Sensor applications, immobilization

The initial hurdle to overcome in the biosensor application of a nucleic acid is that involving its stable attachment on a transducing element which commonly includes a metallic electrode. In the first part of this chapter, we wish to introduce our approach for DNA immobilization (Scheme 1). A detailed characterization of the immobilization chemistry is also presented. In the second part, we follow the development of work from our laboratory on chemical sensor applications of the DNA-modified electrode involving a biosensor for DNA-binding molecules and an electrochemical gene sensor. 

Immobilizing the catalyst on the electrode surface is useful for both synthetic and sensors applications. Monomolecular coatings do not allow redox catalysis, but multilayered coatings do. The catalytic responses are then functions of three main factors in addition to transport of the reactant from the bulk of the solution to the film surface transport of electrons through the film, transport of the reactant in the reverse direction, and catalytic reaction. The interplay of these factors is described with the help of characteristic currents and kinetic zone diagrams. In several systems the mediator plays the role of an electron shuttle and of a catalyst. More interesting are the systems in which the two roles are assigned to two different molecules chosen to fulfill these two different functions, as illustrated by a typical experimental example. 

Stabilization of activated oxidoreductases on time scales of months to years has historically been challenging, and the lack of success in this regard has limited the industrial implementation of redox enzymes to applications that do not require long lifetimes. However, as mentioned in the Introduction, some possibility of improved stability has arisen from immobilization of enzymes in hydrophilic cages formed by silica sol—gels and aerogels, primarily for sensor applications.The tradeoff of this approach is expected to be a lowering of current density because... 

Reflectance measurements provided an excellent means for building an ammonium ion sensor involving immobilization of a colorimetric acid-base indicator in the flow-cell depicted schematically in Fig. 3.38.C. The cell was furnished with a microporous PTFE membrane supported on the inner surface of the light window. The detection limit achieved was found to depend on the constant of the immobilized acid-base indicator used it was lO M for /7-Xylenol Blue (pAT, = 2.0). The response time was related to the ammonium ion concentration and ranged from 1 to 60 min. The sensor remained stable for over 6 months and was used to determine the analyte in real samples consisting of purified waste water, which was taken from a tank where the water was collected for release into the mimicipal waste water treatment plant. Since no significant interference fi-om acid compounds such as carbon dioxide or acetic acid was encountered, the sensor proved to be applicable to real samples after pH adjustment. The ammonium concentrations provided by the sensor were consistent with those obtained by ion chromatography, a spectrophotometric assay and an ammonia-selective electrode [269]. 

Optical sensors for ions use indicators, which exist in two different colors, depending on whether the analyte is bound to them. The use of colored indicators is one of the oldest principles of analytical chemistry, used extensively both in direct analytical spectroscopy and in so-called visual titrations. In their sensing application, the indicator is confined to the surface of the optical sensor or immobilized in the selective layer. In that sense, the oldest and most widespread optical sensor is a pH indicator paper, the litmus paper, which is commonly used for the rapid and convenient semiquantitative estimate of pH of solutions or for endpoint detection in acidobasic titrations. Its hi-tech counterpart is a pH optrode (the name of which is intentionally reminiscent of the pH electrode), which essentially does the same thing (Wolfbeis, 2004). The operation principles and limitations of ion optical sensors are common for all ions. 

X. Sun, P. He, S. Liu, L. Ye and Y. Fang, Immobilization of single-stranded deoxyribonucleic acid on gold electrode with self-assembled aminoethanethiol monolayer for DNA electrochemical sensor applications, Talanta, 47 (1998) 487-495. 

Demas and DeGraff reported the design of highly luminescent transition metal complexes for optical oxygen sensor applications [16]. Table 2 shows the photochemical and photophysical properties of sensing probes using luminescent transition metal complexes in immobilizing polymer films. 

One of the exciting developments associated with ion-selective electrodes has been the fabrication of microelectrodes capable of monitoring an intracellular ion concentration. The history of these developments from the mid-1950s has been reviewed.88 a symposium held in 1996 was devoted to the history of ion-selective electrodes. One paper discussed their development and commercialization,89 another described how the 1970s was the decade in which they really became established,90 a third outlined their industrial applications,91 and a fourth traced the evolution of blood chemistry analyses using them.92 The first attempts to construct biochemical sensors by immobilizing enzymes on electrodes date from the 1960s.93... 

For AW sensor applications, grains of porous powders must be immobilized by some form of thin-film physical support layer on the device surface. This requirement is nontrivial, as it is a complex problem to create a uniform, well-bound layer of tiny, porous particles that is effectively glued to a flat surface without plugging the pores with the glue used for attachment. One class of materials that has been studied as a means to immobilize high-surface-area ains... 

The fractional power in the cladding increases with mode number and capillary length. Thus, for sensor application, excitation of higher-order leaky modes leads to direct illumination of the immobilized fluorophores on the surface. 

The LbL technique is undoubtedly one of the best methods to incorporate biological components into man-made devices. Therefore, sensor applications must be one of the most promising subjects for LbL assemblies of biomaterials. For example, Leblanc and coworkers used several bilayers of chitosan and poly(thiophene-3-acetic acid) as cushion layers for stable enzyme films [187]. The first five bilayers of the cushion layer allowed for better adsorption of organophosphorus hydrolase than the corresponding adsorption on a quartz slide. The immobilized enzyme becomes more stable and can be used under harsher conditions. The assembled LbL films can be used for spectroscopic detection of paraoxon, an organophosphorus compound. This cushion layer strategy provides a well-defined substrate-independent interface for enzyme immobilization, in which the bioactivity of the enzyme is not compromised. This leads to fast detection of paraoxon and quick recovery times. 

For example, Zhang and Cass have also immobilized alkaline phosphatase on a nanoporous nickel-titanium film for sensor applications. 

Samanta, D. and Sarkar, A. (2011) Immobilization of bio-macromolecules on self-assembled monolayers methods and sensor applications. Chem. Soc. 

Table I. Example of Immobilization for Sensor Applications (cont)... 

The sensors are able to detect relevant quantities of agent at levels that are below those that are immediately dangerous, for the following hazards nerve, blood, and blister agents caustic acids or bases reactive aldehydes and potent oxidizers. The covalently attached enzymes make the sensors extremely sensitive and highly specific toward their target chemicals, and the immobilization within the polyurethane matrix provides the necessary robustness for a sensor application outside the laboratory environment. 

---