additionally, the change in cell morphology during the conducted research was observed. Two types of therapeutic procedures were performed with the use of microsystems. These were photothermal therapy (PTT) [4] and electrochemotherapy (ECT). In the case of PTT, after 24 hours from the introduction of cells, a photoactive agent was introduced into the microsystem (gold nanoparticle solutions were used) and incubated for another 24 hours. After this time, the cell medium was passed through the microsystem to wash away the excess of nanoparticles that did not penetrate or bind to the cell membranes, then a laser irradiation was applied. The ECT procedure was carried out in a microsystem equipped with specially designed electrodes. The electrodes were arranged parallelly to each other and tangent to the culture microcells. After introducing the cytostatic compound into the microsystem, the cells were subjected to electric impulse. In this way, drugs were actively introduced into the cells. Results and Conclusions The conducted experiments allowed to evaluate the cytotoxicity of quantum dots for normal and tumor cell lines. As the tests were carried out for both 2D and 3D models, the obtained results were compared. The effectiveness of the PTT procedure carried out in a microsystem with the use of aptamer-modified nanoparticles was assessed. The procedure was found to be more effective in the case of breast cancer cells than for lung cancer cells. A microsystem for ECT was developed and the effectiveness of this method was compared to chemotherapy, which was also carried out in the microsystem. For drugs that poorly penetrate the membranes by endocytosis, the use of electrical impulses increases the amount of drug introduced into the cells. References [1] Cui P., Wang S., “Application of microfluidic chip technology in pharmaceutical analysis: A review”, Journal of Pharmaceutical Analysis, 2019, 9, 238-247. [2] Grabowska-Jadach I., Haczyk M., Drozd M., Fischer A., Pietrzak M., Malinowska E., Brzózka Z., "Evaluation of biological activity of quantum dots in a microsystem", Electrophoresis, 2016, 35, 165-177. [3] Grabowska-Jadach I., Zuchowska A., Olesik M., Drozd M., Pietrzak M., Malinowska E., Brzózka Z., “Cytotoxicity studies of selected cadmium-based quantum dots on 2D: Vs. 3D cell cultures”, New Journal of Chemistry, 2018, 42, 12787-12795. [4] Kalinowska D., Grabowska-Jadach I., Liwinska M., Drozd M., Pietrzak M., Dybko A., Brzozka Z., “Studies on effectiveness of PTT on 3D tumor model under microfluidic conditions using aptamer-modified nanoshells”, Biosensors and Bioelectronics, 2019, 126, 214-221.

Introduction For the last years, low-cost microfluidics and microarrays on polyester substrates have gained applications in a variety of fields, including analytical Po int-of- C are systems. The use of a 2D platforms brings a significant reduction in assay costs, both due to a lower consumption of materials and reagents, as well as a simplicity of manufacturing process in comparison to current approaches [1]. Commercial applicability of immunoassays requires the development of efficient strategies for antibody immobilization in bulk quantities on a wide variety of readily available substrates. Other factors, which influence the usefulness of antibody immobilization approach are scalability, reproducibility and cost-efficiency. Simple, one-step methods such as passive adsorption on hydrophobic surfaces or direct protein immobilization with the use of functionalized silanes are particularly appreciated in fabrication of immunosensing platforms [2]. The use of masking properties of cured toner as an intermediate for passive and covalent immobilization of antibodies on poly(ethylene terephtalate) (PET) seems to be an attractive and still undiscovered path for development of immunoassays on versatile, printable substrates [3]. Fabricatio n of t oner-based platforms for Ab-capturing an d immunosensing Throughout presented research two general approaches for immobilization of antibody and antibody-binding protein (protein A/G) were examined: 1) adsorption on pristine PET@toner foil via hydrophobic interactions, 2) amine-dependent, covalent binding on glycidyloxypropyl tromethoxysilane (GTPMS)- coated surface. Print pattern (at various grayscale levels) was designed in ChemSketch 2.0 and printed on PET surface using HP 100 LaserJet P1006 printer at resolution of 1200 dpi. Pristine PET@toner foils were used for passive adsorption. For GTPMS coating, hydrophilization of toner was performed by a 10-minute treatment with the use of UV/ozone cleaner. Then, 3% GTPMS solution in 50% ethanol was dispensed on freshly-oxidized foil for 30 min. Both multi-purpose substrates characterized by different surface properties were then incubated with rabbit anti-CRP antibody or protein A/G followed by surface blocking. Method To fully characterize the analytical performance in a role of immunosensing platforms, two types of immunoreactions were carried out on prepared 2D Ab/protein arrays. Direct immuno-labelling of rabbit anti-CRP antibody with anti-rabbit IgG-alkaline phosphatase (ALP) conjugate aimed at comparative evaluation of specificity and efficiency of antibody coating. Indirect sandwich immunoassay for C-reactive protein was selected as a model to determine basic working parameters of toner-based immunoassay. Antibody-capturing properties of protein A/G surface were evaluated by determination of monoclonal antibody-ALP conjugate binding capacity. For quantitative analysis, enzymatic reaction was carried out by dispensing p-nitrophenyl phosphate as chromogenic ALP substrate. Results and Conclusions Results of immunolabeling confirmed the usefulness of cured toner as an intermediate for efficient antibody and protein A/G coating via hydrophobicity-driven adsorption. Due to an increase of surface area and a hydrophobicity higher than for pristine PET, the densest layers were observed for fully printed surfaces (100% black level). The differences in the surface properties were also reflected in kinetics of antibody binding. UV/ozone treatment resulted in permanent oxidation and thus hydrophilization of toner surface. The introduction of hydroxyl groups favored reaction with GTPMS. Epoxy-silane coating of oxidized toner enabled a rapid fabrication of protein-reactive surface characterized by a high binding capacity in a single step immobilization. Thanks to good adhesion and thus tight bonding of laser-cut adhesive tape to PET@toner, the microchannel architecture was obtained. It opened up the possibility to carry out immunoassays in microfluidic systems. It was proven that presented methodologies, due to the design and fabrication simplicity, use of commonly available materials and up-scalability, could be successfully applied in immunodiagnostics or flexible microfluidics for Lab-on-a-Foil technology. References [1] E.F.M.Gabriel, B.G.Lucca, G.R.M.Duarte, W.K.T.Coltro, Recent advances in toner-based microfluidic devices for bioanalytical applications, Anal. Methods. 10 (2018) 2952-2962. doi:10.1039/c8ay01095a [2] A.I.Barbosa, A.s.Barreto, N.M.Reis, Transparent, Hydrophobic Fluorinated Ethylene Propylene Offers Rapid, Robust, and Irreversible Passive Adsorption of Diagnostic Antibodies for Sensitive Optical Biosensing, ACS Appl. Bio. Mater. 2 (2019) 2780–2790. 10.1021/acsabm.9b00214 [3] B.L.Thompson, C.Birch, J.Li, J.A.DuVall, D.LeRoux, D.A. Nelson, A-C.Tsuei, D.L.Mills, S.T. Krauss, B.E.Root. J.P.Landers, Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device, Analyst. 141 (2016) 4667-4675 doi:10.1039/c6an00209a Ack nowledgement This work has been financially supported by the National Centre for Research and Development in Poland (grant no. POIR.04.01.04-00-0027/17).

Introduction Nanomaterials due to their physicochemical properties arouse interest of researchers representing various fields of science, including chemical, biological and clinical analysis. Research in these areas has led to a foundation of nanomedicine, in case of which, both diagnosis and therapy of various diseases are based on nanoparticles (e.g. treatment of infectious or cardiovascular diseases and cancer) [1-3]. Nanoparticles made of biodegradable polymers and lipids, gold- and silver-based nanoparticles, carbon nanotubes, semiconductor-based nanoparticles and many others allow the delivery of therapeutic compounds directly to diseased/altered cells. Lab-on-a-chips for cell cultures studies Lab-on-a-chip microsystems are valuable analytical tools that can be used during both in vitro tests of nanomaterials and development of new therapeutic procedures [4]. Lab-on-a-chip systems enable to precisely control the cells’ microenvironment and overcome the limitations of traditional cell culture methods. In microfluidic systems, a transport of necessary substances (oxygen and nutrients) in appropriate concentrations is ensured. In addition, a flow of a culture medium generates shear stresses, the presence of which affects the functioning of cells and can significantly change the diffusion kinetics and interactions of nanomaterials with a cell membrane. By using the microchannel system, it is possible to obtain concentration gradients of the tested compounds, which better mimic conditions found in living organisms. In such systems, the channels and chambers are characterized by a high surface area to volume (SAV) ratio, which reflects the in vivo conditions (gas diffusion, nutrient transport). The consequence of all the mentioned parameters of microsystems is that we can observe phenomena, which do not occur in the macroscale. Method The microsystems that we used during in vitro studies were made of polymer or polymer and glass. The application of biocompatible and transparent materials for microsystems fabrication allows microscopic observation during tests. Microchambers and microchanels in polydimethylsiloxane (PDMS) were obtained using photolithography and molding method. Both elements (PDMS/PDMS or PDMS/glass) of microsystems were permanently bonded after the use of oxygen plasma. Cell culture in the form of a monolayer (2D model) was obtained when a hybrid system was used, while spherical aggregates (3D model) were obtained using a system made of PDMS. Results and Conclusions In our studies, microsystems were used to assess the cytotoxicity of nanomaterials and test the effectiveness of therapeutic procedures. Biological activity of quantum dots was evaluated and their accumulation inside the cells was monitored. It should be underlined, that the application of microsystems made possible to perform and evaluate the effectiveness of two different therapeutic procedures: electrochemotherapy (ECT) and photothermal therapy (PTT). ECT studies were conducted on a cell monolayer (the most commonly used model in biological studies) in a microsystem with integrated electrodes whereas, the PTT studies were conducted on spheroids (model of the early stage of an avascular tumor). References [1] K. Savitsky, X. Yu, Combined strategies for tumor immunotherapy with nanoparticles, Clin Transl Oncol 21 (2019) 1441-1449. doi.org/10.1007/s12094-019-02081-3 [2] J. Xu, W. Han, T. Jia, S. Dong, H. Bi, D. Yang, F. He, Y. Dai, S. Gai, P. Yang, Bioresponsive upconversion nanostructure for combinatorial bioimaging and chemo-photothermal synergistic therapy, Chemical Engineering Journal 342 (2018) 446-457. doi.org/10.1016/j.cej.2018.02.109 [3] C.K.W. Chan, L. Zhang, C.K Cheng, H. Yang, Y. Huang, X.Y. Tian, C.H.J. Choi, Recent Advances in Managing Atherosclerosis via Nanomedicine, Small 14 (2018) 1702793. doi: 0.1002/smll.201702793 [4] D. Kalinowska, I. Grabowska-Jadach, M. Liwińska, M. Drozd, M. Pietrzak, A. Dybko, Z. Brzózka, Studies on effectiveness of PTT on 3D tumor model under microfluidic conditions using aptamer-modified nanoshells, Biosens Bioelectron 126 (2018) 214-221. doi.org/10.1016/j.bios.2018.10.069