Polymer-photosynthetic protein multilayer architectures for herbicide optical detection

Sensors and Actuators B 163 (2012) 69–75

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Polymer-photosynthetic protein multilayer architectures for herbicide optical detection Mauro Giustini a,b,∗ , Mattia Autullo a , Mauro Mennuni a , Gerardo Palazzo b , Antonia Mallardi c,∗∗ a b c

Dipartimento di Chimica, Università “La Sapienza”, P.le Aldo Moro 5, Roma I-00185, Italy Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Firenze, Italy CNR - IPCF, Istituto per i Processi Chimico-Fisici, Via Orabona 4, Bari I-70126, Italy

a r t i c l e

i n f o

Article history: Received 28 November 2011 Received in revised form 3 January 2012 Accepted 4 January 2012 Available online 18 January 2012 Keywords: Herbicide Optical biosensor Photosynthetic reaction center Rhodobacter sphaeroides Layer by layer

a b s t r a c t The design and characterization of an optical biosensor based on a photosynthetic protein deposited on a quartz surface is here presented. The protein reaction center (RC), purified from Rhodobacter sphaeroides, has been immobilized in alternate layers with the cationic polymer poly(dimethyl diallyl) ammonium chloride (PDDA). In this assembly the protein retains its integrity and functionality maintaining its ability to bind herbicides. Upon exposure to continuous light some RC absorbance bands dramatically reduce their intensity (bleaching) and the extent of such a bleaching reflects the amount of bound herbicides. These properties have been exploited for the design of a simple optical biosensor for herbicide. The characterization of the biosensor in detecting the broad family of triazine herbicides is presented. Performance characteristics, such as limits of detection (LOD) and quantification (LOQ), upper determination limit (UDL) and linear range for each herbicide were determined. Among the most striking features of the biosensor are the long lifetime (several months), the high reproducibility and the relatively high sensitivity of detection that can be further enhanced by preconcentrating the samples to be analysed. As a whole, these characteristics coupled to the low demanding instrumental setup, let the RC/PDDA assembly particularly appealing even for the realization of a stand alone analytical apparatus. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Toxic chemicals originating from sources such as agricultural run-off can contaminate soils and both surface and ground water [1,2]. Some toxic compounds with slow degradation rates accumulate in the soil and water [3] and are subsequently stored in plants, animals and human tissues where their toxicity represents a serious health risk [4,5]. The so-called photosystem II (PSII) herbicides act inhibiting the initial steps of photosynthetic processes in plants, leading to their death. They therefore represent an efficient and low cost weed control means. PSII herbicides, belonging to the classes of triazines, diazines, phenols and ureas, represent toxic compounds that are still widely used in agriculture, thus having detrimental effects on the ecosystem and human life [6]. Their wide use in agriculture has resulted often in the herbicide pollution of water and the level of herbicides allowed in

∗ Corresponding author. Tel.: +39 06 49913336. ∗∗ Corresponding author. Tel.: +39 080 5442028. E-mail addresses: [email protected] (M. Giustini), [email protected] (A. Mallardi). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2012.01.009

drinking water is subject to regulation, at least in the industrialized countries. The European Directive for pesticides in drinking water sets maximum concentrations at 0.1 ppb (0.1 ␮g/L) for any individual pesticide or 0.5 ppb (0.5 ␮g/L) for total pesticides [7]. Herbicide detection methods currently used, mainly based on chromatographic techniques (HPLC, LC–MS), are satisfactorily but they require expensive pieces of equipment and pre-concentration steps [8,9] or large-volume direct injection [10]. On this basis a need has arisen for fast and sensitive detection methods to monitor herbicide levels. In recent years, immunoassays [11,12] and immunosensors [13–16] based on antibodies specific for herbicides have been proposed as a valuable alternative. In this respect, also the development of assays exploiting the effect of herbicides on photosynthetic proteins should be very useful. In plants, several herbicides bind to the PSII complex and inhibit the light driven electron transfer system. Photosynthetic biosensors, based on this action mode, provide excellent tools for the detection of herbicides and, in principle, allow a fast and cheap screening of environmental samples. In the last years, different types of photosynthetic materials have been employed as biosensor active layer starting from whole cells to chloroplasts, isolated membranes and photosystems [17–19]. However, cells and organelles loose rapidly their biological activity and also PSII, being a very

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delicate membrane multiprotein complex formed by 25 polypeptide chains and several cofactors, is unstable. The bacterial progenitor of the PSII complex is the photosynthetic bacterial reaction center (RC) present in ancient photosynthetic purple bacteria. It has a high structural homology with the PSII and its functionality is inhibited by the same herbicides that inhibit the PSII in higher plants. The bacterial RC is, however, a very robust, small protein complex (it is formed by only three polypeptide chains). RC can be isolated easily from bacteria and is particularly stable against denaturation, which makes it advantageous compared to materials extracted from plants and algae for the development of herbicide biosensors. The structure of RCs from purple bacteria is well assessed [20–22] being the first membrane proteins to be crystallized. In recent years the RC has gained a central role also in studies dealing with chemical–physical aspects of the membrane proteins such as the interaction with surfactants [23–25], study of protein energy landscape [26–29] and the stability of membrane proteins [24]. Several strategies have been proposed to immobilize the RC on solid substrates [30,31]. The efficient immobilization of proteins on suitable matrices and their integration on precisely engineered nanoscale architectures is often a key step for their use in novel technical applications [32–35]. Lately we have studied the functionality of RC immobilized onto an optically transparent surface by layer-by-layer (LbL) deposition with the cationic polymer poly(dimethyl diallyl) ammonium chloride (PDDA) [36]. In the obtained RC/PDDA multilayers, the protein retains its integrity and functionality maintaining its ability to bind herbicides. In this paper we exploit the properties of RC-containing multilayers for the design of a simple optical biosensor for herbicide determination in water. 2. Experimental 2.1. Reagents N,N-Dimethyldodecylamine-N-oxide (LDAO) was purchased from Fluka as a 30% aqueous solution. nOctyl-␣-d-glucopyranoside (octylglucoside, OG), terbutryn, atrazine, prometon, and terbumeton and ametrine, poly(dimethyldiallylammonium chloride) (PDDA, 20% water solution) were all from Sigma–Aldrich. Unless otherwise stated, the water used throughout this work is bidistilled water from Carlo Erba Reagents. Pre-packed columns of N-vinylpyrrolidone-divinylbenzene copolymer (OASIS HLB Extraction cartridges-6 mL/200 mg of copolymer) were purchased by Waters Inc. 2.2. Instrumentation design The time dependence of bleaching at 870 nm, induced by continuous illumination, was monitored with the instrument of local design previously described [36] and sketched in the inset of Fig. 2B. Briefly, the light beam provided by an 870 nm laser diode pass through the RC/PDDA multilayer and is monitored by a silicon photodiode (UDT 10D) driven in photovoltaic mode. The photocurrent, once converted into a voltage by an operational amplifier (OP637AP, Texas Instr.), is fed to a data acquisition board (DAQ PCI 6013, National Instr.) and then converted into absorbance variation by a software of local design operating in the LabView environment. In these experiments the laser beam has a twofold purpose acting either as photo-excitation source and as measuring beam. 2.3. RC/PDDA multilayer preparation RCs from Rhodobacter sphaeroides were isolated and purified using the detergent LDAO according to Gray et al. [37]. After

purification of the protein, the zwitterionic detergent LDAO was exchanged with the non-ionic detergent OG (see Section 3) and the binding site of the secondary quinone was saturated with exogenous quinone as described in Mallardi et al. [36]. The immobilization of RC in the multilayer with PDDA polymer has been performed on a quartz slide (1 cm × 5 cm, Hellma) as already described by Mallardi et al. [36]. Briefly, substrates were negatively charged by using an oxidative cleaning in “piranha” solution (Oleum H2 SO4 and 30% H2 O2 in a 3:1 ratio by volume) for 15 min on ice (care should be taken in preparing and handling this solution, as the reaction is exothermic and the solution is highly corrosive) and subsequently left in Millipore water for 5 min. The substrates were rinsed with acetone and dried with nitrogen. To adsorb the polycation layer, the slide was immersed in a 2 mg/mL PDDA/water solution. Subsequently, it was washed in Millipore water, and a layer of negatively charged RC was adsorbed by dipping the specimen into 10 mM Tris–HCl buffer, pH = 8.0, containing 0.8% OG and 3 ␮M RC. After rinsing with Millipore water, the above-described steps were repeated in order to obtain the required number of RC/PDDA multilayers. In this way, both sides of the slide are covered with the same number of polyelectrolite multilayers (PEMs). Typical PEMs used in this study consist of 2 × 20 RC layers. Adsorption and washing times were 15 and 5 min, respectively. RC and polycation layers were grown in the dark at 4 ◦ C. 2.4. Herbicide assay The herbicide solutions have been all prepared by water dilution of a concentrated ethanol stock solution of the inhibitors. The utmost care has been posed in keeping in all the samples the concentration of the ethanol constant to 2% (v/v). For natural water sample analysis, after filtration through a 0.2-␮m membrane (Millipore) to eliminate solid particles, well water was spiked with solutions of herbicides in ethanol to a final composition of 2% (v/v) ethanol/water. A 1 cm × 1 cm × 4 cm fluorescence cuvette was filled with 3 mL of the appropriate solution (standard or unknown). The quartz plate supporting the RC/PDDA multilayers was dipped into the solution. A reproducible placement of the plate was achieved by inserting it along the cuvette diagonal. The sample was then illuminated by the laser diode inducing the RC bleaching (see Section 3) and the absorbance values at time 0 and after 6 s of illumination (steady state) were acquired; the difference between these values (Abs870 nm ) was taken as the optical response. For the calibration curve, solution containing the analyte at a randomly chosen concentration was assayed. The very same RC/PDDA multilayer is used to assay the standards and the unknowns. It is thus mandatory to remove herbicides from the previous sample before proceeding with the subsequent measurements. Herbicides are scarcely soluble in water but we found that washing with ethanol in water at 2% by volume allows the complete herbicide removal leaving the RC fully functional. 2.5. Sample preconcentration Preconcentration of herbicide solutions was accomplished by solid phase extraction (SPE) using commercial adsorbent beads of N-vinylpyrrolidone-divinylbenzene copolymers (6 mL/200 mg of copolymer) whose extraction performances have been optimized for our samples. The column was flowed by 2 × 5 mL of methylene chloride followed by 2 × 5 mL of methanol and then dried under reduced pressure (water pump). 10 mL of bidistilled water were then passed through the column in order to remove any trace of methanol. In a typical assay 1 L of solution was loaded onto the column and at the end 200 ␮L of methanol were added to the column in order to facilitate its vacuum drying. The trapped herbicide was

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Fig. 1. LbL assembly of a PDDA–RC multilayer. (A) Multilayer structure scheme; (B) vis–NIR absorption spectra of a multilayer obtained following a different number of RC adsorption cycles (indicated in the labels).

eluted by flowing 7 mL of methanol and the eluate, collected in a glass tube, was vacuum dried and solubilized with 2 mL of a 2% (v/v) ethanol/water solution.

light

DQA Herb  D+ QA− Herb

(2)



3. Results and discussion 3.1. RC/PDDA multilayer features The main driving forces for layer-by-layer (LbL) assembly are electrostatic interactions. Due to their huge charge, polyelectrolytes strongly adsorb on an oppositely charged surface. As a consequence, immersion of a negatively charged surface (clean quartz) into a solution of cationic polyelectrolyte (e.g., PDDA) results in the coverage of the surface by a positively charged polymer layer. Of course, immersion of this newly formed cationic surface in a solution of negatively charged protein (e.g., RC) results in the protein adsorption and in the re-establishment of a negative charge density. By repeating these steps, alternating polyelectrolyte multilayer (PEM) assembly can be obtained (see scheme in Fig. 1A). Between the adsorbing baths, the specimen must be rinsed with water; otherwise, free polyelectrolytes will interact in solution forming neutral RC/PDDA complexes. The efficiency of RC assembly by LbL technique has been estimated during the sequential stages of the adsorption process by recording the Vis–NIR spectrum of RC in water from 500 to 1000 nm. A typical PEM absorption growth is reported in Fig. 1B showing as, in our system, the optical density grows at all the wavelengths with the number of adsorption steps. 3.2. Functionality of RC and effect of herbicides To grasp the principle of functioning of the proposed optical assay a broad description of the functionality of RC is enough (details can be found in several reviews). The core of the RC machinery is a bacteriochlorophyll dimer (D) that upon photon absorption acts as an electron donor transferring an electron to a first ubiquinone molecule (QA ) and subsequently to a second ubiquinone molecule (QB ). The overall process of light-induced charge separation and subsequent recombination, taking place in vitro, is described by the following scheme light

DQA QB  D+ QA QB−

charge separation/recombination involve only the primary quinone as described in the scheme below

(1)



the mechanism of action of herbicides rely on their ability to displace the second quinone from its binding pocket thus hampering the correct functioning of the protein. In such a case the processes of

where Herb denotes the herbicide. The bacteriochlorophyll dimer D is characterized by an absorption band in the near infrared region (max ≈ 870 nm). When D is photo-oxidized to D+ such band shows a dramatic drop in intensity (bleaching). The binding of the herbicide does not influence the extinction coefficients of D and D+ and therefore the herbicide binding is undetectable using classical absorbance (or fluorescence) measurements. Instead, the presence of bound herbicide strongly affects the rates of charge recombination ( and  in Schemes (1) and (2), respectively). Indeed, the rate of D+ reduction when the herbicide occupies the binding site of the secondary quinone (, Scheme (2)) is one order of magnitude faster than that observed in the native state (␭, Scheme (1)). On these bases, the use of RC in optical bioassays was proposed already in 1993 [38]. In that paper the concentration of herbicides was inferred from the deconvolution of kinetics traces obtained from flash-photolysis experiments on RC in solution (i.e. using a setup based on one continuous and one pulsed light sources). Lately, a simplified assay based on the RC response to continuous illumination has been proposed (but the RC was yet in solution and the analysis required yet the deconvolution of kinetic traces) [39]. Under continuous illumination at 870 nm, the simultaneous generation of charge separated states and their disappearance through charge recombination takes place. Eventually, a steady state condition will be reached as a result of the two competing events. The level of bleaching attained in such a steady-state is affected by the presence of bound herbicides. Intuitively, a progressively lower level of bleaching is expected upon increasing herbicide concentrations because the RC with bound herbicide relaxes more efficiently to the neutral state (for a formal description of these kinetics see [40]). On this basis, we proposed an assay based on the ability of light of suitable wavelength (e.g. 870 nm) to simultaneously trigger and probe the RC photochemistry. Representative traces are shown in the left panel of Fig. 2. In these experiments the laser beam has a twofold purpose acting either as photo-excitation source either as measuring beam. It is evident that the illumination triggers a decrease in the sample optical absorbance that eventually reaches a plateau. In the example of Fig. 2, 5 s of illumination are enough to induce a stable (steady-state) drop of −27 × 10−3 in the absorbance of the sensor dipped in pure water, but immersion in a 100 ␮M solution of herbicide terbumeton (TBON) reduces such an absorbance drop to −5 × 10−3 , in agreement with the above discussed considerations. Rigorous kinetic analysis indicates that the value of the

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Fig. 2. Influence of the herbicide terbumeton (TBON) on the bleaching (expressed as mAbs) induced by the measuring light (870 nm). (A) Absorbance kinetics in presence of different TBON concentrations. The reference transmittance was that at time = 0 (Abs(t = 0) = 0). (B) Absorbance change at the steady-state as a function of the TBON concentrations. The ordinate represents the Abs of panel A averaged in the time interval 5.5–6.0 s (rectangle in panel A). The dependence of the steady-state bleaching on the herbicide concentration reflects the binding isotherm of TBON to the RC (Eq. (3)). Inset: simplified scheme of the experimental setup.

As shown in Fig. 2, illumination of the RC/PDDA assembly with a 870 nm light beam results into a decrement of the absorbance at that wavelength. Such a bleaching (Abs870 nm ) depends on the concentration of herbicide in the solution in contact with the PEM, showing the most negative value in the absence of herbicide (pure water) and weakening upon increasing the herbicide concentration. The data were acquired recording the bleaching from measuring light until a steady state condition was reached (traces lasting 6 s were normally acquired, among each measurement the PEM was rinsed as described in Section 2). In the right panel of Fig. 2 the bleaching measured at the steady state (i.e. after 5.5 s of illumination, averaged over 0.5 s) is plotted as a function of the herbicide concentration. The extent of bleaching (Abs870 nm ) decreases with the herbicide concentration and eventually reaches a plateau value. The data Abs870 nm vs herbicide concentration are well accounted for by the classical binding equation (equivalent to the Langmuir’s isotherm) Abs870 nm = Absmin + (Absmax − Absmin )

[h] Kd + [h]

a

-12

ΔAbs870nm

3.3. Sensing performances

-9

UDL ATZ

-15

-18

LOQ

-21

LOD

10

-3

10

-2

10

-1

10

0

10

1

10

2

ATZ concentration /μM

-5

ΔAbs870nm

steady-state bleaching is a linear function of the herbicide bound to the RC [36]. The measure of the bleaching at 870 nm under continuous illumination thus can represent an easy-to-use method to probe the binding of herbicides to RC, allowing for their quantification.

b UDL

-10

TBT

-15 -20 -25

LOQ

-30

LOD

(3)

where [h] denotes the herbicide concentration, Absmin and Absmax are the bleaching value in absence of herbicide and at saturation, respectively, and the dissociation constant Kd is equivalent to the herbicide concentration causing 50% reduction of initial bleaching. The experimental data have been fitted to the above equation and the Kd best-fit data are listed in Table 1 for the five herbicides examined. Fig. 3 shows, on a logarithmic abscissa, two representative calibration curves. The standard deviation  of the bleaching measured for the blank (Absmin , PEM in water) obtained from measurements performed on the same multilayer

10

-4

10

-3

10

-2

10

-1

10

0

10

1

10

2

TBT concentration /μM Fig. 3. Calibration curves for atrazine (ATZ panel A) and for terbutryn (TBT panel B). Steady state bleaching (expressed as mAbs) measurements were made in triplicate; error bars = ±standard deviation. Stars in panel B refer to assay performed on a 10 nM terbutryn solution concentrated by means of solid phase extraction (SPE) to a final concentration of 5 ␮M; the white star denotes SPE performed on a TBT solution in bidistilled water and the gray star denotes SPE performed on a TBT solution in well water.

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Table 1 Analytical performances of PEM towards the atrazine family herbicides. Herbicide

Kd (␮M)

Ametrine (AMT) Atrazine (ATZ) Prometon (PON) Terbumeton (TBON) Terbutryn (TBT)

3.8 13 12 7.1 2.2

± ± ± ± ±

0.7 2 3 0.7 0.3

LOD (␮M)

LOQ (␮M)

0.3 1.3 0.8 0.5 0.17

1.2 5.9 3.2 2.1 0.7

UDL (␮M)

Linear range (␮M)

50 121 168 95 30

1–40 2–100 2–100 1–70 1–25

Measurements performed using four different multilayers have been considered for averages and errors (SD).

or more calibration curves of different herbicides (when not used it was stored in bidistilled water at 4 ◦ C in the dark). Fig. 4A indicates that the extent of maximum bleaching remains essentially constant for the first 50 days: within this time period the reproducibility of the Abs870 nm value measured in water is within 7%. Further use leads to a decrease in this value that drops to 60% of the original value after 70 days from the PEM preparation. This decrease in the RC photoactivity is parallel to the decrease in the absorbance peaks characteristic of this protein (not shown) and this suggests that the extensive rinsing of the specimen over about 2 months induces the desorption of a sizeable fraction of the RC. However, the bleaching achievable after 2.5 months has a signal-tonoise ratio still high enough for herbicide detection at ␮M level (the calibration curve of ATZ in Fig. 3 has been obtained 80 days after 0

ΔAbs°870 nm

A -10

-20

-30

-40 0

10

20

30

40

50

60

70

80

90

time from biosensor fabrication (days) 0,26

B

0,24

N

T

TB O

N

PY

N

TB ON

PY

AM

N

AM T

0,16

PO

0,18

T

0,20

PO N

TB

T

0,22

TB

ΔAbs(100μM herbicide)/ΔAbs°

during the same day (more than five measurements) is below 2%. The main contribution to this fluctuation appears to be related to the repositioning of the plate across the optical path (different parts of the specimen are sampled). Taking into account such an uncertainty on the blank, the limit of detection (LOD) and the limit of quantification (LOQ) have been evaluated as the concentrations corresponding to the mean blank measurement plus 3 and 10, respectively. At high concentration the herbicide saturates the RCs and the biosensor becomes insensitive. Accordingly, an upper determination limit (UDL) has been evaluated as the herbicide concentration for which the measured bleaching deviates from the limiting high concentration value (Absmax in Eq. (3)) by 3. These calibration characteristics are listed in Table 1. It is worth noting how for atrazine the LOD here reported is four times lower than that obtained by means of SPR using RC immobilized on gold [41], demonstrating how a low-cost optical setup can, in this special case, furnish better performances than very expensive SPR equipments. The LOD (0.17–1 ␮M) and UDL (30–170 ␮M) values of different herbicides tested are mainly dictated by the relatively high Kd values, then improvements on the uncertainty of the blank measurements (possible, in principle, using flow setups where the PEM remains immobile) will not change appreciably these figures of merits. Sensitivity and linear ranges (Table 1) have been obtained directly from plots Absmin vs log10 ([h]/M) [42]. While the lower limit of the linear range (around 1–2 ␮M) is almost constant and depends on Absmin , the upper limit depends essentially on the affinity of RC (i.e. 10 × Kd ) for herbicide molecules. The target of PSII herbicides, the RC QB site, is located in the hydrophobic portion of the protein thus allowing the free exchange of the quinone cofactor with the ubiquinone pool located in the hydrophobic core of the membrane. To inhibit the protein function, herbicides must share structure and polarity with that of the natural cofactor. Accordingly to this principle different herbicides have different affinities for photosynthetic proteins. The sensitivity, instead, depends also on the maximum and minimum bleaching attained (Absmax − Absmin , see Eq. (3)) and thus on the amount of RC immobilized on the PEM. Using our standard procedure (2 × 20 RC layers) the initial sensitivity is around 15 mAbs/(concentration decade) but, since upon prolonged use a fraction of the RC is removed from the PEM (see below), the sensitivity decreases with time reaching 7 mAbs/(concentration decade) after 2.5 months.

0,14 0,12

3.4. Lifetime, stability and reproducibility

0

10

20

30

40

50

60

70

80

90

time from biosensor fabrication (days) The RC embedded into the PEM exhibits an exceptional stability and remains photoactive, when stored in distilled water at 4 ◦ C in the dark, for at least 7 months (not shown). In order to test how the exposure to the light and rinsing steps, associated to the herbicide quantification protocol, affect the stability and lifetime of the biosensor, the same PEM have been used over a period of 3 months for several assays on different herbicides; representative examples are shown in Fig. 4. In Fig. 4A is shown the extent of the bleaching obtained in the absence of herbicide on the same PEM. Between each measurement, the specimen has been used to construct one

Fig. 4. Time stability of the biosensor. (A) Time evolution of the bleaching amplitude (expressed as mAbs) measured in the absence of herbicides (blank) for the very same RC/PDDA multilayer. In the periods between two succesive blanks the device has been used to measure calibration curves of different herbicides involving 5–10 different assays and washing steps. (B) The same RC/PDDA multilayer (but different from that of panel A) was used for 3 months to measure calibration curves of different herbicides. Although the maximum bleaching (blank) decreases above 2 months (see Panel A) the ratio between the bleaching in presence of a 100 ␮M herbicide and that measured for the blank remains constant. In the period between two measurement in pure water the device has been used to measure calibration curves of different herbicides involving 5–10 different assays and washing steps.

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been obtained when the dilute terbutryn solution has been prepared using well water (see the stars in Fig. 3B). A by-product of the final vacuum drying step is the removal of potentially interfering volatile organic compounds (toluene, chloroform, etc.).

0 -2

ΔAbs870nm

-4 [TBT]=5μM

-6

4. Conclusions and perspectives

-8 -10

TBT extracted

the multilayer preparation). Actually, the desorption of RC from the PEM results into a decrease of maximum bleaching (i.e. in pure water) but also in a parallel decrease in the minimum bleaching (attained at saturating herbicide concentration), leaving constant the ratio between the maximum and the minimum bleaching (i.e. blank/saturating herbicide content). Fig. 4B reports this ratio over a time window of 3 months; note that between two points at least a calibration curve has been performed and that the specimen is different from that used for Fig. 4A. It is clear from Fig. 4A that the relative response of the biosensor to herbicides is constant over several months (although the maximum bleaching sizeably decrease). Such a stability (astonishing for a polytopic membrane protein) coupled to the inherent robustness of the RC/PDDA multilayer onto quartz plates (we exchanged routinely the specimens between our labs by conventional mail) is very promising for large scale applications.

We present a method to prepare an optical biosensor for the detection of herbicides by alternate layer-by-layer deposition of PDDA and bacterial reaction center on a quartz slide. The analytical performances of this biosensor have been examined. The incorporation of RC into layer-by-layer assemblies presents many advantages. Because the herbicides can be efficiently washed out, the PDDA/RC multilayers can be reused for several samples. This property, coupled with the exceptional stability of the PDDA/RC multilayers assemblies, make possible to perform complete runs (calibration curve plus unknowns) with a small amount of protein (1–2 nmol with our plates). In the present configuration, the PDDA/RC multilayer can be directly used for the screening of substances potentially active in interfering with the photosynthetic apparatus of higher plants, so acting as potentially herbicides. Coupled with SPE the PDDA/RC can be an economic alternative to classical analytical methods for the screening of water samples. The proposed biosensor is sensitive to the whole class of triazines and thus its response will reflect the presence of all the compounds belonging to such a class. This is an advantage for a preliminary screening but precludes the specificity required for a full analytical characterization of the triazine content. All the data here presented deal with the RC extracted from the wild-type strain of the bacterium Rhodobacter sphaeroides, however, there are several mutant strains whose RCs have altered binding properties for some specific herbicides (the so-called herbicide resistant strains) [47]. The comparison among responses obtained (on the same sample) by multilayers made of RCs from different strains might differentiate different herbicides.

3.5. Towards the herbicide limit in drinking water

Acknowledgements

Though banned in EU countries since 2004 (in Italy and Germany since 1991) [43], atrazine is still produced and used in USA (at a rate of 60–80 tons/year) [44], as well as in Third World Countries [45,46]. Other triazine based herbicides, terbutylazine and metribuzine mainly, are currently worldwide used. In this respect, the extensive reusability of the RC/PDDA multilayer and the simplicity of the optical setup (single wavelength absorbance transducer) yield to an almost ideal first-screening biosensor apart from the high detection limit. Actually, the lower herbicides concentration detected by multilayer (∼0.3–1 ␮moles/L) is far exceeding the higher total herbicides concentration allowable in drinking water (fixed in EU countries, as already said, to 0.5 ␮g/L that results into an average concentration of 2 nmoles/L). This limit is strongly related to the reusability of the multilayer core of the biosensor. Actually, although it is conceivable to tailor RCs having very low dissociation constant (e.g. by means of suitable mutagenesis approaches) this should make the regeneration step very difficult. To overcome this intrinsic limit in the RC LOD, the fast preconcentration procedures through solid phase extraction (SPE see Section 2) was tested. 1 L of a 10 nM terbutryn solution in water, was loaded on the column. At the end of the procedure the recovered herbicide was resuspended in 2 mL of a 2% (v/v) ethanol/water solution to obtain a nominal concentration of 5 ␮moles/L. In Fig. 5 the time course of the bleaching obtained from SPE concentrated herbicide solution has been compared with that of a reference 5 ␮moles/L terbutryn solution. As can be clearly seen the two traces are indistinguishable; the same result have

This work was supported by MIUR of Italy (grant PRIN/2008 prot. 2008ZWHZJT) and by the Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI-Firenze).

H 2O

-12 -14 -16 -18 0

1

2

3

4

5

time /s Fig. 5. Validation of the preconcentration protocol RC/PDDA multilayer response to pure water, to a 10 nM terbutryn solution concentrated 500 times through SPE and to a 5 ␮M terbutryn solution control.

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Biographies Mauro Giustini is research professor of chemistry at the University of Rome “La Sapienza”. His research activities are focused on the preparation and characterization of supramolecular aggregates formed by surfactants, polymers and dendrimers as hosting nanocages for proteins, oligo- and poly-nucleotides, drugs and metal complexes. Mattia Autullo received his degree in Natural Science from University of Rome “La Sapienza” in 2009. Present address: Innogenetics s.r.l. Via Vaccareccia 39/a, 00040 Pomezia (Rome), Italy. Mauro Mennuni received his degree in natural science from University of Rome “La Sapienza” in 2007. Presently he is working as writer and consultant in popular science. Gerardo Palazzo is an associate professor in physical-chemistry at the University of Bari. His main research activities of deal with chemical physics of proteins and colloids (researcher ID at http://www.researcherid.com/rid/G-9030-2011). Antonia Mallardi is researcher at the Institute for Chemical and Physical Processes of the National Council of Research (CNR) of Italy. Her main research activities deal with the functionality of photosynthetic proteins and with the development of novel biosensors.