Protein sensors/determination

When compared to fluorescent proteins, fluorophores and quenchers of fluorescence (short quenchers) are small molecules with sizes varying from 1 to 10 A. They are the main building blocks for constructing small molecule FRET probes. As molecular entities, they might influence the performance of the probe to a great extent. Their fluorescent properties will determine the sensitivity and dynamic range of the sensor. The success of the probe for a specific application will depend on the selection of the right fluorophores... 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

The first aspect of biocompatibility is a natural immune response. When a foreign object enters the blood stream, it can be attacked by the body s defense system. The first step is protein adsorption on an object surface. It is believed that the amount and type of protein adsorption is one of the most important steps determining whether the object is tolerated or rejected by the body. The next step is cell adhesion, which may cause aggregation and activation of platelets and triggering of the blood coagulation system with resulting thrombus formation. It may not only lead to sensor failure via surface blocking but directly threatens the patient s health. 

Similar to the work described by Spohn et al. [34], a trienzyme sensor was developed recently for the determination of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine). Leucine dehydrogenase, NADH oxidase, and peroxidase were coimmobilized covalently on tresylate-hydrophylic vinyl polymer beads and packed into a transparent PILL tube (20 cm X 1.0 id), which was used as flow cell. The sensor was free of interferences from protein and NH4+ and it was stable for 2 weeks. The sensor system was applied to the determination of branched-chain amino acids in plasma with recoveries ranging from 98 to 100% [36],... 

Rinsing the sensor surface with buffer results in an irreversible dissociation, because all molecules which dissociate from immobilized protein are removed from the system by the buffer stream, allowing one to determine the rate constant of dissociation separately. 

To give an example both sensitivity coefficients are evaluated for the current sensor (see Sect. 10.3 for details). For the bulk detection of glucose this results in A bulk (rad) = 5.6 x 102 AC (g/ml), whereas for the adsorption of proteins on the sensor surface the overall sensitivity of the sensor is evaluated as A< >layer (rad) = 2.0 x 10 5 Am/A (fg/mm2). Measuring the phase change A< >,-, between any of the two channels i and j can thus give an estimation on the change in analyte concentrations between those two channels. If one channel (e.g., channel N) is used as a reference channel, then ACV = 0 and AmN = 0 and absolute analyte concentrations can be determined. 

The reference 28 authors continue to detail experimental observations that place voltage sensor helices in positions within the membrane. Miller and coworkers conducted site-directed mutagenesis for all residues of helices Sl-S3. ° In these experiments, tryptophan (trp) residues were substituted for each amino acid in turn to determine which residues would be trp-tolerant. These experiments confirmed a-helical conformations for SI and S2 and showed that K+ channel function was altered when trp residues were placed in some (labeled non-trp-tolerant), but not all, positions. The same treatment for helix S3 yielded complex results. At S3 s N-terminal end the distribution of trp-tolerant positions were consistent with an a-helical structure, however, this was not the case at S3 s C-terminal end. Other tests indicated that S3 might be helical for its entire length and that the N-terminal end interfaces with both lipid and protein while the C-terminal end interfaces with water. Comparisons of trp-tolerant or trp-intolerant residues over several different Kv channel... 

Regarding the pH sensor, the carboxy tail length has been demonstrated as a determinant of pH sensitivity [Liu et al., 1993]. Further investigations [Morley et al., 1996] revealed a new model of intramolecular interactions in which the carboxy terminal serves as an independent domain that, under certain conditions, can bind to another separate domain of the connexin protein (e.g. a region including His-95) and close the channel, comparable to the ball-and-chain model for potassium channels. In this receptor (His-95),... 

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