Biosensors already developed

Although a variety of whole-cell-based bacterial sensors have been applied in environmental assays for pollutant monitoring, generally they display a poor response to herbicides (Table 3).

If the electron transfer from the reaction centre to the quinone pool is blocked, such as during the binding of the photosynthetically active pesticides, these parameters change dramatically and can be monitored by electro-optical analysis in a pesticide concentration dependent manner (Figure 6). In this context, an optical biosensor based on the green photosynthetic alga Chlamydomonas reinhardtii described by Tibuzzi and coworkers (2007) was employed to monitor several classes of herbicides, such as atrazine, diuron, ioxynil, terbuthylazine, prometryn and linuron, in a low concentration range (10-8-10-10 M) (Table 3). In particular, a miniaturized optical biosensor instrument was designed and produced for multiarray fluorescence measurements of several biomediators in series, with applications in environmental monitoring and agrofood analysis. In the work by Rea and coworkers (2009), a computational study was performed to design and construct a set of mutant strains from the green photosynthetic alga C. reinhardtii, with higher sensitivity towards several classes and subclasses of herbicides (Table 3).

In this context, an in silico study was performed to predict mutations within the D1-D2 heterodimer which improve its specificity, sensitivity, and binding affinity for atrazine. In detail, taking advantage of the high sequence homology observed between Thermosynecococcus elongatus D1 and D2 proteins and the corresponding proteins from C. reinhardtii (87% and 89% amino acid sequence identity, respectively), the three-dimensional structure of the latter proteins was homology modelled. On the basis of this model, a series of D1 and D2 mutants were generated in silico and the atrazine affinity of wild type and mutant proteins was predicted by binding energy calculations to identify mutations able to increase PSII affinity for atrazine.

DEVELOPED BIOSENSORS

ADVANTAGES

DISADVANTAGES

REFERENCE

Whole-cell-based bacterial biosensors Cyanobacterial PSII-based biosensors

Optical biosensor based on various microalgae or chloroplast and thylakoids membranes Optical multiarray biosensor which employ several mutant strains from C. reinhardtii

Biosensing platform with different biomediators and double detection systems (optical and amperometric)

Amperometric biosensors based on mutant strains from C. reinhardtii

Amperometric biosensors based on thylakoid from

Spinacia oleracea and Senecio vulgaris

- simple and rapid pre-treatment steps

- real samples/complex matrix analyses

- ability to recognize different classes of chemicals

- different recognition elements for various classes of pesticides, insecticides and organophosphorus compounds

- high specificity towards classes and subclasses of herbicides

- low limits of detection in a concentration range from 10-i° to 10-8 M

- high specificity towards classes of herbicides

- low limits of detection in a concentration range from 10-i° to 10-8 M

- high stability of immobilisation biological recognition elements

- real samples/complex matrix analyses

- real samples/complex matrix analyses

- intrinsic instability, short half-life and specificity, poor response

- high limits of detection in a concentration range from 10-8 to 10-3 M or 10-9 to 10-5 M

- low specificity towards specific target analytes

- low specificity towards specific target analytes

Marty et al., 1995 Naessens et al., 2000

Euzet et al., 2005 Giardi et al., 2005 Breton et al., 2006 Tibuzzi et al., 2007 Rea et al., 2009 Giardi et al., 2009 Scognamiglio et al., 2009

Buonasera et al., 2010

- low specificity towards specific target analytes

- low specificity towards specific target analytes

Giardi et al., 2005

Touloupakis et al., 2005

Table 3. Main features of developed biosensors.

New advances in the same context were achieved in amplifying the range of recognition elements and measurement of a significant number of different classes of environmental pollutants. These advances occurred through the development of a biosensing system which uses sets of mutant organisms with different affinities towards pesticides. A library of functional mutations in the unicellular green alga C. reinhardtii for preparing biomediators was presented by Giardi and coworkers (2009). Exploiting bioinformatics to design new mutant strains resulted in the construction of microorganisms which showed different limits of detection for diazines, triazines and urea herbicides, underlined the high potential of bioinformatics and molecular biology in the design of desired biological material suitable for biosensor use.

Fig. 6. (A) Fluorescence profile of C. reinhardtii PSII in the absence and in the presence of atrazine. The binding of the herbicide is related to the main parameters of the profile in a concentration dependent manner. (B) Current profiles of C. reinhardtii PSII in the absence and in the presence of atrazine (10-7 M). The binding of the herbicide decreases the current signal in a concentration dependent manner.

Fig. 6. (A) Fluorescence profile of C. reinhardtii PSII in the absence and in the presence of atrazine. The binding of the herbicide is related to the main parameters of the profile in a concentration dependent manner. (B) Current profiles of C. reinhardtii PSII in the absence and in the presence of atrazine (10-7 M). The binding of the herbicide decreases the current signal in a concentration dependent manner.

A multi-biomediator fluorescence biosensor based on a new versatile portable instrument was assembled by Scognamiglio and coworkers (2009). The biosensor instrument was composed of a 24 cell array configuration able to host different mutant strains for the detection of a variety of herbicide classes such as triazines, diazines and ureas (Figure 7). As we can observe from the described advances in biosensor technology, the main features of a successful biosensor are characterised by the interchangeable recognition elements, which provide the versatility to measure large numbers of analytes. In Buonasera and coworkers (2010), a biosensing platform was constructed to provide an analytical tool applicable to the daily pre-screening of a broad spectrum of samples. The platform combined the most used transduction systems for biosensors, amperometric and optical systems, and used genetically modified microorganisms as versatile biomediators, allowing detection of different subclasses of herbicides.

Fig. 7. Biosensing platform set-up consisting of 24 cells able to host an array of several biomediators.

It represented a sensitive, reliable, and low-cost system able to detect water pollutants such as atrazine, diuron, linuron, and terbuthylazine down to 10-8-10-10 M. Combining the amperometric and optical detection systems, the platform was able to determine the toxicological potential of samples, through the determination of the biomediator physiological activity inhibition. Fluorescence modification and current reduction were related to the concentration of herbicide and quantified by a dedicated data acquisition software. In addition, the opportunity to use a wide range of biological materials made the platform a good candidate for the development of a biosensor with required features. Several other practical aspects seem to be important for the development of biosensors. One of these aspects considers the variability of real samples, whose composition is usually unknown and can vary widely from sample to sample. Suitable biosensors have to demonstrate reliability in field tests followed by validation by standard analytical methodologies. In Giardi and coworkers (2005) a fluorescence multi-biosensor was reported based on the thylakoids activity from different microorganisms used for the determination of several pollutants on real samples from the Tiber river, the Aqua Marcia, the Valle del Sorbo, and the Po river, tested contemporaneously by gas chromatography-mass spectrometry. Similar results were found with both methods. In Touloupakis and coworkers (2005) an amperometric multibiosensor using various photosynthetic preparations as biosensing elements for the detection of herbicides and pollutants on real samples was described. The photosynthetic thylakoid from Spinacia oleracea, Senecio vulgaris and its mutant resistant to atrazine were immobilized on the surface of screen printed electrodes composed of a graphite-working electrode and Ag/AgCl reference electrode deposited on a polymeric substrate (Figure 8). The presence of pollutants was revealed as the effect on PSII due to the sum of various herbicides, mainly triazines (0.210 ^g/l) and phenolic compounds (0.041 ^g/l). The performance of a biosensor is also related to the stability, the operating lifetime and the reusability of the biodevice, which is of critical importance and can affect the success of its use. In biosensor production, the biological material is usually immobilised, entrapped or cross-linked so as to produce an intimate connection or communication between the biomediator and the transducer. Many techniques have been

Fig. 8. Screen printed electrode used for biomediator immobilization.

introduced to overcome manufacturing problems associated with stability. In Buonasera and coworkers (2010) different immobilisation procedures, following physical and chemical approaches, were described as being suitable for several biological recognition elements and to be applied in amperometric and/or fluorescence measurements, some of these biosensors are already commercially available (see www.biosensor.it) (Table 4).

LEAKING

IMMOBILIZATION METHOD

RESIDUAL ACTIVITY %

High

Filter paper disk

56

High

Alumina filter disk

26

High

Glass microfiber filter

49

High

DEAE cellulose

50

Medium

Nitrocellulose

90

Medium

Agar

45

Medium

Agarose

33

Low

Carrageenan

30

Low

Alginate

20

Low

Gelatin

50

High

Lyophilisation

70

Low

Glutaraldehyde

70

Low

Magnetic-beads polymer

60

Low

Binding on gold films

35

Low

Collagen

30

Low

Bovine serum albumine (BSA-GA)

70

Low

Cross-linking on Gold

20

Low

Cross-linking on TiO2

55

Low

Polyacrylamid

41

Low

Polyurethane

28

Low

Photocrosslinkable resin

10

Low

Vinyl

15

Low

Poly(vinylalcohol)

38

Low

Tyrylpyridinium groups

45

Low

Thiophen polymer

57

error less than 10%

Table 4. Immobilization methods applied to several biomediators. The stability of each procedure was evaluated measuring the residual activity and leaking before and after immobilization.

Table 4. Immobilization methods applied to several biomediators. The stability of each procedure was evaluated measuring the residual activity and leaking before and after immobilization.

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