KIT AND METHOD FOR SIMULTANEOUSLY DETECTING DROPLET DRIFT OR DEPOSITION OF MULTIPLE SPRAYS

Abstract

A kit for simultaneously detecting the droplet drift or deposition of multiple sprays includes detection membranes fixed with immobilized probes, transition probes capable of specifically binding to the immobilized probes, and biotinylated chromogenic probes capable of specifically binding to the transition probes. The transition probes are added to the spray liquids as tracers. After spraying, the transition probes specifically bind to the immobilized probes on the detection membranes. The biotinylated chromogenic probes bind to the transition probes through hybridization. After the chromogenic treatment, the droplet volume is determined according to the color depth, and the spray deposition parameters of droplets are determined according to the location and size of colored spots.

Claims

1. A method for simultaneously detecting the droplet drift or deposition volumes of multiple sprays, comprising: (1) adding different transition probes into multiple spray liquids respectively as tracers to give spray liquids containing transition probes, wherein only one transition probe is added to each spray liquid; (2) applying the spray liquids containing transition probes, then the transition probes in the spray liquids specifically bind to the corresponding immobilized probes on the detection membranes, wherein the detection membranes are substrates carrying the immobilized probes; (3) adding biotinylated chromogenic probes, then they binding to the corresponding transition probes through hybridization, after the chromogenic treatment, determining the volume of droplets according to the color depth, and determining the droplet drift or deposition volume according to the location and size of colored spots; wherein the transition probes are not biotinylated, but have nucleotide sequences capable of complementarily pairing with the corresponding immobilized probes and the corresponding chromogenic probes; the immobilized probes do not specifically bind to the chromogenic probes, and different transition probes do not specifically bind to each other.

2. The method according to claim 1, wherein the transition probes and the immobilized probes are single-stranded deoxyribonucleic acids with characteristic sequences; the length of the transition probes is 24-50 nt, and the length of the immobilized probes is 12-25 nt; one end of the immobilized probes is amino-modified and covalently binds to an exposed carboxyl of the substrate.

3. The method according to claim 1, wherein the complementary pairing region of the chromogenic probe and the transition probe is of 15-40 nt; if the immobilized probe is 5-labeled, the chromogenic probe is 3-biotinylated, and if the immobilized probe is 3-labeled, the chromogenic probe is 5-biotinylated.

4. The method according to claim 1, wherein the length of the immobilized probes is 18-20 nt, and the complementary pairing region of the transition probe and the immobilized probe is of 15-25 nt.

5. The method according to any of claims 1-4, wherein the detection membrane in step (2) is prepared according to the following method: acquiring a substrate of a required area, treating the substrate with 0.1-0.3 M HCl, and washing; incubating the substrate in 10-20% EDC solution and washing; incubating the substrate in 0.3-1.0 M NaHCO.sub.3 solution containing 0.025-0.2 M immobilized probe; and incubating the treated substrate in NaOH solution, washing and drying.

6. The method according to claim 5, wherein the detection membrane is prepared according to the following method: acquiring a substrate of a required area, treating the substrate with 0.1 M HCl, and washing; incubating the substrate in 15% EDC for 0.5-1 h and washing; incubating the substrate in 0.5 M NaHCO.sub.3 solution containing 0.03 M immobilized probe for 10-20 min; and incubating the treated substrate in 0.05-0.5 M NaOH solution for 5-15 min, washing and drying.

7. The method according to claim 5, wherein the substrate is a nitrocellulose membrane, a nylon membrane, a carboxylated organic glass film or a carboxylated polypropylene plastic film.

8. The method according to any of claims 1-4, wherein the final concentration of the transition probe in the spray liquid containing the transition probe in step (2) is 0.025-0.1 M.

9. A kit for simultaneously detecting the droplet drift or deposition volumes of multiple sprays, comprising detection membranes, transition probes and chromogenic probes, wherein the numbers of the detection membranes, the transition probes and the chromogenic probes are all >2 and are different; the 3 or 5 end of the chromogenic probes is biotinylated, and the chromogenic probes can specifically bind to the transition probes but cannot specifically bind to the immobilized probes; preferably, the detection membrane is a substrate fixed with the immobilized probe; the length of the immobilized probe is 12-25 nt; one end of the immobilized probe is amino-modified and covalently binds to an exposed carboxyl group of the substrate; the substrate is a material with exposed carboxyl groups; preferably, the length of the transition probe is 24-50 nt.

10. The kit according to claim 9, further comprising a TMB (3,3,5,5-tetramethylbenzidine) single-component solution, and streptavidin-labeled horseradish peroxidase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 is a schematic flow chart of the three-stage reverse dot blot for detecting droplet deposition distribution of multiple sprays.

[0056] FIG. 2A illustrates the images of 5 detection membranes containing immobilized probes, and FIG. 2B illustrates the result of standard curve establishment according to the detection method disclosed herein.

[0057] FIG. 3 illustrates the experiment for simultaneously detecting the droplet properties by two probes in Example 2.

[0058] FIG. 4 illustrates the experiment for simultaneously detecting the droplet properties by three probes in Example 3.

[0059] FIG. 5 illustrates the experiment for simultaneously detecting the droplet properties by four probes in Example 4.

DETAILED DESCRIPTION

[0060] The present invention will be further illustrated below with reference to examples, which should not be construed as limiting the present invention. Modifications or substitutions to the methods, procedures or conditions of the present invention may be implemented without departing from the spirit and scope of the present invention.

[0061] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art.

Example 1. Method for Simultaneously Detecting the Droplet Drift or Deposition Volumes of Multiple Sprays

[0062] As shown in FIG. 1, the process comprises 5 procedures: preparation of detection membrane, preparation of spray liquid, spraying, establishment of standard curve, and chromogenic treatment. The specific process is as follows: based on the binding specificity of single-stranded deoxyribonucleic acids with different characteristic sequences, a series of single-stranded deoxyribonucleic acids with different characteristic sequences were designed as mobilized probes and then fixed on a substrate (a nylon membrane in this example) to prepare a variety of target membranes. Subsequently, the corresponding single-stranded deoxyribonucleic acids with different characteristic sequences were added into different spray liquids as tracers. After spraying, the substrate was retrieved, and information such as droplet size and distribution of the different spray liquids was obtained after signal amplification and chromogenic treatment. Finally, the deposition volume was calculated by computer image processing software.

[0063] 1. Determination of Probes

[0064] The length of the immobilized probes was 12-25 nt, preferably 18-20 nt. One end of the immobilized probes was amino-modified, and the other end covalently bound to an exposed carboxyl of the substrate.

[0065] The transition probes were single-stranded deoxyribonucleic acids with characteristic sequences of 24-50 nt (preferably 36-40 nt) without biotinylation. The complementary pairing region of the transition probes and the immobilized probes was of 15-25 nt.

[0066] The chromogenic probes were single-stranded deoxyribonucleic acids with characteristic sequences of 12-25 nt (preferably 18-20 nt). The complementary pairing region of the chromogenic probes and the transition probes was of 15-40 nt. If the immobilized probes were 5-labeled, the chromogenic probes were 3-biotinylated; and if the immobilized probes were 3-labeled, the chromogenic probes were 5-biotinylated.

[0067] The chromogenic probes could specifically bind to the transition probes but could not specifically bind to the immobilized probes. The sequences of three probes in Table 1 are exemplary probe sequences used in the example. In addition to the nucleotide sequences of the probes in Table 1, any single-stranded deoxyribonucleic acid sequence that satisfies the above requirements can be used as the probes in the present invention.

TABLE-US-00001 TABLE1 1 Immobi- 5-NH.sub.2-ATCAAGAAGGTGGTGAA-3 lized probe1 Transi- 5-TGCTCAGTGTAGCCCATTCACCACCTTCTTGAT- tion 3 probe1 Chromo- 5TGGGCTACACTGAGCA-Biotin-3 genic probe1 2 Immobi- 5-NH.sub.2-ATCAAGAAGGTGGTGAA-3 lized probe1 Transi- 5- tion TGACTGCGAGTAGTAGCCATTCACCACCTTCTTGAT- probe 3 1-2 Chromo- 5TGGCTACTACTCGCAGTCA-Biotin-3 genic probe2 3 Immobi- 5-NH.sub.2-ATCAAGAAGGTGGTGAA-3 lized probe1 Transi- 5-TCTCAGGTACCATTCACCACCTTCTTGAT-3 tion probe 1-3 Chromo- 5TGGTACCTGAGA-Biotin-3 genic probe3 4 Immobi- 5-NH.sub.2-CCACCGTTTTTCCTCAG-3 lized probe2 Transi- 5-TGCTCAGTGTAGCCCACTGAGGAAAAACGGTGG- tion 3 probe2 Chromo- 5TGGGCTACACTGAGCA-Biotin-3 genic probe1 5 Immobi- 5-NH.sub.2-CCACCGTTTTTCCTCAG-3 lized probe2 Transi- 5- tion TGACTGCGAGTAGTAGCCACTGAGGAAAAACGGTGG- probe 3 2-2 Chromo- 5TGGCTACTACTCGCAGTCA-Biotin-3 genic probe2 6 Immobi- 5-NH.sub.2-CCACCGTTTTTCCTCAG-3 lized probe2 Transi- 5-TCTCAGGTACCACTGAGGAAAAACGGTGG-3 tion probe 2-3 Chromo- 5TGGTACCTGAGA-Biotin-3 genic probe3 7 Immobi- 5-NH.sub.2-ATCTTAAATCGCAAGGT-3 lized probe3 Transi- 5-TGCTCAGTGTAGCCCAACCTTGCGATTTAAGAT- tion 3 probe3 Chromo- 5TGGGCTACACTGAGCA-Biotin-3 genic probe1 8 Immobi- 5-NH.sub.2-ATCTTAAATCGCAAGGT-3 lized probe3 Trans- 5- ition TGACTGCGAGTAGTAGCCAACCTTGCGATTTAAGAT- probe 3 3-2 Chromo- 5TGGCTACTACTCGCAGTCA-Biotin-3 genic probe2 9 Immobi- 5-NH.sub.2-ATCTTAAATCGCAAGGT-3 lized probe3 Transi- 5-TCTCAGGTACCAACCTTGCGATTTAAGAT-3 tion probe 3-3 Chromo- 5TGGTACCTGAGA-Biotin-3 genic probe3 10 Immobi- 5-NH.sub.2-ATCCCGAAGGTGGTTAC-3 lized probe4 Transi- 5-GGTACCATCTCAGTAACCACCTTCGGGAT-3 tion probe4 Chromo- 5TGAGATGGTACC-Biotin-3 genic probe4

[0068] 2. Preparation of Detection Membranes

[0069] A nylon membrane enriched with carboxyl groups on the surface was talored into a required size, treated with 0.1 M HCl, and washed; the membrane was incubated in 15% EDC for 1 h and washed; then the membrane was incubated in 0.5 M NaHCO.sub.3 solution containing 0.03 M immobilized probe (probe combination 1 in Table 1) for 20 min; the membrane was then incubated in 0.2 M NaOH solution for 15 min, washed and dried. The thus prepared detection membranes were arranged on the targets to be sprayed for collecting spray droplets and the subsequent detection.

[0070] 3. Preparation and Application of Spray Liquids Formulations to be sprayed (pesticide formulations, liquid fertilizers, other liquid formulations or water) were added into dosing tanks, and then transition probes (in water) were added. The final concentration of transition probes in the spray liquids was 0.60 M. To each spray liquid, only one transition probe was added, and the transition probes were different from each other. Finally, based on the requirements of pesticide formulations, liquid fertilizers or spraying device, a surfactant and ionic buffer were added to prepare transition probe spray liquids (0.02-0.045 mol/L ion buffer and 0.06-0.15% surfactant). The major formulations were water-based formulation and oil-based formulation. In the preparation process of spray liquids in Example 1, the components of the spray liquids were 30 mM trisodium citrate, 3 mM SDS, and 0.06 M transition probe. Probe combination 1 in Table 1 was selected, namely, transition probe 1 was used. After spraying, the detection membranes were retrieved and subjected to chromogenic reaction.

[0071] 4. Establishment of Standard Curve

[0072] 5 detection membranes containing immobilized probes were selected, 0.5 L of spray liquid containing transition probe was applied to 1-5 spots on the 5 detection membranes respectively, wherein the volumes of spray liquid containing transition probe on the 5 detection membranes were 0.5 L, 1.0 L, 1.5L, 2.0L and 2.5L, respectively. Another detection membrane was taken as background. An image file was obtained by photographing or scanning (FIG. 2A). Gray values of unit areas were obtained through an image processing software (for example, Photoshop, Image J), and a total gray value of a selected area was calculated. Finally, a standard curve of total gray value as the ordinate against the volume of spray as the abscissa was plotted, and a corresponding linear equation was calculated. The results are shown in FIG. 2B.

[0073] 5. Chromogenic Treatment

[0074] Detection membranes sprayed with the spray liquids containing the transition probes were incubated in 50 mL of hybridization buffer (an aqueous solution containing 30 mmol/L trisodium citrate and 26 mmol/L SDS) at 37 C. for 40 min; after removing the hybridization buffer, another 50 mL of hybridization buffer was added to wash the detection membranes for 2 min; the detection membranes were then transferred to hybridization buffers containing chromogenic probes at 37 C. for reaction for 15 min, and were washed 3 times with 50 mL of washing buffer (an aqueous solution containing 7.5 mmol/L trisodium citrate and 6 mmol/L SDS) and once with 50 mL of hybridization buffer. 15 L of streptavidin-labeled horseradish peroxidase was added into a hybridization buffer to prepare an enzyme solution; the detection membranes were incubated in the enzyme solution for enzyme-linked reaction at 37 C. for 20 min; the detection membranes were washed with 50 mL of hybridization buffer, and transferred into a TMB single-component solution for chromogenic reaction, wherein the TMB single-component solution was catalyzed by the horseradish peroxidase bound to the detection membrane, and then color developed on the detection membrane; and after 3 min, the membranes were washed with water to terminate the reaction, and dried. Information such as the distribution and size of droplets were directly observed through the chromogenic reaction. Finally, an image file was obtained by photographing or scanning. Gray values of unit areas were obtained through an image processing software (for example, Photoshop, Image J and the like), and a total gray value of a selected area was calculated.

Example 2. Spray Boom Track System-Simulated Field SprayingSimultaneous Detection by Two Probes

[0075] Two detection membranes (hereinafter referred to as detection membrane A and detection membrane B) respectively containing immobilized probe 1 and immobilized probe 2 (combination 1 and combination 4) were prepared according to the method of Example 1. Spray liquids containing transition probes (spray liquid A and spray liquid B) corresponding to the above immobilized probes were prepared according to the method of Example 1 for preparing the spray liquids. Culture dishes were placed on a support just below the middle of the pathway of the spray boom track system, with each culture dish containing 2 detection membranes (one each for A and B). Sprays were applied to the detection membranes under a pressure of 3 bar by the spray crane (speed: 5 km/h, height: 0.5 m) equipped with Lechler ST110-03 standard fan-shaped nozzle, with spray liquids A and B being applied once separately. After spraying, the experimental materials were retrieved, and the detection membranes were subjected to chromogenic reaction with the corresponding chromogenic probes according to the chromogenic treatment described above. The droplet coverage areas on the detection membranes were read by an instrument, and the droplet volume and coverage rate were calculated. Digital images were obtained by approaches such as photographing or scanning. Gray values of unit areas were obtained by an image processing software (for example, Photoshop, Image J and the like), and a total gray value of a selected area was calculated. The deposition volume was calculated from a standard curve. The results are shown in FIG. 3 and Table 2. From the results in FIG. 3, there was no interference between the 2 probe combinations, and the droplets of multiple sprays were simultaneously detected. Furthermore, the results in Table 2 show that the deposition volume of the droplets was accurately quantified.

TABLE-US-00002 TABLE 2 Experiment for simultaneously detecting droplet properties by two probes Probe A B Deposition volume (L/cm.sup.2) 2.40 2.06 Theoretical deposition volume (L/cm.sup.2) 2.86 2.86 Ratio (calculated/theoretical) 0.84 0.72

Example 3. Spray Boom Track System-Simulated Field SprayingSimultaneous Detection by Three Probes

[0076] Three detection membranes (hereinafter referred to as detection membrane A, B and C) respectively containing immobilized probes 1, 2 and 3 (combinations 1, 4 and 7) were prepared according to the method as described above. Spray liquids containing transition probes (spray liquids A, B and C) corresponding to the above immobilized probes were prepared according to the aforementioned method for preparing the spray liquids. Culture dishes were placed on a support below the pathway of the spray boom track system and just in the middle of the pathway of the spray boom track system, with each culture dish containing 3 detection membranes (one each for A, B and C). Sprays were applied to the detection membranes under a pressure of 3 bar by the spray boom track system (speed: 5 km/h, height: 0.5 m) equipped with Lechler ST110-03 standard fan-shaped nozzle, with spray liquids A, B and C being applied once separately. After spraying, the experimental materials were retrieved, and the detection membranes were subjected to chromogenic reaction with the corresponding chromogenic probes according to the chromogenic treatment described above. The droplet coverage areas on the detection membranes were read by an instrument, and the droplet volume and coverage rate were calculated. Digital images were obtained by approaches such as photographing or scanning. Gray values of unit areas were obtained by an image processing software (for example, Photoshop, Image J and the like), and a total gray value of a selected area was calculated. The deposition volume was calculated from a standard curve. The results are shown in FIG. 4 and Table 3. From the results in FIG. 4, there was no interference between the 3 probe combinations, and the droplets of multiple sprays were simultaneously detected. Furthermore, the results in Table 2 show that the deposition volume of the droplets was accurately quantified.

TABLE-US-00003 TABLE 3 Experiment for simultaneously detecting droplet properties by three probes Probe A B C Deposition volume (L/cm.sup.2) 2.23 1.97 1.94 Theoretical deposition volume (L/cm.sup.2) 2.86 2.86 2.86 Ratio (calculated/theoretical) 0.78 0.69 0.68

Example 4. Sprayboom Track System-Simulated Field SprayingSimultaneous Detection by Four Probes

[0077] Four detection membranes (hereinafter referred to as detection membranes A, B, C and D) respectively containing immobilized probes 1, 2, 3 and 4 (combinations 1, 4, 7 and 10) were prepared according to the method as described above. Spray liquids containing transition probes (spray liquids A, B, C and D) corresponding to the above immobilized probes were prepared according to the aforementioned method for preparing the spray liquids. Culture dishes were placed on the iron support below the pathway of the spray boom track system and just in the middle of the pathway of the spray boom track system, with each culture dish containing 4 detection membranes (one each for A, B, C and D). Sprays were applied to the detection membranes under a pressure of 3 bar by the spray boom track system (speed: 5 km/h, height: 0.5 m) equipped with Lechler ST110-03 standard fan-shaped nozzle, with spray liquids A, B, C and D being applied once separately. After spraying, the experimental materials were retrieved, and the detection membranes were subjected to chromogenic reaction with the corresponding chromogenic probes according to the chromogenic treatment as described above. The droplet coverage areas on the detection membranes were read by an instrument, and the droplet number and coverage rate were calculated. Digital images were obtained by approaches such as photographing or scanning. Gray values of unit areas were obtained by an image processing software (for example, Photoshop, Image J and the like), and a total gray value of a selected area was calculated. The deposition volume was calculated from a standard curve. The results are shown in FIG. 5 and Table 4. From the results in FIG. 5, there was no interference between the 4 probe combinations, and the droplets of multiple sprays were simultaneously detected. Furthermore, the results in Table 3 show that the deposition volume of the droplets was accurately quantified.

TABLE-US-00004 TABLE 4 Experiment for simultaneously detecting droplet properties by four probes Probe A B C D Deposition volume 2.06 1.97 2.23 2.40 (L/cm.sup.2) Theoretical deposition 2.86 2.86 2.86 2.86 volume (L/cm.sup.2) Ratio 72% 69% 78% 84% (calculated/theoretical)