Development of a Biosensor for Environmental Monitoring Based on Microalgae Immobilized in Silica Hydrogels

A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU) and various concentrations of the common herbicide atrazine. Microalgae are very sensitive to herbicides and detection of fluorescence enhancement with very good efficiency was realized. The best detection limit was 0.1 μM, obtained with the strain C. reinhardtii after 40 minutes of exposure.


Introduction
Deterioration of the aquatic environment by pollutants is an important problem. Among all of them, heavy metals and herbicides constitute a priority preoccupation: they are frequently found in surface OPEN ACCESS and ground waters and are harmful to aquatic organisms [1][2][3][4][5]. The need for convenient, quick and reliable methods to assess pollutant toxicity is more essential than ever. Environmental monitoring of pollutants with automatic systems applied online and allowing rapid response is one of the best ways to control the quality of the environment. Real time analysis offers the advantage of detecting rapidly the presence of pollutants before they cause any damage. Such a strategy is only possible through biosensors [6].
Photosystem II (PSII)-based biosensors are reported to be able to detect herbicides in the environment [7]. Various herbicides, including the urea, triazine, and phenolic based herbicides, target the vegetal PSII. These substances inhibit photosynthetic electron flow by blocking the PSII quinone binding site and thus modify chlorophyll fluorescence. Many biosensors based on algal chlorophyll fluorescence measurement were developed and have demonstrated the suitability of this technique for herbicide detection [8][9][10][11].
An important point in the design of algal biosensors is the question of cell-binding. One of the most commonly employed procedures, based on the immobilization of microalgae within calcium crosslinked alginate beads is intrinsically biocompatible [12][13][14][15][16], but such matrices are disrupted in water as well as by alginate biodegradation. In the last decade, there have been huge improvements in the development of biocompatible synthetic routes to entrap biological entities within pure inorganic matrices [17][18][19][20][21][22][23]. Among them, routes based on sol-gel chemistry [24] are unique in offering the necessary mild conditions for building composite materials based on robust silica hydrogels and biological entities where the bio-activity gives rise to a wide range of sensing possibilities [25][26][27].
The design of biosensors based on direct silica encapsulation is limited by the restricted cell viability, but recent work has demonstrated the possibility of cell division inside inorganic matrices by means of a two-step encapsulation procedure based on sol-gel chemistry [28][29][30]. This strategy expands the range of possible applications as cells can not only be entrapped within silica hydrogels, but are also able to grow inside, even for periods of months [31]. In addition, the inherent homogeneous and mesoporous texture of the silica hydrogel provides a shield preventing the release of entrapped cells, as well as the contamination of the inner culture by exogenous strains. Moreover, in previous works we have demonstrated the long-term viability of algal cells encapsulated in an alginate-silica hybrid matrix [32].
In this paper, translucent alginate-silica matrices obtained by a sol-gel process have been investigated for entrapment of three algal strains in order to improve biosensor fabrication. After a biocompatibility assay of the inorganic host material, the effects of algal immobilization were tested on their ability to determine herbicides.

Cell Cultures
Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii were used. Algal strains were purchased from The Culture Collection of Algae and Protozoa (Cumbria, United Kingdom). C. vulgaris and P. subcapitata were grown in Lefebvre-Czarda medium [33] whereas C. reinhardtii was grown in trisacetate phosphate (TAP) medium [34] and were transplanted weekly under sterile conditions (autoclaving 20 minutes, 130 °C, 1.3 bars). Algae were maintained in a nycthemeral cycle of 16 hours of illumination at 5,000 lux and eight hours of darkness.

Immobilization Method
In a two-step procedure, cells were previously immobilized in Ca(II)-alginate beads which was subsequently trapped in the inorganic matrix, avoiding any harmful contact of cells with any silica precursors ( Figure 1). This procedure based on inexpensive silica precursors has already been employed for yeast (Saccharomyces cerevisiae), bacteria (Escherichia coli and Bacillus subtilis), algae (Chlorella vulgaris), fungi (Stereum hirsutum) and plant cells (BY2 tobacco and carrot phloematic tissue) [31].

Alginate Beads and Gels
Formation of alginate beads was performed by dropwise addition of a 2% (w/w) Na(I)-alginate cells suspension in a 0.1 M CaCl 2 solution. After 10 min stirring, beads of about 3 mm diameter were easily collected by filtration. The Ca(II)-alginate polymer prevents cell contact with synthesis precursors.
Formation of alginate gel beads in 96 multiwells plate was performed by stirring a 50 μL aliquot of cells suspended with 50 µL of Tris-HCl buffer (10 mM, pH = 7.5) and 100 µL 2% Na(I)-alginate (resp. 100, 100 and 200 µL for 48 multiwell plates). This mixture was introduced in the cavities of the sample holder. Alginate cross-linking was built by immersing the sample holder into a 0.1 M CaCl 2 solution for 10 minutes.

Silica Hydrogels Synthesis
The second step of the immobilization procedure consists of a silicate (sodium silicate, Riedel-de Haën; NaOH 10%, SiO 2 27%) sol-gel process in the presence of commercial silica nanoparticles (LUDOX HS-40, 40% in water, obtained from Aldrich), leading to a nanoporous monolithic structure. Monoliths were prepared at room temperature by mixing volumes of the different precursor solutions A B to obtain a SiO 2 water molar relation of 3.8% with a fixed proportion of polymeric to particulate silica precursors (1:3) at constant pH = 6.5, adjusted with HCl.

Growth Rate of Algae Entrapped within Silicate-Ludox-Based Hydrogels
To evaluate the degree in which the development of algae is affected by the encapsulation in alginate and silica, the algal growth rate as a function of entrapment method was measured. The algal growth inside the voids was studied for individual cavities after 3 days of culture in LC liquid medium. At the initial time, calcium alginate beads with a content of 10 −4 cells/mL were dispensed into 1.5 mL tubes (Eppendorf), and the silica encapsulation procedure was performed. After 3 days, the silica hydrogel was removed and samples were exposed to 0.05% potassium citrate to solubilize the Ca(II)-alginate beads. The total number of cells inside individual cavities was determined by counting cells in a Mallassez counting chamber. To analyze cellular growth, the percentage of inhibition (I) is calculated using the equation: where µ i is the growth rate for test batch i (entrapped cells) and µ c is the mean growth rate for the control batch (free cells). Test and control batches were run in triplicate.

Biosensor Performance
The herbicides (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU, 4 mg/L) and atrazine (10 µM to 0.001 µM) were used as examples of anti-PSII herbicides to evaluate the performance of the fluorescence biosensor (Table 1). Table 1. Experimental design on the microplate Co: control (no herbicide), C1 = 0.001 µM, C2= 0.01 µM, C3=0.1 µM, C4=1 µM and C5 = 10 µM. in free culture medium (in blue), in culture medium added with 2% Na(I)-alginate (sodium alginate, in red), encapsulated in Ca(II)-alginate (calcium alginate, in green), and after the two steps immobilization procedure (calcium alginate/SiO 2 , in purple). 1 2 Response of the algal biosensor was obtained from the chlorophyll fluorescence emission at 682 nm under a 469 nm excitation light with a spectrofluorimeter (Fluostar, BMG). The biosensor response to herbicide was determined by the fluorescence enhancement (E) as soon as a defined amount of herbicide is added to the sample (the herbicide is dissolved in distilled water and added to free and entrapped algae).
For herbicides which inhibit PSII, the enhancement can be determined after 40 minutes using the flowing equation: 100 where F b is the fluorescence for a test batch before contact with the herbicide and F a is the fluorescence for the same test batch after the herbicide exposure. All assays were undertaken with a thickness of silica layer around 1.9 mm and with the following cellular concentrations:

Growth Rate of Entrapped Algae
In the design of biosensors with encapsulated photosynthetic cells, as well as for the design of devices based on optical detection systems it is mandatory to minimize the attenuance of the host matrix in the visible region [35]. The hydrogel properties depend on its microstructure, which can be controlled by sol-gel synthesis variables. In particular, optical properties of silica hosts obtained via a mixed aqueous route based on sodium silicate-silica nanoparticle mixtures can be improved by increasing the relative content of molecular precursors over colloidal silica [36]. By means of the two-step procedure and due to the protection provided by the Ca(II)-alginate pre-encapsulation, silica synthesis can be attempted under more cytotoxic conditions, allowing the use of a higher ionic strength than possible in one-pot encapsulation procedures. In a previous work, we investigated the degree to which the scattering of visible light affects the encapsulated algae growth rate, analyzing the number of Chlorella vulgaris cells developed in voids created inside silicate-Ludox hydrogels as a function of gel thickness [32]. Following the same encapsulation procedure as described in the previous section, except for the proportion of polymeric to particulate silica precursors, which was varied in the range 1:3 to 1:5, it was found that the growth of C. vulgaris was affected by both the attenuance of the matrix and the host thickness. However, for the matrix with higher polymeric precursor content, the growth rate was unaffected, even for gel thicknesses up to 4.0 mm.
In the present study, the optimized ratio of polymeric to particulate silica precursors (i.e., 1:3) was investigated for the two-step encapsulation of C. reinhardtii (CR), P. subcapitata (PS) and C. vulgaris (CV). Silica hydrogel thickness was fixed at 1.9 mm, since in the previous study, regardless of the optical quality of the silica matrix, no detriment in CV growth rate was found for this silica thickness. At the initial time, calcium alginate beads with a content of 10 4 cells/mL were dispensed into acrylic molds, and the silica encapsulation was performed. The algal growth inside the voids was studied for individual cavities after 3 days of culture in AFNOR liquid medium. As shown in Figure 2 and in agreement with previous results, [32] CV growth rate was almost unaffected (growth inhibition ≈ 2%). For the other algal species, we could see a slight difference of sensibility, although in the three cases these results confirm the biocompatibility of the two-step procedure immobilization with a relatively low growth inhibition (6% and 13% for PS and CR, respectively).

Conclusions
In this study, we demonstrate the feasibility of using an inorganic translucent hydrogel to construct an optical biosensor with immobilized algal cells. According to our results and in agreement with former studies [27], the sol-gel immobilization in two steps shows a high biocompatibility with green algae; this encapsulation method is easy to perform and allows one to obtain a matrix with good optical and mechanical performance. Furthermore, the polymeric and particulate silica precursors used in this synthesis are low cost reagents, which is stimulating from the point of view of the scaling up in future applications. The activity of algae whole cells immobilized within the silica hydrogel can be assessed from chlorophyll fluorescence measurements. The translucence of the structure allowed the algal layer to be placed directly in contact with the optical fibers to produce a fluorescence emission detectable by a fluorometer. In addition, this whole-cell reagentless biosensor is economically very interesting because it requires neither substrate nor fluorescent indicator to monitor herbicide levels in the environment.
The use of this entrapment technique is facilitated by the optical transparency, mechanical stability and chemical inertness of the silica host. Optical properties are of crucial importance for both the growing of photosynthetic algal cells inside the biosensing devices and for the detection method based on the determination of the chlorophyll fluorescence enhancement. Since the porosity of the silica matrix (in the range of mesopores) can be tuned by the sol-gel synthesis method used, the transport properties of the host material can be regulated in order to allow free diffusion of aqueous pollutants to make them accessible to algal cells. This technique has been applied to the determination of DCMU and atrazine, as examples of herbicides that are known to inhibit the algal PSII. Among the algal strains tested, Chlamydomonas reinhardtii is the most sensitive specie for atrazine determination, with a limit of detection of 0.1 µM after 40 minutes of exposure.
This optical algal biosensor could be used as an early-warning device to detect those pollutants in effluents. Performance and optimization studies with other herbicides are under way and the results will be compared to other analytical techniques. Before an in situ implementation the limits of detection have to be improved. Concentrations of DCMU and atrazine in controlled effluents are around 0.1 and 10 µg/L and European Environmental Quality Standards (EEQS) are 0.2 µg/L for DCMU and 0.6 µg/L for atrazine [3,37,38].
On previous papers, we have shown that the use of C. vulgaris cells as a bioreceptor allowed the determination of the antiphotosystem II (PSII) herbicides group which target the algal PSII fluorescence [10]. Under the same conditions ([C. vulgaris] = 1.5 × 10 6 cells/mL and phosphate buffer pH 7.0) the limit of detection of atrazine was 1 µg/L (5 × 10 −3 µM) with 1% fluorescence increase. These results were obtained with a high sensitivity fluorometer equipped with single photon counting capabilities (the Spex Fluorolog 2). We believe that using a more accurate fluorometer and by improving the immobilization method to decrease the variability, we could further reduce these detection limits.