CZ33021U1 - Electrochemical cell for determining bacterial drinking water contamination - Google Patents

Description

Technical field

The technical solution concerns the construction of an electrochemical cell for the electrochemical determination of bacterial contamination of drinking water sources. A feature of the invention is the use of a highly sensitive impedimetric biosensor based on a boron doped nanocrystalline diamond (BDNCD) layer modified with sensor molecules as working electrodes.

BACKGROUND OF THE INVENTION

The drinking water quality requirements, which are included in the requirements of the European Drinking Water Directive 98/83 / EC, have been transposed into the Public Health Protection Act No. 258/2000 Coll. And this Decree-related Decree No. 252/2004 Coll., As amended. Microbiological and biological indicators differ in the type of limit determined by the risk factor of the biological agent and the limit itself, which may be 0 CFU / 100 ml for risk organisms, or non-zero for those less severe, indicating rather total organic pollution in water. The main monitored indicators include the coliform bacteria, intestinal enterococci, Escherichia coli, Clostridium perfringens. Pathogenic bacteria contained in drinking water present a persistent serious health risk, often with fatal consequences and with considerable economic impact of epidemic infections. Coliform bacteria are not typical indicators of faecal contamination (rather indicate secondary contamination), the true and plausible indicator of sewage contamination and faecal contamination indicator (according to WHO since 1992) is just Escherichia coli, a thermotolerant coliform bacterium that occurs in large numbers in intestines of humans and animals. Water contaminated with bacteria causes health problems that are most commonly manifested as nausea, diarrhea and vomiting.

The current problem in the detection of microbial contamination in drinking water is mainly that classical microbiological analyzes of drinking water samples are culture-based. The culture procedures (standard operating procedures) are adapted to give an adequate result in the shortest possible time horizon. A culture consisting in applying an appropriate volume of a water sample to the base, specific or selective media. Solid media, poured on petri dishes, containing nutrients that the microorganism, if present in the water, will use for its metabolism, will multiply and become visible on the nutrient medium in the form of KTJ, or colony forming units, after some time , depending on the metabolism of the microorganism (2 to 3 days). In practice, in the case of leakage of bacterial contamination into drinking water, there may be a major problem in that the result of the analysis, ie the detection of the contaminant, is very late. This has initiated considerable research efforts in recent years to find new efficient and especially rapid analytical procedures, including the application of biosensors.

In general, each biosensor consists of two basic components, a recognition element with a specific binding affinity to the target analyte and a transducer that converts the specific binding event into a physically measurable quantity. The recognition element may form anchored sensor biomolecules such as antibodies, peptides, carbohydrates, and DNA oligonucleotides on the transducer surface. From the transducer point of view, biosensors can be categorized into several basic groups. First of all, they are optical biosensors, the most common are surface plasmon resonance sensors. Piezoelectric sensors are based on the observation of changes in the resonance frequency on the quartz crystal and its transformation induced by the mass changed on its surface due to the interaction with the target analyte. Another relatively large group are electrochemical sensors that monitor changes in the electric field caused by interactions at the sensor-sample interface.

One of the limiting factors for the widespread introduction of biosensors into practice is the stability of the recognition

U1 elements, i.e. sensor molecules immobilized on the surface of the transducer, especially proteins. This is particularly dependent on the physicochemical inertness of the transducer and the chemistry used to immobilize sensor biomolecules to its surface. The most commonly used materials for the construction of biosensors are silicon and gold. The disadvantage of these materials is that the chemistry used to attach the sensor molecules to their surface, i.e. the sulfide bridge for gold and the silyl ether bridge for silicon, does not provide anchorage with sufficient stability in view of the long-term and repeated use of biosensors. Another equally significant negative factor is that these materials induce denaturation of the proteins bound to them due to their interaction with their not entirely chemically inert surface. Attempts to suppress this effect by co-immobilizing polyethylene glycol or a lipid bilayer have not been very successful. In relation to the above-mentioned shortcomings, it appears to be a very promising transducer based on a semiconductor boron doped nanocrystalline diamond (BDNCD), since it combines its specific electrochemical properties with unique chemical properties and good biocompatibility of the nanocrystalline diamond surface.

The essence of the technical solution

The essence of the technical solution of the electrochemical cell (see its assembly shown in Fig. 1) is the use of impedimetric biosensors (see its principle shown in Fig. 2) based on a boron doped nanocrystalline diamond (BDNCD) layer modified by sensor molecules as working electrodes. The application of the BDNCD layer as a highly sensitive transducer capable of converting, with high sensitivity, the binding event between the sensor molecule anchored thereon and the bacterial cell surface molecules in an impedance change is an original solution. By using a chemically inert BDNCD layer as a transducer, the above-mentioned drawbacks associated with the limited stability of the recognition element in the case of biosensors based on transducers of not completely inert materials such as silicon or gold are eliminated.

The electrochemical cell consists (as shown in Fig. 1) of a hollow cylindrical vessel of cell 1 made of a non-conductive and chemically inert plastic. The cell vessel is provided with a square hole at the bottom and a recess for locating the working electrode 2 and sealing it with a square seal 3. The electrical connection of the working electrode 2 is provided by a metal lead 4 interposed between the cell and an electrically nonconductive circular seal with screw holes. The upper part of the cell is closed by means of an upper circular flange 8 and screws 7. The upper circular flange is provided with a capillary opening for the inert gas inlet 9, an opening for the auxiliary electrode. The working electrode 2, the auxiliary electrode 10 and the reference electrode 12 are connected to a potentiostat / galvanostat allowing to monitor impedance changes as a result of the coupling response between the sensor molecules. and bacteria.

The key point of the construction of the impedimetric biosensor is the deposition of a semiconducting BDNCD layer on a suitable substrate using the so-called CVD (chemical vapor deposition) technique. The CVD technique is the process of formation (deposition) of a nano-crystalline layer on a suitable support by gas-phase reactions. This technique allows the synthesis of a high-quality BDNCD layer with controlled conductivity by reacting a mixture of methane, hydrogen and diborane in the plasma state. The second key point is the surface chemistry of the nanocrystalline diamine, which will allow the immobilization of sensor molecules (ie molecules that show interaction with the cell surface molecules of bacteria). The applied surface functionalization of BDNCD layers represents a multi-stage process. The CVD-prepared BDNCD layer is first subjected to hydrogen plasma reduction to obtain a hydrogen-terminated layer. Its UV-promoted reaction with trifluoroacetamide allylamine yields a BDNCD layer bearing 3-trifluoroacetamidopropyl groups which is converted by base-catalyzed deprotection of amino groups to a BDNCD layer representing amino groups. This is the starting material for the introduction of functional anchor binding anchors allowing ligation of sensor molecules by so-called click chemistry;

The binding pairs are triple bond and azido). Click chemistry, due to its orthogonality in relation to other functional groups present in the bound biomolecule, was used to oriented covalently anchor bacterial-specific peptide, carbohydrate and DNA oligonucleotide vector molecules. In the case of ligation of recombinant monoclonal antibodies in scFv format, the coiled coil technique was used. The latter technique is based on the intermolecular interaction of two complementary peptides, one of which is covalently anchored to the diamond transducer and the other part of the ligated protein.

Clarification of drawings

Giant. 1 is a perspective view of an electrochemical cell assembly depicted along its vertical axis, showing its individual parts in sequence of their functional interconnection.

Giant. 2 Technical drawing of the electrochemical cell vessel

Giant. 3 is an illustration of the principle of an impedimetric biosensor based on a semiconductor boron doped nanocrystalline diamond (BDNCD) layer modified by sensor biomolecules having an electrochemical cell shown in an exploded configuration in FIG. 1 function electrode (2)

Giant. 4 Schematic representation of BDNCD layers with bonding anchoring surface enabling chemoselective binding of sensor molecules by so-called chemistry handles

Giant. 5 Scheme of preparation of BDNCD layer modified with antimicrobial peptide Magainin with binding affinity for E. colli

Giant. 6 Scheme of preparation of BDNCD layer modified by α-D-mannopyranose with binding affinity to E. colli surface lectin

Giant. 7 Scheme for the preparation of a BDNCD layer modified with a DNA oligonucleotide with binding affinity to E. colli

Examples of technical solutions

Example 1

Design of electrochemical cell

The present electrochemical cell for determining bacterial contamination of drinking water, shown in FIG. 1, when disassembled along a vertical axis, consists of a hollow cylindrical container 1 made of polytetrafluoroethylene (PTFE). The cell container is provided with a square hole and recess at the bottom to accommodate a working square electrode 2 measuring 10 x 10 mm and seal it with a square seal 3. The electrical connection of the working electrode 2 is provided by a 12 mm wide metal cable 4. cells and electrically non-conductive ring gaskets with screw holes 5. Closure of the cell bottom against leakage of the electrolyte solution is secured by tightening with a circular metal flange 6 by means of tightening screws 7. The upper part of the cell is closed by the upper circular plastic flange 8 and screws

The upper flange is provided with a capillary opening for the inlet of inert gas (9), an opening for the auxiliary electrode 10 and an opening for the Luggin capillary with a frit 11 for accommodating the reference electrode 12.

The actual technical solution of the cell container is shown in detail in FIG. 2 (technical drawing), ie.

The outer diameter of the cylindrical vessel of the cell is 60 mm, its height is 45 mm, and the diameter of the inner bore is 25 mm. The cell vessel is provided with six symmetrically spaced holes M6 on the top and bottom of the clamping screws. The inner hole tapers at the bottom and merges seamlessly into a working electrode bed of 10 x 10 mm. It has a square cross-section along the axis of the container of 8x8 mm. At its bottom, the cross-section is increased by 1.05 mm on all four sides to 10.1 mm; see detail 3 in front view 1 and plan view 2. An ethylpropylene rubber seal of the same shape as the opening is inserted from the underside of the bed. The seal has an 8x8 mm hole. A working electrode (2; see Fig. 1) is inserted onto the seal, which is tightened after insertion of a strip conductive lead (4; see Fig. 1) and an electrically non-conductive flexible seal with a diameter equal to the outer diameter of the vessel (5; see Fig. 1) metal flange. Both the lower metal flange 4 and the upper flange 5, made of rigid Delrin plastic (polyoxymethylene), are provided with three symmetrically spaced openings for their bayonet mounting on the clamping screws. To facilitate handling, the flange plows are serrated.

Example 2

Preparation of boron doped nanocrystalline diamond (BDNCD) layer with binding anchoring surface allowing chemoselective binding of sensor molecules by so-called chemistry handles

The impedimetric biosensor shown in FIG. 3, which has the function of a working electrode in the electrochemical cell (2; see Fig. 1), consists of a boron doped nanocrystalline diamond (BDNCD) layer deposited on a 10 x 10 mm Si (silicon) or Nb (niobium) substrate modified by sensor molecules.

BDNCD layers deposited on 10x10 mm Si (silicon) or Nb (niobium) substrates are prepared by so-called chemical vapor deposition (CVD) technology, ie plasma deposition of methane, diborane and hydrogen mixtures. The BDNCD layer prepared by this technology is then subjected to hydrogen plasma reduction to obtain a hydrogen terminated surface.

The hydrogen-terminated BDNCD layer prepared according to Example 2 is transformed in two steps into a layer presenting on the surface of the amino group. The UV-promoted reaction with the reaction with N-allyltrifluoroacetyl allyl amine prepared a surface derivatized with 3-trifluoroacetamidopropyl groups. The success of the modification was demonstrated by XPS (X-ray photoelectron spectroscopy), based on a high proportion of fluorine (11%) and F sl binding band at 689.11 eV indicating the presence of a CF3 group. By basic cleavage of trifluoroacetyl groups, the surface presenting 3-aminoalkyl groups is subsequently obtained. The presence of amino groups was confirmed by the disappearance of the CF3 binding band in XPS as well as subsequent derivatization of 3,5-bis (trifluoromethyl) phenyl isothiocyanate and identification of the F1b binding band at 689.11 eV demonstrating the presence of CF3 groups.

The amino-terminal layers are subsequently modified with linkers carrying functional groups to allow the binding of sensor molecules by the so-called click chemistry based on copper (Cu ⁺ ions) catalysed by Huisgen 1,3-dipolar azide cycloaddition to the triple bond (binding pairs: azido and triple bond). MM / N ', N' - tetramethyl-O- (1 H -benzothnazol-1-yl) uronium hexafluorophosphate (HBTU) promoted N-acylation with propargylacetic acid (pent-4-ynoic acid) prepared a triple bond presentation layer Q; shown in FIG. 4). By analogous A-acylation with azidoacetic acid, the azido-presenting layer (2; shown in Fig. 4) is obtained. The presence of azido groups was demonstrated by its characteristic N 1 band at 404.78 eV in XPS.

Example 3

Preparation of a BainCD layer modified with a Magainin peptide with an affinity for E. colli

-4GB 33021 U1

The BDNCD layer modified with the Magainin peptide with affinity for E. colli (3; schematically shown in Fig. 5) is prepared by monovalent copper (Cu ⁺ ions) catalysed 1,3-dipolar cycloaddition of the azido group of the Magainin peptide extended by azidolysin ( Fig. 4)) with triple bonds of BDNCD layer modified with propargylacetic acid 1 in the presence of tris ((1-hydroxypropyl-1H-1,2,3-triazol-4-yl) methyl) amine (THPTA), which stabilizes Cu ⁺ ions. The reaction is carried out in an aqueous medium wherein Cu ⁺ ions are generated in situ from CuSO4 by treatment with ascorbic acid. The success of the modification was demonstrated by XPS, based on a high proportion of nitrogen (10%) and Nis bond band at 400.04 eV indicating the presence of nitrogen amide bonds in the peptide chain.

Example 4

Preparation of BDNCD layer modified by α-D-mannopyranose with binding affinity to E. colli surface lectin

The BDNCD layer modified with modified α-D-mannopyranose with binding affinity to the surface lectin of E. colli (6; schematically shown in Fig. 6) is prepared under conditions identical to Example 3, i.e. monovalent copper catalysed 1,3-dipolar azido cycloaddition 6-O-DMTr-αD-mannopyranoside with an azide-presenting azide group (5; Fig. 6) with triple bonds of the BDNCD layer modified with propargylacetic acid 7 in the presence of THPTA. The success of the modification was demonstrated by the presence of the N1s band at 400.78 eV in XPS, indicating the presence of nitrogen bound in the triazole. Further evidence was founded so-called. a dimethoxytrityl assay, i.e. a colorimetric determination of 4,4'-Dimethoxytrityl alcohol cleaved from the primary HO-groups of the sacyharide.

Example 5

Preparation of BDNCD layer modified with DNA oligonucleotide with binding affinity to E. colli

A BDNCD layer modified with a DNA oligonucleotide with an E. colli binding affinity (8; schematically shown in Fig. 7) is prepared under conditions identical to Examples 3 and 4, i.e., 1,3-dipolar catalyzed monovalent copper (Cu ⁺ ions) cycloaddition of azido groups of a BDNCD surface modified with azidoacetic acid (2) with a triple bond of an olugonucleotide bearing at the 5 'position a hexynyl group. The success of the modification was demonstrated by XPS by the disappearance of the azido-bonding band at N at 404.78 eV and by the presence of the P2p band at 133.6 eV, which is typical of nucleic acid phosphate.

Example 6

Use of electrochemical cell for determination of bacterial contamination of drinking water

In the assembled electrochemical cell shown in the exploded state in FIG. 1, a 5 ml solution of a suitable buffer is placed. The working electrode 2 is connected to the potentiostat contacts with the frequency analyzer by means of a metal current supply 4. The auxiliary electrode 12 and the reference electrode 11 are connected to the potentiostat contacts with the frequency analyzer. Measure the impedance spectrum in the 1 Hz to 5 MHz frequency range at an open circuit potential with a potential amplitude of 10 mV. The appropriate analyte (in this case water contaminated with the target bacterium) is applied to the PBS buffer solution in the electrochemical cell and the impedance spectrum is measured.

Claims (8)

An electrochemical cell for the electrochemical determination of bacterial contamination of drinking water sources using a working electrode on the principle of a highly sensitive impedimetric biosensor based on a boron doped nanocrystalline diamond layer modified by sensor molecules, characterized in that it consists of a hollow cylindrical vessel (1) made of nonconductive and a chemically inert material with a square hole and a recess in the lower body for positioning the working electrode (2) and sealing it with a square seal (3), a metal lever current (4) to the working electrode (2) wider than the working electrode dimension min . 14 mm, placed between the cell body and an electrically non-conductive ring seal with screw holes (5), metal ring flanges with screw holes (6) to secure the bottom of the cell against leakage of electrolyte solution after tightening the screws (7), top ring flanges (8), provided with screw holes (7), an inert gas capillary hole (9), an auxiliary electrode hole (10), a frit (11) Luggin capillary hole for receiving a reference electrode (12), wherein: the working electrode, the auxiliary electrode and the reference electrode are connected to a potentiostat / galvanostat allowing impedance measurements to be made. Electrochemical cell according to claim 1, characterized in that the working electrode (2) is an impedimetric biosensor consisting of a BDNCD layer on a Si (silicon) substrate modified with sensor molecules. Electrochemical cell according to claim 1, characterized in that the working electrode (2) is an impedimetric biosensor consisting of a BDNCD layer on a Nb (niobium) substrate modified with sensor molecules. Electrochemical cell according to any one of the preceding claims, characterized in that the cell body (1) is made of polytetraflourethylene. Electrochemical cell according to any one of the preceding claims, characterized in that the square seal (3) is made of ethyl propylene rubber. Electrochemical cell according to any one of claims 1 to 4, characterized in that the square seal (3) is made of an elastic non-conductive expanded polytetrafluoroethylene that is resistant to both protic and aprotic polar solvents. Electrochemical cell according to any one of the preceding claims, characterized in that the metal washer (6) is provided with a toothed rim to facilitate handling. Electrochemical cell according to any one of the preceding claims, characterized in that the upper and lower annular closures (8) are provided with a toothed rim to facilitate handling.