WO1997012990A1

WO1997012990A1 - Device for detecting and controlling biofilms

Info

Publication number: WO1997012990A1
Application number: PCT/FR1996/001515
Authority: WO
Inventors: Jean-François LIPSKIER
Original assignee: Thomson-Csf
Priority date: 1995-09-29
Filing date: 1996-09-27
Publication date: 1997-04-10
Also published as: EP0795028A1; FR2739448A1; FR2739448B1

Abstract

A device for detecting and controlling a biofilm being formed on a surface exposed to a moist environment. The device includes at least two microsensors (Co, C1) of which one has a biofilm-neutral surface exposed to the moist environment while another has a biofilm formation-inhibiting surface exposed to the moist environment. The device further includes differential measurement means between the two microsensors. The inhibitory effect of the exposed surface may be provided by antibiotic agents that are active against microorganisms in the biofilm. The device may be used in a water supply system.

Description

BIOFILMS DETECTION AND MONITORING DEVICE

The field of the invention is that of micro-sensors allowing the detection and control of biofilms capable of forming on any type of surface in contact with a wet medium and in particular at the level of the pipes in which an aqueous medium flows.

In general, biofilms are structures formed by bacteria or other microorganisms, which deposit on surfaces exposed to moisture and secrete polysaccharides forming a glycidic matrix extremely resistant to abrasion. This matrix strengthens the adhesion of bacteria to the surface and protects them against chemical agents, in particular disinfectants (Bryers, J.D., Colloids Surf. B: Biointerfaces, 1994, 2 (1-3), 9-23). Such films are formed in particular on the walls of the conduits of drinking water supply networks, and are often responsible for a deterioration in the quality of the water. Thus, the appearance of an unpleasant taste can result from the secretion of soluble organic molecules such as geosmin or isoborneol. In addition, there is generally an increase in the concentration of potentially pathogenic microorganisms, as well as the appearance of a micro-flora and a micro-fauna (protozoa, small crustaceans, worms ...). Finally, these biofilms can be responsible for accelerated corrosion of the installations. This latter problem is encountered not only in drinking water distribution networks, but also in industrial installations such as water injection systems in oil wells (Ferris, FG et al. Can. J. Microbiol , 1992, 38 (2), 1320-1324) or heat exchangers. In the latter, the formation of biofilms also reduces the efficiency of heat transfers, pressure losses are increased, thus significantly reducing the efficiency of the installations (Shelton, DR and Tiedje, JM Appl. Environ. Microbiol., 1984, 48 , 840-848).

The formation of bacterial biofilms is also observed in deionized water distribution systems used in many sectors of industry and research (Richardson Z., Proc. 5th Int. SAMPE Electronics Conf., June 18-20, 19921, 552-558). Many microorganisms have a sufficiently deformable structure to be able to pass through the pores of the filters used in these systems. The problem is particularly acute in the electronics industry. Various chemical species secreted by these microorganisms are sources of impurities which can dramatically affect the performance of semiconductors (JE Martyak, JC Carmody, GR Husted, Microcontamination, 1993, 11 [1], 39-44).

For all these reasons, we are currently seeking to have means of very quickly detecting the formation of biofilms and of measuring their evolution.

Several solutions have been proposed to date: One of the methods used to assess the extent of bacterial contamination and of the formation of a biofilm consists in mechanically taking a sample, then in cultivating the bacteria collected in an agar medium. A count is then carried out by the usual microbiology techniques. This method has a number of drawbacks.

Firstly, the taking of samples is difficult and not very reproducible, due to the excellent adhesion of the bacterial film on most common materials; moreover, such a removal is not always feasible in practice, for example in the case of buried installations or of difficult access.

Second, the bacteria forming the biofilm thrive in an unfavorable environment, poor in nutrients, and are subjected to various aggressions such as thermal shock, osmotic (variable saline concentration, desiccation), or chemical (possible presence of disinfecting agents) . As a result, they develop a number of resistance mechanisms. In particular, their metabolism slows down, and the time required to double their population increases in proportions that are difficult to assess. Finally, these microorganisms lose the ability to develop on synthetic culture media used in the laboratory. For these reasons, the importance of contamination is frequently underestimated.

The method is therefore not only difficult to use, but also unreliable.

Recently, it has been proposed to use a flow cytometry method, based on a fluorescence measurement: a fluorescent marker is synthesized by bacteria from a precursor. The fluorescence intensity measured can, under certain conditions, be correlated with the number of bacterial cells contained in the sample. This technique makes it possible to dispense with the stage of culture on synthetic agar medium, and therefore to take into account the fraction of non-cultivable microorganisms. However, it still requires the taking of representative samples, which is not always easy or even possible. In addition, it requires the addition of a reagent (precursor of the fluorescent label) in relatively large amounts, which can in itself constitute unacceptable contamination.

A miniaturized biofilm sensor, integrated on silicon, has been described (Stenberg M., Stemme G., Kittilsland G. Pedersen K., Sens. Actuators, 1988, 13 (3), 203-221). This sensor actually measures the amount of heat transferred by a moving fluid from a heat source (heating element) to a temperature sensitive diode located downstream. This amount of heat is affected by the presence of a biofilm on the surface of the sensor, forming an insulating barrier opposing heat transfer. Although of undeniable interest, this sensor suffers from the following drawbacks. First, the measurement performed is not specific to the formation of a biofilm. A deposit of another nature (tartar for example), also results in a response from the sensor. Furthermore, a variation in the physical conditions of the measurement (medium temperature, viscosity of the fluid, flow rate, ionic strength, summer, etc.) can give rise to a drift in the detector. Finally, the technology required for manufacturing the device is expensive and still not widely used.

In this context, the subject of the invention is a device for detecting and controlling biofilm forming on a surface S in contact with a wet medium, based on a differential measurement between several micro-sensors, at least one of which has ta property of being neutral with respect to the biofilm, the latter thus being able to form on said microsensor, another microsensor having a contact surface with the wet medium inhibiting the formation of biofilm. This last micro ^: sensor constitutes a reference to which the signal supplied by the first micro-sensor can be compared. More specifically, the subject of the invention is a device for detecting and controlling biofilm forming on a surface S in contact with a wet environment, characterized in that it comprises:

- at least two micro-sensors C ₀ and C- | deposited on the surface S, the micro-sensor C ₀ having a contact surface with the humid medium, neutral with respect to the formation of biofilm, the micro-sensor C- | having a contact surface with the wet environment, provided with a coating R- | inhibiting biofilm formation;

- differential measurement means of the various micro-sensors.

Advantageously, the microsensor C ₀ can have a contact surface with the aqueous medium provided with a coating of the same nature as that of the surface S.

Advantageously, the micro-sensors used in the device of the invention can be sensors sensitive to the mass of material deposited on their surface; it can typically be acoustic wave micro-sensors. In this variant of the invention, the mass linked to the surface of the micro-sensor C ₀ increases as a biofilm is formed. The micro-sensor C- | does not measure an increase in mass resulting from the formation of a biofilm. On the other hand, any physical or chemical phenomenon (in particular the effects linked to variations in temperature or density of liquid) having no causal link with the formation or growth of a biofilm, influences the response of the two micro-sensors C ₀ and C- | in the same way. By comparing the signals from the two micro-sensors, it is thus possible to overcome the above-mentioned disturbances; the response obtained is indeed unequivocally correlated to the formation and growth of the biofilm.

Advantageously, the coating R 1 can comprise an antibiotic agent inhibiting the formation of biofilm which can be enclosed in a host material, suitable for combating a specific type of microorganism. Advantageously, the coating R ₀ can consist of a hydrophobic polymeric material and the coating Ri can comprise an interpenetrating network of macromolecules Mh in a matrix of polymer material identical to that of the coating R ₀ . In order to widen the field of prospecting of the biofilm detection and control device mentioned above, the invention also relates to a device associating a plurality of micro-sensors Cj, ... Cj, ... C _n , provided with coatings Rj, ... Rj, ... R _n in which different antibiotic agents are incorporated, so as to determine the nature of the contaminating species responsible for the formation of biofilm.

The invention will be better understood and other advantages will appear on reading the description which follows, given without limitation and thanks to the appended figures, among which: - Figure 1 illustrates an example of a device according to the invention using two volume acoustic wave micro-sensors;

- Figure 2 illustrates an example of a device according to the invention, using two surface acoustic wave micro-sensors;

- Figure 3 illustrates an example of love acoustic wave micro-sensor, used in an example of a device according to the invention;

- Figure 4 illustrates chemical formulas of agents introduced into an example of coating R- | and inhibiting biofilm formation;

- Figure 5 gives examples of surfactants used in devices according to the invention. We will describe the invention in the context of acoustic wave microsensors, which have the dual advantage of being particularly sensitive and of simple design and suitable for the targeted applications (detection of biofilms in pipes, etc.). However, other types of micro-sensors can be used and in particular micro-sensors such as those described by Stenbeerg et al (mentioned in the prior art cited above).

Generally speaking, there are two types of acoustic wave sensors: volume acoustic wave sensors and surface acoustic wave sensors. The former are often designated by the BAW sign from the English "Bulk Acoustic Waves". The latter are often designated by the acronym SAW ("Surface Acoustic Waves").

BAW sensors are generally made from a plate of piezoelectric material such as monocrystalline quartz, cut along certain orientations corresponding to a strong piezoelectric coupling associated with a low sensitivity to variations in temperature. Pairs of metal electrodes symmetrically deposited on either side of the plate make it possible to excite and detect acoustic waves propagating in the mass of the material. According to the device of the invention, two similar sensors C ₀ and C- | are associated. One, Cj, is provided on one of its electrodes Ei i, with a film of material Rj, inhibiting the formation of biofilms. The other, C ₀ , can be left bare, or can advantageously be provided with a film of material R ₀ on one of its electrodes E ₀ - | , the material R ₀ being identical to the material constituting the installations to be monitored, or at least of composition and very similar chemical structures.

For example, to monitor PVC pipes, it may be advantageous to deposit on the sensor C ₀ , a PVC film of the same kind. Furthermore, in order to minimize the manufacturing and environmental differences between Ci and C ₀ , it may be advantageous to produce the two sensors side by side on the same quartz quartz substrate, as illustrated in FIG. 1, the cons -electrodes E ₀ 2 and E12 ^{of the} two sensors C ₀ and C- | being on the other side of said substrate sq.

In another variant of the invention, the device of the invention comprises surface wave micro-sensors. These sensors include two series of interdigitated electrodes, deposited on the surface of a piezoelectric material. FIG. 2 illustrates an example of a device in which the two sensors are mounted in parallel, the sensor C- | comprises two sets of electrodes SE11 and SE12 separated by a distance d, on the surface of a piezoelectric material, between these two sets of electrodes, the coating R- | is also deposited on the surface of the piezoelectric material. The sensor C ₀ also includes two series of interdigitated electrodes SE ₀ - | and SE ₀ 2, separated by the same distance d as in the sensor Cj. A coating R ₀ can advantageously be deposited between the two series of electrodes SE ₀ ι and SE ₀ 2- All of the sensors C ₀ and Cj can be produced on the same piezoelectric substrate, quartz type, sq.

To perform the function of biofilm formation inhibitor, two types of materials can be used. The first type prevents the adhesion of microorganisms by modifying the surface energy of the substrate; the second inhibits the growth of bacterial colonies by the effect of incorporated antibiotic agents, diffusing slowly towards the surface. a) Coatings reducing cell adhesion:

In this approach, which applies particularly well to polymeric materials, it is sought to reduce the hydrophobic nature of the surface to be protected by incorporating surfactants or surfactants into the substrate sq. Examples of effective surfactants are: poly (ethylene oxide), as well as block copolymers of type A-B-A where A is the poly-

(ethylene oxide) and B is the poly (propylene oxide) known to those skilled in the art under the name "Pluronic" (registered trademark of BASF) or "Synperonic" (registered trademark of ICI); polyionenes such as quaternary polyammoniums or polyphosphonium. These surfactants can be incorporated into the surface of the substrate sq to be protected in different ways: - one can thus simply incubate the substrate sq in an aqueous solution containing the surfactant; this technique applies particularly well in the case of agents of the Pluronic type deposited on hydrophobic surfaces, in particular on polymers such as PVC, polystyrene, PMMA ...: the blocks of type B, hydrophobic, then adhere strongly at the surface, while the hydrophilic type A blocks project towards the aqueous phase, forming a sterically stabilized interface;

- another method, designated by the acronym SPIN (Surface Physical Interpenetrating Networks), consists in placing the surface of the substrate sq to be protected in contact with a surfactant solution (for example poly (ethylene oxide) in a common solvent ; the surfactant is allowed to diffuse in the substrate sq whose surface is swollen by the solvent, then rapid quenching is carried out in a solvent of the surfactant, non-solvent for the material; the surfactant is thus incorporated into the surface of the substrate, in a state metastable;

- A third method consists in using intermediate molecules capable of forming covalent bonds on the one hand with the substrate and on the other hand with the surfactant. This approach applies not only to substrates based on polymer materials, but also to 97/12990 PC17FR96 / 01515

8

metallic surfaces, or inorganic materials, and in particular quartz.

Although all three methods can be used, the second or third are preferred, which have the advantage of better long-term stability. Indeed, the surfactant adsorbed on the surface sq in the first method, can be dissolved in the aqueous phase in the long run. Example 1 of a device according to the invention A detector is produced according to the invention by combining two acoustic wave sensors of volume C ₀ and Cj, coated with a film of poly (vinyl chloride) (PVC) (Aldrich 34, 675-6, high molecular weight) produced by spraying (spray coating) from a 5 gl "1 solution of PVC in tetrahydrofuran (THF), and annealing for 1 h at 60 ° C. The sensor C- | is further incubated, according to a procedure adapted from (Desai, NP et al., biomaterials, 1991, 12 (2), 144-153), in a solution containing: - 17,500 polyethylene glycol (PEG) (Fluka, Mr - 15000-

20000): 80 g.H

- THF: 40%

- deionized water: 60% brought to 50 ° C, for 15 minutes, then soaked in an excess of deionized water. At the end of this treatment, PEG molecules are incorporated in a stable and lasting manner on the surface of the sensor C- | . The hydrophilicity of the latter, compared to the surface of the untreated PVC (such as that of the sensor C ₀ ) can be evaluated by measuring the contact angle of a drop of water on the coating: the angle is significantly lower on C- | (25 °) than on C ₀ (48 °). The method can be adapted for sensors coated with various polymer materials, preferably of the same type as those used in the installations to be monitored: polyurethane, PET, PMMA, polystyrene, etc. The table below summarizes the operating conditions which may be used depending on the material R- _| constituting the coating of the sensor C <| . In this table, Σ 1 denotes the solvent from which this material is deposited, ∑2 the immersion solvent (or solvent mixture) containing the PEG (concentration 80 gl "^''), Σ3 the quenching solvent, H2O denotes deionized water, THF tetrahydrofuran, TFAA trifluoroacetic acid, AC acetone.

The sensors thus treated have a hydrophilic surface inhibiting the attachment of cells capable of forming a biofilm. Example 2 of a device according to the invention

Two sensors C ₀ and C- _| using Love's acoustic waves, are produced according to a process analogous to that described in (Gizeli, E. et al. Sens. Actuators B, 1992, 6, 131-137). These are sensors comprising an intermediate layer between the piezoelectric substrate and the coating R ₀ and / or R- | , in which the propagation speed of the acoustic waves is lower than that of the acoustic waves in the piezoelectric material. FIG. 3 illustrates this type of sensor in which sets of interdigitated electrodes SEj-] and SEJ2 are deposited on the surface of the piezoelectric substrate and covered with the intermediate layer, and more precisely relates to the micro-sensor Ci.

Thus in this type of device according to the invention, these sensors comprise a quartz substrate on which a layer of poly (methyl methacrylate) (PMMA) is deposited. PMMA from sensor C- | is further functionalized in order to obtain a surface inhibiting the formation of biofilm, according to a process described in (Dunkirk, SG et al., J. Biomater, Appl. 6, 131 - 156, 1991), by photochemical reaction with poly (ethylene glycol) (PEG) or with a copolymer of vinylpyrrolidone and of N- (3-amino-propyl) methacrylamide, substituted by a photoactivatable surfactant such as the 4-benzoyl benzoic acid described in FIG. 4. In this example , the functionalized PMMA layer constitutes the coating R- _| . The surface of C ₀ is not modified. Example 3 of a device according to the invention In this example, the sensors C ₀ and Cj have a surface made up of silicon oxide. The surface of Cj, carefully cleaned, is incubated in a solution consisting of (III) NNN-trimethyl-3 (trimethoxysilyl) -l propanaminium chloride (Hϋls America Inc., T2925, FIG. 6) at 10% in methanol for 6 hours, washed with methanol and then with deionized water. At the end of this treatment, the quaternary ammonium groups, which have a bactericidal and fungicidal action, are chemically grafted on the surface of C- | . Other surfactants, such as (IV) (N, N, N-tributyl) -3 (trimethoxysilyl) -l propanaminium bromide, (V) (N-dimethyl N-octadecyl) -3 chloride ( trimethoxysilyl) -l propanaminium, (VI) (N- methyl N, N-didecyl) -3 (trimethoxysilyl) -l propanaminium chloride, capable of inhibiting the formation of biofilms, can be grafted similarly, their chemical formulas are given in figure 5. b) Coatings progressively releasing an antibiotic agent: In this approach, the surface of the sensor C- | is coated with a film made of a polymeric material in which is dispersed an antibiotic agent A (bactericide, fungicide, etc.). Antibiotic A slowly diffuses to the surface and is gradually released into the surrounding aqueous phase.

Example 4 of a device according to the invention The coating of the sensor C- | is produced according to a process inspired by (Golomb, G. and Shpigelman, A., J. Biomed, Mater. Res., 1991, 258), 937-952). A film is deposited by spraying (spray coating) from a solution of polyurethane (approx. 5 g. M) and propylparaben (1 to 2 g. H) in THF. After evaporation of the solvent, the film is annealed at 60 ° C for 1 hour. c) Coatings reducing cell adhesion and gradually releasing an antibiotic agent:

The two previous approaches can be combined to achieve the coating of the sensor C- | , so as to increase its efficiency. Example 5 of a device according to the invention The coating of the sensor C- | is carried out by combining the approaches described in examples 1 and 4: a polyurethane film is produced according to the method described in Example 4. This film is then incubated in a PEG solution as described in Example 1, additionally containing saturated propylparaben. The film is then rapidly soaked in water, as in Example 1. The coating obtained has a hydrophilic surface thanks to the PEG chains incorporated in the polyurethane network. In addition, it gradually releases propylparaben, which has broad-spectrum antibiotic activity.

In general, an additional advantage can be obtained by associating with the sensor C ₀ , not one but n sensors Cj, ... C _n , each of these n sensors being provided with a coating for example of the type described in b) , the n coatings R- | , ... R _n being obtained by incorporating into the same polymer matrix n antibiotic agents having different activity spectra. In the presence of a given species of microorganism, the formation of biofilm is more or less inhibited depending on the coating. The response spectrum of the n + 1 sensors C ₀ , C- | , ... C _n , thus provides an indication of the nature of the contaminating species, analogous to a conventional antibiogram. This makes it easier to select the most suitable disinfection method.

Claims

1. Biofilm detection and control device forming on a surface (S) in contact with a wet environment, characterized in that it comprises:

- at least two micro-sensors (C ₀ ) and (C- |) deposited on the surface (S), the micro-sensor (C ₀ ) having a contact surface with the wet environment, neutral with respect to the formation of biofilm, the microsensor (C-) having a contact surface with the wet environment, provided with a coating (R <|) inhibiting the formation of biofilm;

- means for differential measurement of the various micro-sensors.

2. Biofilm detection and control device according to claim 1, characterized in that the micro-sensor (C ₀ ) has a contact surface with the wet medium provided with a coating (R ₀ ) of a nature identical to that of the surface (S).

3. Biofilm detection and control device according to claim 2, characterized in that the coating (Rj) comprises a host material containing an antibiotic agent, gradually released in the wet environment, the coating (R ₀ ) comprising the same material host without antibiotic agent.

4. Biofilm detection and control device according to one of claims 1 or 2, characterized in that the coating (Rj) comprises material of hydrophobic polymer type and identical to that of the surface (S) and surfactants or surfactants reducing the hydrophobic nature of said material.

5. Biofilm detection and control device according to claim 4, characterized in that the surfactants are copolymers of poly- (ethylene oxide) and poly- (propylene oxide).

6. Biofilm detection and control device according to claim 4, characterized in that the surfactants are poly-ionenes of the quaternary ammonium or phosphonium type.

7. Biofilm detection and control device according to claim 2, characterized in that the coating (R ₀ ) consists of a hydrophobic polymeric material and that the coating (Rj) comprises a interpenetrating network of macromolecules (Mh) in a matrix of polymer material identical to that of the coating (R ₀ ).

8. Biofilm detection and control device according to claim 7, characterized in that the molecules (Mh) are poly- (ethylene glycol) molecules.

9. Biofilm detection and control device according to one of claims 1 to 8, characterized in that the micro-sensors are with acoustic waves.

10. Biofilm detection and control device according to one of claims 1 to 9, characterized in that it comprises a plurality of micro-sensors (Ci), ... (Cj), ... (C _n ) provided with different coatings (R- |), ... (Rj), ... (R _n ) in which are incorporated different antibiotic agents, active with respect to different agents existing in the biofilm.