METHODS FOR TARGET DNA DETECTION USING NON-FUNCTIONALIZED CARBOHYDRATE-CAPPED METALLIC NANOPARTICLES

• US 20200132693A1
• Filed: 03/16/2018
• Published: 04/30/2020
• Est. Priority Date: 03/17/2017
• Status: Active Grant

• Current Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Original Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Inventors: Day, Robert, Baetsen-Young, Amy, Alocilja, Evangelyn C.
• Granted Patent: US 11,226,335 B2
• Time in Patent Office: Days
• CPC Class Codes: B32B 5/16 characterised by features of a layer formed of particles, e.g. chips, powder or granules layer formed of natural mineral particles B32B19/00; layer being formed of wood fibres, chips or particles B32B21/02
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: G01N 33/587 Nanoparticles

Abstract

The disclosure relates to a method for specific detection of a target analyte using probe DNA specific to the target analyte and non-functionalized, carbohydrate-capped metal nanoparticles such as non-functionalized, dextrin-capped gold nanoparticles. A sample mixture including a target DNA analyte and a probe DNA specific thereto is incubated to from a probe DNA-target DNA complex. The non-functionalized, carbohydrate-capped metal nanoparticles and an ionic species such as sodium chloride or other salt are added to the probe DNA-target DNA complex, and the mixture is incubated. Addition of the ionic species creates a detectable distinction, such as color of the resultant mixture, between stabilized metal nanoparticles when the probe DNA-target DNA complex is present and destabilized metal nanoparticles when the probe DNA-target DNA complex is absent. The method can be used for colorimetric detection of plant pathogens and associated diseases in agricultural production systems.

Claims

1. A method for detection of a target analyte, the method comprising:

combining (i) a sample containing or suspected of containing a target DNA analyte with (ii) a probe DNA that is complementary to the target DNA analyte, thereby forming a sample mixture;

incubating the sample mixture under conditions sufficient to bind the probe DNA with any target DNA analyte present in the sample mixture, thereby forming an incubated solution comprising (i) a probe DNA-target DNA complex when the target DNA analyte is present in the sample, and (ii) free probe DNA when the target DNA analyte is not present in the sample;

combining the incubated solution with a non-functionalized, carbohydrate-capped metal nanoparticle and an ionic species, thereby forming an solution-nanoparticle mixture; and

incubating the solution-nanoparticle mixture under conditions sufficient to (i) at least partially stabilize the metal nanoparticle when the probe DNA-target DNA complex is present in the solution-nanoparticle mixture, and (ii) at least partially destabilize the metal nanoparticle when the target DNA analyte is not present in the sample.

2. The method of claim 1, further comprising:

detecting a relative degree of metal nanoparticle stabilization after incubating the solution-nanoparticle mixture.

3. The method of claim 2, wherein detecting a relative degree of metal nanoparticle stabilization comprises detecting a color state of the solution-nanoparticle mixture after incubation.

4. The method of claim 1, wherein the target DNA analyte comprises double-stranded genomic DNA (dsDNAg) characteristic of a target analyte organism.

5. The method of claim 4, wherein the target analyte organism is selected from the group consisting of a virus, a bacterium, a mould, a fungus, and a plant.

6. The method of claim 4, wherein the target analyte organism is a plant pathogen.

7. The method of claim 1, wherein the sample comprises a plant extract and the target DNA analyte comprises a plant pathogen DNA.

8. The method of claim 7, wherein the sample comprises a crude plant extract.

9. The method of claim 1, wherein the probe DNA comprises a single-stranded probe DNA (ssDNAp).

10. The method of claim 1, wherein the single-stranded probe DNA has a length of 5 to 100 nucleotide bases.

11. The method of claim 1, wherein the sample mixture further comprises a buffer.

12. The method of claim 11, wherein the buffer comprises a phosphate-buffered saline (PBS) buffer.

13. The method of claim 1, the sample mixture has a salt concentration of at least 40 mM.

14. The method of claim 1, wherein incubating the sample mixture to form the incubated solution comprises:

denaturing the sample mixture under conditions sufficient to denature any target DNA analyte present in the sample mixture; and

then

annealing the sample mixture under conditions sufficient to hybridize any denatured target DNA analyte present in the sample mixture with the probe DNA, thereby forming the probe DNA-target DNA complex when the target DNA analyte is present in the sample.

15. The method of claim 1, wherein the probe DNA-target DNA complex comprises:

a first region comprising a single-stranded probe DNA (ssDNAp) hybridized to a first strand of a double-stranded target DNA analyte (dsDNA); and

a second region comprising a second strand of the double-stranded target DNA analyte (dsDNA) that is not bound to the first strand of the double-stranded target DNA analyte (dsDNA).

17. The method of claim 1, wherein the non-functionalized, carbohydrate-capped metal nanoparticle comprises a gold nanoparticle and a dextrin capping agent on an outer surface of the gold nanoparticle.

18. The method of claim 1, wherein the non-functionalized, carbohydrate-capped metal nanoparticle is in the form of a non-functionalized, stabilized metal nanoparticle suspension composition comprising:

water in sufficient amount to provide an aqueous medium; and

a plurality of stabilized metal nanoparticles stably suspended in the aqueous medium, each stabilized metal nanoparticle comprising;

(i) a metal nanoparticle core and (ii) a carbohydrate capping agent present as a layer on an outer surface of the metal nanoparticle core in an amount sufficient to stabilize the metal nanoparticle suspension.

19. The method of claim 1, wherein the non-functionalized, carbohydrate-capped metal nanoparticle is free from biomolecules and specific binding pair members which specifically bind to the target DNA analyte.

20. The method of claim 1, wherein the ionic species combined with the incubated solution and the non-functionalized, carbohydrate-capped metal nanoparticle comprises sodium chloride.

16. The method of 15, wherein, after incubation of the solution-nanoparticle mixture, a corresponding probe DNA-target DNA-metal nanoparticle complex comprises:

a first region comprising a single-stranded probe DNA (ssDNAp) hybridized to a first strand of a double-stranded target DNA analyte (dsDNA);

a second region comprising a second strand of the double-stranded target DNA analyte (dsDNA) that is not bound to the first strand of the double-stranded target DNA analyte (dsDNA); and

the metal nanoparticle bound to the second strand of the double-stranded target DNA analyte in the second region.

21. A probe DNA-target DNA-metal nanoparticle complex comprising:

a first region comprising a single-stranded probe DNA (ssDNAp) hybridized to a first strand of a double-stranded target DNA analyte (dsDNA);

a second region comprising a second strand of the double-stranded target DNA analyte (dsDNA) that is not bound to the first strand of the double-stranded target DNA analyte (dsDNA); and

a non-functionalized, carbohydrate-capped metal nanoparticle bound to the second strand of the double-stranded target DNA analyte in the second region.

22. A stabilized complex suspension composition comprising:

water; and

the probe DNA-target DNA-metal nanoparticle complex of claim 21 stably suspended in the water.

23. A kit for detection of a target analyte, the kit comprising:

a probe DNA that is complementary to a target DNA analyte;

a non-functionalized, carbohydrate-capped metal nanoparticle;

optionally a buffer; and

optionally an ionic species.