Reproducibility biosensor

FIGURE 6-14 DNA hybridization biosensors detection of DNA sequences from the E. coli pathogen. Chronopotentiometric response of the redox indicator upon increasing the target concentration in 1.0 pg/ml steps (a-c), in connection with a 2 min hybridization time. (Reproduced with permission from reference 46.)... 

The work in the biosensor industry permitted the testing and proved of stability and reproducibility of enzymes, within the conditions employed in that area. Enzymes with demonstrated stability include lactate oxidase, malate dehydrogenase, alcohol oxidase, and glutamate oxidase. 

MWNTs favored the detection of insecticide from 1.5 to 80 nM with a detection limit of InM at an inhibition of 10% (Fig. 2.7). Bucur et al. [58] employed two kinds of AChE, wild type Drosophila melanogaster and a mutant E69W, for the pesticide detection using flow injection analysis. Mutant AChE showed lower detection limit (1 X 10-7 M) than the wild type (1 X 10 6 M) for omethoate. An amperometric FIA biosensor was reported by immobilizing OPH on aminopropyl control pore glass beads [27], The amperometric response of the biosensor was linear up to 120 and 140 pM for paraoxon and methyl-parathion, respectively, with a detection limit of 20 nM (for both the pesticides). Neufeld et al. [59] reported a sensitive, rapid, small, and inexpensive amperometric microflow injection electrochemical biosensor for the identification and quantification of dimethyl 2,2 -dichlorovinyl phosphate (DDVP) on the spot. The electrochemical cell was made up of a screen-printed electrode covered with an enzymatic membrane and combined with a flow cell and computer-controlled potentiostat. Potassium hexacyanoferrate (III) was used as mediator to generate very sharp, rapid, and reproducible electric signals. Other reports on pesticide biosensors could be found in review [17],... 

The second-generation 02" biosensors are mainly based on the electron transfer of SOD shuttled by surface-confined or solution-phase mediators, as shown in Scheme 2(b). In 1995, Ohsaka et al. found that methyl viologen could efficiently shuttle the electron transfer between SOD and the glassy carbon electrode and proposed that such a protocol could be useful for developing 02 biosensors [125], Recently, Endo et al. reported an 02, biosensor based on mediated electrochemistry of SOD [148], In that case, ferrocene-carboxaldehyde was used as the mediator for the redox process of SOD. The as-developed 02 biosensor showed a high sensitivity, reproducibility, and durability. A good linearity was obtained in the range of 0 100 pM. In the flow cell system, tissue-derived 02 was measured. 

FIGURE 12.6 Cyclic voltammograms at a laccase biosensor in the absence (a) and presence (b) of 02 Sparging of 02 through the electrolyte between scans (2 min) yields reproducible and increased catalytic reduction currents (C-F) compared to ambient 02 levels. Scan rate lOmVs 1 in 0.05M acetate buffer of pH 4.5. (From [55], with permission from Wiley.)... 

Among various enzyme immobilization protocols, entrapment in polymer membranes is a general one for a variety of transducers. Formation of a membrane from a solution of already synthesized polymer is simpler and reproducible compared to chemical polymerization. The simplicity of this immobilization procedure should provide reproducibility for the resulting biosensors the latter is strongly required for mass production. 

Biosensors normally offer highly specific molecular recognition reactions like enzyme/substrate-, antigen/antibody-, DNA/DNA-, or protein-interactions [67]. Due to their specific sensing principles and set-up they are limited to special applications and boundary conditions. The limited stability and reproducibility of these devices requires higher standards of maintenance and recalibration. 

Figure 1.26 Scheme of immuno-biosensor developed by Liu and Gooding, exploiting the size of proteins and the space that a protein takes up to block ion access to the redox probe. (Reproduced by permission of The Royal Society of Chemistry from [142].)... 

Figure 3.1 — (A) Configuration of p-nitrophenyl phosphate biosensor (a) common end of bifurcated bundle b) retaining 0-ring (c) inner nylon mesh with enzyme (d) outer nylon mesh (not drawn to scale). (B) Processes occurring at the biosensing tip a enzyme/scatter layer S enzymatic substrate P light absorbing product. (Reproduced from [34] with permission of the American Chemical Society).

Figure 3.6 — (A) Fibre-optic biosensor system a septum b needle guide c thermostated reaction vessel d fibre bundle e enzyme membrane f screw cap g stirring bar h reaction medium i black PVC jacket j 0-ring. (B) Continuous-flow fibre-optic sensor system for the bioluminescence determination of NADH. (Reproduced from [41] with permission of Marcel Dekker, Inc.)...

Figure 3.8 — (A) Biosensors used in different FI manifolds to perform reaction-rate measurements (I) stopped-flow manifold (II) iterative flow-reversal system (III) open-closed configuration S sample B buffer P pump IV injection valve PC personal computer IMEC immobilized enzyme cell D detector W waste SV switching valve. (B) Types of recordings obtained by using the three types of biosensors and measurements to be performed on them in order to develop reaction-rate methods. (Reproduced from [50] with permission of Elsevier Science Publishers).

Figure 3.10 — Flow manifolds for implementation of flow-through biosensors. (A) Flow injection merging-zones manifold for the bioluminescence detennination of ATP. ATP standards (30 fiL) and luciferin (30 fiL) are injected into the buffered carrier streams, each pumped at 0.7 mL/min and synchronously merged 12.5 cm downstream. Distance from merging point to immobilized enzyme coil, 2.2 cm. (Reproduced from [59] with permission of Elsevier Science Publishers). (B) Completely continuous flow manifold for the determination of NADH. (Reproduced from [71] with permission of the Royal Society of Chemistry). (C) Segmented-flow manifold for the determination of L-(+)-lactate. (Reproduced from [65] with permission of Marcel Dekker, Inc.). (D) Single-channel flow injection manifold with immobilized reagent for the detennination of glucose. (Reproduced from [77] with permission of Elsevier Science Publishers).

Figure 3.18 — Microbiosensor types. (A) Oxygen electrode. (B) CO, (C) L-lysine, (D) hypoxanthine, and (E) glucose biosensors. (Reproduced from [109] and [111] with permission of Marcel Dekker, Inc., and Elsevier Science Publishers).

Figure 3.20 — Experimental set-up for implementation of chemoreceptor-based biosensors. (Reproduced from [125] with permission of Elsevier Science Publishers).

Biochemical oxygen demand (BOD) is one of the most widely determined parameters in managing organic pollution. The conventional BOD test includes a 5-day incubation period, so a more expeditious and reproducible method for assessment of this parameter is required. Trichosporon cutaneum, a microorganism formerly used in waste water treatment, has also been employed to construct a BOD biosensor. The dynamic system where the sensor was implemented consisted of a 0.1 M phosphate buffer at pH 7 saturated with dissolved oxygen which was transferred to a flow-cell at a rate of 1 mL/min. When the current reached a steady-state value, a sample was injected into the flow-cell at 0.2 mL/min. The steady-state current was found to be dependent on the BOD of the sample solution. After the sample was flushed from the flow-cell, the current of the microbial sensor gradually returned to its initial level. The response time of microbial sensors depends on the nature of the sample solution concerned. A linear relationship was foimd between the current difference (i.e. that between the initial and final steady-state currents) and the 5-day BOD assay of the standard solution up to 60 mg/L. The minimum measurable BOD was 3 mg/L. The current was reproducible within 6% of the relative error when a BOD of 40 mg/L was used over 10 experiments [128]. 

Figure 5.15.C shows the auxiliary manifold used for application of this sensor to the determination of hydrogen peroxide. The sample is injected into an appropriate buffer that is merged with a stream of substrate. The mixture then reaches the biosensor, where the enzymatic reaction and retention of the product formed take place. On switching the valve, the eluting stream flushes the reaction product retained in the biosensor, which is thus made ready for the next sample. In this way, H2O2 can be determined at the parts-per-billion level over a wide linear concentration range with excellent reproducibility.

The selection of appropriate microorganisms is a possible way to improve the correlation between BOD and BODj [16,53]. The prerequisite for the use of microorganisms for BOD-sensors is a wide substrate spectrum. Therefore several samples of activated sludge from different wastewater plants were investigated [ 13,14]. One problem with an activated sludge based biosensor is the variability of sensor response with time. These BOD-sensors with an undefined variety of microbial species revealed no reproducible results. For that reason, BOD-sensors were developed using various types of defined cultures of microorganisms (Table 1). 

Further progress of ECL probes immobilization methods should result in new robust, stable, reproducible ECL sensors. Especially, the use of electrochemilumi-nescent polymers may prove to be useful in this respect. There are also good prospects for ECL to be used as detection in miniaturized analytical systems particularly with a large increase in the applications of ECL immunoassay because high sensitivity, low detection limit, and good selectivity. One can believe that miniaturized biosensors based on ECL technology will induce a revolution in clinical analysis because of short analysis time, low consumption of reactants, and ease of automation. 

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