Sensor Technology in Neuroscience
By Michael Thompson, Larisa-Emilia Cheran, Saman Sadeghi
The Royal Society of Chemistry Copyright © 2013 M Thompson, L-E Cheran and S Sadeghi
All rights reserved.
Introduction to Biosensor Technology
1.1 Sensor Anatomy, Signaling and Properties
The notion of a sensor device is common knowledge to all. The range of these structures in modern times is immense, ranging from simple physical measurements such as temperature to complex devices that incorporate human cells in their design. The number of applications is also numerous including industrial processing, pharmaceutical analysis, automotive operation, military technology and environmental signaling to name just a few areas of use. In this section we introduce the basics of a special branch of sensor technology that deals with the detection of chemicals, with relevance to the research in neuroscience described later in the chapter. The emphasis is on devices, which constitute the main structures employed in biosensor technology, rather than a comprehensive review of the field.
Devices that detect and signal the presence of chemicals have evolved through two different pathways, although the distinction between the two is somewhat arbitrary. The structure is composed of a chemical recognition site attached to a substrate surface which, in turn, is in close proximity and union with a transducer. Such a system can be used to respond selectively to the presence of chemicals we term the target or analyte, either in the gas or liquid phase. The chemical recognition site is often referred to as the receptor or probe. The technology relies on the ability of such a configuration to 'recognize' chemicals through selective binding at the substrate surface of the device with such surface presence being converted into an electrical signal via the particular physics of a transducer. Chemical sensors are generally considered to involve non-biological/biochemical probes in their design and the same is often regarded to be the case for the analyte. A simple example is the well-known tin oxide sensor which responds to gases such as carbon monoxide. In contrast, biosensors are composed of a union between a transducer and biological/ biochemical receptor. A schematic of the structure including transduction types is depicted in Figure 1.1. Note that the probe can consist of a variety of biological entities ranging from antibodies to live biological cells Given that nucleic acids and proteins are chemicals, as are the targets that cells as probes are designed to detect, it is clear that the distinction between chemical- and biosensors is artificial.
Crucial aspects of biosensor technology are the nature of the response of the device with respect to time and whether ancillary chemistry is required in addition to the basic probe in order to achieve a signal. With respect to the former point, there are sensor specialists who take the view that such a device must respond to its analyte in real time. Conversely, the field is often considered to include 'one-test' disposable structures where there is no attempt to conduct a measurement over a period of time, except in a repeated dipstick fashion. The ubiquitous pregnancy and glucose test strips that are widely commercially available constitute examples of this approach to biodetection. The use of an adjunct chemical in addition to the receptor to achieve a transducer signal is often termed tagging or labeling. An example of this strategy is the use of dyes in conjunction with nucleic acid probes in order to produce fluorescent signals. In certain cases, there is insufficient intrinsic fluorescence in nucleic acid molecular probes to allow the direct possibility of detection. The same is true for electrochemical methods where organometallic complexes (of Ru) have to be employed for work with nucleic acids in order to detect redox chemistry. Technology where such an approach is avoided is called 'label-free detection' and is often regarded to be attractive in view of the fact that sensor fabrication becomes a somewhat simpler process.
Additional important technical factors are the possibilities for incorporation of the device in flow-through automation, sensor miniaturization and prevention of non-specific adsorption. Such automation involving standalone systems avoids time-consuming personal intervention and allows rapid data collection and validation. Microfluidic systems offer speed and saving of reagent costs. Nonspecific adsorption of unwanted components on the device surface poses something of an Achilles' heel for biosensor technology. The selectivity and limit of detection of the sensor when used, for example, in blood, serum, urine and tissue will clearly be influenced strongly by interfering components of the biological sample. It appears to be the case that wide scale use of biosensors in, for example, clinical biochemistry, has not occurred primarily because of this issue.
The placement of a solid transducer–probe combination into a biological sample will result in a signal originating from a composite response, Xn, according to the following matrix:
Xn = Sn1C1 + Sn2C2 + ···· SnnCn (1.1)
where C and S values represent the concentration of analyte and interferants in proximity to the device, and the response sensitivities, respectively.
An enormous number of components would be expected to be involved in this equation in terms of biological samples. A sensitive response (e.g. volts or amps) implies maximization of one S value and, for a selective signal, a minimization of all other S values. To a first approximation, it is necessary to trap the analyte on the device surface to allow the sensor response and, as mentioned above, to repel or avoid such binding of all other elements. Accordingly the sensor signal will be a composite of the chemistry of the attachment process and the physical perturbation caused by the probe–analyte complex. This leads to some interesting aspects concerning the nature of the couple between physical chemistry and the transduction process. In certain cases, as will be seen in later sections, the mere presence of the analyte can influence the transducer and the resulting signal is often referred to as a 'mass response'. However, a situation can be envisaged where a structural shift, such as a probe conformational change, and regardless of whether the response is related to final state effects or the change itself, is required for detection to take place. The physical chemistry of transduction in this case is reminiscent of agonist versus antagonist interactions and will be familiar to the biochemist community. All these mechanisms will obviously be an intrinsic component of the sensitivity parameter, Snn, outlined above.
In summary, there are a number of key desirable properties that a biosensor should possess, although some features are of course more important for some applications than others:
Selectivity or even specificity (see ref. 4 for an excellent definition of these parameters) with respect to the response to the analyte, as described above, is a given. Non-specific binding through adsorption to the device surface by interferants will clearly have a strong influence on selectivity, especially if such components are at a high concentration in the sample under analysis.
High sensitivity is required with a resulting low value of limit of detection.
High accuracy is required in terms of concentration measurement for the analyte. Signaling must be conducted with high reproducibility — a precision issue.
The dynamic range with respect to response should be as high as possible for certain targets (for example, clinical values of an analyte may vary considerably).
With regard to real-time detection, speed of response is crucial. For the onetime determination device this aspect may not be so important.
Device response calibration in terms of concentration is mandatory and especially important for the real-time sensor. This feature has constituted an extremely difficult problem when it comes to, for example, the operation of a corporeal implantable structure. Early solutions to this issue may well lie in a strategy of device self referencing.
User ease of use may well be a factor especially where expertise in device understanding is lacking.
There are ancillary issues such as robustness and cost which may be crucial from a more commercial point of view.
A bibliography of texts that deal with biosensor technology and related subject matter is provided at the end of this chapter.
1.2 Genesis of Biosensor Technology
The advent of biosensor technology has been widely attributed to the work of Clark and Lyons, which involved an electrochemical amperometric mechanism for the measurement of glucose concentrations in biological samples. Prior to this system (1950s), Clark et al. were working on the electro-chemical detection of oxygen dissolved in blood and tissue via O2 reduction, and this research served as something of a precursor to the subsequent developments with glucose.
The basis of the original glucose measurement was an enzyme electrode (Figure 1.2) in which a layer containing the enzyme, glucose oxidase (GO) was placed in close proximity to a platinum electrode polarized at a potential of +0.6 V. The substrate reacts with the enzyme according to:
[MATHEMATICAL EXPRESSION OMITTED]
The Pt electrode is designed to respond to the hydrogen peroxide produced by the enzyme–glucose reaction. This progress led to a large number of O2 mediated devices being developed which included a variety of enzymes and substrates. A further early example connected to glucose detection was the work of Updike and Dicks who immobilized a gel impregnated with the oxidase on a semi-permeable membrane. In this experiment, the amperometric signal resulted from changes in the partial pressure of oxygen as it diffused through the membrane to a sensor. Not surprisingly other components of the basic oxidase reaction have been studied such as pH changes associated with the production of gluconic acid; in this situation the electrochemistry involved potentiometry.
Although a large number of enzyme-based systems have been developed over many years, it is fair to say that by far the most research has centered on glucose because of the obvious significance to the diabetic patient. Literally hundreds if not thousands of scientific papers have been devoted to this substrate. In modern times, O2 as a redox mediator is avoided and other species capable of electron transfer are employed to serve the same purpose. Indeed such a strategy is very much a component of the commercial test strips that are so familiar to individuals suffering from diabetes.
We now turn to a concise look at contemporary biosensor technology using a device outside-to-inside approach.
1.3 Probe Attachment to the Sensor Structure
In order for a biosensor to function it is mandatory to couple the probe or receptor to the solid surface of the particular transducer being employed, and if not bound at the device interface, it must generally be arranged be in close proximity. Over the years a plethora of methods have been developed for this purpose, and although some of these are common to various probes, a large number have been designed for a specific receptor type. The overall goal of much of this chemistry is to achieve the maximum signal with respect to operation of the device in use. In this respect there are a number of key factors at play.
It is obviously critical to retain the binding activity of the probe for its target. It is anticipated that certain proteins, for example, may be partially denatured on attachment to the sensor surface. Others such as antibodies may be more robust. Such is generally the case for DNA and oligonucleotides whereas RNA moieties are considered to be more prone to alteration in structure. When it comes to cells it is clearly important for them to be active and alive; this area is discussed in detail in the following chapter.
The particular orientation of the probe with respect to the plane of the device surface is an important element. As an example, if the Fab region of an antibody is 'hidden' from the target because of binding of this region to the solid surface instead of the Fc component, a true immunochemical interaction cannot be expected.
In terms of sensitivity it is crucial to maximize the density of probe molecules per surface unit area with the caveat specified in the next point. Such density may well be influenced by the roughness and surface area of the device. Furthermore, there are cases where polymer films are employed rather than plane surfaces but the same criterion holds in terms of overall surface density. In this situation a key factor is the ability of the analyte to diffuse into the film in order to bind to probes attached to polymer strands.
The spatial characteristics of the probe with regard to the surface plane are a factor that is important but not studied widely. If probes are in close molecular proximity on the surface this may result in steric hindrance to target binding. Another consideration is the possibility that the chemistry of probe attachment may result in 'island' formation. In this case the overall number of binding sites will be significantly reduced. As would be anticipated, control of this chemistry is very difficult to achieve.
There are other more mundane factors such as the cost of reagents and the potential longevity of the modified sensor surface. These are clearly critical from a commercial standpoint.
We take a concise look at the myriad of possible strategies for probe attachment to devices. The particular surface modifications necessary to impose cells on a device is considered in the following chapter.
1.3.1 Direct and Linker Adsorption
This constitutes the simplest method for placing a biomolecule onto a substrate, although there are inherent dangers for the technique in terms of possible undesirable conformational changes. The probe of interest is chemically or physically adsorbed from solution directly, in most cases, on the substrate surface of the device in use. Various protocols are employed to achieve such an effect such as dip casting, painting, spraying and spin coating — these terms will be self-explanatory to the reader. The interaction of the probe with the surface is characterized primarily by hydrogen bonding, van der Waals forces and various dipole electrostatic interactions. The energy of the attachment process is of the order of 10–40 kJ mol-1 and is highly dynamic in nature. The latter may lead to a lack of stability in terms of longevity of sensor performance.
It has been considered that a major aspect of this type of chemistry, when it comes to protein molecules, is the hydrophobic interaction with a hydrophobic substrate. In this case protein molecules can change their conformation in order to expose more hydrophobic domains. This sort of effect will clearly be deleterious if the native tertiary structure of the molecule is altered with respect to target binding. Although hydrophobic effects can be damaging as indicated, this sort of effect is by no means restricted to non-polar surfaces. Our laboratory showed conclusively that the well-known protein, avidin, is compromised with regard to its structure by hydrophilic interfaces. Presumably, the reason for this lies in the instigated interactions of surface polar functional groups with the carbohydrate moieties of the protein molecule.
The last comment leads to a look at the use of intervening adsorbed molecules as a linker for eventual biomolecule attachment. A system which is employed extremely widely is avidin/streptavidin/neutravidin chemistry. Avidin is a tetrameric protein that contains four binding sites for the ligand, biotin. Interaction of the protein with this molecule results in a particularly strong bond (Kd = 10-15). Accordingly, various biomolecules can be modified with relative facility by biotin addition in order to link them to surface-attached avidin, or a sister molecule such as the deglycosylated version, neutravidin. With respect to device operation, particularly where on-line detection is involved, it is common practice to introduce the protein into the system where it is allowed to simply adsorb to the device surface. Although four biotin binding sites are present, there is no doubt that at least two of these are expected to be unavailable on steric grounds for the reasons outlined above.
An analogous chemical system is the use of protein A. This is a polypeptide (molecular weight 42 kD) isolated from Staphylococcus aureus which is capable of binding specifically to the Fc region of various antibody molecules and has thus been employed in immunosensor technology. One strategy similar to the case of avidin outlined above is to introduce this protein (for surface adsorption) in a system prior to antibody addition. (Continues...)
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