Cell-based Sensor Research Articles

Cell-based sensor for analysis of EGFR biomarker expression in oral cancer

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. To address this clinical need, we have developed a cell-based sensor to detect oral cancer biomarkers, such as the epidermal growth factor receptor (EGFR) whose over-expression is associated with early oral tumorigenesis and aggressive cancer phenotypes. The lab-on-a-chip (LOC) sensor utilizes an embedded track-etched membrane, which functions as a micro-sieve, to capture and enrich cells from complex biological fluids or biopsy suspensions. Once captured, "on-membrane" immunofluorescent assays reveal the presence and isotype of interrogated cells via automated microscopy and fluorescent image analysis. Using the LOC sensor system, with integrated capture and staining technique, EGFR assays were completed in less than 10 minutes with staining intensity, homogeneity, and cellular localization patterns comparable to conventional labeling methods. Further examination of EGFR expression in three oral cancer cell lines revealed a significant increase (p < 0.05) above control cells with EGFR expression similar to normal squamous epithelium. Results obtained in the microfluidic sensor system correlated well with flow cytometry (r(2) = 0.98), the "gold standard" in quantitative protein expression analysis. In addition, the LOC sensor detected significant differences between two of the oral cancer cell lines (p < 0.01), accounting for disparity of approximately 34 000 EGFR per cell according to quantitative flow cytometry. Taken together, these results support the LOC sensor system as a suitable platform for rapid detection of oral cancer biomarkers and characterization of EGFR over-expression in oral malignancies. Application of this technique may be clinically useful in cancer diagnostics for early detection, prognostic evaluation, and therapeutic selection. Having demonstrated the functionality of this integrated microfluidic sensor system, further studies using clinical samples from oral cancer patients are now warranted.

Cellular impedance biosensors for drug screening and toxin detection.

Cell-based impedance biosensing is an emerging technology that can be used to non-invasively and instantaneously detect and analyze cell responses to chemical and biological agents. This article highlights the fabrication and measurement technologies of cell impedance sensors, and their application in toxin detection and anti-cancer drug screening. We start with an introduction that describes the capability and advantages of cell-based sensors over conventional sensing technology, followed by a discussion of the influence of cell adhesion, spreading and viability during cell patterning on the subsequent impedance measurements and sensing applications. We then present an electronic circuit that models the cell-electrode system, by which the cellular changes can be detected in terms of impedance changes of the circuit. Finally, we discuss the current status on using cell impedance sensors for toxin detection and anti-cancer drug screening.

Genetic parts to program bacteria

Genetic engineering is entering a new era, where microorganisms can be programmed using synthetic constructs of DNA encoding logic and operational commands. A toolbox of modular genetic parts is being developed, comprised of cell-based environmental sensors and genetic circuits. Systems have already been designed to be interconnected with each other and interfaced with the control of cellular processes. Engineering theory will provide a predictive framework to design operational multicomponent systems. On the basis of these developments, increasingly complex cellular machines are being constructed to build specialty chemicals, weave biomaterials, and to deliver therapeutics.

Self-organization of yeast cells on modified polymer surfaces after dewetting: newperspectives in cellular patterning

In recent years, biological micro-electro-mechanical systems (commonly referred to asBioMEMS) have found widespread use, becoming increasingly prevalent in diagnostics andtherapeutics. Cell-based sensors are nowadays gaining increasing attention, due to cellularbuilt-in natural selectivity and physiologically relevant response to biologically activechemicals. On the other hand, surrogate microbial systems, including yeast models, havebecome a useful alternative to animal and mammalian cell systems for high-throughputscreening for the identification of new pharmacological agents. A main obstacle inbiosensor device fabrication is the need for localized geometric confinement ofcells, without losing cell viability and sensing capability. Here we illustrate anew approach for cellular patterning using dewetting processes to control celladhesion and spatial confinement on modified surfaces. By the control of simplesystem parameters, a rich variety of morphologies, ranging through hexagonalarrays, polygonal networks, bicontinuous structures, and elongated fingers, can beobtained.

Cells on chips

Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. Further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as for cell-based sensors with biochemical, biomedical and environmental functions. Highly integrated microdevices show great promise for basic biomedical and pharmaceutical research, and robust and portable point-of-care devices could be used in clinical settings, in both the developed and the developing world.

Functionalized Surface Arrays for Spatial Targeting of Immune Cell Signaling

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.

Rapid localized cell trapping on biodegradable polymers using cell surface derivatization and microfluidic networking

Spatial control over cell attachment is essential for controlling cell behavior and engineering cell-based sensor arrays. Here we report on a patterning procedure that can be utilized on a wide range of adherent and non-adherent cell types without the need to identify the exact peptide sequence or extracellular matrix (ECM) necessary for optimal cell attachment. This is achieved by converting native sialic residues present on the surface of most cells into non-native aldehydes using a mild sodium periodate treatment. The aldehyde groups are then reacted with biotin hydrazide to produce biotinylated cells. Avidin is patterned onto the surface of a biotinylated biodegradable block copolymer, polylactide–poly(ethylene glycol)–biotin (PLA–PEG–biotin) by microfluidic networking using a PDMS stamp. The biotinylated cells then bind specifically to the patterned avidin regions. The PEG that is presented from the PLA–PEG–biotin copolymer in the regions without avidin immobilization minimizes cell binding in the non-patterned regions.

Field-effect transistor with recombinant potassium channels: fast and slow response by electrical and chemical interactions

Electrical interfacing of semiconductor devices with ion channels is the basis for a development of neuroelectronic systems and of cell-based biospecific electronic sensors. To elucidate the mechanism of cell–chip coupling, we studied the voltage-gated potassium channel Kv1.3 in HEK 293 cells on field-effect transistors in silicon with a metal-free gate of silicon dioxide. Upon intracellular depolarization there is a positive change of the effective extracellular voltage on the transistor with an amplitude that correlates with the gating of Kv1.3 channels, but with a dynamics that is far slower than channel gating. After repolarization there is a fast negative change of the transistor signal followed by a slow relaxation dynamics without any membrane current. To rationalize the involved transistor response, we propose a concept that combines the electrodiffusion of ions in the cell–chip junction with selective ion binding in the electrical double layer of silicon dioxide. The model implies (i) an electrical charging and discharging of the cell–chip capacitance within a microsecond, (ii) a changing K+ concentration in the cell–chip junction within a millisecond and (iii) a changing adsorption of K+ and Na+ ions within tens of milliseconds. The total transistor signal is a superposition of the changed electrical potential in the extracellular space between cell and chip and of the changed surface potential at the chip surface.

Microfluidic Gradient-Generating Device for Pharmacological Profiling

We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cell-based sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K+ channels (hERG) and ligand-gated GABA(A) receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC50 and EC50 values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.

Biochip sensors for the rapid and sensitive detection of viral disease

Recent advances in DNA and protein microarray methodology and the emerging technology of cell-based sensors have massively increased the speed and sensitivity with which we can detect viral infections.

Cell-based CMOS sensor and actuator arrays

In recent years, increasing knowledge about in vitro cell handling and culturing has encouraged a variety of CMOS-based approaches to stimulate and detect electrical activity of biological cells. This paper outlines in a topical review the scope of cell-based biosensors and actuators for in vitro applications ranging from single-cell detection to multisite probing of complex neural tissue. Recent examples are selected to demonstrate how standard CMOS processes have been used to engineer arrays with different functionality.

A fractal analysis of pathogen detection by biosensors

A fractal analysis is presented for the detection of pathogens such as Franscisela tularensis, Yersinia pestis (the bacterium that causes plague), Bacillus anthracis, Venezuelan equine encephalitis (VEE) virus, Vavcinia virus, and Escherichia coli using a cellular analysis and notification of antigens risks and yields (CANARY) biosensor [T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213–215, T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D. Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T. Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213–215. Science Online, www.sciencemag.org/cgi/content/full/031/5630/213/DC1]. In general, the binding and dissociation rate coefficients may be adequately described by either a single- or a dual-fractal analysis. An attempt is made to relate the binding rate coefficient to the degree of heterogeneity (fractal dimension value) present on the biosensor surface. Binding and dissociation rate coefficient values obtained are presented. Due to the dilute nature of the analyte(s) present, in some cases, a triple-fractal analysis is required to adequately describe the binding kinetics. It should be noted, and this is not entirely unexpected, that there is a lot of variation in the original experimental data when dilute concentrations of the analyte were analyzed by the CANARY biosensor [T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213–215, Science Online, www.sciencemag.org/cgi/content/full/031/5630/213/DC1]. The data analyzed in this manuscript appears smoother since only discrete points at different time intervals were analyzed. The kinetics aspects along with the affinity values presented are of interest and should along with the rate coefficients presented for the binding and the dissociation phase be of significant interest in help designing better biosensors for an application area that is bound to gain increasing importance in the future.

Respiratory response of cell-based sensors to toxicants measured by using pseudo-random signals

Cell-based sensors using such as Pseudomonas putida and Bacillus subtilis were applied to examine the toxicity of chemicals. Both toxicant and substrate solutions were introduced into the sensor system according to M-series and anti-symmetric M-series pseudo-random binary signals, X n ( n = 15, minimal pulse width 4 min, period 60 min) and Y n ( n = 30, minimal pulse width 4 min, period 120 min). Toxicants such as Ag +, formaldehyde, azide, and hypo-chlorite were used to demonstrate the proposed method. The individual responses of substrate and toxicant were obtained at a time by calculating the cross-correlation function between the input and the output signals. Effects of toxicant on the response of substrate and the response of toxicant itself can be observed at a time, so that the method appears to be useful in studying the behavior of microorganisms in the presence of toxicants.

Novel cell patterning using microheater-controlled thermoresponsive plasma films.

A novel approach is reported for cell patterning based on addressable microheaters and a poly(N-isopropyl acrylamide) (pNIPAM) themoresponsive coating. This thermoresponsive coating is created by a radio frequency NIPAM plasma and is denoted as plasma polymerized NIPAM (ppNIPAM). Films of ppNIPAM with a good retention of monomer side-chain functionality are produced using low-power continuous plasma deposition. Cell adhesion and cell detachment tests indicate that the surface switches between adhesive and nonadhesive behaviors as a function of temperature. The use of a photolithographically fabricated microheater array allows the ppNIPAM transition to occur spatially under the control of individual heaters. This localized change in the surface adhesive behavior is used to direct site-specific cell attachment. Patterned adhesion of two types of cells has been visualized on the array through fluorescent markers. Applications for diagnostic devices, cell-based sensors, tissue engineering, and cell transfection are envisioned.

In Situ Peptide-Modified Supported Lipid Bilayers for Controlled Cell Attachment

The control of cellular interactions with engineered materials is critical for the development of cell-integrated biochips used in cell-based sensors, “lab-on-a-chip” bioanalytical systems, and artificial neuronal networks, as well as medical implants and functional biomaterial scaffolds for tissue engineering. Supported lipid bilayers offer efficient reduction of nonspecific cell and protein binding and, if selectively functionalized, constitute one attractive approach to surface modification strategies of materials used in such devices. The present work describes the in situ modification of supported lipid bilayers through the coupling of a cysteine-terminated peptide to thiol-reactive maleimido lipids incorporated in the bilayer. The accumulation of peptide at the lipid bilayer interface was monitored by the quartz crystal microbalance technique with dissipation monitoring (QCM-D). Coupling of the peptide could be detected by QCM-D with a high signal-to-noise ratio despite its low molecular weight (2 kDa), primarily because the mass uptake included both peptide and the water associated to it. Lipid bilayers that were modified with the cysteine-terminated IKVAV-containing peptide promoted the binding of anti-IKVAV antibodies, as well as the attachment of PC12 cells, which express a membrane receptor for the IKVAV sequence. Very low nonspecific binding of peptides, proteins, and the cells was observed on nonfunctionalized lipid bilayers. Similarly, IKVAV-functionalized lipid bilayers were resistant to serum protein adsorption as well as the binding of non-IKVAV-specific antibodies. QCM-D and fluorescence recovery after photobleaching revealed that the lipid bilayers persisted under all the experimental conditions used for cell attachment, including staining and fixation. Thus, the described lipid-based surface modification is highly relevant for the development of controlled cell-attachment substrates and can even be applicable for patterning cell attachment because lipid-bilayer formation by vesicle fusion is material-specific.

Tuning into Pathogens

The need for rapid detection of pathogens, such as in clinical samples and in food, has acquired a new urgency with the threat of bioterrorism and the emergence of familiar and unfamiliar infectious diseases. Harnessing the exquisite capacity of B cells for pathogen recognition, Rider et al. created a biosensor, named CANARY, that detects low levels of viruses and bacteria in a variety of sample formats. Expression of the calcium-sensitive bioluminescent protein, aequorin, in a B cell line permitted a rapid and highly specific luminescent signal to be generated after pathogen-specific cross-linking of membrane antibodies. T. H. Rider, M. S. Petrovick, F. E. Nargi, J. D. Harper, E. D. Schwoebel, R. H. Mathews, D. J. Blanchard, L. T. Bortolin, A. M. Young, J. Chen, M. A. Hollis, A B cell-based sensor for rapid identification of pathogens. Science 301 , 213-215 (2003). [Abstract] [Full Text]

A B cell-based sensor for rapid identification of pathogens.

We report the use of genetically engineered cells in a pathogen identification sensor. This sensor uses B lymphocytes that have been engineered to emit light within seconds of exposure to specific bacteria and viruses. We demonstrated rapid screening of relevant samples and identification of a variety of pathogens at very low levels. Because of its speed, sensitivity, and specificity, this pathogen identification technology could prove useful for medical diagnostics, biowarfare defense, food- and water-quality monitoring, and other applications.

An improved polymer electrolyte-based amperometric hydrogen sensor

An improved polymer electrolyte membrane (PEM) fuel cell-based amperometric hydrogen sensor that operates at room temperature has been developed. The electrolyte used in the sensor is a PVA/H3PO4 blend, which is a proton-conducting solid polymer electrolyte. A thin film of palladium is used as the anode and platinum supported on carbon as the cathode. The sensor functions as a fuel cell, H2/Pd//PVA-H3PO4//Pt/O2, and the short-circuit current is found to be linearly related to the hydrogen concentration. The basic principle, details of assembly, response behaviour of the sensor and its application are discussed.

Cholera toxin-induced modulation of gene expression: elucidation via cDNA microarray for rational cell-based sensor design

Cell-based biosensors utilize functional changes in cellular response to identify the biological threats in a physiological relevant manner. Cell-based sensors have been used for a wide array of applications including toxicological assessment and drug-screening. In this paper, we utilize DNA arrays to identify differential gene expression events induced by toxin exposure for the purpose of developing a reporter gene assay system compatible with insertion into a cell-based sensor platform. HT29, an intestine epithelial cell line, was used as a cell model to study the cholera toxin (CT)-induced host cell modulation using DNA array analysis. A false positive model was generated from analysis of housekeeping genes in untreated control experiments to characterize our system and to minimize the number of false positives in the data. Threshold probability scores (−3.72), which gives <0.02% false positives for up/down regulation from the false positive model, were used to identify 73 and 25 known genes/expression tag sequences (ESTs) that were up- and down-regulated, respectively, in cells exposed 23 nM of CT. Using quantitative multiplex PCR assay, the gene expression levels for several genes shown to be modulated according to the microarray experiments, such as apolipoprotein D ( Apol D), E-cadherin, and cyclin A2, were confirmed. The differential expression of genes encoding cytochrome P450, glutathione transferase (GST), and MGAT2 were noteworthy and consistent with previous studies. Our study provides an approach to analyze cDNA microarray data with defined false positive rates. The utility of cDNA microarray information for the design of cell-based sensor using a reporter gene approach is discussed.

Effects of p-nitrophenol and organophosphorous nitroaromatic insecticides on the respiratory activity of free and immobilized cells of strains S-11 and BA-11 of Pseudomonas putida

The possibility of using the respiratory activity (RA) of microbial cells (of strains S-11 and BA-11 of Pseudomonas putida) as an instrument for quantitative determination of organophosphorous nitroaromatic insecticides, metaphors and sumithion, and their hydrolysis product, p-nitrophenol (PNP), has been explored. The dependences of RA on the concentrations of the three compounds were linear within the range 0.5-2.5 microM. The cells of the strain BA-11 exhibited maximum selectivity in the determination of the compounds. The RA of microbial cells differing in the modes of immobilization (adsorption to carrier surfaces vs. incorporation into gels) have been compared. Prospects of development of the microbial cell-based sensor system for determining metaphors, sumithion, and PNP in aqueous media are discussed.