The active microzone

Active flow-through (bio)chemical sensors include a microzone where a (bio)chemical reaction, a separation or both takes place. The active microzone may be located in the flow-cell itself (Figs 2.6.B and 2.6.C) or built into a probe sensor for insertion into a continuous-flow analytical system (Fig. 2.6.A). The external appearance of a sensitive microzone can be as widely different as the type of detector and process concerned. This is discussed in greater detail in the following section. 

The way in which the active microzone is retained also depends on its relationship to the detector (Fig. 2.6) and the type of interaction with the analyte or its reaction product. If the microzone is an integral part of the probe, an additional support (usually a membrane) is often required, so contact with the sample is hindered to some extent. On the other hand, a microzone located in a flow-cell can be retained in various ways. Thus, if the microzone consists of a porous solid or particle, the flow-cell is simply packed with two filters in order to avoid washing out (e.g. see [21]). Too finely divided solids (viz. particle sizes below 30-40 pm) should be avoided as they require pressures above atmospheric level, which complicates system design and precludes use of microzones with a high specific surface. Placing a separation membrane in a flow-cell is 

When the active species is to be reused many times, they must be immobilized permanently at the active microzone, which is how the reagent and catalyst are usually immobilized. An immobilized reagent must act in a reversible manner or be regenerable (usually by analyte removal or elution) on the other hand, a catalyst is self-regenerating, so no external action is required to make the sensor reversible. 

It should be noted that immobilization on the active microzone can occasionally be both permanent and temporary such is the case when two reagents (e.g. see [23]) or a catalyst plus the reaction product (e.g. see [24]) are to be immobilized. Double immobilization is also common practice when the inunobilized reagent retains the analyte and gives rise to a detectable alteration (a colour, fluorescence, mass or heat energy change) of the sensitive microzone (e.g. see [19]) all three processes (reaction, separation and detection) take place simultaneously rather than sequentially (see Chapter 5). 

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One other, very descriptive classification of flow-through sensors is based on the location of the active microzone and its relationship to the detector. Thus, the microzone can be connected (Figs 2.6. A and 2.6.B) or integrated (Fig. 2.6.C) with the measuring instrument. Sensors of the former type use optical or electric connections and are in fact probe sensors incorporated into flow-cells of continuous analytical systems they can be of two types depending on whether the active microzone is located at the probe end (e.g. see [17]) or is built into the flow-cell (e.g. see [18]) — in this latter case. 

Figure 2.6 — Classification of flow-through sensors according to the location of the active microzone relative to the measuring instrument (A,B) connected (C) built-in. (Reproduced from [1] with permission of the Royal Society of Chemistry).

Other possible classifications of flow-through sensors have been excluded from Fig. 2.4 because they are either of little consequence or dealt with in other sections below. Such is the case with the classification based on whether one or more of the active reaction ingredients (analyte, reagent, catalyst, reaction product) is immobilized temporarily or permanently on the active microzone. In addition, the immobilization process may involve one or several active components. 

Figure 2.7 — Types of species retained and immobilization at the active microzone of a flow-through sensor.

Depending on the type of detection involved and the process taking place at the active microzone (reaction and/or separation), the flow-cell that contains the microzone can exist in a variety of configurations [1], all... 

Figure 2.13 — Continuous configurations coupled on-line to flow-through biochemical sensors involving permanent immobilization of the reagent (R) at the active microzone. Symbol meanings are given in Fig. 2.12. For details, see text.

One alternative regenerating procedure involves the sequential aspiration of the sample and regenerating carrier (Fig. 2.17.B.2), which requires actuating the switching valve in order to restore the sensor. It allows larger sample volumes to be used in order to raise the analyte concentration at the active microzone when highly dilute samples are to be processed (e.g. see [19]). 

One temporal concept to be borne in mind in this context is whether the (bio)chemical reactions and mass transfer separations taking place at the active microzone (one or both of which, by definition, take place simultaneously with detection) are simultaneous or sequential relative to each other. Whether such processes take place at the same or a different time has a marked effect on the sensor performance and type of transient signal obtained. 

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