Polymer and surface bound sensors

If practically useful sensors are to be developed from the boronic acid sensors described above, then they will need to be integrated into a device. One way to help achieve this goal is to incorporate the saccharide-selective interface into a polymer support. 

Nagasaki and Kimura have used polymers of poly(lysine) with boronic acids appended to the amine residue as saccharide receptors [158-160]. On saccharide com-plexation these polymers are converted from neutral sp boron into anionic sp hybridized boron. The anionic polymer thus formed interacts with added cyanine dye. Saccharide binding can then be read-out by changes in the absorption and induced circular dichroism (ICD) spectra of the cyanine dye molecule. 

Wang has employed the template approach using monomer 84 to prepare a fluorescent polymer with enhanced selectivity towards D-fructose [161,162]. Appleton has used a similar approach using monomer 85 to prepare a D-glucose selective polymer [77]. The Appleton polymer clearly shows the value of the imprinting technique. Here, the selectivity of the monomer for D-fructose over D-glucose has been reversed in the polymeric form. 

James and co-workers have developed polymer sensors by grafting a solution based D-glucose selective receptor 27d to a polymer support [78]. The major difference between the polymer-bound system 86 and solution-based system 27d is the D-glucose selectivity, which drops for polymer 86 (whereas the selectivity with other saccharides is similar to those observed for compound 27d). However, the polymeric system still has enhanced D-glucose selectivity (nine times) over the monoboronic acid model compound. The reduced binding of 86 for D-glucose has been attributed to the proximity of the receptor to the polymer backbone. 

Wolfbeis has prepared a polyaniline with a near-infrared optical response to saccharides. The film was synthesized by copolymerization of aniline and 3-amino-phenyl boronic acid. Addition of saccharides at pH 7.3 led to changes in absorption at 675 nm [167]. 

Smith has prepared a grafted polymer containing a ribonucleoside 5 -tripho-sphate selective sensor. The polymers were prepared using poly(allylamine) (PAA) to which 10% of boronic add monomer unit 202 was grafted.Also, a library of potential sialic acid receptors was prepared. In this case, the polymers were prepared using PAA to which 2% of the boronic acid monomer unit 202 was grafted. The final polymers also contained various amounts of 4-hydroxybenzoic acid, 4-imidazolacetic acid, octanoic acid and/or succinic anhydride. 

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Redox enzymes have been assembled in a monolayer on the solid surface by a potential-assisted self-assembling method as well as a thiol-gold selfassembling method. These enzymes are electronically communicated with the solid substrate through a molecular interface of conducting polymer and a covalently bound mediator. Electron transfer type of enzyme sensors have been fabricated by the self-assembling methods. 

The sensors discussed so far are based on ligands covalently bound to the polymer backbone. Other methods of detection - often referred to as mix and detect methods - work by simple noncovalent incorporation of the polymer with the ligand of interest. Reichert et al. generated liposomes of polydiacetylene with sialic acid for the same purpose of detection as Charych s surface-bound polymers, but realized that covalent functionalization of the polymer was not necessary [17]. Through simple mixing of the lipid-bound sialic acid with the polymer before sonication and liposome formation, they were able to form a functional colorimetric recognition system (Fig. 8). 

This result shows that DNA-conjugated TA-polyallylamine can be immobilized on a surface via self-assembly. To verify that this result is due to self-assembly, DNA-conjugated TA-polyallylamine lacking TA side chains was used as a control. This polymer was also retained on the surface after washing (Fig. 6A, curve II) the residual response (1900 response unit (RU)) was probably due to nonspecific interaction between the DNA side chains of the polymer and the gold surface of the sensor. However, the amount of bound polymer was less than that for the DNA-conjugated TA-polyallylamine. The SPR response in the presence of polyallylamine alone (without DNA) was low (only 780 RU) after washing (Fig. 6A, curve III). 

Reproducibility problems in the production of polymer layers as described above can sometimes occur. Since washing steps and drying procedures are often incorporated to remove loosely bound material, and changes in intensity or duration of these steps can result in different layers. Thus the amoimt of the immobilised protein will be different and reduce the reproducibility of the sensors response. The use of thick organic films produced by this method can also lead to frequency instabihty and loss of sensitivity. It is also difficult to control film thickness and homogeneity, which also effect the reproducibility of the surface produced. Weak adhesion between the polymer and the substrate can also be a problem. Methods, such as plasma polymerisation and electropolymerisation, have been developed to overcome these problems and to gain more control over the layer parameters. 

Attempts to construct simple and direct affinity sensors have been under way for about 15 years. Janata [303] has shown that the antigen-antibody complex is charged differently from the antigen and antibody themselves. When an antibody is bound to the surface of a hydrophobic polymer located on a metal conductor then the surface charge between... 

Functionalized conducting monomers can be deposited on electrode surfaces aiming for covalent attachment or entrapment of sensor components. Electrically conductive polymers (qv), eg, polypyrrole, polyaniline [25233-30-17, and polythiophene/23 2JJ-J4-j5y, can be formed at the anode by electrochemical polymerization. For integration of bioselective compounds or redox polymers into conductive polymers, functionalization of conductive polymer films, whether before or after polymerization, is essential. In Figure 7, a schematic representation of an amperomethc biosensor where the enzyme is covalendy bound to a functionalized conductive polymer, eg, P-amino (polypyrrole) or poly[A/-(4-aminophenyl)-2,2 -dithienyl]pyrrole, is shown. Entrapment of ferrocene-modified GOD within polypyrrole is shown in Figure 7. 

Glutamate Sensors. The glutamate sensors were constructed using graphite rods (Ultracarbon, Bay City, MI), which were polished to a smooth finish and coated with a small amount of redox polymer (0.033 Mmole of polymer-bound ferrocene) dissolved in chloroform. After evaporation of the solvent, a 5 m1 aliquot of glutamate oxidase (Yamasa Shoyu Co., Japan) solution (100 units/ml in pH 7.0 phosphate buffer) was added to the surface and allowed to dry at room temperature. The electrodes were stored under dry conditions at 5°C. 

An early attempt to make a real electrochemical sensor based on a molecularly imprinted methacrylate polymer utilised conductometric measurements on a field-effect capacitor [76]. A thin film of phenylalanine anilide-imprinted MAA-EDMA copolymer was deposited on the surface of semiconducting p-type silicon and covered with a perforated platinum electrode. An AC potential was applied between this electrode and an aluminium electrode on the back side of the semiconductor and the capacitance measured as a function of the potential when the device was exposed to the analyte in ethanol. The print molecule could be distinguished from phenylalanine but not from tyrosine anilide and the results were very variable between devices, which was attributed to difficulties in the film production. The mechanism by which analyte bound to the polymer might influence the capacitance is again rather unclear. 

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