Methods for target DNA detection using non-functionalized carbohydrate-capped metallic nanoparticles

Patent metadata

Field	Value
Publication number	US-11226335-B2
Application number	US-201816494380-A
Country	US
Kind code	B2
Filing date	Mar 16, 2018
Priority date	Mar 17, 2017
Publication date	Jan 18, 2022
Grant date	Jan 18, 2022

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.

First claim

What is claimed is:

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 free from negatively charged polymer capping agents and an ionic species, thereby forming a 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 , wherein 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).

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.

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.

21. The method of claim 1 , wherein the carbohydrate-capped metal nanoparticle is free from negatively charged capping agents.

22. The method of claim 1 , wherein the carbohydrate-capped metal nanoparticle is free from capping agents other than carbohydrates.

23. The method of claim 1 , wherein:
(i) a maintained color state between initial and final solutions corresponds to the presence of the target DNA, and
(ii) a changed color state between initial and final solutions corresponds to the absence of the target DNA.

Assignees

• Univ Michigan State

Inventors

• Day Robert
• Baetsen-Young Amy
• Alocilja Evangelyn C

Classifications

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q2563/155

  Particles of a defined size, e.g. nanoparticles · CPC title

• G01N33/587 Primary

  Nanoparticles · CPC title

• B32B5/16

  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 title

• C12Q1/6825 Primary

  Nucleic acid detection involving sensors · CPC title