17 research outputs found
Microengineered analytical tools for cell-based studies in environmental monitoring, pharmacology and neuroscience
Monitoring cells’ Tehavior in-vitro and evaluation of their response to different types of stimulations are the major oTjectives in cell-Tased studies. The results of these studies answer questions on the physiology of the cells, cell-to-cell signaling and impact of external stimulations on the cells. Exploiting cells for oTtaining such information has Teen applied for detection of toxicants in the environment, studying the pathology of diseases, development of new drugs and exploring cellular mechanisms and functions of living organisms. This thesis is mainly focused on investigation of novel tools with simple working principles and innovative approaches for studying cells in-vitro. The developed tools are designed with an uncomplicated faTrication procedure, and are intended to Te integrated in an automated standalone system. In environmental monitoring using cell-Tased systems, cultured cells are exposed to samples and their response is monitored to detect the samples’ cytotoxicity. These samples need to Te pretreated Tefore introducing them into the cell culture. This pretreatment includes adjustment of temperature, osmolality and pH. On-site automatic adjustment of osmolality and pH is not reported in the literature. One of the developed tools in this thesis is a fully automatic device for in-line adjustment of these two parameters ruling out the drawTacks of the conventional methods including particularly dilution of the samples and denaturation of their ingredients. In order to monitor the metaTolism of the cells Tefore and after stimulation, measuring the concentration of glucose and lactate in a few microliter sample oTtained from the culture medium is studied. For this measurement, an enzyme-Tased SU-8 microreactor cartridge with amperometric detection of hydrogen peroxide is designed and faTricated. In comparison to previously reported approaches in which the Tiosensors are implemented inside the cell culture, measuring the metaTolites in an extracted sample from the culture medium prevents contamination of the cells’ microenvironment and crosstalk Tetween the adjacent Tiosensors. The characterization of the cartridge and the role of SU-8 as a suTstrate for immoTilization of enzymes are investigated. It is revealed that the aging and hard Taking of the SU-8 suTstrate Tefore the enzyme immoTilization as well as the sample flow-rate have an impact on the cartridge response. In environmental or pharmaceutical samples applied to cell-Tased systems, the presence of toxicants with inhiTitory effects on enzymes, or electroactive agents that can interfere with the amperometric measurement can limit the application of enzyme-Tased Tiosensors for detecting the metaTolites in the culture medium. Therefore, a protocol for sample preparation is investigated in this thesis that enaTles the application of enzymes and amperometric methods in such measurements. The Tenefits of using the cartridge and the protocol are demonstrated in monitoring the effect of Triton X-100, CuCl2 and acetaminophen on glucose and lactate metaTolism of Caco-2 epithelial cells. The cartridge design enaTles measuring analyte concentrations as low as a few nanomolars using a stopped-flow protocol. Thus, it can Te an appropriate tool for detection of neurotransmitters in a neuronal cell-culture. ImmoTilizations of glutamate oxidase and choline oxidase enzymes in the SU-8 microreactors are investigated. The cartridge caliTration curves for measuring glutamate and choline over a period of 7 days are presented. Furthermore, a compartmentalized neuronal cell-culture device that enaTles the sampling from the regions where the extrasynaptic neurotransmitters are released is presented. In addition to the capaTility of stimulation of the cells chemically, a suTstrate with microelectrodes is faTricated to provide the possiTility of applying electrical stimulations to the cells. A sampling microchannel is structured in the device where the synaptic clefts form. The liquid content of this microchannel will Te transferred to the cartridge for detection of the neurotransmitters realized upon electrical or chemical stimulation of the cultured neuronal populations. Culturing rat’s hippocampal neurons on this device, revealed the TiocompatiTility of the culture device over a period of 20 days. Furthermore, Ty immunostaining of the cells, the formation of synapses containing glutamate and GABA neurotransmitter vesicles in the sampling microchannel was oTserved. The compartmentalized neuronal cell-culture and the microreactor cartridge for in-vitro monitoring of neurotransmitters can Te applied for studying neuromudulation and synaptic plasticity as well as for neurotoxicological or neuropharmacological assessments
Increasing the sensitivity of enzyme-based biosensors using copper-chloride and EDTA
The effect of CuCl2 and EDTA on increasing the sensitivity of enzyme biosensors was evaluated. The experiments revealed that successive exposure of these solutions to our biosensors can increase the sensitivity of the glucose and lactate biosensors up to 88% and 50%, respectively
Microfluidic approach for simultaneous measurement of choline and glutamate neurotransmitters in in-vitro monitoring of neural cells
Sample preparation microfluidic cartridge for on-line adjustment of osmolarity in miniaturized cell-based analysis systems
Cell based biosensors are increasingly used as detection tools for tracing toxins in environmental samples. In such biosensors the reaction of cells cultivated in vitro is monitored upon exposing them to different samples. As the sample will be in close contact with living cells, controlling the sample properties, especially osmolarity, is necessary. Osmolarity difference across the cell membrane can lead to cell death upon exposure to the sample [1]. Herein, we present a microfluidic cartridge designed to realize the adjustment of the sample osmolarity to any desired level without the need for an external osmometer. This cartridge is easy to fabricate, and can be simply integrated in a cell-based microfluidic system for regulating the osmolarity on-line
Enzyme SU-8 microreactors: simple tools for cell-culture monitoring
Monitoring metabolism fluctuations inside a cell culture is a valuable method for assessment of the cells vitality. Enzyme-based biosensors can provide selective measurement of metabolites such as glucose, lactate, glutamate and choline. However, integration of these biosensors inside a cell culture is a challenging issue that can disrupt the properties of the cells microenvironment or influence the biosensors' enzyme functioning. Herein, a technique for measuring the abovementioned metabolites in a cell culture without affecting the enzymes or the cells is presented. In this study, SU-8 is investigated as a suitable substrate for a simple enzyme immobilization. Two SU-8 microreactors are designed inside a microfluidic cartridge and functionalized with different enzymes. The implemented microreactors are used for detection of two metabolites simultaneously in a few microliters of a sample extracted from the cell-culture medium. Sub-micromolar concentrations are detectable using this device. The results of measuring variations in glucose and lactate concentration inside a cell culture, before and after exposing the cells to three different toxicants, are presented. In order to eliminate the enzymes disruption by the toxicants present inside the medium, a protocol for a toxicant-free sampling is investigated
Cell metabolism monitoring using a microfluidic cartridge with enzyme-based microreactors
We present a novel microfluidic approach for on-line analysis of cell metabolism by simultaneous measurement of two important indicators (glucose and lactate) in a small volume (100 µl) of cell-culture medium. The in-vitro study of cell metabolism is especially valuable for evaluating the effect of toxins on physiochemical state of living organism
Portable automated osmolality and pH adjusting apparatus for pretreatment of environmental water samples delivered into a cell-based biosensor
Using our portable in-line fluidic pretreatment apparatus we have adjusted the osmolality and pH of Swiss river samples automatically without any dilution or degradation of the samples. The samples will be tested by means of a cell-based water quality biosensor for detection of toxins. For cell-based biosensors, the osmolality and pH of the sample are critical parameters that must be precisely controlled to prevent cell lysis. Using forward osmosis (FO), we developed a device for a low-cost adjustment of these parameters to the standard values of a cell-culture medium (pH: 8.5, Osmolality: 330 ±10 mmol/kg)
Forward osmosis in a portable device for automatic osmolality adjustment of environmental water samples evaluated by cell-based biosensors
Forward osmosis (FO) is a well-established process that has been used for different applications like desalination of water, concentration of foods or drugs, and energy harvesting. We exploited this process in a fully automatic system to adjust osmolality of environmental water samples that are to be tested by cell-based biosensors. In cell-based biosensors, samples are brought into contact with living cells. Therefore, the samples osmolality and should be in a range that is tolerable for the cells. Controlling these parameters has been a significant challenge especially in environmental monitoring, where the biosensors are required to work on-site. In this paper, we introduce a low-cost portable fluidic system that works automatically, and adjusts the osmolality and pH of environmental samples without diluting or denaturizing the ingredients of the samples. We report the performance of this system in adjusting the osmolality and pH of Swiss environmental waters with a natural osmolality of 4 1 mmol/kg and a pH of 7.84 +/- 0.02. (c) 2013 Elsevier B.V. All rights reserved
Forward osmosis in a portable device for automatic osmolality adjustment of environmental water samples evaluated by cell-based biosensors
Forward osmosis (FO) is a well-established process that has been used for different applications like desalination of water, concentration of foods or drugs, and energy harvesting. We exploited this process in a fully automatic system to adjust osmolality of environmental water samples that are to be tested by cell-based biosensors. In cell-based biosensors, samples are brought into contact with living cells. Therefore, the samples' osmolality and pH should be in a range that is tolerable for the cells. Controlling these parameters has been a significant challenge especially in environmental monitoring, where the biosensors are required to work on-site. In this paper, we introduce a low-cost portable fluidic system that works automatically, and adjusts the osmolality and pH of environmental samples without diluting or denaturizing the ingredients of the samples. We report the performance of this system in adjusting the osmolality and pH of Swiss environmental waters with a natural osmolality of 4±1 mmol/kg and a pH of 7.84±0.02