RESUMEN
Dementia is one of the big medical challenges of our time with Alzheimer's, Huntington's and Parkinson's disease among its most common forms. In year 2000, 4.5 million people were diagnosed with Alzheimer's disease in the United States. In the case of Alzheimer's disease one of many contributing factors is a metabolic imbalance that leads to elevated oxidative stress levels. Consequences of this imbalance can be symptoms like apraxia, agnosia or sundowning. The use of field-effect transistors is a novel approach to study the effects of external stimuli on cells in vitro to provide researchers with a new tool for high resolution and high throughput studies to better understand cellular interaction and the effects of pharmacological compounds. In our study we use ion-sensitive field-effect transistors (FETs) to analyze the apoptosis inducing effects of hydrogen peroxide treatment on primary cells obtained from the subventricular zone of postnatal BALB/c mice. Upon apoptosis, the cell-substrate adhesion of the neurons is gradually weakened until complete detachment. In former studies we used our FET devices to conduct Electrical Cell-substrate Impedance Sensing (ECIS) experiments on the single cell level using morphologically different cell lines. Here we demonstrate that our novel approach of ECIS using FET devices can be expanded to primary neuronal tissue with high prospects for further studies in the field of pharmacological research.
Asunto(s)
Apoptosis/efectos de los fármacos , Técnicas Biosensibles , Ventrículos Laterales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Espectroscopía Dieléctrica , Peróxido de Hidrógeno/administración & dosificación , Ventrículos Laterales/citología , Ratones , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Cultivo Primario de CélulasRESUMEN
We introduce a novel technique of impedimetric sensing of cellular adhesion, which might have the potential to supplement the well-known technique of Electrical Cell-substrate Impedance Sensing (ECIS) in cell culture assays. In contrast to the already commercialized ECIS method, we are using ion-sensitive field-effect transistor (ISFET) devices. The standard gold microelectrode size in ECIS is in the range of 100-250 µm in diameter. Reason for this limitation is that when downscaling the sensing electrodes, their effective impedance governed by the metal-liquid interface impedance is becoming very large and hence the currents to be measured are becoming very small reaching the limit of standard instrumentation. This is the main reason why typical assays with ECIS are focusing on applications like cell-cell junctions in confluent cultures. Single cell resolution is barely reachable with these systems. Here we use impedance spectroscopy with ISFET devices having gate dimensions of only 16 × 2 µm(2), which is enabling a real single cell resolution. We introduce an electrically equivalent circuit model, explain the measured effects upon single cell detachment, and present different cellular detachment scenarios. Our approach might supplement the field of ECIS with an alternative tool opening up a route for novel cell-substrate impedance sensing assays with so far unreachable lateral resolution.
Asunto(s)
Espectroscopía Dieléctrica , Análisis de la Célula Individual , Transistores Electrónicos , Adhesión Celular , Técnicas de Cultivo de Célula/instrumentación , Células Cultivadas , Espectroscopía Dieléctrica/instrumentación , Impedancia Eléctrica , Oro/química , Células HEK293 , Humanos , Microelectrodos , Análisis de la Célula Individual/instrumentaciónRESUMEN
In this study, impedance spectroscopy measurements of silicon-based open-gate field-effect transistor (FET) devices were utilized to study the adhesion status of cancer cells at a single cell level. We developed a trans-impedance amplifier circuit for the FETs with a higher bandwidth compared to a previously described system. The new system was characterized with a fast lock-in amplifier, which enabled measuring of impedance spectra up to 50 MHz. We studied cellular activities, including cell adhesion and anti-cancer drug induced apoptosis of human embryonic kidney (HEK293) and human lung adenocarcinoma epithelial (H441) cells. A well-known chemotherapeutic drug, topotecan hydrochloride, was used to investigate the effect of this drug to tumor cells cultured on the FET devices. The presence of the drug resulted in a 20% change in the amplitude of the impedance spectra at 200 kHz as a result of the induced apoptosis process. Real-time impedance measurements were performed inside an incubator at a constant frequency. The experimental results can be interpreted with an equivalent electronic circuit to resolve the influence of the system parameters. The developed method could be applied for the analysis of the specificity and efficacy of novel anti-cancer drugs in cancer therapy research on a single cell level in parallelized measurements.