Radoslaw Kwapiszewski

Microfluidic devices as tools for mimicking the in vivo environment

One of the major branches of microfluidic development is cell engineering. A number of devices for cell cultivation, lysis, single-cell analysis and cell-based toxicity tests have been reported in the literature. The variety of structures that can be created leads to devices more closely mimicking the in vivo environment than classic cell cultures. Studies on this topic will have an effect on the evaluation of methods that can replace animals in biomedical research. The aim of this review is to present latest advancements of “lab-on-a-chip” for cell cultivation and engineering. The authors focus on the achievements leading to in vivo-like methods. The materials and fabrication methods in silicon, glass, PDMS and other polymers were briefly characterized. Microfluidic devices were applied for mimicking the in vivo environment at various levels of mammalian body organization—from the surroundings of single cells to interactions between functional organs. Solutions for “human-on-a-chip”, perfusion cell cultures, extracellular matrix analogues, microscaffolds, spheroid formation and co-cultures were reviewed in this paper. The presented solutions have the potential to become new cellular models for toxicology, drug development and biomedical research.

Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip.

In this work, we present a microfluidic array of microwells for long-term tumor spheroid cultivation and anticancer drug activity evaluation. The three-dimensional microfluidic system was obtained by double casting of poly(dimethylsiloxane). Spheroids of HT-29 human carcinoma cells were cultured in the microsystem for four weeks. After two weeks of the culture growth slowdown and stop were observed and high cell viability was determined within next two weeks. The characteristics of a homeostasis-like state were achieved. A cytostatic drug (5-fluorouracil) was introduced into the microsystem with different frequency (every day or every second day) and different concentrations. The geometry and construction of the microsystem enables flushing away of unaggregated (including dead) cells while viable spheroids remain inside microwells and decreasing spheroid diameter can be observed and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concentrations of 5-fluorouracil. It was also observed, that higher frequency of drug dosing resulted in more rapid spheroid diameter decrease. The presented microfluidic system is a solution for cell-based studies in an in vivo-like microfluidic environment. Moreover, observation of decreasing spheroid dimensions is a low-cost, label-free and easy-to-conduct mean of a quantitative determination of a 3D cellular model response to a applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture.

Miniaturized tools and devices for bioanalytical applications: an overview

This article presents an overview of various miniaturized devices and technologies developed by our group. Innovative, fast and cheap procedures for the fabrication of laboratory microsystems based on commercially available materials are reported and compared with well-established microfabrication techniques. The modules fabricated and tested in our laboratory can be used independently or they can be set up in different configurations to form functional measurement systems. We also report further applications of the presented modules e.g. disposable poly(dimethylsiloxane) (PDMS) microcuvettes, fibre optic detectors, potentiometric sensors platforms, microreactors and capillary electrophoresis (CE) microchips as well as integrated microsystems e.g. double detection microanalytical systems, devices for studying enzymatic reactions and a microsystem for cell culture and lysis.

An Enzymatic Microreactor Based on Chaotic Micromixing for Enhanced Amperometric Detection in a Continuous Glucose Monitoring Application

The development of continuous glucose monitoring systems is a major trend in diabetes-related research. Small, easy-to-wear systems which are robust enough to function over many days without maintenance are the goal. We present a new sensing system for continuous glucose monitoring based on a microreactor incorporating chaotic mixing channels. Two different types of chaotic mixing channels with arrays of either slanted or herringbone grooves were fabricated in poly(dimethylsiloxane) (PDMS) and compared to channels containing no grooves. Mixing in channels with slanted grooves was characterized using a fluorescence method as a function of distance and at different flow rates, and compared to the mixing behavior observed in channels with no grooves. For electrochemical detection, a thin-film Pt electrode was positioned at the end of the fluidic channel as an on-chip detector of the reaction product, H(2)O(2). Glucose determination was performed by rapidly mixing glucose and glucose oxidase (GOx) in solution at a flow rate of 0.5 microL/min and 1.5 microL/min, respectively. A 150 U/mL GOx solution was selected as the optimum concentration of enzyme. In order to investigate the dependence of device response on flow rate, experiments with a premixed solution of glucose and GOx were compared to experiments in which glucose and GOx were reacted on-chip. Calibration curves for glucose (0-20 mM, in the clinical range of interest) were obtained in channels with and without grooves, using amperometric detection and a 150 U/mL GOx solution for in-chip reaction.

Substrate inhibition of lysosomal hydrolases: α-Galactosidase A and β-glucocerebrosidase

OBJECTIVE We have investigated the kinetics of α-galactosidase A and β-glucocerebrosidase deficient in Fabry and Gaucher diseases, respectively. DESIGN AND METHODS We have performed spectrofluorymetric measurements of the activity of enzymes using a derivative of 4-methylumbelliferone as a substrate and a human T-cell line as a source of enzymes. RESULTS We have observed the substrate inhibition effect, which is related to temperature. CONCLUSIONS The diagnostic procedures for Fabry and Gaucher diseases used now in laboratory practice neglect temperature-dependent substrate inhibition, which may significantly reduce the sensitivity of enzyme activity determinations.

Effect of a high surface-to-volume ratio on fluorescence-based assays.

AbstractIn the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale. FigureDependence of the linear range on surface-to-volume ratio in microfluidic detection.

Three-layer poly(methyl methacrylate) microsystem for analysis of lysosomal enzymes for diagnostic purposes

Lysosomal storage diseases are chronic, progressive and typically have a devastating impact on the patient and the family. The diagnosis of these diseases is still a challenge, however, even for trained specialists. Accurate diagnostic methods and high-throughput tools that could be readily incorporated into existing screening laboratories are urgently required. We propose a new method for measuring the activity of lysosomal enzymes using a microfluidic device. The principle of the method is the fluorometric determination of a protonated form of 4-methylumbelliferone directly in the enzymatic mixture. Compared to the standard diagnostic protocols, the method eliminates the necessity to add alkaline buffer to stop the enzymatic reaction, and thus, the number of analytical steps is reduced. The system allows for on-chip short-term incubation of the enzymatic reagents, leading to a much simplified analytical procedure and a significantly shortened processing time. We measured the activity of β-galactosidase in RPMI-1788 human B lymphocytes and in isolated leukocytes from healthy adults. The method shows a good agreement with the standard diagnostic method. The agreement was confirmed by statistical analysis including construction of a Bland-Altman plot. The approach presented can be an alternative for the currently used diagnostic procedures. The method developed has a potential for the implementation into complex microfluidic devices thus becoming a powerful tool for a high-throughput and multiplex screening of newborns.

Effect of downscaling on the linearity range of a calibration curve in spectrofluorimetry

AbstractInterest in the microfluidic environment, owing to its unique physical properties, is increasing in much innovative chemical, biological, or medicinal research. The possibility of exploiting and using new phenomena makes the microscale a powerful tool to improve currently used macroscopic methods and approaches. Previously, we reported that an increase in the surface area to volume ratio of a measuring cell could provide a wider linear range for fluorescein (Kwapiszewski et al., Anal. Bioanal. Chem. 403:151–155, 2012). Here, we present a broader study in this field to confirm the assumptions we presented before. We studied fluorophores with a large and a small Stokes shift using a standard cuvette and fabricated microfluidic detection cells having different surface area to volume ratios. We analyzed the effect of different configurations of the detection cell on the measured fluorescence signal. We also took into consideration the effect of concentration on the emission spectrum, and the effect of the surface area to volume ratio on the limit of linearity of the response of the selected fluorophores. We observed that downscaling, leading to an increase in the probability of collisions between molecules and cell walls with no energy transfer, results in an increase in the limit of linearity of the calibration curve of fluorophores. The results obtained suggest that microfluidic systems can be an alternative to the currently used approaches for widening the linearity of a calibration curve. Therefore, microsystems can be useful for studies of optically dense samples and samples that should not be diluted. FigureMicrofluidic systems as a tool to increase the dynamic range of fluorophores

7.1.2 A novel tool for biochemical diagnostics of rare genetic disorders: an integrated microfluidic system with optical detection

In this work, an integrated microfluidic system for biochemical diagnostics of lysosomal storage disorders is presented. The polymeric microsystem consists of a zone for hydrodynamic focusing of cell suspension, a mixing microchannel, and an optical detection module. The system was used for determination of the activity of enzymes deficient in Fabry and Gaucher diseases. On the contrary to the currently used protocols of determination of lysosomal enzymes’ activities, the microdevice enables significant reduction of the time of analysis. Moreover, the experimental set enables to avoid termination of enzymatic reaction and sample dilution, what increases sensitivity of the developed method. Due to easy fabrication steps and their low cost, the system seems to be a prospective tool for a point-of-care approach.