RU202193U1 - A device for immunochromatographic highly sensitive and out-of-laboratory detection of the phytopathogenic bacterium Clavibacter michiganensis, based on an increase in the intensity of the recorded colorimetric signal due to the catalytic properties of the nanomarker - Google Patents

Abstract

The claimed device is intended for immunochromatographic highly sensitive, out-of-laboratory and express detection of the phytopathogenic bacteria Clavibacter michiganensis in extracts of leaves and tubers of potatoes. The device is a multi-membrane composite with pre-applied and dried immunoreagents (test strip). The composition of the multimembrane composite includes a working nitrocellulose membrane with preliminarily applied immunoreagents, fixed on a solid polystyrene base, a glass fiber membrane coated with a conjugate of porous core-shell nanoparticles - Au-Pt, stabilized with polyvinylpyrrolidone with rabbit cells, polyclonal antibodies against C. membrane for absorbing and separating the components of the test sample and the final adsorbent membrane for absorbing the components of the sample after the reaction and maintaining the migration of liquid along the test strip due to capillary forces. A distinctive feature of the claimed device is that a conjugate of porous core-shell nanoparticles - Au-Pt, stabilized with polyvinylpyrrolidone with polyclonal antibodies against C. michiganensis, is used as a label. Au-Pt particles possess both optical and catalytic (peroxidase-like properties), which makes it possible to use them in immunochromatographic analysis (ICA) as a nanosized label with their own optical signal, and to reduce the ICA detection limit based on an increase in the color intensity upon low concentrations of antigen in the sample due to the peroxidase-like properties of nanoparticles, leading to the accumulation of colored products of substrate oxidation. The high resistance of the particles to chemical inhibitors of peroxidases (sodium azide) and temperature (up to 100 ° C) allows the use of Au-Pt nanoparticles for signal amplification even in samples with high endogenous peroxidase activity after its leveling. In the control zone (K.Z.) applied immunoglobulin-binding protein A. In the test zone (T.Z.) applied rabbit polyclonal antibodies specific to bacterial cells of C. michiganensis. A conjugate of polyclonal rabbit antibodies specific to detectable bacterial cells with porous Au-Pt core-shell nanoparticles stabilized with polyvinylpyrrolidone is applied to the glass fiber membrane. For the analysis, no additional stationary devices are required. The duration of the analysis is 15 minutes, it includes the stages of preparing the analyzed sample, carrying out an immunochromatographic analysis using a test strip, adding a substrate solution to increase the intensity of the recorded colorimetric signal. The technical task of the claimed utility model is to increase the intensity of the recorded staining of T.Z. due to a reaction catalyzed by porous Au-Pt core-shell nanoparticles directly on the test strip. The technical result of the claimed utility model is to reduce the detection limit of the phytopathogenic bacterium C. michiganensis by using a simple one-step procedure provided by adding a substrate solution containing a mixture of 3,3'-diaminobenzidine, hydrogen peroxide and Ni2 + ions to the test strip. 4 tbl, 3 ex, 2 dwg

Description

The utility model relates to the detection of the phytopathogenic bacterium Clavibacter michiganensis (C michiganensis) and can be used as an express laboratory and out-of-laboratory tool for carrying out phytosanitary control by specialized services, customs control of seed, monitoring of infection by farms.

Phytopathogenic microorganisms affect a wide range of crops. Infection with phytopathogenic microorganisms leads to significant economic losses due to a decrease in plant productivity (up to 100%), deterioration of their storage capacity and resistance to pests.

The bacterial phytopathogen C. michiganensis, an aerobic, non-spore-forming gram-positive bacterium, is widespread in the Russian Federation. The main plant affected by C. michiganensis is the potato. The defeat of the potato leads to rotting of its tubers (the so-called ring rot of potatoes) and the emergence of foci of pitted rot. Infection can occur during the harvest season due to the penetration of bacteria through areas with damaged skin. The detection of low concentrations of bacteria that do not cause the development of severe symptoms (latent infection) is of particular interest for practical use, since when tubers with a low concentration of bacteria are planted, symptoms develop only during the flowering process. Thus, isolating infected plants will result in significant economic losses. To prevent large crop losses and significant losses, it is necessary to diagnose latent infections in the seed. According to regulatory documents - GOST 33996-2016 “Seed potatoes. Technical conditions and methods for determining the quality "- C. michiganensis is included in the list of controlled phytopathogenic microorganisms of potatoes. According to the recommendations of the regulatory documentation, the detection limit for immunochemical methods of analysis is 10 ⁴ cells / ml. However, to diagnose latent infections, it is necessary to achieve lower detection limits.

For the detection of C. michiganensis, various variations of the polymerase chain reaction method, enzyme immunoassay, and immunochromatographic analysis are used. Despite such advantages as specificity and low detection limit, the application of methods based on polymerase chain reaction requires expensive stationary equipment, conditions that ensure the absence of contamination, and qualified personnel. The duration of the analysis (at least one hour excluding sample preparation) also limits the applicability of these methods in out-of-laboratory conditions.

For the highly sensitive detection of C. michiganensis, actively developing methods of immunochemical analysis can also be used - enzyme immunoassay (ELISA) and immunochromatographic (ICA) analysis. The capabilities of immunoassay are associated with its methodological simplicity, as well as high productivity, providing simultaneous testing of multiple samples for 2-3 hours. However, the performance of ELISA includes several additional stages (adding reagents and washing), associated with the need for additional consumables (microplates, pipette tips, etc.) and equipment for recording the analysis results. ICA, unlike ELISA, does not require additional equipment and allows mass screening in non-sterile out-of-laboratory conditions using minimal sample preparation. In the classical format, ICA is a one-step analysis and allows for qualitative (presence / absence of colored TZ) and quantitative analysis (registration of the intensity of the colorimetric signal in TZ, determination of the number of bacteria by comparing the intensity of the colorimetric signal of the sample and samples with a known number of bacteria ) by accumulation of the colored marker.

However, the detection limit of a one-stage ICA is often insufficient for highly sensitive detection. One of the approaches to lowering the ICA detection limit is based on increasing the intensity of the recorded colorimetric signal due to the catalytic conversion of substrate molecules into a colored product. Signal amplification is achieved due to the fact that a single catalyst nanoparticle leads to the conversion of a large number of substrate molecules into a registered product.

The increase in the recorded color intensity is associated with the accumulation of the product of the enzymatic reaction on the test strip. Metal nanoparticles can be used as a catalytic label, since they have unique optical properties, can be easily conjugated to receptor molecules, and show high catalytic activity and stability in the presence of enzyme inhibitors. The most widely used catalytically active nanomarkers are Au, Pt, Pd, Ir particles of various shapes and morphologies. However, the high cost of noble metal catalysts limits their use in analytical test systems.

A feature of catalysis using nanoparticles is the many active centers located on the surface of the particles. Thus, most of the noble metal atoms are screened (inside the particles) and do not participate in catalysis. To reduce the consumption of noble metals in the synthesis of catalytically active particles, an approach was proposed based on the formation of a thin layer of a catalytically active metal on nanoparticles, so that all surface exposed metal atoms are available for the substrate and catalytic conversion. This approach was used in a number of works to obtain catalytically active Au-Pt nanoparticles. For example, Zhang et al used a conjugate of Au-Pt nanoparticles with anti-rabbit IgG antibodies for ICA. Gao et al used nanoparticles consisting of a gold core and a monolayer platinum coating as a label in the ICA of the prostate specific antigen.

The synthesis protocols used are based on the formation of a platinum coating on gold nanoparticles; however, they do not allow reaching the maximum exposure of the catalytically active platinum coating. The formation of a porous platinum coating on seed gold nanoparticles will increase the surface area of catalytically active particles. The literature describes the synthesis of porous Au-Pt nanoparticles. Thus, Loynachan et al. Used gold nanoparticles stabilized with polyvinylpyrrolidone as seed particles to form a porous platinum shell. High concentrations of platinum (up to 3 mM) were used for the synthesis of porous particles; the high cost of particles limits their use for routine screening. For the synthesis of porous platinum particles, Jiang et al. Used the reaction of joint reduction of platinum and gold salts in the presence of the Pluronic F127 polymer. However, in this case, high concentrations of noble metal salts (10 mM) were also used, and joint reduction does not allow achieving exposure of catalytically active platinum particles on the surface.

Thus, the known developments of immunochromatographic test systems are focused on the use of non-optimal from the point of view of catalysis, non-porous particles of the core-shell type, and the protocols for the synthesis of porous particles require high concentrations of expensive salts of noble metals, and also do not allow achieving maximum exposure of catalytically active platinum particles on surface.

A distinctive feature of the proposed utility model is the use for ICA of core-shell nanoparticles - Au-Pt, which have a porous structure of a catalytically active platinum shell and require 3-10 times less (1 mM) amount of expensive platinum for synthesis. Polyvinylpyrrolidone (molecular weight 10 kDa) forms a dense adsorption layer around gold nanoparticles (20 nm) and leads to the formation of a porous platinum shell upon reduction of a platinum salt (Na ₂ PtCl ₆ , 1 mM) in the presence of sodium ascorbate (10 mM). The resulting porous Au-Pt nanoparticles can be bound to antibodies through physical adsorption.

ICA is based on the migration of a liquid sample along the test strip under the action of capillary forces without the use of additional equipment. When the sample passes through the immunoreagents immobilized on the test strip, double immune complexes are formed in different parts of the membrane when the sample comes into contact with the conjugate (C. michiganensis cells with conjugated antibodies with porous Au-Pt nanoparticles), triple immune complexes when the sample passes through the test zone ( T.Z.) (C. michiganensis cells with conjugate of antibodies with porous Au-Pt nanoparticles - antibodies immobilized in T.Z.) and binding of excess free conjugate in the control zone (K.Z.) (conjugate of antibodies with porous nanoparticles Au- Pt - immunoglobulin-binding protein A in K.Z.) A marker conjugated with specific antibodies (porous Au-Pt nanoparticles) is distributed over the working membrane due to the formation of immune complexes, and its presence or absence in certain areas of the membrane leads to staining. To reduce the ICA detection limit, a substrate solution (10 μl) is applied to the test strip, consisting of 0.05% 3,3'-diaminobenzidine, 0.03% hydrogen peroxide and 0.05% nickel sulfate in 50 mM phosphate buffer (pH = 7.2). After a five-minute incubation of the substrate solution n of the test strip under the action of Au-Pt nanoparticles, the unstained substrate is converted into an insoluble product, the accumulation of which in the T.Z. and K.Z. significantly increases the intensity of the colorimetric signal.

The use of ICA for the detection of phytopathogenic microorganisms provides a number of advantages - effective parallel screening of a large number of samples in out-of-laboratory conditions, rapid analysis (15-20 min) with minimal sample preparation, ease of detection and interpretation of results.

Despite the fact that ICA is being actively developed and used for the diagnosis of phytopathogens and other toxic contaminants, the development of immunochromatographic test systems for highly sensitive detection of C. michiganensis for the purpose of out-of-laboratory detection of latent infections of potato ring rot has not been described.

The closest analogs of the claimed utility model are immunochromatographic test systems using Au-Pt core-shell nanoparticles, presented in the works:

Jiang et al., Sensitive detection of Escherichia coli 0157: H7 using Pt-Au bimetal nanoparticles with peroxidase-like amplification)), Biosensors and Bioelectronics, 2016, v. 77, p. 687-694 .;

Loynachan et al., Platinum nanocatalyst amplification: redefining the gold standard for lateral flow immunoassays with ultrabroad dynamic range)). ACS Nano, 2018, V. 12, p. 279-288.

The technical task of the claimed utility model is to increase the intensity of coloring T.Z. via a reaction catalyzed by porous Au-Pt core-shell nanoparticles on a test strip.

The technical result of the claimed utility model is to reduce the detection limit of the phytopathogenic bacterium C. michiganensis by using a simple one-step procedure, provided by adding a substrate solution containing a mixture of 3 3'-diaminobenzidine, hydrogen peroxide and Ni ²⁺ ions to the test strip. The porous structure of Au-Pt core-shell nanoparticles (Figure 1 A), which distinguishes them from standard core-shell particles (Figure 1 B), is achieved due to the adsorption of polyvinylpyrrolidone on the surface of the seed gold nanoparticles, which prevents the formation of a plugin monolayer around the seed particles. The porous morphology provides a large surface area for active particles, significantly increasing their specific catalytic activity as compared to non-porous core-shell particles. Porous Au-Pt nanoparticles retain up to 80% of peroxidase-like activity in the presence of sodium azide, a peroxidase inhibitor, which makes it possible to use the proposed scheme to increase the intensity of the colorimetric signal even in plant samples with high endogenous peroxidase activity.

A device for express immunochromatographic laboratory and out-of-laboratory highly sensitive detection of C. michiganensis in potato leaves and tubers extracts is proposed. The device is a multi-membrane composite with pre-applied and dried immunoreagents (test strip). The composition of the composite (Fig. 2) includes a working nitrocellulose membrane with preliminarily applied immunoreagents fixed on a solid polystyrene base, a glass fiber membrane coated with a conjugate of porous core-shell nanoparticles - Au-Pt. stabilized with polyvinylpyrrolidone with rabbit polyclonal antibodies specific to C. michiganensis bacterial cells, a membrane for the absorption and separation of the components of the test sample and the final adsorbent membrane for the absorption of sample components after the reaction and maintenance of liquid migration along the test strip due to capillary forces.

The specified technical result is achieved by:

- Immunoglobulin-binding protein A was applied to the control zone (K.Z.);

- rabbit polyclonal antibodies specific to C. michiganensis cells were applied to the test zone (T.Z.);

- a conjugate of porous Au-Pt nanoparticles with polyclonal rabbit antibodies specific to C. michiganensis is applied to the glass fiber membrane

- when adding a mixture of a substrate solution containing a mixture of 3,3'-diaminobenzidine, hydrogen peroxide and Ni ^{2+ ions,} Au-Pt nanoparticles catalyze the oxidation reaction of 3,3'-diaminobenzidine with hydrogen peroxide, nickel ions provide the formation of an insoluble dark-colored reaction product, working nitrocellulose membrane.

Table 1 shows the characteristics of the materials from which the elements of the claimed device are made.

The analysis is carried out as follows:

1. 100 μl of the analyzed plant extract (extract of leaves or tubers of potatoes) is introduced into a plastic test tube with a capacity of 1.5 ml.

2. The test strip is immersed vertically with the lower end of the membrane to absorb the sample into the extract to be analyzed and kept at room temperature for 5 minutes.

3. Remove the test strip and place it on a dry horizontal surface for 5 minutes.

4. In 10 minutes after the beginning of the movement of the liquid along the test strip, the result of the analysis is recorded visually or using a detector with video digital recording.

5. Add 20 µl of substrate solution and incubate for 3 minutes. The result of the analysis is recorded visually or using a detector with video digital registration.

The claimed device operates as follows (see Fig. 1). If C. michiganensis cells are present in the sample, then under the action of capillary forces they move along the absorbent membrane (5) with a liquid flow, reach the glass fiber membrane (4) and react with polyclonal antibodies on the surface of conjugates of porous Au-Pt nanoparticles to form double immune complexes (C. michiganensis cells - antibodies labeled with porous Au-Pt nanoparticles). The formed double immune complexes under the action of capillary forces move along the working nitrocellulose membrane (2) and interact with those immobilized in T.Z. polyclonal antibodies to C. michiganensis with the formation of ternary complexes (antibodies immobilized on the membrane in T.Z. - C. michiganensis cells - antibodies labeled with porous Au-Pt nanoparticles). The excess of unconnected in T.Z. conjugates of porous nanoparticles with antibodies continue to move along the working nitrocellulose membrane (2) and interact with protein A immobilized in the K.Z. (A), with the formation of double complexes (anti-species antibodies immobilized on the membrane - antibodies labeled with colloidal gold).

The interpretation of the analysis results is based on the presence of stained T.Z. and K.Z .:

1. If after 10 minutes two dark-colored zones (TZ and KZ) appear on the working membrane of the test strip, then the test result is considered positive, i.e. the plant sample contains C. michiganensis cells. In this case, an additional stage of signal amplification is not required.

2. If after 10 minutes one dark-colored short circuit appears on the working membrane of the test strip. while staining T.Z. is not observed, then the result of the analysis is considered negative, i.e. the plant sample does not contain C. michiganensis cells or their number is less than the ICA detection limit. In this case, an additional stage of signal amplification is performed. To do this, add 20 μl of the substrate solution to the test strip. After adding the substrate, the strip is kept in an upright position for 3 minutes. The presence of dark-colored T.Z. indicates the presence of C. michiganensis bacterial cells in the analyzed sample, the absence of stained T.Z. and the presence of stained K.Z. indicates the absence of C. michiganensis in the analyzed sample.

3. If, after 10 minutes, no colored lines are formed on the working membrane of the test strip or only T.Z. (without color formation K.Z.), then the result of the analysis is considered invalid (Fig. 2e). If this happens, retest with a new test strip.

The effectiveness of this approach is confirmed by the following examples:

Example 1 (the effect of sodium azide on the peroxidase activity of potato tuber extracts and the conjugate of porous Ai-Pt nanoparticles with polyclonal antibodies against C. michiganensis cells)

The choice of the sodium azide concentration, which ensures the inhibition of endogenous peroxidases in the plant extract while maintaining the high pericosidase-like activity of porous Au-Pt nanoparticles, makes it possible to use this nano-sized marker for ICA without additional washes. For 5 min, various concentrations of sodium azide (0-0.15%) were incubated with 100 μl of potato tuber extract and 95 μl of phosphate buffer containing 5 μl of porous nanoparticles. Then, 100 μl of a substrate solution containing 50 mM 3,3'5,5'-tetramethylbenzidine, 3 mM hydrogen peroxide in 50 mM citrate buffer, pH = 5.0 was added and kept for 10 min. The enzymatic reaction was stopped by the addition of 50 μl of 1 M sulfuric acid. The intensity of staining at a wavelength of 450 nm was recorded using a photometer in triplicate. The results are shown in Table 2.

Au-Pt nanoparticles retain up to 82% of the peroxidase-like activity under conditions that ensure complete inactivation of endogenous peroxidases. Thus, the addition of 0.05% sodium azide makes it possible to amplify the signal using the catalytic activity of porous nanoparticles even when analyzing samples with high endogenous peroxidase activity.

Example 2 (detection of C. michiganensis cells in potato tuber extract without performing a signal amplification step).

Using the proposed device, the analysis of potato tubers extracts is carried out, with the addition of 103-106 cells / ml (cells / ml) of C. michiganensis and an extract of potato tubers that does not contain bacterial cells. An aliquot to be analyzed (100 μl of the extract) is added to a test tube, after which the test strip is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm into the sample and incubated at room temperature for 5 minutes. Remove the test strip and place it on a horizontal surface. The analysis result is evaluated after 5 minutes using the video digital detector software. The analysis is carried out in triplicate using test strips from different batches. The analysis results are shown in Table 3.

The limit of ICA detection was the number of C. michiganensis cells providing a colorimetric signal in T.Z. exceeding the sum of the mean of the colorimetric signal and three standard deviations for the negative sample (healthy extract). For ICA without signal amplification, the sum of the mean value of the colorimetric signal and three standard deviations for the negative sample was 0.85. Thus, the detection limit for this system was 104 cells / ml C. michiganensis. It can be seen from the data presented that the recorded intensity of the colorimetric signal in the test zone makes it possible to reliably detect 104 cells / ml of C. michiganensis in the extract of potato tubers within the stated analysis time (10 min).

Example 3 (detection of C. michiganensis cells in potato tuber extract without carrying out the signal amplification step).

Using the proposed device, an analysis of potato tubers extracts is carried out, with the addition of 10 ³ -10 ⁶ cells / ml (cells / ml) C. michiganensis and an extract of potato tubers that does not contain bacterial cells. An aliquot to be analyzed (100 μl of the extract) is added to a test tube, after which the test strip is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm into the sample and incubated at room temperature for 5 minutes. Remove the test strip and place it on a horizontal surface. After 5 minutes, 20 μl of the substrate solution is added. The analysis result is evaluated after 5 minutes using a video digital detector. The analysis is carried out in triplicate using test strips from different batches. The analysis results are shown in Table 4.

The limit of ICA detection was the number of C. michiganensis cells providing a colorimetric signal in T.Z. exceeding the sum of the mean of the colorimetric signal and three standard deviations for the negative sample (healthy extract). For ICA without signal amplification, the sum of the mean value of the colorimetric signal and three standard deviations for the negative sample was 1.63. Thus, the detection limit for this system was 103 cells / ml C. michiganensis. From the experimental data presented, it can be seen that the recorded intensity of the colorimetric signal in the test zone makes it possible to reliably detect 10 ³ cells / ml C. michiganensis in the extract of potato tubers within the stated analysis time (10 min).

Compared to the system without signal amplification, a tenfold decrease in the detection limit was obtained, which was achieved in a short time (additional 5 min) and did not require complex methodological solutions (adding a drop of a ready-to-use substrate solution to the test strip).

Brief description of the drawings.

In fig. 1 shows micrographs of nanoparticles obtained by transmission electron microscopy. A - Au-Pt core-shell nanoparticles. B - porous Au-Pt core-shell nanoparticles stabilized with polyvinylpyrrolidone.

In fig. 2 shows a diagram of the proposed device for immunochromatographic express laboratory and out-of-laboratory detection of C. michiganensis. 1 - solid polystyrene base of a working nitrocellulose membrane; 2 - working nitrocellulose membrane; 3 - final adsorbent membrane for absorbing sample components after the reaction; 4 - glass fiber membrane coated and dried with a conjugate of porous Au-Pt nanoparticles with polyclonal rabbit antibodies specific to C. michiganensis; 5 - membrane for absorption and separation of the test sample; 6 - T.Z. with applied polyclonal rabbit antibodies to C. michiganensis; 7 - K.Z. with applied immunoglobulin-binding protein A.

Claims (1)

A device for express immunochromatographic out-of-laboratory detection of a phytopathogenic bacterium - Clavibacter michiganensis, including a test strip consisting of a polystyrene substrate, on which a nitrocellulose membrane with pre-applied and dried antibodies is fixed, a fiberglass membrane coated with a nucleus and dried antibodies with nucleated antibodies and dried with conjugated antibodies - Au-Pt, stabilized by polyvinylpyrrolidone, a membrane for absorption and separation of the test sample and a final adsorbent membrane for absorption of sample components after the reaction and maintenance of liquid migration along the test strip due to capillary forces, control and test zones are applied to the nitrocellulose membrane, control zones are applied to the control immunoglobulin-binding protein A is immobilized in the zone, polyclonal rabbit antibodies specific to C. michiganensis bacterial cells are immobilized in the test zone, a polyclonal conjugate is applied to the glass fiber membrane rabbit antibodies specific to C. michiganensis, with porous Au-Pt nanoparticles, which form immune complexes as a result of migration of the sample in the test zone, lead to the development of staining, and the intensity of which is proportional to the number of C. michiganensis cells in the sample, an increase in the intensity of staining at low concentrations of C .michiganensis is based on the accumulation of an insoluble product of the catalytic oxidation of 3,3'-diaminobenzidine in the presence of hydrogen peroxide and nickel ions and is achieved due to the peroxidase-like properties of porous Au-Pt nanoparticles used as a catalytic and colorimetric label.

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