13.1.1 Thick-film biosensors

Screen-printing is a proven technology used for the manufacture of devices in a wide range of industries, such as electronics and solar cells, through to the graphic arts. The technique of screen-printing is capable of producing electrodes in the range of a few μm to 100 μm in thickness; the sensors can hence be defined as thick-film electrodes. Various geometries can be easily made by simple alteration of the screen design, and other features, such as reference, counter electrodes and dielectric layers can be readily added in further printing steps. Carbon is a low-cost material and when coupled with screen-printing manufacturing can produce sensors very economically in large numbers, allowing these to be viewed as disposable, ‘one shot’ devices. This offers the possibility of decentralised testing, particularly in medical applications, exemplified by their application in the blood glucose monitoring market. Screen-printed electrodes (SPEs) offer comparable electrochemical behaviour to more expensive electrode materials, such glassy carbon, but can be used once and discarded, hence removing the need for regeneration or pre-treatment between each measurement. This also allows for their use by relatively untrained personnel, as no knowledge of the more in-depth aspects of electrochemistry and preparation is needed.

Reagents such as enzymes, antibodies, mediators, catalysts and chelating agents can be readily immobilised to the surface of the carbon or mixed with the ink itself. Because of this, and the economic manner in which SPEs can be manufactured, they offer an attractive approach for the construction of many different types of biosensors. These advantages have led to a large, active interest, both in research into and for the commercial application of these devices. A number of reviews have recently reported on the application of these sensors (Domínguez-Renedo et al., 2007; Hart et al., 2004, 2007; Kalcher et al., 2009; Mahbubur Rahman et al., 2010; Pohanka and Skládal, 2008; Privett et al., 2010; Reyes Plata et al., 2010).

Figure 13.1 shows a representation of a screen-printed sensor, illustrating the general components that can be readily laid down by this technique. As can be seen, all the components required for the biosensor can be readily realised by screen-printing. Arrays of sensors can also be made, such as the ‘comb’ or array of screen-printed carbon electrodes (SPCEs), which have recently been developed for the determination of a number of organophosphate pesticides (OPs) at nM concentrations by Crew et al. (2011).

The following section gives a short overview of the underlying electrochemistry of these sensors and a brief history of the development of these devices. This is then followed by a review of the current applications of thick-film biosensors reported in the past four years. Emphasis is placed on sensor fabrication methods, operating details and performance characteristics for selected applications.

13.1.2 Definition of a biosensor

Biosensors can be defined in a number of ways (Ronkainen et al., 2010). Figure 13.2 shows a generalised scheme for the components of a thick-film or screen-printed electrode.

For the purposes of this review, biosensors have been defined using a version of the definition given by Théavenot et al. (1999). Hence, they are analytical devices that incorporate some form of biological material, such as tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc. These biological components are intimately associated with or integrated within a physicochemical transducer or transducing microsystem. Such devices usually produce a digital electronic signal that is proportional to the concentration of a specific analyte or group of analytes.

13.1.3 Practical experimental considerations

13.1.4 Electrochemical techniques

Common methods employed in the development of thick-film biosensors and their applications are amperometry, chronoamperometry, potentiometry, conductimetry and impedance spectroscopy. More recently, techniques such as stripping voltammetry have also been employed in conjugation with nanoparticle-based immunoassays, the application of which is described later. More detailed discussions dealing with the underlying mechanisms and parameters that can be probed and calculated by these techniques have been given (Adams, 1969; Compton and Banks, 2011; Galus, 1976; Grieshaber et al., 2008; Kissinger and Heineman, 1983, 1984; Mabbott, 1983; Pletcher, 1991; J. Wang, 2006). The following scheme gives a summary of the basic relationships:

13.1.5 History of thick-film biosensors

Several very good reviews of the history and development of biosensor technology have already been given (D’Orazio, 2003; Newman and Turner, 2005; Vaddiraju et al., 2010; J. Wang, 2001, 2008). The first reported biosensor was made by Clark and Lyons in 1962 based on a modification of the laboratory-based oxygen electrode (Clark and Lyons, 1962). Their innovative idea was to modify working electrodes with glucose oxidase and measure the oxygen consumed by the enzyme as an indirect measurement of the glucose present in the sample. Utilising this approach, it was found possible to analyse a range of other biomedically important compounds by employing other oxidase enzymes (Chistiansen and Jakobsen, 1995; Guilbault, 1968; Mascini and Guilbault, 1986). In 1975, Yellow Springs Instrument Company put this concept on the market, making the first commercially available dedicated blood glucose sensor. However, these first-generation biosensors were found to suffer from variations in oxygen tension and stoichiometric limitations that can occur in the sample.

In the mid-1970s, non-physiological electron acceptors were introduced to overcome these issues (Schlapfer et al., 1974). Examples of such mediators include quinones and conductive organic salts, such as ferricyanide and ferrocene derivatives. A large volume of work occurred on these second-generation biosensors in the 1980s (Cass et al., 1984; Frew and Hill, 1987). This decade also saw the introduction of the first modified disposable thick-film screen-printed glucose biosensors (Hilditch and Green, 1991; Matthews et al., 1987), an important advance allowing for the decentralised testing and development of cheap single-shot devices, such as that used in the commercial ExacTech blood glucose meter (Cardosi and Turner, 1991), launched by Medisense Inc. in 1987. The introduction of this device led to a sea change in biosensors technology with the introduction of pen-sized (Cardosi and Turner, 1991) and mobile phone (Heller and Feldman, 2010) amperometric-based devices. The current operation of most commercial glucose biosensors does not differ significantly from the ExacTech meter.

A number of drawbacks have been highlighted with these second-generation biosensors. The potential toxicity of the mediator and its possible leaching from the electrode means that the possibility of in vivo usage is problematic. It is also possible that even when utilising a mediator the presence of oxygen can still result in the oxidation of the reduced enzyme, leading to errors in quantification. In the 1990s, a number of investigations into co-immobilising the enzyme and the mediator at the electrode surface were made to address some of these issues. This can be achieved by immobilising the mediator and enzyme in a conductive redox polymer on the electrode surface (Gregg and Heller, 1990; Ohara et al., 1993). This offers other advantages; surrounding the enzyme in this way helps to transport electrons from the redox centre of the enzyme to the electrode in an array of rapid electron relays, so giving faster responses and higher current densities. More recently, further advances have been obtained by the introduction of nanotechnology. Due to the similar size of these materials and the enzyme, it is possible to ‘wire’ the redox centre of the enzyme directly to the electrode (Gilardi and Fantuzzi, 2001). Reportedly, this can overcome problems associated with oxygen dependence. In these third-generation biosensors, the enzyme redox centre is bound to the electrode surface. This allows for electrons to flow to and from the electrode, directly monitoring the enzyme’s redox behaviour and its interaction with the substrate.

Commercially, the biosensor market is still dominated by glucose biosensors, accounting for approximately 85% of the world biosensors market in 2004, which was then estimated to be US$5 billion. According to one recent report, this will reach US$11.5 billion by 2012 (Yoo and Lee, 2010), and grow to an estimated US$14.42 billion by 2016 (Anon, 2010). Predictions show that security and bio-defence have exhibited the largest increase in annual revenue growth rates.

Nevertheless, market penetration can be difficult (Luong et al., 2008). Recent reviews highlight the problems and issues that commercialisation faces (Luong et al., 2008; Siontorou and Batzias, 2010). The reported cost of introducing the FreeStyle glucose biosensor to the commercial biosensor market was US$100 million, a sum beyond the means of most small companies (Heller and Feldman, 2010). At present, there are 18 companies in the USA producing 56 different glucose biosensors; however, only four major companies, Abbott, Bayer, LifeScan, and Roche, account for over 90% of the USA market (Yoo and Lee, 2010).

Future developments will properly focus on the further application of nanotechnology and techniques such as plastic electronics (Sokolov et al., 2009) and synthetic biology (Keasling, 2008). Other new markets will be identified, such as coronary heart disease, which at present represents the main cause of death throughout the world (World Health Organisation, 2008) and consequently presents an opportunity for the development of biosensors capable of its early diagnosis.

13.2.1 Screen-printed glucose biosensors

Usiu and Ozkan (2007) have reviewed the application of SPEs and carbon electrodes for the determination of a number of pharmaceuticals. Arguably, the most successful both in terms of commercial successes and related research activity is the glucose biosensor (Hu, 2009; Lee, 2008; J. Wang, 2001, 2008). As can be seen from Table 13.1, the majority of these utilise glucose oxidise, EC 1.1.3.4 (GOx) with a suitable mediator for the measurement of the enzymatically generated hydrogen peroxide. GOx offers several advantages as it is a stable enzyme, allowing for the manufacture of sensors that are durable and reproducible. In the presence of oxygen, GOx catalyses via its redox centre flavin adenine dinucleotide (FAD) the formation of gluconolactone and hydrogen peroxide (Eq. [13.7]). Consequently, glucose concentrations can be determined by the consumption of oxygen or by the concentration of hydrogen peroxide produced, as described earlier. The former approach suffers from a number of problems, as the ambient level of oxygen must be controlled and needs to be constant, a situation that is not readily obtainable in real blood samples. Oxygen also exhibits a fairly high reduction potential in which other sample components may interfere, and its redox reaction is not readily reversible.

[13.7]

The latter approach, of monitoring the hydrogen peroxide generated, offers advantages over the measurement, allowing for simpler and more selective amperometric measurements to be utilised. The direct electrochemical determination of hydrogen peroxide requires high applied potentials, leaving the approach open to interference from a number of common blood components, such as uric acid and ascorbic acid. However, it is possible to employ some form of mediator or catalyst to lower the applied potentials required. This also has the added advantage of lowering the background currents and lessens the extent of interference of other sample components.

13.2.2 Glucose dehydrogenase-based screen-printed biosensors

Compared to GOx-based systems, glucose dehydrogenase (GDH) enzymes can offer certain advantages, such as oxygen independence, allowing for their use in low oxygen conditions such as fermentation. However, problems have been reported with the utilisation of the pyrroloquindine quinone (PQQ)-dependent form of GDH as this has a high relative activity in the presence of maltose, presenting a serious problem in the determination of blood glucose levels and consequent misdiagnoses. Again, as with H₂O₂, the direct determination of nicotinamide adenine dinucleotide (NADH) requires high applied potentials for its determination. However, it is possible to monitor this with the use of a suitable mediator.

Figure 13.7 illustrates that the underlying mechanism utilised for one such mediated approach is that made by Piano et al. (2010a) as part of a flow injection system for the determination of glucose in serum. The SPCE was modified with the electrochemical mediator Meldola’s Blue–Reinecke Salt (MBRS), coated with GDH (from Bacillussp.), and NAD⁺. A cellulose acetate layer was deposited on top of the device to act as a perm-selective membrane. The developed prototype biosensors were found to be stable for at least 240 days at a temperature of 37 °C. Using an applied potential of + 50 mV, the response was found to be linear over the range of 0.075–30 mM glucose, with the former representing the detection limit.

Screen-printing technology has focused on the production of two-dimensional sensors, planar sensors, which can be then integrated with devices produced by other production techniques, such as plastic injection moulding, if further functionality is required. Nevertheless, alternative approaches have been recently investigated, such as that reported recently by Santiago et al. (2010). They have utilised screen-printing to fabricate three-dimensional carbon micro-devices. These screen-printed microsystems consisted of a microchannel structure formed by two electrodes printed in parallel on a polyester film substrate; the sides of these act as the walls of the microchannel electrochemical cell. The working electrode was a graphite ink, and the reference/counter electrode was Ag/AgCl. The screen-printed carbon-exposed edge area was reported to be c. 0.1 cm² with a final volume of the microchannel being in the region of 20 μl. The developed sensor was applied to the ultrasensitive electrochemical detection of alkaline phosphatase, obtaining a detection limit of below 10^− 12 M for GOx-and of 10^− 15 M for GDH-modified systems using a 15 minutes of incubation time. A potential pulse polypyrrole electropolymerised generated film was used to entrap either GOx or GDH to the working carbon electrode. This is reported to be superior to common methods of entrapment, as it is more reproducible and more stable.

13.2.3 Lactate

The monitoring of lactate is important for disciplines such as sport science, as it is produced in increasing amounts during physical exercise. Its major use is in the food and beverage industries, where it is used in cider and wine manufacture, and as an acidulant and for the manufacture of bread additives. It is also used as a chemical intermediate in textile finishing and in leather tanning. Recently, its determination has been utilised for monitoring toxic events in cell culture media at a screen-printed biosensor (Rawson et al., 2009).

Principally, two enzymes are used for the production of lactate biosensors; lactate oxidase (LOX) and lactate dehydrogenase (LDH). The combination of these with SPCEs has been shown to be a highly effective method and has been recently been reviewed (Hart et al., 2004, 2007; Kalcher et al., 2009; Nikolaus and Strehlitz, 2008; Privett et al., 2010; Zhou et al., 2009). A number of these devices are summarised in Table 13.2. LOX is one of the most commonly reported for biosensor construction; in the presence of oxygen it catalyses the production of pyruvate and hydrogen peroxide (EQ. [13.8]):

[13.8]

As with GDH, described in the previous section, alternatively LDH can be utilised (Eq. [13.9]). The enzyme does not require the presence of oxygen for the detection of lactate and so can be used in anaerobic conditions.

[13.9]

Piano et al. (2010b) have recently reported on the use of SPCEs modified with MBRS complex for the flow injection analysis of lactate with LDH. This was found to be an advantage over the previously reported MBRS-mediated electrodes, as the Reinecke salt was found to be insoluble and not leached from the SPCE, allowing for flow injection analysis to be successfully performed. The screen-printed biosensors were stable for at least 17 days when stored at 4 ° C.

13.2.4 Ethanol biosensors

Previous investigations on screen-printed alcohol biosensors have been reviewed (Hart et al., 2004; Kalcher et al., 2009). More recently, alcohol screen-print-based sensors have been developed for use in the field of forensic science investigations. Luo et al. (2008) have utilised such an approach for the forensic determination of blood alcohol utilising alcohol dehydrogenase (ADH) (Luo et al., 2008). This was fabricated by cross-linking ADH with glutaraldehyde and NAD⁺ on a screen-printed electrode previously modified with nafion. The electrode was modified with MBRS via repeated voltammetric scanning of a 1 mM solution in 0.1 M phosphate pH 8 buffer. The developed biosensor was able to determine serum alcohol concentrations over the range 1 to 5 mM. This could be used to determine alcohol concentrations in intoxicated drivers. However, post-mortem analysis of blood alcohol appears impossible, since the biosensor cannot differentiate between common alcohols and aldehydes found in these samples.

Mediator-free screen-printed alcohol and acetaldehyde biosensors have recently been reported for their determination in breath (Gessei et al., 2009). These ‘bio-sniffers’ were formed by screen-printing a carbon working electrode on one side of a filter paper substrate and on the reverse side, the Ag/AgCl electrode. The working electrodes were modified by either immobilizing alcohol oxidase (AOx) or aldehyde dehydrogenase (ALDH). AOx oxidises ethanol to acetaldehyde and hydrogen peroxide. The latter was detected directly by monitoring the amperometric oxidation peak as observed at + 0.9 V. In the presence of NAD⁺, acetaldehyde is oxidised to acetic acid, NADH and proton by ALDH. Hence by monitoring amperometric oxidation of NADH at + 0.65 V, the concentrations of acetaldehyde could be determined. Good responses to ethanol and acetaldehyde vapour were recorded over the ranges 1.0 to 100 ppm and 0.2 to 4.0 ppm, respectively, covering the ranges expected to be seen in human breath after consumption of alcoholic beverages.

13.2.5 Cholesterol

The term ‘total cholesterol’ refers to the total concentration of the two forms of cholesterol present in blood: the free form and the esterified form. To measure total cholesterol, two enzymatic reactions have been utilised. Cholesterol esterase (ChE) is employed to convert ester to cholesterol, which together with the original cholesterol can then be measured using cholesterol oxidase (ChOx), which in the presence of oxygen converts cholesterol to cholest-4-en-3-one and hydrogen peroxide. A number of previous applications of SPCEs for the determination of cholesterol have been reviewed by Hart et al. (2004, 2007).

Using a similar approach to that described for glucose and lactate above, the hydrogen peroxide generated can be monitored as an indirect way to quantify the concentration of cholesterol present in the sample. Shih et al. (2009) have used a similar approach to this, however, in utilising the catalytic reduction of hydrogen peroxide at a SPCE modified with Fe₃O₄, ChOx and ChE. Using a 10 μl sample volume, the screen-printed sensor was able to gain a detection limit of 19.4 mg/dl with a linear range from 100 to 400 mg/dl for total cholesterol.

Utilising electrodes fabricated from metals such as Au or Pt, it is possible to measure hydrogen peroxide at relatively low applied potentials. Shen and Lui (2007) have used an Au electrode with a self-assembled ChOx monolayer for the determination of cholesterol. The Au working was part of a screen-printed three-electrode cell, consisting of a screen-printed Ag/AgCl reference and Au counter electrode. Gold was found to be superior to Pt as a working electrode, giving better sensitivity.

Alternatives to ChOx have been also investigated at SPEs, such as a cholesterol biosensor based on cytochrome P450scc (Carrara et al., 2008). P450scc is highly specific towards cholesterol and its metabolites, catalysing the side-chain cleavage of C27 cholesterol to C21 pregnenolone in the presence of molecular oxygen and NADPH-ferri hemoprotein reductase. This approach has been investigated at a rhodium–graphite SPE modified with multi-walled carbon nanotubes (MWCNT) and cytochrome P450scc as catalytic enzyme. Recently its activity has also been shown to be enhanced at SPEs modified with gold nanoparticles (AuNP; Shumyantseva et al., 2005).

13.2.6 Miscellaneous proteins, peptides and amino acids

The amylases group of enzymes break down starches and glycogen to maltose and are widely distributed in both animal and plant tissues. The main biological function of salivary alpha-amylase (sAA) is the enzymatic digestion of carbohydrates, converting these to maltose and maltotriose. Consequently, during periods of energy expenditure and stress, elevated concentrations are seen and can be utilised as a method of evaluating and identifying such periods. Mahosenaho et al. (2010) have utilised a three-enzyme SPCE biosensor for its determination of sAA activity, but enzymatic products of this reaction are not readily determinable. However, utilising α-glucosidase, maltose, which can be hydrolysed to α-glucose, once generated, this can be covered by mutarotase into β-D-glucose, the substrate of GOx. The generated β-D-glucose is then hydrolysed to gluconic acid and hydrogen peroxide by GOx. The resulting hydrogen peroxide generated can then be measured via chronoamperometry utilising its Prussian blue-mediated reduction.

13.2.7 Nucleic acids and purines

SPCEs have recently been utilised for the monitoring of genotoxicity (Rawson et al., 2010). Investigations were made using differential pulse voltammetry (DPV) to identify the oxidation products generated from the interaction of Cr^6 + with double-stranded DNA. Using such an approach, the reaction products could be readily screened in a single voltammetric scan. In a separate study, Ferancová et al. (2007) have investigated the effects of Sn^2 + and As^3 + at a DNA multi-walled nanotube modified SPCE using [Co(phen)₃]^3 + as an electrochemical DNA cleavage marker. Here, by utilising DPV, it was found possible to readily identify the DNA damage.

A number of drugs are known to interact with DNA and recently these interactions have been monitored voltammetrically at a SPCE. Ravera et al. (2007) has demonstrated that it possible to monitor covalent binding at N7 of guanine, electrostatic interactions, and intercalation resulting from interactions of antitumoral Pt-, Ru-, and Ti-based metallodrugs at DNA immobilised on screen-printed electrodes.

13.2.8 Food pathogens

13.2.9 Viruses

Screen-printed biosensors have been developed for the detection of a number of important viruses and recently been reviewed (Yadav et al., 2010). Mahmoud and Luong (2010) have reported the development of a screen-printed gold electrode (SPGE) surface modified with either AuNPs or thiolated single-walled carbon nanotubes/gold nanoparticles (SWCNT/AuNPs) for the determination of human immunodeficiency virus 1 (HIV-1) in sera. DPV investigations showed a linear relationship between shifts in peak potential and increasing numbers of HIV-1 viruses over the range 6.8 × 10² to 1.11 × 10⁶ copies/mL in HIV-infected serum samples.

13.3.1 Phosphate

The determination of inorganic phosphate (Pi) is important both environmentally and medicinally. It is an important parameter for both environmental and potable water quality. In the presence of inorganic phosphate, oxygen and cofactors, pyruvate can be decarboxylated by pyruvate oxidase to give H₂O₂, acetyl phosphate and CO₂. It is possible to utilise the enzymatically generated H₂O₂ to quantify the corresponding concentration of Pi. Gilbert et al. (2009, 2010) have utilised this approach for the determination of Pi in human urine and water samples at a CoPC-modified SPCE. Utilising the CoPC-mediated electrocatalytic oxidation of the generated peroxide, a linear response for Pi was gained over the range 2.27 × 10^− 5 to 1.81 × 10^− 4 M, with a corresponding limit of detection of 4.27 × 10^− 6 M. The principles underlying the mechanism of this biosensor are shown in Fig. 13.8.

13.3.2 Nitrates and nitrites

Nitrates are of both commercial and environmental importance, widely used in both explosives and fertilisers. One particular area of concern is pollution of the environment from agricultural run-off of nitrates to rivers, lakes and potentially to drinking water. Consequently, there is increasing interest in the development of sensors capable of determining nitrate levels in the environment and water-courses. Recently, Albanese et al. (2010) have utilised SPCEs modified with nitrate reductase obtained for Escherichia coli. The two mediators, Azure A or methyl viologen, were investigated. However, Azure A was found to be less effective, open to problems such as leaching into the sample solution. Using the optimised methyl viologen-based sensor as part of a flow injection system, detection limits of 0.1 mM with a linear to 10 mM nitrate were obtained.

Quan et al. (2005) have immobilised nitrate reductase derived from yeast using a polymer (poly(vinyl alcohol))-entrapment method to both a glassy carbon electrode and a SPCE. Utilising the electron-transfer mediator, methyl viologen, at an applied potential of − 0.90 V (vs. Ag/AgCl), a linear range of 15–250 μM and a detection limit of 5.5 μM was reported. The utility of the proposed sensor system was demonstrated by determining nitrate in Han River water samples, before and after treatment. Good agreement was obtained between the results gained by both ion chromatography and spectroscopic determination and the SPCE biosensor. The activity of nitrate reductase was affected by the presence of oxygen; however, this effect was readily overcome by the addition of a sulphite to the sample as an oxygen scavenger.

13.3.3 Pesticides and chemical warfare agents

A number of SPCE-based applications for the determination of OPs and chemical warfare agents have previously been reviewed (Amine et al., 2006; Andreescu and Marty, 2006; Arduini et al., 2010a; Hart et al., 2004, 2007; Kalcher et al., 2009; Miao et al., 2010; Wanekaya et al., 2008) and Table 13.3 summarises some recent developments made in this area. OPs and warfare agents act by inhibiting the enzyme acetylcholinesterase (AChE) required for normal nervous function, catalysing the hydrolysis of the neurotransmitter acetylcholine into acetate and choline. The inhibition of AChE results in the build-up of acetylcholine, which interferes with muscular responses, leading to a number of symptoms and eventually death due to respiratory failure (Miao et al., 2010).

Amperometric measurement of enzyme inhibition can be made in a number of ways, such as by the measurement of thiocholine produced by the action of AChE on acetylthiocholine, or via the production of hydrogen peroxide formed by the oxidation of choline by choline oxidase (Wu et al., 2009). Similarly, p-aminophenol formed from the hydrolysis of p-aminophenyl acetate by AChE can also be readily determined. However, AChE activity can be inhibited by neurotoxins other than Ops, such as carbamates and heavy metals, and hence selectivity can suffer.

Organophosphate hydrolase (OPH) has also been utilised in such sensors (Joshi et al., 2006) exhibiting broad substrate specificity hydrolysing a number of OPs and chemical warfare agents such as sarin (O-isopropy lmethylphosphonofluoridate) and soman to p-nitrophenol, which can be detected electrochemically. Reportedly, this approach, utilising OPH, offers advantages for the biosensing of OPs as they act as the substrates for the enzyme rather than an inhibitor.

Arduini et al. (2007) have utilised a SPCE modified with an in-situ generated layer of Prussian blue formed from the addition of potassium ferricyanide in 10 mM HCl and 0.1 M ferric chloride in 10 mM HCl directly on the surface of the working electrode. Butyrylthiocholine (BChE) or AChE was then immobilised on the SPCE surface with glutaraldehyde and a nafion/BSA membrane was added across the surface. Sarin gas measurements were made by the addition of a 5 μl of a pH 7.4 phosphate buffer, the resulting depletion in enzyme activity was found to be dependent on sarin gas exposure and, for a 30 s incubation time, inhibitions of 43% and 34%, were obtained for 0.1 and 0.5 mg m^− 3, respectively.

13.3.4 Whole cell photosynthetic pesticide biosensors

A contrasting approach is the application of whole cell photosynthetic-based biosensors for the determination of pesticides (Tibuzzi et al., 2008), recently reviewed by Scognamiglio et al. (2010). Several herbicides have been investigated in this manner utilising a screen-printed carbon transducer. Interestingly, screen-printing of actual algal cells has also been shown possible (Shitanda et al., 2009). The algal ink was prepared by mixing unicellular microalga Chlorella vulgaris cells, carbon nanotubes and a sodium alginate solution. The modified ink was screen-printed directly onto the surface of a SPCE. Photosynthetically generated oxygen was monitored amperometically as part of a flow injection system and exposures to atrazine (12 μM) and Duiron (DCMU) (1 μM) resulted in a 50% depletion in oxygen production.

13.3.5 Polychlorinated biphenyls

An important part of many assays is the isolation and separation of the target analyte from the sample and a wide number of procedures have been developed to achieve this goal. Recently, attention has focused on the development and application of magnetic separation techniques, which employ small magnetic particles (Šafařik et al., 1999, 2004; Saiyed et al., 2003). These consist mainly of nano-sized iron oxide particles (Fe₃O₄ or γ-Fe₂O₃), which are suspended in a carrier liquid. Frequently referred to as magnetic, the majority of the particles presently used are in fact superparamagnetic, consequently requiring an external magnetic field to be magnetic, and once this is removed they redisperse. This allows for a simple method for concentrating and isolating the particles from the sample matrix. It is possible to modify these particles with different ligands (streptavidin, Protein A, etc.) and chemically derivatised particles with specific recognition groups such as monoclonal and polyclonal antibodies. This allows for the possibility of using these modified particles to isolate analytes to the magnetic bead, which can then be easily isolated by the application of a magnetic field.

Magnetic bead-based screen-printed biosensors have been reported assays for the determination of several polychlorinated biphenyls (PCBs) (Centi et al., 2007). These are a group of highly stable and persistent industrial pollutants. They have been shown to undergo bioaccumulation and as a result are responsible for a number of environmental issues and poisonings.

Centi et al. (2006) have utilised magnetic beads modified with protein G for subsequent coating with the PCB antibodies, immunoglobalin G (IgG) anti-PCB28 or IgG anti-PCB77. A 50 mL suspension of these antibody-coated beads was then mixed with 940 mL of sample and 10 mL of alkaline phosphatase-labelled antibodies. After incubation for 20 minutes, the beads were magnetically separated down onto the SPCE surface and the supernatant removed.

Then the enzymatic substrate (α-naphthyl phosphate) in buffer was deposited on the SPCE to close the electric circuit. After five minutes, the enzymatic product was determined by DPV. Detection limits were found to be between 0.3 and 0.8 μg/l. The developed immunosensors were successfully applied for detection of PCBs in marine sediment extracts. Lin et al. (2008a) have utilised a similar approach with antibody-labelled magnetic beads. However, this approach utilised a HRP-labelled PCB (HRP–PCB) and an alkali phosphate-labelled PCB to compete with the native sample PCB. Once the competitive assay was complete, the beads were magnetically isolated and the enzyme substrate added for determination by square wave voltammetry at the SPCE.

13.3.6 Polyaromatic hydrocarbons

Polyaromatic hydrocarbons (PAHs) are known to interact with DNA producing a number of differing products, some of which result in strand cross-linking and eventual mutations. However, it is possible to utilise there phenomena as a means to determine their presence and concentration. Del Carlo et al. (2008) have used DNA electrochemical biosensors based on four different types of DNA (calf thymus ssDNA, calf thymus dsDNA, salmon testis ssDNA and salmon testis dsDNA). Using benzo(a)anthracene and phenanthrene as model compounds, they showed that variation in the guanine signal revealed the interaction of PAHs with the immobilised DNA. The salmon ssDNA modified device was found to be the most successful and was further evaluated for benzo(a)anthracene, fluorene, indeno(1,2,3-cd)pyrene, anthracene and phenanthrene in 5–40 ng/mL solutions, and for benzo(a)pyrene (5–50 ng/mL). A concentration-dependent variation of the DNA guanine oxidation peak was observed for all compounds. Using the salmon ssDNA-modified device, the PAH concentrations present in samples of contaminated mussels (Mytilus galloprovincialis) were successfully investigated.

13.3.7 Biosensors for metal ion detection

A large volume of research has focused the determination of metal ions utilising stripping voltammetry at either at SPEs modified with Bi or Hg or at plain unmodified SPEs. These are not classed as biosensors and are consequently outside the scope of this review. However, a number of extensive reviews on these sensors have been recently reported (Arduini et al., 2010a; Honeychurch and Hart, 2003; Kokkinos and Economou, 2008; Stozhko et al., 2008; Svancara et al., 2010). Nevertheless, as mentioned earlier in the pesticide section above, other toxins, such as heavy metals, can also readily inhibit enzyme activity (Andreescu and Marty, 2006), an effect that can be harnessed to develop biosensors for their determination (Amine and Mohammadi, 2007). One such recent example utilises the inhibition effect that Hg^2 + ions exhibit on the enzymatic activity of urease, which can be monitored at AuNP-modified SPCEs. (Domínguez-Renedo et al., 2009). Urease was immobilised to the SPCE surface by cross-linking with bovine serum albumin and glutaraldehyde. Metallic AuNPs were found to enhance the sensitivity of the sensor. These were electrochemically deposited on the modified carbon working electrode surface, using a 0.1 mM solution of HAuCl₄ in 0.5 M H₂SO₄. Mercury measurements were undertaken once a steady-state current was obtained after the addition of urea. Additions of Hg^2 + resulted in a current decrease proportional to the amount of Hg^2 + added. Using this approach, a detection limit for Hg^2 + of 4.2 μM was obtained. The sensor was found capable of successfully determining human plasma samples fortified with 1.0 μM Hg^2 +.

A similar approach has been utilised by Sanllorente-Méndez et al. (2010) for determining arsenite (AsO₃^3 −) as, in this case, an AchE-modified SPCE. Arsenite concentrations were measured utilising the inhibitory effect this species has on the activity of AChE on the conversion of acetylthiocholine iodide to thiocholine iodide and acetic acid. As thiocholine is electrochemically active, the product of this reaction can be readily monitored amperometically utilising the optimised applied potential of + 0.6 V. As additions of As^3 + inhibit the enzymatic reaction, a corresponding drop in the amperometric signal is gained with increasing As^3 + concentration. Using such an approach, a detection limit of 1.1 × 10^− 8 M with a corresponding linear range up to 1 × 10^− 7 M As^3 + was obtained. The developed sensor was capable of determining 1.0 μM As^3 + in tap water. Further investigation on a certified As^5 + water sample was possible on adding sodium thiosulphate to facilitate the reduction of the AChE inert As^5 + to As^3 +.

The interaction of arsenate with L-cysteine has been successfully utilised as the basis of a biosensor for the determination of As in a number of environmental water samples (Sarkar et al., 2010). L-cysteine reduces arsenate to arsenite and is in the process oxidized to L-cystine. The reaction involves electron transfer at the working electrode and thus the response can be monitored amperometically. By immobilising L-cysteine to the surface of the SPCE, it was found possible to gain a limit of detection of between 1.2 and 4.6 ng/mL. Interferences for oxidising agents such as nitrate were investigated, and no effects were found from nitrate concentrations commonly present in drinking water.

SPCE biosensors based on the well understood glucose oxidase enzyme system have also been described for the determination of a number of metal ions, including; Hg^2 +, Ag⁺, Cu^2 +, Cd^2 +, Co^2 + and Ni^2 + (Guascito et al., 2009). As part of a flow injection system the sensor was able to obtain detection limits in the low ppm with Ag⁺ detection limits in the ppb region.

Shewanellasp. is known for its role in biogeochemistry, as a metal-reducing bacterium involved in the cycling of iron, manganese, trace elements and phosphates. Prasad et al. (2009) have demonstrated the use of these bacteria as the electron transfer material for electrochemical sensors in the determination of arsenite, hydrogen peroxide and nitrite. Shewanellasp. CC-GIMA-1 bacterial suspension (prepared in 0.1 M, pH 7.4 phosphate-buffered saline solution) was drop-coated on the electrode surface and allowed to settle under room temperature for 1 hour. This simple approach was found better than growing bacterial biofilm on electrode material, which was laborious and time consuming. Cyclic voltammetric investigations showed the bacteria exhibit electron exchange with the SPE surface, behaving similarly to a bacterial biofilm. It is well known that graphite electrodes, containing a carbon–oxygen functional group similar to humates, provide a natural habitat for the bacteria. The bacteria films were found to be able to sustain themselves by using carbon–oxygen functionalities of SPCE as electron acceptors for respiration. The effects of arsenite (50–500 μM) hydrogen peroxide (50 μM–2.5 mM), and nitrite (100–500 μM) were studied by cyclic voltammetry and the reduction peak current of the Shewanellasp. modified electrode was found to increase in the case of As from c. 6 μA–10 μA for an addition of 500 μM arsenite. Additions of Fe^3 + were also found to exhibit similar voltammetric behaviour.

13.3.8 Immunoassay based on stripping voltammetry of metal nanoparticle labels

Nanoparticles, owing to their similarity in sizes to a number of important biomolecules such as antibodies or DNA strands, make good candidates for labels in immunoassays. This is practically true if Au or other heavy metal nanoparticles are utilised, allowing for sensitive electrochemical techniques such as stripping voltammetry to be utilised. This approach has recently been reviewed by Escosura-Muniz and Merkoci (2010). One of the first reports using such an approach was made by Dequaire et al. (2000), who used AuNPs as labels for the determination of IgG. Quantification of the AuNPs was achieved by anodic stripping voltammetry (ASV) at a screen-printed electrode, gaining a detection limit of c. 450 pg/mL (3 pM). Similarly low detection limits (20 pg/mL) were reported using Cd nanoparticles labels for the determination of prostate-specific antigen (PSA) by Liu et al. (2007, 2008) and Lin et al. (2008b), again utilising ASV as the measurement step at a SPCE, a detection limit of 0.02 ng/mL being reported. Brainina et al. (2010) have utilised Fe nanoparticles for the determination of Salmonella typhimurium (strain SL 7207) utilising a magnetic-based separation, followed by digestion of the Fe nanoparticles in a mixture of 0.36 M H₂SO₄ and 0.28 M HNO₃. The concentration of Fe^3 + liberated was quantified by the cathodic adsorptive stripping voltammetry of its catechol complex in 0.1 M sodium acetate.

Silver nanoparticles have been used in a similar manner by Chikae et al. (2010). These have been utilised as part of a sandwich-type immunoassay for human chorionic gonadotropin (hCG). The biosensor comprises a primary antibody immobilised on the surface of a SPCE and an Ag nanoparticle-labelled secondary antibody. After the immunoreaction, the concentration of Ag captured on the SPCE surface was measured by ASV. To achieve this, the immobilised Ag metal nanoparticle was first electrochemically oxidised to form Ag⁺ ions by applying by a potential of + 1.6 V. The concentration of these Ag⁺ was then determined by a differential pulse voltammeter. Utilising this approach, a detection limit of 7.2 pg/mL hCG was obtained with a linear range from 10 to 1000 pg/mL.

13.3.9 Food

The application of SPCEs and biosensors for the determination of a wide range of analytes in food has recently been reviewed (Cock et al., 2009; Ricci et al., 2007a; Tudorache and Bala, 2007; Viswanathan et al., 2009). Romanazzo et al. (2009, 2010) have used an electrochemical competitive enzyme-linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain at a SPCE as a sensing platform. Their developed sensor was able to determine the total amount of the mycotoxins, HT-2 and T-2. The magnetic beads were first coated by immobilizing a HT2-KLH conjugate toxin, and after a blocking step to avoid non-specific absorption, a monoclonal antibody was then added allowing for competition between the immobilised HT-2 and free HT-2 or T-2 present in the sample. Hence at the end of the competition step, the amount of the monoclonal antibody linked to the immobilised HT-2 at the magnetic beads is inversely proportional to the amount of toxin that was present in the sample solution. A secondary antibody labelled with alkaline phosphatase is then added to bind with the immobilised HT-2. The final measurement step was performed by dropping an aliquot of magnetic bead suspension onto the surface of a screen-printed working electrode. The magnetic beads are immobilised and concentrated by means of a magnet placed precisely under the SPE. After two minutes of incubation between magnetic beads and a substrate for alkaline phosphatase, the enzymatic product is detected by DPV, giving a voltammetric signal proportional to the original mycotoxins’ sample concentration.

Recently, the inhibition of alkaline phosphatase by caffeine has been utilised for its determination in tea, coffee and cola at the Au SPE biosensor (Akyilmaz and Turemis, 2010). By measuring the decrease in conversion of the substrate, p-nitrophenol phosphate by alkaline phosphatase, a linear relationship with caffeine concentration was found from 0.1 μM to 10 μM, with a corresponding limit of detection of 8 nM.

Acetaldehyde is commonly found in alcoholic beverages, resulting from oxidation of ethanol by ADHs and from the fermentation of pyruvate by pyruvate decarboxylase. It is usually present in wine at concentrations between 60 μM and 300 μM. Its determination is important in such beverages as it interferes with the antioxidant and antiseptic function of sulphur dioxide. Noguer et al. (2004) have utilised a MBRS-modified SPE, modified with a sol-gel matrix containing AILDH and NAD⁺ for the determination of acetaldehyde in wine as part of a flow injection system. Acetaldehyde can be determined in beverages using enzymatic oxidation by an NAD⁺-dependant ALDH according to the reaction shown in equation (Eq. [13.10]).

[13.10]

The NADH formed is stoichiometric with the amount of acetaldehyde present and can be readily amperometically determined via its interaction with MBRS as described in the section describing glucose determination with GDH using an applied potential of − 150 mV.

The ability to monitor the fertility of animals is an important parameter in animal husbandry. Recently, Pemberton and Hart (2007) have developed a biosensor for the determination of progesterone in bovine milk. Oestrus onset is indicated by a rapid fall in the concentration of milk progesterone to below 2–5 ng/mL. The biosensor was based on competitive immunoassay either in batch mode (chronoamperometric measurement) or in stop–flow mode in a continuous flow system (amperometric). Utilising the chronoamperometric batch mode, a detection limit of around 5 ng/mL was obtained suitable for monitoring the onset of oestrus, giving a result within 40 minutes. However, this approach requires the use of a blank measurement step to cater for any variations due to electroactive interferents or surface-adsorbed species present in the milk sample. This problem can be overcome by utilising DPV instead of chronoamperometry. The stop–flow amperometric approach has the advantage of requiring no blank subtraction, but requires 60 minutes to complete and lends itself more to off-site rather than on-site analysis.

13.3.10 Aflatoxins

Aflatoxins produced by moulds Aspergillus flavus and Aspergillus parasiticus are carcinogenic to humans. Aflatoxin has an inhibitory effect on AchE, allowing for its measurement using a choline oxidase amperometric biosensor. Ben Rejeb et al. (2009) have utilised a choline oxidase enzyme-modified SPE for the determination of aflatoxin. The residual activity of the enzyme was calculated after application of the sample, hence giving an indirect detection of the aflatoxin that may be present. The AChE activity is highly pH-dependent with best results obtained at pH 7.4. The amperometric method allowed for the detection of low aflatoxin concentrations that cannot be detected by the classical spectrophotometry assay because of the omission of the dilution step commonly used for this latter approach.

13.3.11 Antibiotics

Sulphonamide antibiotics such as sulfamethoxazole (SMX) and sulfapyridine (SP) are widely used and have been employed for the control of diseases such as American foulbrood (Bacillus larvae) and European foulbrood (Streptococcus pluton) seen in the honey-bee. Consequently, it is necessary to monitor their residues in the honey produced. Centi et al. (2010) have utilised a screen-printed eight-electrode array for their determination. Each working electrode was printed with its own screen-printed silver pseudo-reference electrode and graphite counter electrode. An eight-hole methacrylate box was fixed onto the strip, so giving an eight-cell electrochemical array. Each array was then placed in a holding block with eight magnet bars. The assay was based on a competitive scheme, in which sulphonamides (competitors) compete with a fixed concentration of 2-(4-aminobenzensulfonylamino)ethanoic acid–alkaline phosphatase labelled (SA1-(CH2)5-AP), against antibodies immobilised on magnetic beads. Once the affinity reaction occurred, the electrochemical measurement was carried out by DPV to evaluate the extent of the immunochemical reaction. It was found necessary to utilise a sample clean-up step using solid phase extraction, as one of the sulphonamides was found to react with the reducing sugars in honey.

Tsekenis et al. (2008) have utilised a labelless immunosensor for the antibiotic ciprofloxacin in milk using AC impedance. Polyaniline was electrodeposited onto the SPE to immobilise a biotinylated antibody for ciprofloxacin using classical avidin–biotin interactions. The antibody-modified electrodes were exposed to solutions of antigen in milk and interrogated using AC impedance. The faradaic component of the impedance of the electrodes was found to increase with the growing concentration of antigen. Control sensors were fabricated using antibodies specific to species not found in milk. Calibration curves were obtained by subtraction of the responses for specific and control antibody-based sensors, so eliminating the effects of non-specific adsorption of antigen. Sensors exposed to ciprofloxacin in milk gave increases in impedance, whereas ciprofloxacin in phosphate buffer led to decreases, indicating the possibility of developing sensors that can both detect and differentiate between free and chelated antigen. A linear dependence of impedance current with log₁₀ of concentration was obtained between 0.1 and 100 ng/mL ciprofloxacin.

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