Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.
The underlying sensing mechanism of single-cell-based integrated microelectrode array (IMA) biosensors was investigated via experimental and modeling studies. IMA chips were microfabricated and single-cell-level manipulation was achieved through surface chemistry modification of IMA chips. Individual fibroblast cells (NIH3T3) were immobilized on either lysine-arginine-glycineaspartic acid (KRGD) short peptide-modified or fibronectin extracellular-cell-adhesion-moleculemodified microelectrodes to record the impedance variations of cell-electrode heterostructure over a frequency range of 1 to 10 kHz. By fitting experimental data to an application-specific single-celllevel equivalent circuit model, important sensing parameters, including specific cell membrane capacity, cell membrane resistivity, and averaged cell-to-substrate separation, were determined. It was demonstrated that biofunctionalization of planar microelectrode surface by covalently linking short peptides or fibronectin molecules could achieve strong or tight cell adhesion (with an estimated averaged cell-to-substrate separation distance of 11-16 nm), which, in turn, improves the transduced electrical signal from IMA chips. Analyses on frequency-dependent characteristics of single-cellcovered microelectrode impedance and of IMA sensor circuitry response have revealed an addressable frequency band wherein electrical properties of single cells can be distinctively determined and monitored for cellular biosensing applications. The presented work addresses some major limitations in single-cell-based biosensing schemes, i.e. the manipulation of a single cell, the transduction of weak biological signals, and the implementation of a proper model for data analysis, and demonstrates the potential of IMA devices as single-cell biosensors.
The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that hosts individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation.
We report on a cell-based biosensor application that utilizes patterned single-cell arrays combined with confocal Raman spectroscopy to observe the time-dependent drug response of individual cells in real time. The patterned single-cell platform enables individual cells to be easily located and continuously addressable for Raman spectroscopy characterization of biochemical compositional changes in a non-destructive, quantitative manner so that discrete cellular behavior and cell-to-cell variations are preserved. In this study, human medulloblastoma (DAOY) cells were exposed to the common chemotherapeutic agent etoposide, and Raman spectra from patterned cells were recorded over 48 hours. It was found that 87.5% of cells monitored exhibited a sharp decrease in DNA and protein associated peaks 48 hours after drug exposure, corresponding to cell death. The remaining 12.5% of cells showed little to no reduction in key Raman biomarkers, indicating their drug resistance. Furthermore, the patterned cell population showed a very similar response to etoposide as confluent cell cultures as confirmed by flow cytometry. Finally, patterned cells were assessed with TUNEL assay for apoptosis due to DNA fragmentation after etoposide exposure. The results agree well with those from the Raman spectroscopy analysis. This combined biosensor-Raman platform provides a quick, simple way to assess cell responses to chemical and biological agents with high throughput and can be potentially used for a wide variety of biomedical applications such as pharmaceutical drug discovery, toxin tests, and biothreat detection.
We present in this communication a hybrid polymer/nanocrystal photovoltaic device architecture wherein a net poly(3-hexylthiophene) (P3HT) light-absorbing film is inserted underneath the blended layer of P3HT and PbSe nanocrystal quantum dots in the active region. Such a design features the vertical integration of planar and bulk heterojunctions, which allows for the employment of a thinner bulk heterojunction for more efficient carrier collection without an excessive reduction of the overall light absorption by the photovoltaic cell. The measured device performance represents a significant improvement over previously reported hybrid cells containing bulk heterojunctions of P3HT and Pb(S,Se) nanocrystal quantum dots.
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