Indirect immunosensors

Fig. 9. Immunosensor approaches where A is the analyte, is the labeled analyte, and Y is the antibody, (a) Direct immunosensors where the actual antigen—antibody interaction is measured (b) indirect immunosensors 1 and 2 which utilize formats similar to competitive and displacement...

There are mainly three types of transducers used in immunosensors electrochemical, optical, and microgravimetric transducers. The immunosensors may operate either as direct immunosensors or as indirect ones. For direct immunosensors, the transducers directly detect the physical or chemical effects resulting from the immunocomplex formation at the interfaces, with no additional labels used. The direct immunosensors detect the analytes in real time. For indirect immunosensors, one or multiple labeled bio-reagents are commonly used during the detection processes, and the transducers should detect the signals from the labels. These indirect detections used to need several washing and separation steps and are sometimes called immunoassays. Compared with the direct immunosensors, the indirect immunosensors may have higher sensitivity and better ability to defend interference from non-specific adsorption. 

Immunosensors have been designed which use both direct and indirect immunoassay technology to detect specific analytes within a minute or less in a variety of matrices (see Fig. 9). Indirect immunosensors may employ ELA, FLA, or CLIA principles whereby enzyme-, fluorophore- or chemiluminescent-labeled analyte competes with the target (nonlabeled) analyte for binding sites on the immobilized antibody. Unbound (free) labeled analyte is then quantitated using an electrochemical, optical, or electromechanical transducer and compared to the amount of target analyte in the sample. 

In contrast to direct immunosensors, indirect immunosensors, using as a label an enzyme [307-309] or an ionophore [310], have been more developed. This technique is derived from well established and widely utilized enzyme immunoassays evaluated by photometric methods. Enzyme immunoassay with electrochemical detection (EEIA) represents an important innovation of EIA by the replacement of optical detection with voltammetric or potentiometric measurements. It may also be adapted to flow-through systems [311, 312]. 

A.F. Chetcuti, D.K.Y. Wong, and M.C. Stuart, An indirect perfluorosulfonated ionomer-coated electrochemical immunosensor for the detection of the protein human chorionic gonadotrophin. Anal. Chem. 71, 4088-4094 (1999). 

The assays were carried out using monoclonal antibodies in the direct and indirect formats. OTA working range, /50 and LODs were 0.05-2.5 pg/L and 0.1-7.5 pg/L, 0.35 ( 0.04) pg/L and 0.93 ( 0.10) pg/L, 60 pg/L and 120 pg/L in the direct and indirect assay formats, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I50 in real samples was 0.2 pg/L corresponding to 1.6 pg/ kg in the wheat sample with an LOD of 0.4 pg/kg (calculated as blank signal—3er). Within- and between-assay variability were less than 5%... 

Currently, immunosensors provide a powerful tool for monitoring biospecific interactions in real time or for deriving information about the binding kinetics of an immunoreaction or the structure/function relationships of molecules. Amperometric, potentiometric, conductive, and impedimetric transducers have been applied in direct and indirect electrochemical immunoassays. 

Labeled immunosensors typically make use of a reporter molecule attached to the respective antibody. Apart from the electrochemical detection schemes discussed below, immunosensors employ fluorescent labels, radioactive labels, or nanoparticles, among other reporter systems [407—410]. Labeled immunosensors normally operate using either a direct or indirect sandwich procedure or a competitive format as depicted in Figure 1.18. 

---