BIOLOGICAL NANOPARTICLE DETECTING METHOD WITH HIGH SENSITIVITY

Abstract

The present disclosure discloses a biological nanoparticle detection method with high-sensitivity in which the biological nanoparticle is reacted with a corresponding aptamer-modified copper compound nanoparticle for a period of time; then a surfactant is added to prevent the reactant particles from agglomeration; next, the reaction solution is passed through a filter membrane to enrich the biological nanoparticle-copper compound conjugate, during which small-sized molecules including proteins and uric acid pass directly through the filter membrane; then the filter membrane is washed with PBS, and silver nitrate is added for reaction; and finally a mixed solution of triethylamine hydrochloride, 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide are added for development, and the color change of the filter membrane is visually observed by naked eyes or by means of a camera.

Claims

1. A biological nanoparticle detection method with high-sensitivity, comprising the following steps: Step S1: reacting a copper compound nanoparticle with a surface membrane protein aptamer having a sulfhydryl group of the biological nanoparticle to obtain a copper compound-membrane protein aptamer conjugate; Step S2: filtering a biological nanoparticle solution containing the biological nanoparticle through a first filter membrane, and adding the copper compound-membrane protein aptamer conjugate to the filtered solution, to obtain a biological nanoparticle-copper compound conjugate after reaction; Step S3: adding a surfactant to the reaction solution obtained in Step S2, filtering through a second filter membrane, and washing the second filter membrane with PBS to obtain a third filter membrane containing the biological nanoparticle-copper compound conjugate; and Step S4: adding a AgNO.sub.3 solution to the third filter membrane obtained in Step S3 and reacting; and then adding a mixed solution of triethylamine hydrochloride, hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine, reacting for development, and observing the color change of the filter membrane visually by naked eyes or by means of a camera.

2. The biological nanoparticle detection method according to claim 1, wherein the biological nanoparticle is an exosome or a virus.

3. The biological nanoparticle detection method according to claim 1, wherein in Step S1, the copper compound nanoparticle is one or more selected from a group consisting of: cupric sulfide, cupric oxide, cuprous oxide, and cuprous sulfide, the size of the copper compound nanoparticle is 5 to 50 nm, and the surface membrane protein aptamer having a sulfhydryl group is one or more selected from a groups consisting of: CD63 aptamer, CD81 aptamer, CD9 aptamer, EpCAM aptamer, HER2 aptamer, MUC1 aptamer, and PSMA aptamer; the reaction time of the copper compound nanoparticle with the surface membrane protein aptamer is 8 to 24 hrs; and the pore size of the first filter membrane is 200 nm.

4. The biological nanoparticle detection method according to claim 1, wherein the reaction time of the biological nanoparticle solution with the copper compound-membrane protein aptamer conjugate in Step S2 is 0.5 to 10 hrs.

5. The biological nanoparticle detection method according to claim 1, wherein a volume of the biological nanoparticle solution in Step S2 is adjustable, when the concentration of the biological nanoparticle solution is low, the volume of the biological nanoparticle solution is increased to improve the sensitivity.

6. The biological nanoparticle detection method according to claim 1, wherein the surfactant in Step S3 is one or more selected from a group consisting of: sodium dodecyl sulfate, cetyltrimethylammonium bromide, and polyvinylpyrrolidone, and the concentration of the surfactant is in the range of 0.1 to 2.0%.

7. The biological nanoparticle detection method according to claim 1, wherein the AgNO.sub.3 solution in Step S4 has a concentration of 10.sup.−5 to 10.sup.−3 M and a volume of 10 to 50 uL, the second filter membrane in Step S4 has a pore size in the range of 20 to 200 nm, and the reaction time of substance on a surface of the third filter membrane with the AgNO.sub.3 solution is in the range of 5 to 10 min, the concentration of triethylamine hydrochloride is 0.05 to 0.2 M, the concentration of hydrogen peroxide is 0.1 to 0.5 M, the concentration of 3,3′,5,5′-tetramethylbenzidine is 0.1 to 1.0 mM, and the reaction time is 5 to 30 min.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly explain the technical solutions in the embodiments of the present disclosure or in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Evidently, the drawings depicted below are merely some embodiments of the present disclosure, and those skilled in the art can obtain other drawings based on the structures shown in these drawings without any creative efforts.

[0024] FIG. 1 is a diagram showing a comparison of the changes in color between the filter membrane in the control group and the filter membrane obtained after the reaction with a solution of an exosome of 10.sup.9 counts/mL in a first Example of the present disclosure;

[0025] FIG. 2 is a diagram showing a comparison of the changes in color between the filter membrane in the control group and the filter membranes obtained after the reaction with various concentrations of exosome solutions in a second Example of the present disclosure; and

[0026] FIG. 3 is a diagram showing a comparison of the changes in color between the filter membrane in the control group and the filter membrane obtained after the reaction with a solution of an exosome of 1×10.sup.7 counts/mL in a third Example of the present disclosure.

[0027] The objects, functional characteristics and advantages of the present disclosure will be further described in combination with the embodiments and with reference to the accompanying drawings.

DESCRIPTION OF THE EMBODIMENTS

[0028] The technical solutions in the embodiments of the present disclosure will be described clearly and fully with reference to the accompanying drawings in the embodiments of the present disclosure. Apparently, the embodiments described are merely some, rather than all of the embodiments of the present disclosure. All other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present disclosure shall fall within the protection scope of the present disclosure.

[0029] It should be noted that if there are directional indications (such as on, below, left, right, front, back . . . ) involved in the embodiments of the present disclosure, these directional indications are only used to explain the relative positional relationship and movement of various components in a specific posture (as shown in the figures). If the specific posture changes, the directional indications will change accordingly.

[0030] In addition, if there are descriptions “first”, and “second”, etc. in the embodiments of the present disclosure, the descriptions “first” and “second” are used herein merely for the purposes of description, and are not intended to indicate or imply the relative importance or implicitly point out the number of the indicated technical feature. Therefore, the features defined by “first”, and “second” may explicitly or implicitly include at least one of the features. In addition, the technical solutions in various embodiments can be combined with each other, on the condition that the combinations can be accomplished by those of ordinary skill in the art. When a combination of technical solutions is contradictory or cannot be achieved, it is considered that such a combination of technical solutions does not exist, and does not fall within the protection scope of the present disclosure.

[0031] The present disclosure provides a method for high-sensitivity detection of an exosome.

First Example

[0032] In this example of the present disclosure, 1 mL of a CuS nanoparticle solution with a concentration of 10.sup.13 counts/mL was added to 20 uL of a solution of CD63 aptamer having a sulfhydryl group. Then, a 2 M sodium chloride solution was gradually added to give a final sodium chloride concentration of 0.1 M in the solution system. After 8 hrs of reaction, the reaction solution was centrifuged and washed to obtain CuS nanoparticles bearing CD63 aptamer.

[0033] 20 uL of CuS nanoparticles bearing CD63 aptamer was added to 1 mL of an exosome solution with a concentration of 10.sup.9 counts/mL and reacted for half an hour. Then 0.1% sodium dodecyl sulfate was added, and the solution was filtered through a filter membrane with a pore size of 50 nm and washed three times with PBS, to obtain a filter membrane containing exosomes-CuS. 10 uL of a AgNO.sub.3 solution having a concentration of 1.0×10.sup.−3 M was added to the filter membrane and reacted for 5 min.

[0034] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 5 uL of a newly prepared 10 mol/L hydrogen peroxide solution were added to 500 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0035] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without exosomes) and the filter membrane containing exosomes of 10.sup.9 counts/mL. After standing for 5 min, the change in color between the filter membranes was observed and detected visually by naked eyes or by taking photos with a camera. The changes in color between the control group (the filter membrane obtained by performing the above experiment with the control solution without exosomes) and the filter membrane containing exosomes of 10.sup.9 counts/mL is shown in FIG. 1. As shown in FIG. 1, the filter membrane with exosomes has a very significant difference in color compared to the filter membrane without exosomes.

Second Example

[0036] In this example of the present disclosure, 1 mL of a CuS nanoparticle solution with a concentration of 10.sup.13 counts/mL was added to 20 uL of a solution of CD63 aptamer having a sulfhydryl group. After 8 hrs of reaction, the reaction solution was centrifuged and washed to obtain CuS nanoparticles bearing CD63 aptamer.

[0037] 20 uL of CuS nanoparticles bearing CD63 aptamer was added respectively to 1 mL of an exosome solution with a concentration of 1×10.sup.7 counts/mL, 5×10.sup.7 counts/mL, 1×10.sup.8 counts/mL, 5×10.sup.8 counts/mL, and 1.0×10.sup.9 counts/mL and reacted for half an hour. Then 0.1% cetyltrimethyl ammonium bromide was added, and the solution was filtered through a filter membrane with a pore size of 50 nm and washed three times with PBS, to obtain a filter membrane containing exosomes-CuS. 10 uL of a AgNO.sub.3 solution having a concentration of 5.0×10.sup.−4 M was added to the filter membrane and reacted for 10 min.

[0038] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 5 uL of a newly prepared 10 mol/L hydrogen peroxide solution were added to 500 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0039] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without exosomes) and the filter membranes containing different concentrations of exosome (1×10.sup.7 counts/mL, 5×10.sup.7 counts/mL, 1×10.sup.8 counts/mL, 5×10.sup.8 counts/mL, and 1.0×10.sup.9 counts/mL). After 5 min of reaction, the changes in color between the filter membranes was detected by visual colorimetric method or by taking photos with a camera. The change in color is shown in FIG. 2. As shown in FIG. 2, as the exosome concentration in the sample solution continues to increase, the color of the filter membrane continues to deepen, such that direct observation with the naked eyes is achieved.

Third Example

[0040] In this example of the present disclosure, 50 mL of a CuS nanoparticle solution with a concentration of 10.sup.13 counts/mL was added to 1 mL of a solution of CD63 aptamer having a sulfhydryl group. After 8 hrs of reaction, the reaction solution was centrifuged and washed to obtain CuS nanoparticles bearing CD63 aptamer.

[0041] 40 mL of CuS nanoparticles bearing CD63 aptamer was added to 200 mL of an exosome solution with a concentration of 1.0×10.sup.7 counts/mL and reacted for half an hour. Then 0.3% sodium dodecyl sulfate was added, and the solution was filtered through a filter membrane with a pore size of 50 nm and washed three times with PBS, to obtain a filter membrane containing exosome-CuS. 10 uL of a AgNO.sub.3 solution having a concentration of 5.0×10.sup.−4 M was added to the filter membrane and reacted for 5 min.

[0042] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 5 uL of a newly prepared 10 mol/L hydrogen peroxide solution were added to 250 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0043] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without exosomes) and the filter membrane containing exosomes of 1.0×10.sup.7 counts/mL. After 5 min of reaction, the change in color between the filter membranes was detected by visual colorimetric method or by taking photos with a camera. The changes in color are shown in FIG. 3.

Example 4

[0044] In this example of the present disclosure, 1 mL of a CuS nanoparticle solution with a concentration of 10.sup.13 counts/mL was added to 20 uL of a solution of CD63 aptamer having a sulfhydryl group. Then, a 2 M sodium chloride solution was gradually added to give a final sodium chloride concentration of 0.1 M in the solution system. After 8 hrs of reaction, the reaction solution was centrifuged and washed to obtain CuS nanoparticles bearing CD63 aptamer.

[0045] 100 uL of CuS nanoparticles bearing CD63 aptamer was added to 5 mL of an exosome solution with a concentration of 10.sup.7 counts/mL and reacted for half an hour. Then 0.5% sodium dodecyl sulfate was added, and the solution was filtered through a filter membrane with a pore size of 50 nm and washed three times with PBS, to obtain a filter membrane containing exosome-CuS. 10 uL of a AgNO.sub.3 solution having a concentration of 1.0×10.sup.−3 M was added to the filter membrane and reacted for 5 min.

[0046] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 10 uL of a newly prepared 10 mol/L hydrogen peroxide solution were added to 400 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0047] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without exosomes) and the filter membrane containing exosomes of 10.sup.7 counts/mL. After standing for 5 min, the changes in color between the filter membranes in the control group and the experiment group was detected by visual colorimetric method or by taking photos with a camera.

Example 5

[0048] In this example of the present disclosure, 0.01 g of sodium dodecyl sulfate (SDS) was added to 1.0 mL of a CuS solution, and then 30 uL of 100 uM Thiol-Virus Aptamer and 10 uL of 2.5 mM tris(2-carboxyethyl)phosphine (TCEP) were added and reacted for 30 min to obtain a mixed solution. A 2 M NaCl solution was gradually added to give a final NaCl concentration of 0.1 M in the mixed solution. After 12 hrs of reaction, excess Thiol-Virus Aptamer was removed by centrifugation and washing three times with PBS, to obtain CuS-DNA complex particles, which was made up to 1.0 mL with PBS and stored in a freezer at 4° C.

[0049] 1.0 mL of a solution containing a certain concentration of highly pathogenic H5N1 avian influenza virus was added to 20 uL of the above-mentioned CuS-DNA solution. After mixing and reacting for 1 hr, 0.5% SDS was added, and the solution was passed through a filter membrane having a pore size of 60 nm to obtain a filter membrane containing virus-CuS complex particles. Then the filter membrane was taken out and 20 uL of 10.sup.−4 M AgNO.sub.3 was added and reacted for 5 min.

[0050] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 10 uL of a newly prepared 10 mol/L hydrogen peroxide solution were added to 400 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0051] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without viruses) and the filter membrane containing viruses of 10.sup.7 counts/mL. After standing for 5 min, the changes in color between the filter membranes in the control group and the experiment group was detected by visual colorimetric method or by taking photos with a camera.

Example 6

[0052] In this example of the present disclosure, 0.01 g of sodium dodecyl sulfate (SDS) was added to 1.0 mL of a CuS solution, and then 30 uL of 100 uM Thiol-Virus Aptamer and 10 uL of 2.5 mM tris(2-carboxyethyl)phosphine (TCEP) were added and reacted for 30 min to obtain a mixed solution. A 2 M NaCl solution was gradually added to give a final NaCl concentration of 0.1 M in the mixed solution. After 12 hrs of reaction, excess Thiol-Virus Aptamer was removed by centrifugation and washing three times with PBS, to obtain CuS-DNA complex particles, which was made up to 1.0 mL with PBS and stored in a freezer at 4° C.

[0053] 1.0 mL of a solution containing a certain concentration of highly pathogenic H5N1 avian influenza virus was added to 20 uL of the above-mentioned CuS-DNA solution. After mixing and reacting for 1 hr, 0.5% SDS was added, and the solution was passed through a filter membrane having a pore size of 70 nm to obtain a filter membrane containing virus-CuS complex particles. Then the filter membrane was taken out and 20 uL of 10.sup.−4 M AgNO.sub.3 was added and reacted for 5 min.

[0054] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 10 uL of a newly prepared 10 mol/L hydrogen peroxide solution were added to 400 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0055] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without viruses) and the filter membrane containing exosomes of 10.sup.8 counts/mL. After standing for 5 min, the changes in color between the filter membranes in the control group and the experiment group was detected by visual colorimetric method or by taking photos with a camera.

Example 7

[0056] In this example of the present disclosure, 0.01 g of sodium dodecyl sulfate (SDS) was added to 1.0 mL of a CuS solution, and then 30 uL of 100 uM Thiol-Virus Aptamer and 10 uL of 2.5 mM tris(2-carboxyethyl)phosphine (TCEP) were added and reacted for 30 min to obtain a mixed solution. A 2 M NaCl solution was gradually added to give a final NaCl concentration of 0.1 M in the mixed solution. After 12 hrs of reaction, excess Thiol-Virus Aptamer was removed by centrifugation and washing three times with PBS, to obtain CuS-DNA complex particles, which was made up to 1.0 mL with PBS and stored in a freezer at 4° C.

[0057] 1.0 mL of a solution containing a certain concentration of highly pathogenic H5N1 avian influenza virus was added to 20 uL of the above-mentioned CuS-DNA solution. After mixing and reacting for 1 hr, 1.0% SDS was added, and the solution was passed through a filter membrane having a pore size of 70 nm to obtain a filter membrane containing virus-CuS complex particles. Then the filter membrane was taken out and 20 uL of 10.sup.−4 M AgNO.sub.3 was added and reacted for 5 min.

[0058] A newly prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution and 10 uL of a newly prepared 20 mol/L hydrogen peroxide solution were added to 400 uL of a triethylamine hydrochloride solution, and mixed uniformly to prepare a detection solution.

[0059] The detection solution was added to the control group (a filter membrane obtained by performing the above experiment with a control solution without viruses) and the filter membrane containing viruses of 5*10.sup.8 counts/mL. After standing for 5 min, the change in color between the filter membranes in the control group and the experiment group was detected by visual colorimetric method.

[0060] The preferred embodiments of the present disclosure have been described above, which, however, are not intended to limit the scope of the present disclosure. Equivalent structural transformations, directly/indirectly applied to other related technical fields, made on basis of the disclosure of the description and drawings of the present disclosure without departing from the concept of the present disclosure, are included in the scope of protection of the present disclosure.