DETECTION TECHNOLOGIES
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Figure 1. (click to enlarge)The electrochemical immunosensor platform. (a) A flow injection system is used in which standards or samples, enzyme-analyte conjugates, and hydrogen peroxide substrates are introduced simultaneously to the electrode located inside a thin-layer flow cell. The flow cell is connected to a potentiostat and computer. (b) The electrode is a disposable screen-printed device with layers of silver and carbon onto which conducting polymers and antibodies are immobilized. (c) A monolayer of antibody (yellow) is immobilized at the conducting-polymer surface (green). The mixture of analyte (blue circle), conjugate (blue/red), and substrate (pink circles) passes over the electrode surface. Competitive binding of conjugate to the electrode results in an increase in the real-time current response (green arrow).
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Why Use Electrochemical Immunosensors?
Sensor-related technologies have shown a great deal of promise in developing IVD devices capable of simple analytical measurements. A lot of success has been achieved with physical sensors, which are reliable and have been applied to nearly every conceivable physical measurement task. There has also been much success with chemical sensors in measuring analytes such as ions and gases.1 However, less success has been achieved in developing biosensors that rely on biomolecular interactions for transduction. Much of this lack of success has been related to the properties of biomolecules and to finding ways to detect their interactions with the environment.2
Two transduction processes have received the most attention: optical and electrochemical. Optical methods are sensitive and reliable, and have been used in various biosensor applications. However, electrochemical methods have achieved the most success in developing commercial biosensor devices for point-of-care and personal use, a primary example of which is the glucose biosensor.3 This is partly due to the simplicity and low cost of electrochemical transduction instrumentation compared with its optical counterpart.
An essential element for taking electrochemical measurements is the electrode. Electrodes are composed of conducting but electrochemically inactive materials. The most widely used material is carbon in a range of physical forms. The mass production of electrodes has required high-throughput, low-cost techniques, which are also compatible with the materials necessary for electrode fabrication, such as screen-printing. Ink formulations can be made that can be easily printed and are also conductive and electrochemically inert. Carbon inks and pastes are excellent electrode platforms. Research has investigated the physical processes involved in producing screen-printed carbon electrodes and the best way to optimize their application in electrochemical immunosensors.4,5
Analytically and diagnostically, no assay platform has achieved the widespread use that one immunoassay has. This platform is inherently flexible, making it nearly universally applicable. It is also difficult to compete with its sensitivity and selectivity. Based on a review of the field of electrochemical immunosensors, combining the benefits of immunoassays with electrochemical technologies to create an immunosensor would seem logical.6
However, combining these two systems does not necessarily translate into a useful diagnostic system.7 Several elements of the immunoassay process conflict with the simplicity of the sensor format. The immunoassay is a multistep platform, and although many of these steps have been removed in many commercial systems for end-users, they are still a necessary part of the assay. While homogeneous assays appear simple and elegant, they have never achieved the levels of sensitivity and wide applicability of heterogeneous assays. The latter technique requires separating bound and unbound species, which is also required of assays performed electrochemically.
Challenges of Electrochemical Immunosensors
The interaction of an antibody with an antigen is not readily detectable, resulting in only small changes in enthalpy and physical conformation. From an electrochemical standpoint, several processes have been investigated to detect this interaction directly, such as potentiometric and impedimetric methods.8,9 However, due to the highly ionic and electrically noisy environment in which such interactions take place, many of these methods are unreliable.
One indirect detection technique that can be applied to electrochemical systems and optical immunoassays is enzyme labeling. A range of enzymes with some redox properties (e.g., oxidases, peroxidases, reductases) has been used in electrochemical platforms. In particular, horseradish peroxidase has been widely used. This peroxidase has a high substrate turnover rate and is tolerant of its substrate. Its active site is also close to the external surface of the protein, thereby creating direct and mediated electrochemical transduction processes that are more efficient than with many other enzymes and that allow sensitive detection of hydrogen peroxide.10
Direct electrochemical transduction between a bare electrode surface such as carbon and an enzyme active site is inefficient, even with peroxidase. Many strategies can be applied to rectify such inefficiency, such as soluble electron-transfer mediators. However, these mediators do not remain localized at the electrode. By applying conducting polymers to electrode surfaces that remain localized, they can act as a point for biomolecular attachment, increase the efficiency of electron transfer, and serve as useful materials in electrochemical immunosensors.11
Approaches to Developing Electrochemical Immunosensors
A research team at the National Centre for Sensor Research (NCSR), based at Dublin City University (Dublin, Ireland), has been developing a technology that may enable users to derive quantitative analytical information with an electrochemical immunosensor assay. One of the technology’s key features is the use of low-cost, single-use, screen-printed electrodes as the sensor platform that are modified with a highly conducting polymer layer and a biorecognition molecule such as an antibody. Measurements are performed in a traditional way with a competition- or inhibition-based immunoassay involving the analyte to be measured and a peroxidase-labeled analog. In the presence of a hydrogen peroxide substrate, peroxidase binding at the surface transduces a signal to the electrode through a flow of electrons (see Figure 1). This system has several key differences from other similar technologies.
First, the modified electrode surface and the immobilized antibody have been optimized to yield a surface that requires no additional blocking treatments. Second, the presence of unbound peroxidase label in the vicinity of the electrode surface does not transduce an electrochemical signal. No separation steps need to be performed, allowing the prospect of single-step immunodiagnostic measurements. Third, because separation steps are not required, binding interactions can be monitored in real time. With a flow injection system and a subsaturation measurement concept, several measurements can be performed on a single sensor surface, including calibration and sample measurements, in a matter of seconds. With these properties, this system is a potential technology for taking quantitative immunodiagnostic measurements in a range of in situ applications.
The electrochemical immunosensor uses polyaniline, with the addition of suitable dopants. This conducting polymer is electrochemically deposited onto the electrode surface. Biological molecules can be immobilized by electrostatic interactions, resulting in efficient electron-transfer processes. Electrodeposition of the polymers is the best way to deposit them. However, this method restricts the rate at which the devices can be produced. The research team is looking at physical deposition of conducting coatings to develop formulations that are capable of competing with electrochemical deposition techniques.
One promising method, the use of conducting-polymer nanoparticles, is being developed at the Intelligent Polymers Research Institute at the University of Wollongong (New South Wales, Australia). These nanoparticles have improved handling characteristics compared with the carcinogenic bulk solution monomer aniline. Their nanoscale also allows ultrathin films to be deposited onto electrodes. Such well-controlled thin films are essential for effective behavior of the polymer layers (e.g., conductance, electron transfer rates, quality of biomolecular immobilization).12 The films are also potentially amenable to high-throughput processes such as ink-jet printing.
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Figure 2. (click to enlarge) Gold-labeled antibody layer on polyaniline-modified carbon paste electrodes. Increasing the antibody concentration increased the density of immobilization on the polymer surface: (a) 1.25 mg/ml antibody, (b) 0.6 mg/ml antibody, (c) 0.1 mg/ml antibody.
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Electron transfer between horseradish peroxidase and the electrodes was efficient using conducting-polymer nanoparticles. Research also showed that the binding of peroxidase at the electrode surface with antibodies resulted in mediated electron transfer.13 While more information about the processes involved is needed, what seems to be apparent is that intermolecular distances are important. Adequate proximity of the enzymes to the electrode surface is essential, and introducing any additional distance between the enzyme active site and the electrode surface reduces electron-transfer efficiency. The size of the antibody and its orientation on the electrode may also play a crucial role. The research team, in collaboration with the school of biotechnology at Dublin City University, is investigating this by using smaller antibody fragments (e.g., single-chain antibodies) and developing methods to orientate recombinant antibody fragments at the electrode surface.
The characteristics of antibody-modified conducting-polymer surfaces have been carefully optimized. Control of the antibody solution used for immobilization could result in a surface with excellent interference rejection properties. At the ideal concentration, antibodies are deposited in a monolayer or submonolayer. This process maximizes the amount of capture reagent on the surface, minimizes inhibition of electron transfer by surface overcrowding, and prevents nonspecific interaction with bulk solution enzyme materials. The monolayer nature of the surface has been characterized electrochemically and colorimetrically.14 More-recent studies using gold-labeled antibodies and scanning electrochemical microscopy confirm this distribution of antibodies on the electrode surface (see Figure 2).
The Atrazine Immunosensor
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Figure 3. (click to enlarge) Electrochemical inhibition immunoassay of atrazine. (a) A series of electrode surfaces were preincubated with free atrazine of various concentrations for 15 minutes before atrazine-HRP and hydrogen peroxide were simultaneously introduced to the electrode, resulting in a real-time binding curve that was inversely proportional to the free atrazine concentration. (b) Calibration curve based on the real-time binding data showing detection of atrazine as low as 0.1 ppb (0.5 nM).
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The platform described above has taken quantitative measurements of several analytes. Initial measurements used biotin as a model analyte.15 Measurements were extended to analyzing atrazine. A principal drawback of the system in this configuration was that the electrodes could only be used for a single measurement. Interelectrode variability meant that errors were much greater than desired, which made calibration difficult. In addition, the immunoassay was performed using the traditional method of sequential incubations.
However, in combination with a flow injection system, and in the presence of peroxidase-labeled antigens and a hydrogen peroxide substrate, the interaction of the antibodies with the conjugate could be measured in real time.16 With such a conceptual breakthrough, assay components could now be handled in a convenient manner. All of the immunoassay components (i.e., free analyte, peroxidase conjugate, substrate) could be applied simultaneously to the sensor surface, and a proportional response could be derived directly (see Figure 3).
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Figure 4. (click to enlarge). Multicalibrant analysis technique. Binding at an antibody-modified surface follows a saturation profile. Early in the saturation curve, the relationship between bound concentration and time is linear, so several measurements can be performed within this linear region.
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Performing a single measurement on an electrode still did not solve the problem of assay calibration. In immunoassays, a large proportion of the assay is devoted to establishing calibration for the measurements. This is also necessary for electrochemical immunosensors. However, an important phenomenon of antibody binding had not yet been exploited. Mass transport–limited binding of antibodies and antigens on a finite surface produces a saturation-binding curve. In the early phase of this binding process, the relationship between the binding rate and time is linear. As long as measurements were performed within this linear region, several measurements could theoretically be performed on a single surface, which would allow calibration standards and samples to be analyzed on a single electrode surface (see Figure 4).
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Figure 5. (click to enlarge) (a) A real-time, multicalibrant binding curve showing the sequential interaction of the antibody-modified electrode with hydrogen peroxide, atrazine-HRP, and a series of concentrations of free atrazine. (b) A plot of the slope of the electrode response vs. free atrazine concentration.
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This phenomenon was tested with a sensor using single-chain antibody fragments to atrazine that were supplied by Haptogen Ltd. (Aberdeen, Scotland). It was possible to apply at least four standards to a single electrode and derive a linear calibration plot based on the rates of the binding interactions (see Figure 5). Normalized interelectrode variability was less than 1.5%, and the assay could be performed in about 150 seconds.17 This methodology has also been tested by the school of biotechnology at Dublin City University, where researchers applied the principle to an optical immunosensor device and found that at least 15 measurements could be performed on a single, unregenerated chip surface. This principle could increase assay throughput and allow extended use of the chip surface, yielding benefits in time and cost.18
Conclusion
The electrochemical immunosensor platform is reaching a level of maturity such that it is ready for commercial development. A three-year technological development plan for this system has been launched with Enterprise Ireland to develop a self-contained disposable device that could allow quantitative diagnostic measurements. The NCSR is continuing to develop ways to improve the mass production of the modified electrode surfaces by using a range of material formulations and deposition techniques. The NCSR is also looking at nanomaterials and nanostructuring techniques to bring about further improvements in the electron transduction process used to detect the antibody-antigen interaction.19
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Anthony J. Killard, PhD, is a research fellow, and Malcolm R. Smyth, PhD, is executive dean of the faculty of science and health, and the principal investigator of the sensors and separations group at the National Centre for Sensor Research, Dublin City University (Dublin, Ireland). The authors can be reached at tony.killard@dcu.ie and malcolm.smyth@dcu.ie, respectively.
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2. AJ Killard and MR Smyth, “Biosensors,” in Biomolecular Films, ed. JF Rusling (New York: Marcel-Dekker, 2003), 451–497.
3. AJ Killard et al., “Electroanalysis and Biosensors in Clinical Chemistry,” in The Encyclopedia of Analytical Chemistry, ed. RA Meyers (New York: Wiley, 2000), 173–207.
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8. SQ Hu et al., “A Label-Free Electrochemical Immunosensor Based on Gold Nanoparticles for Detection of Paraoxon,” Talanta 61, no. 6 (2003): 769–777.
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10. JT Schumacher et al., “Direct Electron Transfer Observed for Peroxidase to Screen-Printed Graphite Electrodes,” Electroanalysis 13, nos. 8–9 (2001): 779–785.
11. YF Yang and SL Mu, “Bioelectrochemical Responses of the Polyaniline Horseradish Peroxidase Electrodes,” Journal of Electroanalytical Chemistry 432, no. 1 (1997): 71–78.
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14. A Morrin et al., “Characterisation of Horseradish Peroxidase Immobilisation on an Electrochemical Biosensor by Colorimetric and Amperometric Techniques,” Biosensors & Bioelectronics 18, nos. 5–6 (2003): 715–720.
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16. AJ Killard et al., “Development of an Electrochemical Flow-Injection Immunoassay (FIIA) for the Real-Time Monitoring of Biospecific Interactions,” Analytica Chimica Acta 400 (1999): 109– 119.
17. K Grennan et al., “Atrazine Analysis Using an Amperometric Immunosensor Based in Single-Chain Antibody Fragments and Regeneration-Free Multi-Calibrant Measurement,” Analytica Chimica Acta 500 (2003): 287–298.
18. PP Dillon et al., “Novel Assay Format Permitting the Prolonged Use of Regeneration-based Sensor Chip Technology,” Journal of Immunological Methods 296, nos. 1–2 (2005): 77–82.
19. X Luo et al., “The Application of Nanoparticles in Electrochemical Sensors and Biosensors,” Electroanalysis 18, no. 4 (2006): 319–326.
