Dynamic multiphase reaction in one-pot for CRISPR/Cas-derived ultra-sensitive molecular detection

1 . An aqueous, miscible, multiphase, one-pot detection system, comprising a first phase comprising a solution comprising a nucleic acid detection system; and a second phase in diffusive communication with the first phase, the second phase comprising a solution having a higher density than the first phase, and a nucleic acid amplification system.

2 . The system of claim 1 , wherein the solution of the first phase and/or the solution of the second phase is a sucrose solution, a polysucrose solution, a glycerol solution, a sorbitol solution, a copolymerized sucrose and epichlorohydrin solution, a dextran solution, or a combination thereof.

3 . The system of claim 1 , wherein the nucleic acid amplification system comprises Nucleic Acid Sequence Based Amplification (NASBA), Transcription Mediated Amplification (TMA), Helicase Dependent Amplification (HDA), Recombinase polymerase amplification (RPA), Strand Displacement Amplification (SDA), Loop-mediated Isothermal Amplification (LAMP), Chimera Displacement Reaction (RDC), Isothermal Chimeric Amplification of Nucleic Acids (ICAN), SMart Amplification Process (SMAP), Linear Isothermal Multimerization Amplification (LIMA), Dual-Priming Isothermal Amplification (DAMP), isothermal multiple-self-matching-initiated amplification (IMSA) or Self Extending Amplification (SEA).

4 . The system of claim 3 , wherein the recombinase polymerase amplification system comprises a single-stranded deoxyribonucleic acid (DNA)-binding protein (SSB), a recombinase, and a strand-displacing polymerase.

5 . The system of claim 1 , wherein the nucleic acid detection system comprises a Type V clustered regularly interspaced short palindromic repeats (CRISPR)/Cas detection system, a colorimetric detection system, a bioluminescence detection system, or an electrochemical detection system.

6 . The system of claim 1 , wherein the nucleic acid detection system is a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas detection system comprising a Cas12a endonuclease, a CRISPR ribonucleic acid (crRNA) comprising a complementary sequence to a target sequence, and a single-stranded reporter deoxyribonucleic acid (DNA) comprising a detectable label.

7 . The system of claim 1 , wherein the volume ratio of the first phase to the second phase is 1:10 to 10:1.

8 . The system of claim 1 , further comprising a third phase, the third phase comprising a solution having a higher density than the second phase, wherein the third phase is in diffusive contact with the second phase.

9 . The system of claim 8 , wherein the third phase comprises a nucleic acid preparation system.

10 . A multiwell plate and/or a device comprising the system of claim 1 .

11 . A method of detecting a target nucleic acid in a nucleic acid sample suspected of containing the target nucleic acid, the method comprising providing the system of claim 1 , wherein the first phase comprises a detectable label; and amplifying the target nucleic acid in the second phase for a time sufficient to provide an amplified target nucleic acid and to allow the amplified target nucleic acid to diffuse to the first phase, wherein diffusion of the amplified target nucleic acid to the first phase activates the nucleic acid detection system and turns on the detectable label providing detection of the target nucleic acid in the nucleic acid sample.

12 . The method of claim 11 , wherein the solution of the second phase is a sucrose solution, a glycerol solution, or a polysucrose solution.

13 . The method of claim 11 , wherein the nucleic acid amplification system comprises Nucleic Acid Sequence Based Amplification (NASBA), Transcription Mediated Amplification (TMA), Helicase Dependent Amplification (HDA), Recombinase polymerase amplification (RPA), Strand Displacement Amplification (SDA), Loop-mediated Isothermal Amplification (LAMP), Chimera Displacement Reaction (RDC), Isothermal Chimeric Amplification of Nucleic Acids (ICAN), SMart Amplification Process (SMAP), Linear Isothermal Multimerization Amplification (LIMA), Dual-Priming Isothermal Amplification (DAMP), isothermal multiple-self-matching-initiated amplification (IMSA), or Self Extending Amplification (SEA).

14 . The method of claim 13 , wherein the recombinase polymerase amplification system comprises a single-stranded deoxyribonucleic acid (DNA)-binding protein (SSB), a recombinase, and a strand-displacing polymerase.

15 . The method of claim 11 , wherein the nucleic acid detection system comprises a Type V clustered regularly interspaced short palindromic repeats (CRISPR)/Cas detection system, a colorimetric detection system, a bioluminescence detection system, or an electrochemical detection system.

16 . The method of claim 11 , wherein the nucleic acid detection system is a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas detection system comprising a Cas12a endonuclease, a CRISPR ribonucleic acid (crRNA) comprising a complementary sequence to a target sequence, and a single-stranded reporter deoxyribonucleic acid (DNA) comprising a detectable label.

17 . The method of claim 11 , further comprising providing a third phase, the third phase comprising a solution having a higher density than the second phase, wherein the third phase is in diffusive contact with the second phase, and wherein the third phase comprises an unpurified sample comprising the nucleic acid sample, wherein the nucleic acid sample is cell-free nucleic acids and diffuses from the third phase to the second phase.

18 . The method of claim 17 , wherein the unpurified sample is a blood sample, a saliva sample, or a tissue sample.

19 . The method of claim 11 , wherein the target nucleic acid is a pathogen deoxyribonucleic acid (DNA), a pathogen ribonucleic acid (RNA), or a biomarker of disease.

20 . The method of claim 11 , wherein amplifying is done at a temperature of 30° C. to 65° C.