4. Potentiometric immunoassays
4.3. Electrochemical immunoassays
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using quantum dot labels in the electrochemical immunoassays, and enabled the simultaneous multiplexed measurement of protein targets utilizing different semiconductor nanoparticle label traces (ZnS, CdS, PbS, CuS)[199]. Each biorecognition label yielded to a distinct voltammetric peak whose peak potential and current reflected the identity and concentration, respectively, of the corresponding analyte.
The application of colloidal gold/silver as amplification in immunology dates back to the early 1970s when the development of silver-enhanced methods allowed gold labels to provide high-resolution, specific and sensitive immunocytochemistry[194]. The use of colloidal gold as electrochemical immunoassay label, however, for voltammetric monitoring of protein interactions was pioneered only in 2000[200] [201]. To further enhance the sensitivity of gold nanoparticle label-based immunoassays various techniques have been developed. For example, the nanoparticle-promoted precipitation of silver on gold nanoparticle labels[202][203] [204], where the silver ions were detected by voltammetric[202][204] or potentiometric[203] method after dissolution of the metallic silver. A common problem in Ag enhancement can be the high background signal as a result of non-specific precipitation of silver onto the substrate.
Various techniques have been developed to reduce this non-specific deposition to further increase the sensitivity of this method.
35 immunoassays based on the radioisotopic label[208] (radioimmunoassay, RIA) gaining very low detection limits, and thus resulting in an enormous growth rate in the use of RIA in both the clinical and research laboratories. RIA, however, has the disadvantages that accompany radioisotope handling, together with an inability to distinguish label which is bound from that which is not. There has therefore been an enormous effort to find suitable replacements for the radiolabel. The most successful of these have been enzyme linked immunosorbent assay (ELISA)[162][163]. The popularity of the method started to grow tremendously after, allowing the application of such assays also by unspecialized users and in field applications.
Electrochemically-based immunoassays have also mostly used ELISA and thus enzyme amplification. Enzyme labels are easy to connect with electrochemical detection because of their ability to yield an electroactive product following the catalytic conversation of a non-electroactive substrate. The most common substrate ‒ enzyme combinations utilized were NAD/NADH and glucose-6-phosphate dehydrogenase[209]; phenyl phosphate/phenol or later aminophenyl phosphate (APP)/aminophenol and alkaline phosphatase[210][211], respectively[165].
These early assays used mostly amperometric techniques for detection, quantitatively relating the magnitude of current, arising under controlled potential conditions, from the redox reaction of the product to the amount of analyte present. Later, several other suitable alternative substrates were also investigated to replace the common APP, such as 1-naphthyl phosphate[212] or ascorbic acid 2-phosphate[213]. These substrates increased the efficiency of the readout and minimized non-specific interferences by possessing lower detection potentials; hence improved the sensitivity; ensured lower-assay cost; and enabled faster enzymatic reaction. Amperometric detection methods remained up to now the most commonly used electrochemical transduction techniques, due to their fast detection, broad linear range, and low detection limit[205]. They also have the advantage that unspecific binding of molecules other than the labelled immunoreactant will not contribute to the signal. The main drawback is, however, when using any of the above mentioned substrates that they can cause passivation of the surface of the sensing electrode, leading to small amperometric responses and poor reproducibility.
Voltammetric techniques, which are extremely sensitive, were also shown to be the electrochemical methods of choice in enzyme immunoassays very early on. In these methods the potential is varied while measuring the current change related to the analyte. An interesting example is the use of interdigitated microelectrode arrays (IDAs), consisting of pairs of microelectrode fingers that are held at different potentials to achieve redox “cycling” of the electroactive species to be detected[214]. Voltammetric sensing, however, possesses several drawbacks as well, unless ultramicroelectrodes are used, the current signal is strongly dependent on mass transport conditions.
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Upon the specific molecular recognition of the antigen by the immobilized antibody, there are changes in the interfacial charge, capacitance, resistance, mass, and thickness at the sensor surface. Thus there is lately an emerging interest in exploring electrochemical techniques that follow these interfacial changes enabling direct, label-free, and sensitive real-time monitoring of the antibody-antigen interactions[170]. Electrochemical impedance spectroscopy (EIS) can be an effective method for probing the features of an electrode surface modified by immunocomplexes[215]. These impedimetric methods relay on measuring the change in impedance as a result of the antibody-antigen interaction[170]. Another technique, based on similar principles, is conductimetric immonosensing, where the change in conductance is measured upon the interaction[216]. Capacitance detection of immunoassays exploits the change in dielectric properties or thickness of dielectric layer, i.e. the inorganic insulator and the immobilized molecules, at the electrolyte-dielectric interface due to the antibody-antigen interaction. During the interaction the thickness of the immobilized bioactive layer changes, causing an alteration in the capacitance[217].
4.3.1. Potentiometric immunoassays
Potentiometry has the simplest measurement technique and instrumentation from the electrochemical detection approaches, and furthermore it is free of the problems mentioned before concerning other electrochemical techniques. In potentiometric detection principle, the change in the membrane potential of an ion-selective electrode, occurring after the specific antibody-antigen binding, is measured. A logarithmic relationship between the electrode potential and the concentration of the detected species exists, as given by the Nernst equation. The research in potentiometric immunoassays started already in the 1970s, with pioneering contribution by the group of Rechnitz[218][219][220] and Janata[221][222].
Direct, label-free potentiometric detection of antibody-antigen interactions is also possible with this method, by immobilizing one of the participants of the immunoreaction on the indicator electrode. In a so-called ionophore modulation immunoassay[223] a K+-selective ionophore was covalently linked to the target antigen, and the presence of the antibodies in the sample was found to alter the EMF of the electrode. Another alternative is the use of polycation-selective electrodes for competitive homogenous assays[224], where the potentiometric response of a synthetic polycation-analyte substrate is suppressed when binding to the antibody occurs. A third direct potentiometric method[225], not using ion-selective electrodes though, is based on the change of the charge when the binding of an antigen at the surface of an Ag-electrode with immobilized antibody occurs. Using the potentiometric transduction capabilities of single-walled carbon nanotubes in combination with the recognition capabilities of protein specific RNA aptamers disease-related proteins were as well measured with direct potentiometry from blood[226]. Label-free
37 potentiometric detection is rapid and simple, with no separation steps required. A main disadvantage is, however, that the change in potential due to the antibody antigen interaction is relatively small, and interferences from the sample matrix may prevent this small signal from being successfully detected, therefore the reliability and sensitivity of these assays have been limited[217].
Early efforts about the potentiometric detection of labelled immunoreagents mainly focused on using potentiometric gas sensors, such as ammonia or CO2 electrodes, in conjugation with urease[227][228][229]-, asparaginase[228]-, adenosine deaminase[228][230] -, and chloroperoxidase[231]-label. There were also a few attempts to measure the pH change caused by the urease enzyme label[232][233]. Another research direction aimed at generating I- and F- ions, by enzymatic reaction of the commercially more available horseradish peroxidase[234][235] and alkaline phosphatase[236], to detect them with all-solid-state iodide[234]- and fluoride[235] [236]-selective electrodes. Although these detection methods seemed promising, the limited availability of commercial reagents needed for the unconventional ELISA systems, as well as the poor detection limits and dynamic range of the early potentiometric ion or gas sensors clearly limited their practical utilization.
Recent improvements on the field of solid-contact electrodes and the improvements in the lower detection limit of the ion-selective electrodes motivated researchers to revisit potentiometry as a detection method in immunoassays. The few interesting approaches published in the past decade used nanoparticle-label. In the first one a miniaturized Ag+-selective electrode was successfully used to detect silver ions, released by the oxidative dissolution of silver enhancement of gold nanoparticle labels of a sandwich immunoassay[203]. Following the same principle, potentiometric immunoassays were built on the detection of Cd2+ released from CdSe quantum-dot-labelled tracer antibodies[237] or aptamers[238]. The most recent works in the field reported lately, i.e.
after the research described in this thesis was published, will be briefly summarized in the respective outlook part.
Although ion-selective potentiometry is not affected by mass transport limitations, passivation of the electrode surface or limited selectivity that concerns other electrochemical detection techniques, it seems that little follow-up work was done on most of the early approaches. Potentiometric immunoassay detection provides a simple measurement method with widely available instrumentation, and establishes miniaturization and low-cost microfabrication of the electrodes. Since ion-selective electrodes are already extensively used in commercial point-of-care sensors and automatic blood-gas analysers the limited number of potentiometric immunoassays reported up to now is rather surprising.
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