Single-cell-based sensors and synchrotron FTIR spectroscopy: a hybrid system towards bacterial detection

Microarrays of single macrophage cell-based sensors were developed and demonstrated for potential real-time bacterium detection by synchrotron FTIR microscopy. The cells were patterned on gold electrodes of silicon oxide substrates by a surface engineering technique, in which the gold electrodes were immobilized with fibronectin to mediate cell adhesion and the silicon oxide background was passivated with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. Cell morphology and IR spectra of single, double, and triple cells on gold electrodes exposed to lipopolysaccharide (LPS) of different concentrations were compared to reveal the detection capability of this cell-based sensing platform. The single-cell-based system was found to generate the most significant and consistent IR spectrum shifts upon exposure to LPS, thus providing the highest detection sensitivity. Changes in cell morphology and IR shifts upon cell exposure to LPS were found to be dependent on the LPS concentration and exposure time, which established a method for the identification of LPS concentration and infected cell population. Possibility of using this single-cell system with conventional IR spectroscopy as well as its limitation was investigated by comparing IR spectra of single-cell arrays with gold electrode surface areas of 25, 100, and 400 microm2 using both synchrotron and conventional FTIR spectromicroscopes. This cell-based platform may potentially provide real-time, label-free, and rapid bacterial detection, and allow for high-throughput statistical analyses, and portability.     

Fibroblast growth and H-7 protein kinase inhibitor response monitored in microimpedance sensor arrays

Functional genomic studies and drug candidate testing both require high throughput, parallel experimentation strategies to screen for variable cellular behaviors. In this article we describe the use of an impedance sensing electrode array that is capable of sensing cell "presence" as well as the extent of cell (focal) attachment to the substrate. The signals provided by mouse fibroblasts on a sensing structure containing four different sized electrodes are reported. In the absence of cells, each electrode's impedance was found to depend as expected on electrode size and frequency. The impedance increased by several-fold when fibroblasts attached and spread out over time. More notably, the sensors also detected the cellular response to the protein kinase C inhibitor, H-7. H-7 inhibits actomyosin contractility; thereafter, the loss of focal adhesion complexes occurs. The sensors, in turn, detected an impedance decrease after H-7 addition and an increase in impedance after H-7 removal.     

Impedance studies of bio-behavior and chemosensitivity of cancer cells by micro-electrode arrays

Impedimetric analysis on adherently growing cells by micro-electrodes provides information related to cell number, cell adhesion and cellular morphology. In this study, cell-based biosensor with micro-electrode arrays (MEAs) was used to monitor the culture behavior of mammalian cancer cells and evaluate the chemosensitivity of anti-cancer drugs using electrochemical impedance spectroscopy. The platinum electrode arrays were fabricated by semiconductor technology to a 10 x 10 pattern, with diameter of 80 microm of each electrode. The human oesophageal cancer cell lines (KYSE 30) were cultured on the surface of the electrodes with the pre-coated fibronectin, the connecting protein for tumor cells metastasis and adhesion in extracellular matrix. Morphology changes during cells adhesion, spreading, and proliferation can be detected by impedimetric analysis in a real time and non-invasive way. Cisplatin was added to cells for potential drug screening applications. The experimental results show that this well-known anti-cancer drug has characteristic chemosensitivity effects on KYSE 30 cells which can be detected by MEA. Thus, this cell-based chip provides a useful analytical method for cancer research.     

Effect of cell senescence on the impedance measurement of adipose tissue-derived stem cells

Label-free and real-time monitoring of stem cells based on electrical impedance measurement is increasingly utilized for the quality control of the isolated stem cells to be used in stem cell-based tissue therapy or regenerative medicine. In spite of that the proliferative capacity and multipotency of stem cells are dependent on the type and age of the source tissue, however, the effect of the cell senescence on the impedance measurement of stem cells has not yet been studied. We investigated whether the senescence of adipose tissue-derived stem cells (ADSCs) can be detected by electrical impedance spectroscopy. For this, ADSCs at passage 9 and 31 were prepared and those genetic characteristics and growth kinetics were evaluated by quantitative polymerase chain reaction and cell counting. While the identified ADSCs were grown on the indium tin oxide electrodes, the impedance spectra were measured and interpreted by fitting analysis with an equivalent circuit model. ADSCs at passage 9 adhered on the electrode were small and spindle-shaped whereas the cells at passage 31 were flattened and larger than younger cells. At the beginning of culture time when the cell adhesion occurred, the resistance at 4.6 kHz of passage 31 cells was higher than passage 9 due to the larger size of older cells. Afterwards, the value of passage 9 cells increased higher than passage 31, since younger cells proliferated more than old cells. Therefore, the impedance measurement could characterize the proliferative capacity of ADSCs during expanded culture.     

A bio-inspired support of gold nanoparticles-chitosan nanocomposites gel for immobilization and electrochemical study of K562 leukemia cells

A novel nanocomposites gel was prepared by neutralizing a designer nanocomposites solution of chitosan encapsulated gold nanoparticles formed by reducing in situ tetrachloroauric acid in chitosan. The bio-inspired gel was designed for immobilization and electrochemical study of cells and monitoring adhesion, proliferation, and apoptosis of cells on electrodes. Using K562 leukemia cells as a model, an impedance cell sensor was constructed. The methods for preparation of the gel and immobilization of cells were simple and "green". The nanocomposites gel showed improved immobilization capacity for cells and good biocompatibility for preserving the activity of immobilized living cells. The living cells immobilized on glassy carbon electrode exhibited an irreversible voltammetric response and increased the electron-transfer resistance with a good correlation to the logarithmic value of concentration ranging from 1.34 x 10(4) to 1.34 x 10(8) cells mL-1 with a limit of detection of 8.71 x 10(2) cells mL-1 at 10sigma. This work implied that the nanocomposites gel based on biopolymer and nanoparticles possessed potential applications for biosensing and provided a new avenue for electrochemical investigation of cell adhesion, proliferation, and apoptosis.     

Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment

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 host 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.     

Patterning of cells on functionalized poly(dimethylsiloxane) surface prepared by hydrophobin and collagen modification

Controllable cell growth on poly(dimethylsiloxzne) (PDMS) surface is important for its potential applications in biodevices. Herein, we developed a fully biocompatible approach for patterning of cells on the PDMS surface by hydrophobin (HFBI) and collagen modification. HFBI and collagen were immobilized on the PDMS surface one after another by using copper grids as a mask. HFBI self-assembly on PDMS surface converted the PDMS surface from hydrophobic to hydrophilic, which facilitated the following immobilization of collagen. Collagen had admirable ability to support cell adhesion and growth. Consequently, the HFBI/collagen-modified PDMS surface could promote cell adhesion and growth. What is more, the native PDMS surface did not support cell adhesion and growth. Patterning of cells was achieved by directly culturing 293T cells (the human embryonic kidney cell line) on the PDMS surface patterned with HFBI/collagen. Further studies by means of gene transfection experiment in vitro showed that the patterned cells were of good bioactivities. Herein, the biocompatible preparation of cell patterns on the PDMS surface could be of many applications in biosensor device fabrication.     

A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells

Uniform patterning of cells is highly desirable for most cellular studies involving cell-cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6x6 PIRE array show that cells patterned within minutes with good uniformity (48+/-6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1+/-0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.     

Analysis of the sensitivity and frequency characteristics of coplanar electrical cell-substrate impedance sensors

A PDMS-glass based micro-device was designed and fabricated with 12 coplanar impedance sensors integrated for electrical cell-substrate impedance sensing (ECIS). The sensitivity and frequency characteristics of the sensors were investigated both theoretically (equivalent circuit model) and experimentally for the commonly used micro-electrode dimension scale (20-80 microm). The experimental results matched well with the theoretical model analysis and revealed that, within this micro-electrode dimension scale, as the electrode width decreased or as the total electrode length decreased the sensitivity of sensor increased over the whole sensing frequency range, whilst electrode to electrode distance had no influence on sensitivity. Through our frequency characteristics analysis, the whole frequency range could be divided into four parts. New functions describing the dominant components in each frequency range were defined and validated experimentally, and could be used to explain the phenomenon of an ECIS sensing frequency window. The contribution to the impedance measurement of cells growing on the edges of the electrodes was determined for the first time. Finally, novel proposals for ECIS sensor design and ECIS measurements were presented.     

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis.     

High spatial resolution impedance measurement of EIS sensors for light addressable cell adhesion monitoring

In this paper, impedance measurement of electrolyte-insulator-semiconductor (EIS) structure with high spatial resolution was proposed to monitor cell adhesion. The light addressing ability of this work overcomes the geometrical restrict of cell culture on conventional impedance detection devices such as interdigitated electrode (IDE) and electric cell-substrate impedance sensing (ECIS). Instead of studying cells on predetermined sites of IDE and ECIS, cells cultured anywhere on EIS sensor surface can be addressed and selected as target cells. Principle and primary models for high resolution impedance detection were described and tested by experiments. The EIS sensor was investigated in terms of its intrinsic characteristics, like impedance behavior, voltage characteristic, frequency dependency and photovoltaic effect. Optimized working condition was studied for cell experiments. Cell adhesion under treatment of 0.1% Triton X-100 was monitored using rat kidney cells as the source. Results showed good sensitivity (10% change of impedance) and resolution (40 μm) for cell adhesion impedance detection and suggested this work should be suitable for monitoring cell impedance. Further improvements on sensitivity, spatial resolution were discussed as well as the further applications for single cell monitoring and cell adhesion imaging.     

Highly stable enzyme precipitate coatings and their electrochemical applications

This paper describes highly stable enzyme precipitate coatings (EPCs) on electrospun polymer nanofibers and carbon nanotubes (CNTs), and their potential applications in the development of highly sensitive biosensors and high-powered biofuel cells. EPCs of glucose oxidase (GOx) were prepared by precipitating GOx molecules in the presence of ammonium sulfate, then cross-linking the precipitated GOx aggregates on covalently attached enzyme molecules on the surface of nanomaterials. EPCs-GOx not only improved enzyme loading, but also retained high enzyme stability. For example, EPC-GOx on CNTs showed a 50 times higher activity per unit weight of CNTs than the conventional approach of covalent attachment, and its initial activity was maintained with negligible loss for 200 days. EPC-GOx on CNTs was entrapped by Nafion to prepare enzyme electrodes for glucose sensors and biofuel cells. The EPC-GOx electrode showed a higher sensitivity and a lower detection limit than an electrode prepared with covalently attached GOx (CA-GOx). The CA-GOx electrode showed an 80% drop in sensitivity after thermal treatment at 50°C for 4 h, while the EPC-GOx electrode maintained its high sensitivity with negligible decrease under the same conditions. The use of EPC-GOx as the anode of a biofuel cell improved the power density, which was also stable even after thermal treatment of the enzyme anode at 50°C. The excellent stability of the EPC-GOx electrode together with its high current output create new potential for the practical applications of enzyme-based glucose sensors and biofuel cells.     

Topographical and physicochemical modification of material surface to enable patterning of living cells.

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Fabrication of a sensing module using micromachined biosensors

Micromachining is a powerful tool in constructing micro biosensors and micro systems which incorporate them. A sensing module for blood components was fabricated using the technology. The analytes include glucose, urea, uric acid, creatine, and creatinine. Transducers used to construct the corresponding sensors were a Severinghaus-type carbon dioxide electrode for the urea sensor and a Clark-type oxygen electrode for the other analytes. In these electrodes, detecting electrode patterns were formed on a glass substrate by photolithography and the micro container for the internal electrolyte solution was formed on a silicon substrate by anisotropic etching. A through-hole was formed in the sensitive area, where a silicone gas-permeable membrane was formed and an enzyme was immobilized. The sensors were characterized in terms of pH and temperature dependence and calibration curves along with detection limits. Furthermore, the sensors were incorporated in an acrylate flow cell. Simultaneous operation of these sensors was successfully conducted and distinct and stable responses were observed for respective sensors.     

Stepwise formation of patterned cell co-cultures in silicone tubing

Here, we describe a method for producing patterned cell adhesion inside silicone tubing. A platinum (Pt) needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with layer-by-layer deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The utility of this approach was demonstrated by patterning co-cultures of hepatocytes or endothelial cells with fibroblasts. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.     

Rapid detection of Bacillus stearothermophilus using impedance-splitting

An impedance splitting method was used to detect Bacillus stearothermophilus in suspension and attached to stainless steel surfaces. The effects of bacterial metabolism on the impedance of the culture medium and the ionic layers of the measuring electrodes were recorded using the BacTrac 4000 microorganism growth analyser. Impedance changes were measured at 55 degrees C. Seven of the eight media produced changes in the electrode impedance (E-value) and all media produced negligible changes in the impedance of the culture medium (M-value). Good correlations were obtained between cell numbers and the E-value measured over 18 h (r > 0.9) for the two strains of B. stearothermophilus tested in trypticase soy broth. The E-value correlations were used to estimate the numbers of both vegetative and spore forms of B. stearothermophilus as either planktonic or adhered cells. For the detection of B. stearothermophilus using impedance, only methods where the E-value impedance is recorded, can be used.     

Topographical and Physicochemical Modification of Material Surface to Enable Patterning of Living Cells

ABSTRACT:?

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor

A strategy for patterned cell adhesion based on chemical surface modification is presented. To confine cell adhesion to specific locations, an engineered surface for high-contrast protein adsorption and, hence, cell attachment has been developed. Surface functionalization is based on selective molecular-assembly patterning (SMAP). An amine-terminated self-assembled monolayer is used to define areas of cell adhesion. A protein-repellent grafted copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), is used to render the surrounding silicon dioxide resistant to protein adsorption. X-ray photoelectron spectroscopy, scanning ellipsometry and fluorescence microscopy techniques were used to monitor the individual steps of the patterning process. Successful guided growth using these layers is demonstrated with primary neonatal rat cardiomyocytes, up to 4 days in vitro, and with the HL-1 cardiomyocyte cell line, up to 7 days in vitro. The advantage of the presented method is that high-resolution engineered surfaces can be realized using a simple, cost-effective, dip-and-rinse process. The technique has been developed for application on a CMOS cell-based biosensor, which comprises an array of microelectrodes to extracellularly record electrical activity from cardiomyocytes.     

Dielectrophoretic registration of living cells to a microelectrode array

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 Ipm diameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device.In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20,000 cells onto aI cm2 grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70 +/- 1% fidelity versus 17 +/- 1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.     

Dielectrophoretic registration of living cells to a microelectrode array

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 micromdiameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20000 cells onto a 1cm(2) grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70+/-1% fidelity versus 17+/-1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.