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Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan

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52 MIHRIMAH OZKAN ET AL.

unstable, thus necessitating packaging approaches to limit degradation of biosensor performance.

3.6. CELL BASED BIOSENSORS

Cell based biosensors on the other hand offer a broad spectrum detection capability. Moreover by using cells as the sensing elements provides the advantage of in-situ physiological monitoring along with analyte sensing and detection. A cell by itself encapsulates an array of molecular sensors. Receptors, channels, and enzymes that may be sensitive to an analyte are maintained in a physiologically stable manner by native cellular machinery. In contrast with antibody-based approaches, cell-based sensors are expected to respond optimally to functional, biologically active analytes. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Sensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cell sensor signals are available these approaches include measures of cell fluorescence, metabolism, impedance, intracellular potentials, and extracellular potentials. Finally the technique of using extracellular sensing on single mammalian cells to detect specific chemical analytes is discussed in detail. The associated signal analyses that results in a unique identification tag associated with each cell type for a specific chemical agent also called “Signature Patterns” is described. The advantages of this technique in comparison to other cell based techniques namely stems from speed of response as well as accuracy and reliability in analyte identification. This is also verified with the conventional fluorescent techniques. The technique of chemical detection based on individually patterned cell’s extracellular potential variations is also known as electrical sensing. The viability and the reliability of this technique are discussed.

3.7. CELLULAR MICROORGANISM BASED SENSORS

Microorganism pathways are activated by some analytes, such as pollutants. These pathways are involved in metabolism or nonspecific cell stress that result in the expression of one or more genes (Belkin et al., 1993) Immobilized yeast his one of the most commonly used sensor. It has been used in the detection of formaldehyde [69] and toxicity measurements of cholanic acids [13]. The changes in metabolism indicative of the analyte were detected via O2 electrode measurements or extracellular acidification rates. One of the areas where microbial biosensors are widely used is in environmental treatment processes. This is done by detecting the biochemical oxygen on demand (BOD) [65, 66, 73, 133]. Most of the BOD sensors consist of a synthetic membrane with immobilised microorganisms as the biological recognition element. The bio-oxidation process is registered in most cases by means of a dissolved O2 electrode. A wide variety of microorganisms have been screened during the construction of BOD sensors. The microbial strains selected are chosen for their ability to assimilate a suitable spectrum of substrates. BOD sensors based on a pure culture have the advantages of relatively good

MICROARRAY AND FLUIDIC CHIP FOR EXTRACELLULAR SENSING

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Principle of cellular micro-organism based biosensor

 

Cathode (Pt)

 

 

 

 

 

Anode (Ag)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Oxygen electrode

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Electrolyte

 

 

 

 

 

 

 

 

 

 

 

 

Phosphate buffer

 

 

 

 

 

 

 

 

 

 

Teflon membrane

 

Rubber O-ring

 

 

 

 

 

 

 

 

 

 

 

 

Dialysis membrane

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

An immobilised mixed culture of microorganisms

FIGURE 3.1. Schematic of the most commonly used cellular micro organism based sensor-Biochemical Oxygen Demand (BOD) sensor. An immobilized mixed culture of microorganisms in combination with a Clark-type oxygen electrode is used for analyzing the components of waste water. The BOD sensor is aimed at being highly capable of analyzing a sample of complex constituents with relatively low selectivity. A Clark-type probe for dissolved oxygen is used as the physical transducer, which consistsof a platinum cathode as the working electrode, a silver anode as the reference electrode, and a 0.1 M potassium chloride (KCl) electrolyte. The Teflon side of the synthetic microbial membrane is attached to the cathode of the oxygen probe. The electrolyte is filled in the space between the synthetic biomembrane and those two electrodes [75].

stability and longer sensor lifetime, but are restricted by their limited detection capacity for a wide spectrum of substrates. The general schematic of a microbial biosensor is shown in figure 3.1 [75]. Modified bacteria have served as whole cell sensor elements for the detection of napthalene and its metabolic product salicylate [68] benzene [2] toluene [10] mercury [121] and middle chain alkanes such as octane.139. The touted advantage of the microorganism based sensors is that generic detection is possible as any alteration of a microorganism-based biosensor response is important and that insufficient selectivity actually offers the identification advantage [29]. The crux being that if generic detection is the ultimate goal then cell-based sensors that can give physiologically relevant information along with detection would offer a better advantage and capability.

3.8. FLUORESCENCE BASED CELL BIOSENSORS

Optical assays rely on absorbance, fluorescence or luminescence as read-outs. Instruments are available that can conveniently and rapidly measure light from standard 56 and 384 well micro titer plates. More customized systems have been developed to detect signals from very high density plates containing over a thousand individual wells. The migration to miniaturized assays (10 µL volume or less) and higher density formats favor non-invasive assay methods with the largest and brightest signals [72, 59]. Furthermore, genetically encoded probes offer the possibility of custom engineered biosensors for intracellular biochemistry,

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MIHRIMAH OZKAN ET AL.

specifically localized targets, and protein-protein interactions. Fluorescence imaging has proven to be an invaluable tool for monitoring changes in the concentrations of ions and protein expression related to cellular signaling [23, 138].

Recently, fluorescence based technologies have been implemented in high-throughput screening [30]. New fluorescent reagents based on the combination of molecular biology, fluorescent probe chemistry, and protein chemistry is being developed for cell-based assays. Reporter gene constructs, such as green fluorescent protein, have been implemented in genetically engineered mammalian and nonmammalian cell types [150] to achieve measures of cell function rather than radioligand binding. (Giuliano et al., 1994). The fluorescent detection is generally based on fluorescence resonance energy transfer (FRET). The most basic use of fluorescent biosensors is the collection of photons from a cell or tissue to detect the occurrence of a process with temporal resolution.

Spatial information expands the usability of biosensors by adding subcellular and supracellular information. On the subcellular level, the read-out of the biosensor can be sampled with spatio-temporal resolution, enabling the morphological dissection of the studied process. An advantage of spatial resolution is the possibility to integrate data from different biosensors or other cell-state parameters to gain additional information— for example, on causal connections. The biological machinery inside cells can be investigated by various microscopic techniques and biosensors using fluorescent techniques [143]. The idea behind fluorescence detection is largely related to FRET. Figure 3.2 gives the concept of using FRET in a simple biosensor design consisting of a minimal protein

Principle of fluorescence based cell biosensors

Protein

Acceptor-labeled

 

 

protein

GFP

Sensitized

emission

FRET

Donor

Donor

emission

emission

FIGURE 3.2. Fluorescence spectroscopy approaches, are progressively making their way into the field of cell biology. This novel development adds an aspect other than spatial resolution to microscopy—detection of protein activity in the cell. Due to the availability of an ever-increasing range of intrinsically fluorescent proteins that can be genetically fused to virtually any protein of interest, the area of their application as fluorescent biosensors has reached the inner workings of the living cell. The most basic use of fluorescent biosensors is the collection of photons from a cell or tissue to detect the occurrence of a process with temporal resolution. This can be achieved using fluorescence energy transfer techniques (FRET). The principle of FRET is shown in figure 3.2. FRET is a photophysical phenomenon where energy is transferred non-radiatively from a donor fluorophore to an acceptor fluorophore [147].

MICROARRAY AND FLUIDIC CHIP FOR EXTRACELLULAR SENSING

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domain fused to green-fluorescent protein (GFP) that interacts specifically with molecules that are transiently generated at specific sites in cells. This allows monitoring of second messenger generation by imaging translocation of the fluorescent protein molecule [147]. In spite of the obvious utility of fluorescent techniques, there are three important considerations:

First, in mammalian cell systems, the cells that are readily transfected are those belonging to the tumor derived type as a result the cell types that can be detected using this technique is limited. Second, cell loading with fluorescent dyes is generally considered to be a potentially invasive technique. Third, analytes of interest must be examined for autofluorescence to determine the feasibility of cellular fluorescent assays for the resolution of small effects.

3.9. CELLULAR METABOLISM BASED BIOSENSORS

Another category of cellular biosensors relies on the measurement of energy metabolism, a common feature of all living cells. This is especially useful in testing drugs as in cancer research. The combined application of microfabrication technology to microfluidics have aided in the development of portable sensors. The changes in the cell metabolism due to the effect of a chemical reagent are transduced into electrical signals that are read out and analyzed. McConnel et al. 1992, developed and described a microsensorbased device, called the Cytosensor Microphysiometer [71] that was reported to be useful in the assessment of chemosensitivity of different human tumor cell lines [20, 26]. The instrument integrates up to eight channels, and detects sensitively and continuously the rate of extracellular acidification of cellular specimens. According to published results, it appears to be suited for clinical applications. Figure 3.3 shows the cross section of the

Principle of cellular metabolism based biosensor

Culture

 

 

 

Culture

 

 

medium

 

 

 

medium

 

Electric

in

 

 

 

out

 

contacts

 

 

 

 

 

 

 

 

Fluidic body

 

 

 

 

Cell culture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

200– 800 m

Microsensor chip

 

 

 

Membrane

 

 

 

 

 

(silicon, glass)

 

 

 

filter insert

 

 

FIGURE 3.3. Two microsensor-based test systems for the dynamic analysis of cellular responses have been developed by the Henning group based on the principle of Cytosensor Microphysiometer R . One of them is equipped with transparent glass chips (GC), the other one with silicon chips (SC). The systems accommodate both adherent cell types and cell suspensions/tissue explants. The above schematic shows a cross-section of the chip and culture area found in both prototype versions [53].

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MIHRIMAH OZKAN ET AL.

general schematic of a morphological sensor used in cancer drug testing applications [53]. The disadvantage of this technique is that proper interpretation of data derived using this approach requires parallel experiments in the presence of known receptor antagonists that eliminate specific receptor responses.

3.10. IMPEDANCE BASED CELLULAR SENSORS

The membranes of biological materials including cells exhibit dielectric properties. By culturing cells over one or more electrode contacts, changes in the effective electrode impedance permits a noninvasive assay of cultured cell adhesion, spreading, and motility 41,98. By combining microfludics with microelectronics it has been shown that the physiological and morphological changes in primary mammalian cell cultures can be monitored [87]. It has been shown that cultures prepared from neonatal rat cerebral cortex were placed in a confined channel containing a balanced salt solution, and the electrical resistance of the channel was measured using an applied alternating current. If the volume of the cells increases, then the volume of the solution within the channel available for current flow decreases by the same amount, resulting in an increase in the measured resistance through the channel. If the volume of the cells decreases, a decrease in resistance would be recorded. This method allows continuous measurements of volume changes in real time [94]. Figure 3.4 shows the schematic of an impedance sensor. Electric cell—substrate impedance sensing (ECIS) is the technique that is used to monitor attachment and spreading of mammalian cells quantitatively and in real time. The method is based on measuring changes in AC impedance

Principle of impedance based cellular biosensors

oscillator

Lock-in

Amplifier

1 M

Photoresist

gold film

FIGURE 3.4. The schematic of an impedance sensor. Electric cell–substrate impedance sensing (ECIS) is the technique that is used to monitor attachment and spreading of mammalian cells quantitatively and in real time. The method is based on measuring changes in AC impedance of small gold-film electrodes deposited on a culture dish and used as growth substrate. The gold electrodes are immersed in the tissue culture medium. When cells attach and spread on the electrode, the measured electrical impedance changes because the cells constrain the current flow. This changing impedance is interpreted to reveal relevant information about cell behaviors, such as spreading, locomotion and motility. They involve the coordination of many biochemical events [38].

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of small gold-film electrodes deposited on a culture dish and used as growth substrate. The gold electrodes are immersed in the tissue culture medium. When cells attach and spread on the electrode, the measured electrical impedance changes because the cells constrain the current flow. This changing impedance is interpreted to reveal relevant information about cell behaviors, such as spreading, locomotion and motility. They involve the coordination of many biochemical events. They are extremely sensitive to most external parameters such as temperature, pH, and a myriad of chemical compounds. The broad response to changes in the environment allows for this method to be used as a biosensor. The measurements are easily automated, and the general conditions of the cells can be monitored using a computer controlling the necessary automation (Keese and Giaever, 1986). Impedance techniques are theoretically capable of dynamic measurements of cellular movement at the nanometer level, a resolution above that of conventional time lapse microscopy [38]. Impedance measurements have been used to assess the effect of nitric oxide on endothelin-induced migration of endothelial cells (Noiri et al., 1994). From standpoint of biosensors, changes in cell migration or morphology tend to be somewhat slow; marked changes in impedance in the presence of cadmium emerged only after 2–3 h of exposure [25]. Thus for real time tracking and monitoring the effects of analytes a sensing technique based on impedance measurements would be slow and cumbersome.

3.11. INTRACELLULAR POTENTIAL BASED BIOSENSORS

An important aspect of the information that can be derived from cell-based biosensors relates to the functional or physiologic significance of the analyte to theorganism. To this end, bioelectric phenomena, characteristic of excitable cells, have been used to relay functional information concerning cell status [43] Membrane excitability plays a key physiologic role in primary cells for the control of secretion and contraction, respectively. Thus, analytes that affect membrane excitability in excitable cells are expected to have profound effects on an organism. Furthermore, the nature of the changes in excitability can yield physiologic implications for the organism response to analytes. Direct monitoring of cell membrane potential can be achieved through the use of glass microelectrodes. Of particular interest was whether or not cell-based sensors could be used to rapidly detect chemical warfare agents such as VX and soman (GD). In bullfrog sympathetic ganglion neurons, both VX and soman have been shown to increase membrane excitability in a manner consistent with voltagegated Ca2+channel blockade [54, 55]. The basic principle behind intracellular measurements is that tissue slices are prepared and are exposed to chemical analytes under test and the electrical activity from excitable cells are measured using patch clamp technique. In this technique a giga ohm seal is created by inserting a microelectrode into the cell under study. Figure 3.5 shows electrical activity being measured from striatal cholinergic interneurons in young adult rats using recording pipettes. Figure 3.5A shows the control electrical activity, whereas figure 3.5B shows the electrical activity recorded from cells exposed to Ca2+ ions blockade agents [88]. This technique illustrates the utility of excitable cells as sensors with sensitivity to chemical warfare agents; however, the invasive nature of intracellular recording significantly limits the robustness of this approach for biosensor applications. Another drawback is that excitable cells assemble into coupled networks rather than acting as isolated elements; As a result, for certain sensing applications the ability to simultaneously

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MIHRIMAH OZKAN ET AL.

Principle of intracellular potential based biosensors

A P21

B P53

a

 

a

 

30 m

b

b

50 mV

200 ms

150 pA

FIGURE 3.5. Measurement of extracellular potentials of striatial cholingeric internurons in young and adult rats. 5(A) IR DIC images of cholingeric interneurons in the dorsolateral striatium obtained from P. . . .Aa. and P. . . . . . rats. Ba. Recording pipettes were attached on the cell surface. Ab and Bb, membrane potential recorded fro neuronsin Aa and BA, respectively in response to applied chemical reagents namely magnesium chloroide (MgCl2). 5(B) Voltage sags during hyperpolarization, characteristic of cholingeric neurons are indicated [88].

monitor two or more cells is essential as it permits measurements of membrane excitability and cell coupling. This is not possible using intracellular techniques. The advantage of the technique is that the physiological state can be assessed. Due to the invasiveness of the technique it is not possible to apply it for long term measurements. This can be rectified by using the exrtracellular potential as the sensing indicator. Extracellular sensing from excitable cells relies on microelectrode technology. This makes the technique non-invasive and in cases where the sensing from a network of cells is required the microelectrodes function as an array of sensing elements. Also long term sensing is possible using this technique.

3.12. EXTRACELLULAR POTENTIAL BASED BIOSENSORS

In recent years, the use of microfabricated extracellular electrodes to monitor electrical activity in cells has been used more frequently. Extracellular microelectrode arrays offer a noninvasive and long-term approach to the measurement of biopotentials [19]. Multielectrode arrays, typically consisting of 16–64 recording sites, present a tremendous conduit for data acquisition from networks of electrically active cells. The invasive nature of intracellular recording, as well as voltage-sensitive dyes, limits the utility of standard electrophysiological measurements and optical approaches. As a result, planar microelectrode arrays have emerged as a powerful tool for long term recording of network dynamics. Extracellular recordings have been achieved from dissociated cells as well; that is more useful in specific chemical agent sensing applications. The current microelectrode technology comprises of 96 microelectrodes fabricated using standard lithography techniques as shown in figure 3.6A

MICROARRAY AND FLUIDIC CHIP FOR EXTRACELLULAR SENSING

Principle of extra cellular potential based biosensors

(A)

A C

B1

B

D

 

 

 

B2

 

Vdd

 

 

W/L=36/3

W/L=36/3

W/L=20/3

 

 

 

 

 

W/L=600/9

W/L=600/9

 

 

Vin

 

 

 

 

 

 

 

36 kQ

20pF

 

 

 

 

 

W/L=36/3

W/L=36/9

W/L=36/9

W/L=12/3

 

 

 

 

 

 

 

Vss

59

W/L=80/3

Vout

W/L=231/3

FIGURE 3.6(A). Extra cellular multiple-site recording probes. A: 6-shank, 96-site passive probe for 2-dimensional imaging of field activity. Recording sites (16 each; 100 µm vertical spacing) are shown at higher magnification. B: 8-shank, 64-site active probe. Two different recording site configurations (linear, B1 and staggered sites, B2) are shown as insets. C : close-up of on-chip buffering circuitry. Three of the 64 amplifiers and associated circuits are shown. D: circuit schematic of operational amplifier for buffering neural signals) [22].

[22]. More detailed work by Gross and colleagues at the University of North Texas over the past 20 yr have demonstrated the feasibility of neuronal networks for biosensor applications [43, 46]. In this work, transparent patterns of indium–tin–oxide conductors, 10 µm wide, were photoetched and passivated with a polysiloxane resin [42, 44]. Laser de-insulation of the resin resulted in 64 recording “craters” over an area of 1 mm2, suitable for sampling the neuronal ensembles achieved in culture. Indeed, neurons cultured over microelectrode arrays have shown regular electrophysiological behavior and stable pharmacological sensitivity for over 9 months [45]. Figure 3.6B shows neuronal cultures on a 64 microelectrode array [99]. In fact, their precise methodological approach generates a co culture of glial support cells and randomly seeded neurons, resulting in spontaneous bioelectrical activity ranging from stochastic neuronal spiking to organized bursting and long-term oscillatory activity (Gross et al., 1994). Microelectrode arrays coupled with “turnkey” systems for

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Principle of extra cellular potential based biosensors

FIGURE 3.6(B). Neuronal cultures on a 64 microelectrode array. Laser de-insulation of the resin resulted in 64 recording “craters” over an area of 1 mm2, suitable for sampling the neuronal ensembles achieved in culture. neurons cultured over microelectrode arrays have shown regular electrophysiological behavior and stable pharmacological sensitivity for over 9 months [99].

signal processing and data acquisition are now commercially available. In spite of the obvious advantages of the microelectrode array technology for biosensing for determining the effect of chemical analytes at the single cell level it becomes essential to pattern the dissociated cells accurately over microelectrodes. Single cell sensing forms the basis for determining cellular sensitivity to wide range of chemical analytes and determining the cellular physiological changes. Also analyses of the extracellular electrical activity results in unique identification tags associated with cellular response to each specific chemical agent also known as “Signature Patterns”.

3.13. CELL PATTERNING TECHNIQUES

There are three cell patterning methods that are currently in use. The first is a topographical method, which is based on the various microfabrication schemes involved in developing microstructures that enable the isolation and long-term containment of cells on the substrates [15, 76]. Other fabrication techniques used for cell patterning and the formation of ordered networks involves the development of the bio-microelectronic circuits, where the cell positioning sites function as field effect transistors (FET). This provides

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a non invasive interface between the cell and the microelectronic circuit [60, 95, 150]. These multi-electrode designs incorporating the topographical method have become increasingly complex, as the efficiency of cell patterning, has improved and hence fabrication has become more challenging and the devices are unsuitable for large-scale production. The other drawback is the need for an additional measurement electrode for determining the electrical activity from the electrically excitable cells. The second method is based on micro-contact printing (µCP) where simple photolithography techniques are coupled with the use of some growth permissive molecules (e.g., an aminosilane, laminin-derived synthetic peptide, Methacrylate and acrylamide polymers or poly-L-Lysine) that favor cell adhesion and growth and anti permissive molecules like fluorosilanes to form ordered cell networks [117, 118, Scholl et al., 2002, Wyrat et al., 2002, 146]. The disadvantage of this technique is the presence of multiple cells on a single patterned site that results in formation of a dense network of cell processes along the patterned areas. This in turn results in difficulties in measurement as well as determination of the electrical activity associated with a specific cell. The third method is based on using biocompatible silane elastomers like polydimethylsiloxane (PDMS). Cell arrays are formed using microfluidic patterning and cell growth is achieved through confinement within the PDMS structure. This technique is hybrid in the sense that it also incorporates µCP for promoting cell adhesion [47, 137]. The drawback of this technique is its complexity. As of today no single technique has been developed that (1) efficiently isolates and patterns individual cells onto single electrodes

(2) provides simultaneous electrical and optical monitoring (3) achieves reliable on-site and non-invasive recordings using the same electrode array for both positioning as well as recording.

3.14. DIELECTROPHORESIS FOR CELL PATTERNING

The method that satisfies the above mentioned requirements for trapping cells is the use of dielectrophoretic forces [62, 80], it was determined that cells under the influence of low AC fields can be manipulated based on the variance in their dielectric properties and this process is termed as dielectrophoresis (DEP) [107]. Dielectrophoretic cell separation works on the principle of dielectrophoretic forces that are created on cells when a non-uniform electrical field interacts with the field induced electrical polarization on the cells. Depending on the dielectric properties of the cells relative to the suspending medium, these forces can be either positive or negative and can direct the cells toward strong or weak electrical fields respectively. Next to the (frequency-dependent) electrical properties of particle and medium the DEP force is determined by the particle dimensions and the gradient of the electric field. Field strengths between two and several hundred kV/m are required for trapping particles [35] shown that several types of living cells are capable to survive the rather high electric fields over longer periods of time up to two days. This was shown for red blood cells, mouse fibroblasts (3T3, L929), suspensor protoplast, bacteria, and yeast [3, 33, 80]. However, high temperatures, which can be caused by an electric field in a medium of high conductivity, can be disastrous [3, 36]. Previous research in the field of DEP has already shown that small particles and living cells can be manipulated by DEP [34, 80, 89, 104].