COMMON ELECTROCHEMICAL DETECTION SYSTEMS FOR DNA BIOSENSORSBIOSENSORS

Each biosensor is composed of a biological receptor, as described in the former section, and of a transducer.

The receptor, which is generally located on the surface of the biochip, captures the target molecule, inducing a consequent modification in a property of the solution close to the sensor surface (e.g. dielectric constant, or availability of electrochemically active molecules). The transducer is the unit that detects such property change and converts it into a readable electrical signal. A number of technologies are used for the Table 7.2 Summary of the advantages and disadvantages of conventional laboratory analysis,

immunosensors, and DNA biosensors.

Method Characteristics & advantages Limitations & disadvantages Conventional

laboratory analysis

Well established Precise

Long response time, mainly due to the detection step, which generally requires the cultivation of the microbe.

The analysis must be performed in a biological laboratory.

Usually unable to detect dead organisms.

Immunosensors Compatible with portable devices, and the assay can be performed by untrained personnel

Typical response time inferior to one hour, without considering sample pretreatment.

Micromanufacturing techniques allow testing of several organisms in a single assay, on a single biochip. and the assay can be performed by untrained personnel.

Possibility to integrate with DNA amplification established techniques.

Sensitive, selective and specific enough for water pathogens.

Typical response time of 2 to 4 hours, including sample amplification.

Micromanufacturing techniques allow testing of several sequences in a single assay, on a single biochip.

The sample needs to be pretreated for DNA extraction.

It is difficult to achieve absolute quantification.

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transduction, each using a particular physicochemical property change that can be electrochemical, optical, and mechanical (D’Orazio, 2003; Rodriguez-Mozazet al., 2004). Each technique has specific advantages, for example, the use of surface plasmons in Surface Plasmon Resonance (SPR) devices allows for real-time label free kinetic measurements, colorimetric sensors give a response within minutes that can be read by the naked eye, and electrochemical sensors are the most miniaturizable and integrable.

In this section, we present the basic principles of DNA biosensors based on electrochemical transduction.

This class of sensors offers several advantages over other technologies, mainly related to its intrinsic capabilities of miniaturization and integration with electronic circuits. The strong connection with electronics both at the level of the control circuitry and of the biochip fabrication technology, allows to take advantage and incorporate knowledge from the very well established field of information technology. Not only the response signal is already electrical and can be directly processed by conventional electronics in a cheap and fast manner, but also the biochip itself can be considered part of the electrical circuit, so that electronic architectures typical of sensing devices, such as the Wheatstone bridge, can be replicated with the inclusion of the biochip as one of the sensing impedances (Luong et al., 2008). The high sensitivity, simplicity, and cost competitiveness gave a wide popularity to this class of technologies, so that a large fraction of the market and of the research publications on biosensors are based on electrochemical transducers (Meadowset al., 1996).

In summary, electrochemical detectors have fast response, high sensitivity, small dimensions, low cost, easy signal integration, and are compatible with microfabrication technology. All this makes such class of transducer technologies the best candidate for the integration in microchip devices.

In the next sections, the most used electrochemical detection techniques are presented: amperometry and voltammetry.

7.3.1 Amperometry

Amperometric biosensors are based on the measurement of an oxidation or a reduction process occuring at the measuring electrode (Miret al., 2007). Such redox processes take place at a rate that depends on the voltage– called the excitation voltage–which is applied and kept constant by the measuring system. The signal measured is the oxidation or the reduction current as a function of time. The detection process generally involves the use of suitable enzymes which convert substrate molecules to measurable electroactive species. The majority of amperometric biosensors use receptor molecules immobilized on the surface of the measuring electrode, as in (Liuet al., 2004), but this is not necessary, as the electroactive product can be produced in the bulk by the enzyme, and then diffuse to the electrode, where it is detected.

The first amperometric biosensor developed for a commercial application is the glucose biosensor (1962 by L. C. Clark), which still has huge medical implications in the treatment and follow-up of diabetic patients.

(Harvey, 2000) The selectivity to glucose is guaranteed by the presence of a set of isolating membranes: a polycarbonate membrane, which is permeable to glucose and dissolved oxygen, a second membrane bound to glucose oxidase molecules, which catalyzes glucose oxidation to gluconolactone and hydrogen peroxide, and finally a cellulose acetate membrane, through which H2O2diffuses and reaches the platinum or gold electrode. This system makes use of an amperometric chemical sensor of hydrogen peroxide that Clark developed few years earlier.

The hydrogen peroxide reaches a system of two electrodes positioned behind the cellulose acetate membrane (Figure 7.5a). The two electrodes and the measurement setup are designed so that the system applies a constant voltage at the interface between a platinum electrode (called the working electrode) and the sample. In this case, the second electrode (called the counter electrode) is a silver ring treated with a surface coating of silver chloride.

At the electrode the hydrogen peroxide is oxidized by an excitation potential, following the reaction:

H2O2(aq)+2OH⁻(aq)O2(g)+2H2O(l)+2e⁻

The working scheme of the biosensor is represented in Figure 7.5b.

Recently, electrochemical mediators have been introduced to substitute hydrogen peroxide as a charge carrier. They operate at lower electrode potential, and give the advantage of decreasing the interferences by other electrochemically active species found in complex matrices (Farréet al., 2005).

In another configuration, amperometry can be used in DNA biosensors by employing electroactive indicators to monitor the hybridization event (Mikkelsenet al., 1996). Several biochemical strategies can be used: for example the electrochemical indicator can be a molecule that has higher affinity towards DNA in the double helix form, compared to the affinity for single strands. After exposing the biosensor to the sample, a certain amount of probe strands on the surface of the biosensor hybridizes with the target, with a consequent increase in the amount of detectable indicator bound to DNA on the electrode.

Another strategy uses a sandwich configuration (Figure 7.6), where a secondary reporter probe DNA hybridizes a free sequence of the target. If the reporter probe is tagged with a redox enzyme, such as horseradish peroxidase, a mediator can be continuously oxidized by the enzyme, and then reduced at the electrode, with a resulting reduction current measured by the amperometric electronics (Wang, 2000).

Figure 7.5 The glucose biosensor. In (a) the structure of the biosensor, which consists of a set of membranes deposited on the two measuring electrodes, is represented. In (b) the conceptual use of the membranes to pre-filter the sample, to host glucose oxidation, and to further filter the solution that reaches the electrodes for the measurement, is shown.

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7.3.2 Voltammetry

Voltammetry reveals the amount of redox molecules reduced or oxidized when the electrochemical cell is excited with a controlled potential. In voltammetric experiments, the potential is changed with time in a predetermined way, while the resulting current is measured. Herein, the working principles and the measurement interpretation of linear sweep voltammetry (LSV) and cyclic voltammetry (CV) are introduced.

LSV and CV are well established techniques, as they were developed in the first half of the 20th century (Matheson & Nichols, 1938), and described theoretically about ten years later by Randles and Sevcik (1948).

In LSV measurements, the excitation voltage is ramped at a constant rate while the current is monitored (Christensen & Hamnett, 1994). The resulting signal is displayed in a voltammogram (Figure 7.7a), where the current is represented as a function of the applied voltage. In CV, the voltage is ramped linearly as well, but the potential is swept back and forth one or several times between a minimum and a maximum value. The response is displayed in a voltammogram (Figure 7.7b), like for LSV signals.

In typical voltammograms of LSV and of CV, a peak-shaped current stands on top of a background current that increases with the voltage. The background current can be associated to the movement and collection of charges on the surface of theworking electrode(charging of the double layer capacitance), and in normal conditions it does not contain useful information. On the other hand, the informative parts of the curves are the peaks, whose shape depends on the concentration of the electroactive molecule, as well as on other features, such as the reversibility of the redox reaction.

In the special case of electroactive molecules bound to the surface, the rise of the redox peaks can be easily interpreted as the oxidation (or reduction) which starts at a sufficiently high potential, as defined Figure 7.7 Typical results of LSV (A) and CV (B) measurements. The voltammograms represent the current generated at varying excitation potential.

Figure 7.6 Example of a DNA sandwich configuration for an enzymatic detection of a target sequence.

by the Nernst equation. After some time, at a higher potential, most of the molecules initially available have reacted, thus the reaction drops off because the molecules already oxidized (or reduced) cannot oxidize (or reduce) again. When there are no more reduced (or oxidized) molecules available to react, the reaction stops, and the current goes back to the background value, completing the peak-shaped curve in Figure 7.7.

In the case of electroactive species floating in the bulk, the number of available redox molecules is not limited, but still the shape of the voltammetric response curve is very similar to the former case. In fact, even if the bulk contains a large quantity of redox molecules, only the ones that are close enough to the electrode so that they can diffuse to it in the timescale of the experiment are able to react with the electrode. In other words, distant molecules cannot reach the electrode. Also in this case, the availability of redox species close to the surface decreases as the reaction goes along. The shape of the oxidation and reduction peaks depends not only on the concentration of redox agents in solution, but also on the rate at which the voltage scan is performed. Also, if more than one redox molecules are used, the many signals can be identified and separated because they happen at different potentials.

Both for LSV and for CV setups, the most relevant operative feature used for DNA detection is the quantification of the redox agent that has reacted at the electrode. This amount is correlated with the total area of the peak. Both LSV and CV are very precise in this measurement with respect to other electrochemical techniques including amperometry, also because the measuring instrument, called the potentiostat, does not simply apply a voltage across two electrodes, but it uses a three electrodes setup to precisely control the potential drop across the interface between the one measuring electrode (called the working electrode) and the bulk (Figure 7.8). The electronics and the other two electrodes are specifically designed so that the result depends only on the molecules reacting at the working electrode, while it is independent of the reactions occurring elsewhere. The other electrodes are the reference electrode, which maintains a constant potential with respect to the bulk, and sets the reference for the control of voltages, and thecounter electrode, which closes the electrical circuit to allow the electrons exchanged between the working electrode and the sample to travel through the potentiostat to the sample again, and so it keeps electroneutrality.

Figure 7.8 Conventional electrochemical cell and schematization of the electrical circuit used for the measurement.

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7.4 THE ON-CHIP SIMPLIFIED ELECTROCHEMICAL TECHNIQUE