Biosensor Technology and the Clinical Biochemistry Laboratory – Issue of Signal Interference from the Biological Matrix

MICHAEL THOMPSON, SONIA SHEIKH, CHRISTOPHE BLASZYKOWSKP AND ALEXANDER ROMASCHIN

1.1 Laboratory Clinical Biochemical Assays

Assays for biochemicals of clinical interest in biological fluids, tissues or feces can be arbitrarily divided into tests performed in either the clinical biochemistry laboratory or via a point-of-care device. The latter technology often takes the form of a one-use structure that yields a result in a localized environment such as the home, hospital bedside or general practitioner's facility. The blood glucose and pregnancy assay devices, which involve discardable test strips, will be familiar to many. This type of structure has attracted a high level of interest in recent years, not least from the commercial standpoint. Often quoted advantages are said to lie in convenience, speed of analysis and cost savings. There are of course attractive features in certain cases for procuring a measurement in a particular location. Obviously, biosensor devices might be expected to play a major role in this arena. However, the present discussion concentrates on the potential for use of biosensors in the dedicated central biochemistry laboratory, which is typically located in a major hospital for obvious reasons. Assays performed in this type of facility are very wide ranging in scope with samples originating from the areas of diagnosis of medical conditions, toxicology or monitoring of drug therapy. Sera or plasma dominate the types of samples analyzed but many assays also involve various tissues, feces and other bodily fluids such as urine.

Test technology associated with all the various aspects of medicine is a large and expensive operation. In terms of the cost of performing assays, the Province of Ontario, for example, spends well over 1 billion Canadian (CDN) dollars annually, the tests being conducted in both private laboratories and hospital facilities. A large hospital such as St. Michael's in Toronto performs a vast array of tests such as those involving immunology, hematology, bacteriology, mycology, cytology, genetics and various aspects of pathology in addition to biochemical measurements. With respect to the latter, some 3 million tests are performed annually with reagent costs alone being in the region of 2 million CDN dollars. Although not intended to be exhaustive, Table 1.1 provides an overview of the type of biochemical and immuno-assays conducted by the clinical biochemistry on an annual basis together with total numbers comprised of both in- and out-patient tests. The "winner" is the thyroid stimulating hormone (TSH) test, which alone costs several million dollars annually. The equipment employed in the portfolio of clinical measurements ranges from conventional chromatography to sophisticated mass spectrometry, and combinations thereof. As would be expected given the sheer volume of samples, the laboratory is highly automated and robotized. Typically, a blood sample will be bar-coded then centrifuged to extract serum before entering an automated analytical train. Samples may then be directed automatically to their various analytical stations. A good example of a subsequent analysis would be the prevalent magnetic-bead enzyme-linked immunosorbent assay (ELISA) method for targets where immuno-assay is feasible. Despite the high level of automation incorporated into the analytical train, it should be emphasized that measurements are still performed in a batch-based fashion. Having said that, it is certainly the situation that a number of protocols offer multi-analyte capability, the aforementioned ELISA assay being a case in point.

Biosensor technology - in principle - offers rapid, label-free measurements, which can be conducted in highly automatable configurations. Furthermore, the possibility for multiplying tests using generic sensing physics is certainly an attractive strategy. However, the clear reality is that biosensor devices do not figure prominently in the clinical biochemistry laboratory. Use of ion-selective electrode technology for certain cations might represent a contradiction to this statement, but it could justifiably be argued that these devices do not constitute biosensors anyway! The dearth of biosensor devices in the clinical biochemistry laboratory also appears to be matched by the lack of employment of so-called "lab-on-a-chip" devices, which are generally characterized by microfluidic sample handling. Time will tell but at present neither of these technologies appears to have fulfilled the promise offered. This chapter attempts to evaluate possible reasons for this including a detailed look at one major problem – that of interference by adsorbed species from the biological matrix. In order to provide a backcloth to discuss the issues at stake, we take a prior concise look at biosensor technology. The chapter has something of an emphasis on label-free methodology.

1.2 Biosensor Technology

1.2.1 Biosensor Architecture

A biosensor is composed of chemical recognition sites (probes) attached to a substrate surface that, in turn, is in close proximity and union with a transducer. Ideally, this configuration responds sensitively and selectively to the presence of (bio)chemicals, we term the target or analyte, usually present in the liquid phase. The technology is based on the recognition of species through selective binding of the target to the probe at the substrate surface of the device with such surface presence being converted into an electrical signal. The overall architecture is depicted in Figure 1.1. Note that probes are invariably composed of biological entities such as cells or biochemicals such as antibodies. The sample itself may or may not be of biological origin. An important aspect of biosensor technology is the nature of the response of the device with respect to time. The field is often considered to include the aforementioned "one-test" disposable systems, where there is no attempt to conduct a measurement over a period of time. This type of device is more often than not at the heart of the point-of-care assay. From the clinical biochemistry point of view with regard to the central laboratory, both time-responsive and one-shot structures might be applicable. This issue will be considered in more detail later in the chapter.

The signal obtained from a selective biosensor response will take the following form (Eq. (1.1)):

[MATHEMATICAL EXPRESSION OMITTED] (1.1)

where C and S values represent the concentration of analyte and interferents 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 samples such as serum or urine. A sensitive response implies maximization of one S value and, for a selective signal, a minimization of all other S values. Clearly, the sensor signal will be a composite of the chemistry of the attachment process and the physical perturbation caused by the probe–analyte complex. In this respect, it is crucial that, when designing a sensor for particular applications, the physics of the complete transduction process be thoroughly characterized and understood. In fact, some devices simply respond to the presence of the analyte, whereas others detect structural shifts caused by target–probe binding. The distinction between these mechanisms is not always evident.

1.2.2 Probe Attachment to Device Surfaces

A mandatory aspect of biosensor fabrication is the attachment of the biochemical probe to the surface of the transducer of choice. Over the years, a host of methods have been developed with this goal in mind. A number of criteria are relevant to this component of sensor production:

• It will be vital to retain the binding activity of the probe in terms of its ability to bind the target. It is anticipated that certain proteins, for example, may be partially denatured and lose affinity upon attachment to the sensor surface, whereas others such as antibodies may be more robust.

• The proper spatial orientation of the probe with respect to the plane of the device surface will be an important element to ensure exposure of binding sites for target capture.

• In terms of sensitivity, it is crucial to maximize the density of probe molecules per surface area unit. Simply stated, the more probe available on the device surface, the greater will be the sensor signal.

• 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 too close proximity on the surface, this may result in steric hindrance to target binding. In practice, there exists a maximal (optimal) effective probe loading.

• There are other more mundane factors such as the cost of reagents and the potential longevity of the modified sensor surface. These will clearly be critical from a commercial standpoint.

Some examples of methods for probe attachment follow. This short compendium is not intended to be an exhaustive treatment.

1.2.2.1 Noncovalent Attachment

The probe of interest can be chemically or physically adsorbed from solution directly, in most cases, on the substrate surface of the device. Various protocols are employed to achieve such an effect such as dip casting, painting, spraying and spin coating, where these terms will be self-explanatory to the reader. The interaction of the probe with the surface will be characterized primarily by hydrogen bonding and/or hydrophobic interactions. Adsorbed molecules can also act as a linker for eventual biomolecule attachment. A system that is widely employed 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 binding (Kd = 10-15 M). Accordingly, various biomolecules can be modified with relative facility by biotin incorporation in order to link them to surface-attached avidin or a sister molecule such as the aglycosylated version, neutravidin. An analogous strategy uses protein A. The latter is a polypeptide (MW~42kDa) isolated from Staphylococcus Aureus that is capable of binding specifically to the Fc region of various antibody molecules and has thus been employed in immunosensor technology.

Another noncovalent approach is the trapping of the recognition molecule within the three-dimensional structure of a specific chemical matrix. The matrix may simply act as a "holding" moiety or be modified in some way to take part in the transduction process. For the former, polymers such as polyacrylamide have been employed in a number of experimental protocols. However, there are a number of potential disadvantages to this type of approach for placing the probe on the device surface including the possibility of biomolecule leakage and/or denaturation. An additional consideration is the necessity for the target to diffuse into the polymer matrix in order for the biochemical interaction to take place. An analogous procedure uses encapsulation by sol-gel technology, wherein a solution of a monomer such as an alkoxide (the sol) is induced to polymerize into a biphasic configuration (the gel) that incorporates both liquid and solid. A typical monomer among many would be tetraethylorthosilicate (TEOS – EtO4Si), which is readily hydrolyzed by water to produce a siloxane-based polymeric structure with a gel consistency. A cavity can be formed around the probe of interest in somewhat the same manner as for the polymers mentioned above.

1.2.2.2 Covalent Binding

Attachment via covalent bonds has been by far the most used approach. A very wide variety of chemistries have been employed with modest success in terms of the criteria outlined above. Many functional groups, whether directly present on the device substrate or obtained by modification, have been utilized to form a probe partial monolayer. Groups are available on biomolecules to instigate the surface link and examples of these for proteins are presented in Table 1.2.

A common protocol to bind enzymes, antibodies or molecular receptors to the substrate is to initially functionalize the surface, which is then followed by a second reaction of activation. In essence, this process simply allows a convenient, highly reactive "linker" moiety to be introduced to the system. The resulting interface is then normally allowed to react in turn with one of the protein nucleophilic groups mentioned above. The literature is replete with many examples of this sort of approach and, in Figure 1.2, we provide a schematic of one strategy. If groups already evident on the device surface are not used directly, functionalization of surfaces is often achieved with species such as aminopropyltriethoxysilane (APTES), which reacts with interfacial hydroxyl groups on whatever substrate they are present.

1.2.2.3 Self-Assembled Monolayer Chemistry

The introduction of close-packed monolayers has been used widely in biosensor development. Two very different strategies are employed the first being the Langmuir–Blodgett film technique. In this experiment, close-packed mono-layers are imposed on a surface by transferring lipid or lipid-like (amphiphilic) films from the Langmuir trough, under the correct surface pressure conditions, by a dipping process. In principle, several layers can be deposited in sequence using this dipping approach. The very important advantage offered by this technique is the possibility to combine artificial lipid membrane configurations directly and in situ with integral membrane proteins (IMPs). Such membrane systems are, of course, widely prevalent as signaling devices in biology.

An important chemistry, which has been significantly developed in recent years, is the self-assembled monolayer (SAM). This approach relies on the use of linking molecules that are engineered to spontaneously form ordered molecular assemblies on solid substrates. In this case, the most common strategy has been the assembly of relatively long chain thiols on the surfaces of clean gold substrates, as shown in Figure 1.3. The distal end of bifunctional thiols can then be employed for conventional covalent binding of proteins and oli-gonucleotides as described above (Figure 1.2). An alternative technique is the use of trichlorosilanes rather than thiols, which can be bound to surfaces that have been functionalized with or naturally possess –OH groups. Examples of substrates in this case would be silicon dioxide (e.g. quartz – Figure 1.4) and indium-tin oxide (ITO).

1.2.3 Devices and Transduction

A very large amount of work has been performed on a variety of biosensor structures. A bibliography detailing this effort up to the end of 2012 is provided herein. Three distinct areas of physics have been employed with respect to the transduction process, these being electrochemistry, acoustic wave technology and electromagnetic radiation. There is considerable variety in terms of the nature of measurement, for example, possible label-free operation and secondly dipstick or response with time methodology. Moreover, a number of adjunct techniques have been employed in order to enhance the level of information obtained from the sensor determination.

1.2.3.1 Electrochemical Devices

A number of different approaches are possible via electrochemistry, which involves the transfer of electrons or charge at solid/liquid or liquid/liquid interfaces. Those involving solid structures unsurprisingly dominate the field, which is summarized in Table 1.3. A particularly useful feature of many of these techniques is that they can be combined "naturally" with various forms of integrated electronic circuitry. Indeed, the necessary chemistry can be imposed directly on the surface of such an electronic device (see below).

Historically, one of the earliest devices generated from the world of electrochemistry was the sensor based on potentiometry. In this technique, an indicating electrode for the analyte of interest is incorporated in an electrochemical cell (with reference electrode) for which minimal or no current is passed. In this category, the ion-selective (indicating) electrode (ISE) forms the basis of systems, which are capable of detecting ions and other species of biochemical interest. By far the most common in use is the glass electrode, which is sensitive to changes in hydronium-ion concentration. Other systems available for selective ion detection include electrodes that employ crystalline matrices, e.g. LaF3 for F-, or incorporate liquid ion exchangers in polymer matrices such as systems for sensing Ca2+. The ISE has also been the basic structure used to develop enzyme-based molecular sensors where analytes are allowed to interact with immobilized enzymes and transform into products detected by the electrode.