WO2004040004A1 - Multienzymatic conductimetric or potentiometric biosensor with unicellular algae, detection method using same - Google Patents

Abstract

The invention concerns a multienzymatic biosensor for detecting specific pollutants potentially present in an aqueous liquid, comprising a conductimetric or potentiometric sensor, with a reference electrode and a measuring electrode whereon living unicellular algae are immobilized, said algae comprising different reactive membrane-bound enzymes, the detectable pollutants being capable of inhibiting the enzymatic activity of one of the membrane-bound enzymes, as well as detection methods using said biosensors.

Description

CONDUCTIMETRIC OR POTENTIOMETRIC MULTI-ENZYMATIC BIOCAPTOR WITH UNICELLULAR ALGAE, DETECTION METHOD USING SUCH A BIOCAPTOR

The present invention relates to the technical field of biosensors. In particular, the subject of the invention is a multi-enzymatic electrochemical biosensor with unicellular algae, as well as a method for detecting the possible presence of pollutants within an aqueous liquid using said biosensor.

A biosensor is a tool that transforms a biochemical phenomenon into an electrical signal. It is composed of a biological element also called a bioreceptor, as well as a transducer which converts into an electrical signal the biochemical modification occurring at the level of the bioreceptor. There are different types of biosensors which differ in the nature of their bioreceptor, but also of their transducer.

In the area of the environment, it is very important to have tools capable of detecting the presence of pollutants or toxic substances within aqueous media, such as aquatic ecosystems, water from treatment plants, industrial effluents. Indeed, the problem of pollution of water, for example, by pesticides and heavy metal ions is becoming more and more critical. To limit the aggression of pollutants on ecosystems, it is necessary to develop early detection tools capable of detecting them quickly, which are also easily transportable to sites to be monitored and which have relatively low cost prices.

The detection of low concentrations of pollutants in the environmental field requires precise and sensitive methods of analysis. Chromatographic or spectroscopic methods are widely used, but these methods are expensive, long to implement and above all have the major drawback of detecting only certain species which are specifically sought. As a result, toxic products may still be present in the samples without being able to identify them. Other approaches for controlling pollution of aqueous liquids have been developed. Ecotoxicity on living organisms ranging from rainbow trout to bioluminescent bacteria is widely used. These methods are implemented works not to identify particular pollutants, but to give an idea of the toxicity of a mixture vis-à-vis the living organism used as a bioindicator. In this case, the response time is sometimes long, that is to say it can reach several days, and the measurements are confined to the laboratories. It would therefore be interesting to have other more appropriate techniques. The inventors came up with the idea of developing new biosensors capable of detecting different types of pollutants in an aqueous liquid. Among the biosensors for detecting pollutants, there are so-called mono-enzymatic biosensors. A single enzyme is immobilized on these biosensors, so that they are sensitive only to a single class of specific inhibitors of the said enzyme and therefore cannot be used to detect the presence of many pollutants present simultaneously in the liquids aqueous to study. In addition, the enzymes that can be used in this type of application are limited in number because of their instability and their prohibitive price. This type of mono-enzymatic biosensors are therefore not suitable for environmental applications.

Other biosensors use whole cells as a bioreceptor such as different microorganisms such as bacteria or algae. We can refer to SENSOR & ACTUATORS, 1996, 1334, 270-275 which describes optical biosensors based on measurements of disturbance of algal metabolism at the level of its general functions such as photosynthesis and fluorescence. In this case, the measurements made are not based on the enzymatic activity of the whole cells used.

Claude DURRIEU et al describe in Ecotoxicology and Environmental Safety, 2002, 51, 206-209, a biosensor with optical detection on which unicellular algae are immobilized. The enzymatic activity of alkaline phosphatase is measured using a fluorometer and makes it possible to detect the presence of heavy metals. However, these measurements require the use of a fiuorimeter making the sensor difficult to transport. In addition, such a biosensor is not suitable for the detection of heavy metals present in low concentration, for example less than 10 ppb. In this context, one of the objectives of the present invention is to provide a new device capable of detecting the possible presence of pollutants in an aqueous liquid, this device having to meet the following requirements: to be able to detect and to identify, selectively, different pollutants present in the aqueous media to be controlled, to be able to carry out instant and in situ measurements, to be presented in a miniaturized form easy to implement in the field and easily transportable, to be stable and capable of carrying out continuous measurements, - being easy to manufacture and at a relatively low cost price, being able to detect small quantities of pollutants, in particular of the order of ppb. The subject of the invention is therefore a multi-enzymatic biosensor, intended to detect the possible presence of specific pollutants within an aqueous liquid, comprising a conductimetric or potentiometric sensor with a reference electrode and a measurement electrode on which algae living single-cell cells are immobilized, these algae comprising different reactive membrane enzymes, the detectable pollutants having the power to inhibit the enzymatic activity of one of the membrane enzymes. Another aspect of the invention relates to a method for detecting the possible presence of pollutants within an aqueous liquid, characterized in that it comprises the following steps: a) bringing a biosensor as defined above into contact with the aqueous liquid, then b) measure the possible inhibition of the enzymatic activity of a first membrane enzyme in the unicellular algae to deduce the presence of specific pollutants responsible for the inhibition detected, c) repeat step b) to a second membrane enzyme. The inventors have shown that it is possible to obtain particularly efficient biosensors by associating a conductimetric or potentiometric sensor with living unicellular algae. Indeed, unicellular algae have a high sensitivity to pollutants linked to their power large accumulator. Furthermore, these organisms are very easy to handle and cultivate. In addition, unicellular algae are particularly robust organisms whose size and shape facilitate their immobilization on the electrochemical sensor. Above all, unicellular algae naturally have different types of membrane enzymes that can be used to detect different specific pollutants, even present in low concentrations. These enzymes being in their natural environment, they therefore have very good stability.

Furthermore, the use of electrochemical sensors, of the conductimetric or potentiometric type, allows rapid measurement which can be carried out directly in the field. In addition, the conductimetric or potentiometric sensors used are commercially available at low cost.

The biosensor according to the invention therefore comprises living unicellular algae immobilized on the surface of the biosensor intended to be brought into contact with the aqueous liquid to be studied. It is possible, for example, to use single-cell algae from the family of chlorophyceae, for example algae Scenedesmus subspicatus, Pseudokirchneriella subcapitata or preferably chlorella vulgaris, or even from the family of euglenophyceae or cyanophyceae. These unicellular algae are immobilized on the measurement electrode of an electrochemical sensor according to the invention. This immobilization can be done by any means, for example by adsorption then crosslinking. In particular, a membrane can be produced by combining the algal cells with a substance capable of polymerizing, adsorbing this membrane on the electrode, then adding a crosslinking or polymerizing agent in order to crosslink the membrane and therefore immobilize the algae. As a substance capable of polymerizing, it is possible for example to use bovine serum albumin (BSA) or calcium alginate in combination with glutaraldehyde or calcium chloride respectively as crosslinking agent. The use of BSA is particularly preferred. Advantageously, a homogeneous mixture of algal cells and BSA containing 5 to 15% of BSA will be deposited on the electrode. In particular, it is best to deposit from 1.10 ³ to 10.10 ³ algae / mm ² of electrode surface, and preferably from 1.10 ³ to 3.10 ³ algae / mm ² of electrode surface.

The unicellular algae have different membrane enzymes capable of reacting with different specific pollutants present in the aqueous liquid to be studied. In particular, mention may be made of the alkaline phosphatase enzymes whose enzymatic activity is inhibited by the presence of heavy metals, the esterase enzymes whose enzymatic activity is inhibited by the presence of organophosphorus derivatives and the nitrate reductase enzyme whose activity enzyme is inhibited by NH ⁺ ions. The biosensors according to the invention are therefore perfectly suited for detecting the presence and variations in the concentration of heavy metals (Cd, Zn, Pb ...) or of organophosphorus pesticides in aqueous liquids.

The principle of detection consists in monitoring the influence that the liquid to be analyzed has on the enzymatic activity of the enzymes present at the level of the membrane of the algal cells. For example, it is known that heavy metals such as Cd, Zn, Pb behave like inhibitors of the enzymes alkaline phosphatases. Consequently, by measuring the enzymatic activity obtained with the sensor in the presence of a substrate specific for alkaline phosphatase at saturation, before contact of the sensor with the medium to be checked and by comparing it with the enzymatic activity obtained after contact with the medium, it is deduced therefrom if the medium contains heavy metals responsible for the inhibition of the enzymatic activity of the alkaline phosphatase observed. Of course, the membrane enzyme must be reactive, that is to say capable of reacting with a specific substrate. In the case of the alkaline phosphatase enzyme, present in particular in chlorella vulgaris, it is necessary to prepare it to make it reactive. To do this, the algae are kept in a phosphate-free medium for a minimum of 15 days, in order to exhaust their phosphate reserve.

The enzymatic activity is measured using the conductimetric or potentiometric sensor. Indeed, the enzymatic reaction causes chemical changes such as the formation of phosphate ions or variations in pH directly measurable thanks to the conductimetric or potentiometric sensors by detection of the conductance or the pH of the solution. Unlike others electrochemical sensors, of the amperometric type, conductimetric or potentiometric sensors do not require the presence of electroactive species.

The electrochemical sensor comprising a reference electrode and a measurement electrode on which living algal cells are immobilized, makes it possible to detect the chemical reactions occurring at the level of the enzymes in the form of a measurable electrical signal. This electrochemical sensor is, for example, connected, by means of electronic means capable of processing the electrical signal emitted, to a tracer table making it possible to follow the evolution of the signal. The types of sensors or electrodes that can be used are: - potentiometric sensors making it possible to determine the potential difference which is established between a first measurement electrode on which the algal cells are deposited and a second reference electrode or,

- conductimetric sensors; the principle of conductimetric sensors is based on the measurement of conductance at the level of the detection cells located at the end of the sensor. This conductance varies when highly mobile charge carriers are generated or consumed by the enzymatic reactions.

It is possible to use a conductimetric transducer made up of two pairs of interdigitated platinum electrodes deposited on a Si / SiO ₂ substrate. One of the electrodes is used to make the measurement while the other plays the role of reference. Such a transducer is immersed in the solution to be tested. The measured electrical signal is amplified and transferred to a plotter. It is then easy to study the variations in conductance during the injection of a reagent. The signal traced corresponds to the difference of the two signals emitted at the level of the two detection electrodes. Potentiometric sensors measure local variations in pH directly related to the concentration of H ^{4 *} ions. During certain enzymatic reactions, the release of H ⁺ or OH ^" ions can locally modify the pH. Electrodes may be used on the surface of which SiOH groups are deposited. During an enzymatic reaction, the variation in pH in the membrane on which the algae are immobilized will cause the transformation of SiOH into

SiO ^" or SiO ⁺ H _2. These accumulations of charges on the surface of the ionosensitive electrodes then cause a variation in potential which can be measured. The simpler and less expensive conductimetric sensors are preferred to potentiometric sensors.

The advantages of such electrochemical transducers are numerous. First of all, they do not have a complicated reference electrode: it is sufficient to immobilize on the reference electrode unicellular algae devoid of enzymatic activity. For this, use will be made, for example, of unicellular algae previously heated to 100 ° C. or a mixture of AFNOR T90-304 medium, immobilized under the same conditions as for the measurement electrode. These electrochemical transducers make it possible to obtain miniaturized biosensors, easily transportable and therefore usable for carrying out measurements directly in the field. In addition, they are insensitive to light.

The method according to the invention is based on the measurement of the inhibition of the enzymatic activity of algal membrane enzymes, inhibition due to the presence of pollutants within the aqueous liquid to be controlled behaving as inhibitors. According to a preferred embodiment, a series of reference measurements is carried out, during which the electrical signal emitted by the biosensor is detected when the latter is brought into contact with a variable concentration of suitable substrate of the reactive membrane enzyme of which we want to measure activity. From this series of measurements, a concentration of substrate is deduced therefrom resulting in the saturation of the enzyme activity of the enzyme. The evolution of the electrical signal dS as a function of the substrate concentration is, for example, plotted on a plotter. This electrical signal is representative of the enzymatic activity. Saturation corresponds to the level highlighted in Figs. 1 and 2.

This series of reference measurements is carried out before carrying out the detection measurement on the liquid to be checked. For this, the electrodes of the biosensor are brought into contact with the aqueous liquid, also called an analyte, to be checked. The contacting of the biosensor with the analyte to be checked can, for example, be carried out in one of two ways:

- Either by direct contact: in this case, the electrodes are immersed in the analyte to be checked and immediately a concentration of substrate corresponding to the saturation of the enzyme whose activity is to be measured is introduced; - Either by prior contact: the electrodes are immersed in the analyte to be checked for a time t, preferably less than 60 minutes, included for example in the range from 15 to 30 minutes, the electrodes are removed, then put in contact with the substrate at the saturated concentration. The contact time should not be too long to prevent pollutants from diffusing through the algae membrane, which could cause the death of the algae.

Then, the enzymatic activity obtained is measured at this concentration of substrate. The possible inhibition of the enzymatic activity of this enzyme is measured by comparing the electrical signal dS obtained, before contact and after contact of the biosensor with the controlled aqueous liquid, at this same concentration.

In the case of direct contact, the biosensor is immersed in the analyte until the signal stabilizes to determine its amplitude dSi „iti _a ι, the desired volume of substrate is injected to determine the signal dSfi _na ι after stabilization, then the value of dS = dSinitiai-dSfi _na ι, used for the comparison, is calculated. According to another embodiment, the comparison can be carried out at a concentration slightly lower than the minimum concentration resulting in the saturation of the enzymatic activity of the enzyme during the series of reference measurements (this concentration is, for example, located a little before the turn presented in FIGS. 1 and 2), which makes it possible to highlight higher percentages of inhibition.

According to a first advantageous mode, the inhibition of the alkaline phosphatase enzymes is measured, then the percentage of inhibition obtained is correlated with the concentration of inhibitors contained in the controlled aqueous liquid, and in particular with the concentration of heavy metal ions. In particular, the inhibition of alkaline phosphatase enzymes is measured using> αrα-nitrophenyl-phosphate as a substrate.

According to a second advantageous mode, the inhibition of the acetylcholinesterase enzymes is measured, then the percentage of inhibition obtained is correlated with the concentration of inhibitors contained in the controlled aqueous liquid, and in particular with the concentration of organophosphorus derivatives. . In particular, the inhibition of the acetylcholinesterase enzymes is measured using acetylcholine chloride as a substrate. According to a third advantageous mode, the inhibition of alkaline phosphatase enzymes and the inhibition of acetylcholinesterase enzymes, as mentioned above, die independently.

The series of reference measurements are, in this case, carried out successively and the measurements after contact use the prior contacting defined above.

The detection method according to the invention makes it possible to detect the presence of very low concentrations of pollutants in an aqueous liquid, and in particular the presence of heavy metal ions and / or organophosphorus derivatives at a concentration of the order of ppb or lower. Indeed, the biosensors according to the invention are multi-enzymatic due to the natural presence of different membrane enzymes in unicellular algae. These membrane enzymes are accessible by the pollutants contained in the aqueous liquid to be controlled. Measuring the enzymatic activity of these different enzymes before and after contact of the biosensor with the aqueous liquid to be analyzed makes it possible to deduce the inhibition of certain enzymatic activities and therefore the presence of specific inhibitors in the controlled aqueous liquid. In addition, the percentage of inhibition observed can be directly related to the concentration of inhibitors, qualified as pollutants.

Furthermore, these biosensors are inexpensive, are in a miniature form which is easily transportable and manipulable and can be used directly in the field for rapid measurements, since their response time is a few minutes. In addition, unlike bacteria, for example, unicellular algae are not very sensitive to bacterial contamination and, unlike isolated enzymes, they do not need to be kept cold. Consequently, due to this great stability, the biosensors of the invention can be stored for at least twenty days without special conditions, in particular in the case of chlorella vulgaris. The shelf life of the biosensors is of course linked to the lifespan of the immobilized algal cells and to the stability of the enzymatic response. The biosensors according to the invention can be used in many fields related to the environment where it is important to continuously monitor aqueous media. These biosensors may in particular be used for the monitoring industrial effluents and detecting pollutants with a view to triggering alarm systems and thus contributing to the preservation of aquatic environments. The examples below illustrate the invention without, however, limiting it.

EXAMPLES

Preparation of membranes and sensors

We use algal strains Chlorella Vulgaris purchased from the Culture Collection of Algae and Protozoa Cumbria, Great Britain.

To have a satisfactory level of alkaline phosphatase activity, algae must be subjected to a phosphorus deficiency period. For this, the cultures sown in AFNOR T90-304 medium and placed in bubbling (150 liters of air / hour) in the culture chamber make it possible to obtain algae in their exponential growth phase (approximately 4 days after transplanting). These algae are then transferred to an AFNOR T90-304 medium without phosphate by centrifugation (4000 revolutions / minute for 10 minutes) then resuspended at a concentration of 3.75 × 10 ⁵ algae / ml. Maximum activity is then obtained for a starvation period of 21 days in a culture chamber and in bubbling. At the end of this period, the algae can be stored, for one month, at 4 ° C. and in the dark without noticeable attenuation of their alkaline phosphatase activity. Two mixtures are prepared from 10 mg of BSA per 100 μl of algae: one contains living algal cells and BSA, the other dead algal cells (passing at 100 ° C for approximately 15 minutes) devoid of activity phosphatase and BSA in the same proportions (mixture deposited on the reference electrode). Each mixture is deposited on one of the two detection electrodes at the end of a conductimetric sensor. Finally, the sensors are placed in contact with glutaraldehyde vapors for 20 minutes. EXAMPLE 1 Measurement of the Alkaline Phosphatase Activity

The buffer, the MgCl and the water are mixed beforehand. The biosensor is immersed in this reaction medium until the signal stabilizes, then a volume of substrate is injected for each measurement. After stabilization of the response signal, the amplitude of the variation dS (μS) before / after injection is calculated. The curve representing dS as a function of the substrate concentration, called enzymatic kinetics, can then be plotted and makes it possible to deduce the concentration of substrate resulting in the saturation of the enzymatic activity of the enzyme. Two substrates were used: methyl umbelliferyl phosphate (MUP) and αra-mtrophenyl phosphate (pNPP). The enzymatic kinetics of alkaline phosphatase obtained with pNPP is presented in Fig. 1.

Then, before contact with the polluted aqueous solution, the enzymatic activity of the alkaline phosphatase is measured, by injecting a volume of substrate corresponding to the saturation of the enzyme, then the sensor is brought into contact with the polluted aqueous solution for a time t. The measurement of enzyme activity is then repeated by injecting the same volume of substrate corresponding to the saturation of the enzyme and the inhibition observed is calculated relative to that before contact.

Such measurements of the inhibition of the activity of alkaline phosphatase were carried out on aqueous solutions containing cadmium ions Cd ²⁺ at concentrations of between 0.1 ppb and 10 ppm.

The volume of pNPP injected is 100 μl, corresponding to 0.86 mM, the contact time between the algae and the Cd ²⁺ ions is 45 minutes. The TABLE below indicates the inhibition rates obtained for Cd ²⁺ concentrations of 1.10 and 100 ppb.

EXAMPLE 2 Measurement of the Acetylcholinesterase Activity

The principle of the measurement is the same as that described in Example 1 for alkaline phosphatase. The substrate used is acetylcholine chloride (AChCl). The enzymatic kinetics of acetylcholinesterase obtained with AChCl is presented in Fig. 2.

Measurements of the inhibition of the activity of acetylcholinesterase were carried out on aqueous solutions containing paraoxon-methyl at concentrations of 50 and 100 ppb.

The volume of AChCl injected is 100 μl, corresponding to 10 mM and the contact time between the algae and paraoxon-methyl is 15 minutes.

For a paraoxon-methyl concentration of 100 ppb, an inhibition rate of acetylcholinesterase of 100% is obtained. EXAMPLE 3 Measurement of the Alkaline Phosphatase Activity Then of the Acetylcholinesterase Activity

The inhibition of the activity of alkaline phosphatase and then of acetylcholinesterase is successively measured, in accordance with Examples 1 and 2 respectively, observed by immersing the biosensor in an aqueous solution containing 25 ppb of cadmium Cd ²⁺ ions and 50 ppb of paraoxon.

The volumes of substrate injected are 100 μl, ie 0.86 mM for pNPP and

100 μl or 10 mM for AChCl. The contact time between the algae and the mixture

Cd ²⁺ / paraoxonmethyl is 30 minutes. A rate of inhibition of acetylcholinesterase of 100% is obtained and a rate of inhibition of alkaline phosphatase of

80%.

Claims

1 - Muti-enzymatic biosensor, intended to detect the possible presence of specific pollutants within an aqueous liquid, comprising a conductimetric or potentiometric sensor with a reference electrode and a measurement electrode on which living unicellular algae are immobilized, these algae comprising different reactive membrane enzymes, the detectable pollutants having the power to inhibit the enzymatic activity of one of the membrane enzymes. 2 - Biosensor according to claim 1, characterized in that the unicellular algae are Chlorella Vulgaris algae. 3 - Biosensor according to claim 2, characterized in that, before immobilization, the Chlorella Vulgaris algae are maintained in a phosphate deficiency medium for a minimum period of 15 days. 4 - Biosensor according to one of claims 1 to 3, characterized in that the algal cells are immobilized at a rate of 1.10 ³ to 10.10 ³ algae / mm ² of electrode surface, preferably from 1.10 ³ to 3.10 ³ algae / mm ² of electrode area. 5 - Biosensor according to one of claims 1 to 4, characterized in that the unicellular algae are immobilized with polymerized bovine serum albumin and glutaraldehyde as crosslinking agent. 6 - Method for detecting the possible presence of pollutants in an aqueous liquid to be controlled, characterized in that it comprises the following successive steps: a) bringing a biosensor according to one of claims 1 to 5 into contact with the aqueous liquid to be checked, then b) measure the possible inhibition of the enzymatic activity of a first membrane enzyme in the unicellular algae to deduce therefrom the presence, in the controlled aqueous liquid, of specific pollutants responsible for the inhibition detected, c ) repeat step b) for a second membrane enzyme. 7 - detection method according to claim 6 characterized in that, prior to step a), a series of reference measurements is carried out during which detects the electrical signal emitted by the biosensor when it is brought into contact with a variable concentration of suitable substrate of the reactive membrane enzyme whose activity is to be measured, to deduce in particular a concentration of substrate causing saturation of the enzyme activity of the enzyme, and in that in step b), the possible inhibition of the enzyme activity of this enzyme is measured, by comparing the electrical signal obtained, before contact and after contact with the biosensor with the controlled aqueous liquid, at the same concentration, corresponding to the saturation of the enzymatic activity of the enzyme during the series of reference measurements. 8 - detection method according to claim 6 characterized in that, prior to step a), a series of reference measurements is carried out during which the electrical signal emitted by the biosensor is detected when the latter is brought into contact with a variable concentration of suitable substrate of the reactive membrane enzyme whose activity is to be measured, to deduce therein in particular a concentration of substrate resulting in the saturation of the enzyme activity of the enzyme, and in that in step b), the possible inhibition of the enzymatic activity of this enzyme is measured, by comparing the electrical signal obtained, before contact and after contact of the biosensor with the controlled aqueous liquid, at the same concentration, corresponding to a concentration slightly lower than the minimum concentration leading to saturation of the enzyme activity of the enzyme during the series of reference measurements. 9 - Detection method according to one of claims 6 to 8 characterized in that the inhibition of the enzymes alkaline phosphatases is measured, then the percentage of inhibition obtained is correlated with the concentration of inhibitors contained in the aqueous liquid controlled, and in particular the concentration of heavy metal ions. 10 - Detection method according to claim 9 characterized in that the inhibition of alkaline phosphatase enzymes is measured using para-nitrophenyl phosphate as substrate. 11 - Detection method according to one of claims 6 to 8 characterized in that one measures the inhibition of the acetylcholinesterase enzymes, then correlates the percentage of inhibition obtained at the concentration of inhibitors contained in the controlled aqueous liquid, and in particular at the concentration of organophosphorus derivatives. 12 - Detection method according to claim 11 characterized in that the inhibition of acetylcholinesterase enzymes is measured using acetylcholine chloride as substrate 13 - Detection method according to one of claims 6 to 12 characterized in that, independently, the inhibition of the alkaline phosphatase enzymes is measured, then the percentage of inhibition obtained is correlated to the concentration d inhibitors contained in the controlled aqueous liquid, and in particular at the concentration of heavy metal ions, and the inhibition of the acetylcholinesterase enzymes is measured, then the percentage of inhibition obtained is correlated with the concentration of inhibitors contained in the controlled aqueous liquid, and in particular at the concentration of organophosphorus derivatives. 14 - Detection method according to one of the preceding claims for detecting the presence of heavy metal ions and / or organophosphorus derivatives at a concentration of the order of ppb or lower in an aqueous liquid.