Methods of macromolecular analysis using nanochannel arrays

• US 8,722,327 B2
• Filed: 03/28/2008
• Issued: 05/13/2014
• Est. Priority Date: 03/28/2007
• Status: Active Grant

• Current Assignee: BioNano Genomics, Inc.
• Original Assignee: BioNano Genomics, Inc.
• Inventors: Cao, Han, Deshpande, Parikshit A., Austin, Michael D., Boyce-Jacino, Michael
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US12/057,987
• Publication Number:
  US 20080242556A1
• Time in Patent Office: 2,237 Days
• Field of Search: 435/6.11, 435/287.2, 506/9, 536/23.1, 977/704
• US Class Current: 435/6.1
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/041 Connecting closures to device or container
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2600/172 : Haplotypes
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 : Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

Methods of analyzing features such as the physical size of macromolecules or biomarkers along large genomic DNA molecules were disclosed as well as the devices for carrying out such high throughput analysis in a massively parallel fashion. Methods of fabricating such devices are also disclosed.

Claims

1. A method of characterizing one or more macromolecules using a nanofluidic device, comprising:

labeling regions of a sample macromolecule at either end of a nucleotide zone of interest and regions of a control macromolecule at either end of a nucleotide zone of interest, wherein the sample macromolecule and the control macromolecule each comprise a nucleotide zone of interest having at least two labels thereon;

translocating at least a portion of at least one region of the labeled macromolecules through a fluidic nanochannel segment disposed substantially parallel to a surface of a substrate, wherein the fluidic nanochannel segment contains and elongates at least a portion of a region of the macromolecule, and wherein the fluidic nanochannel segment has a characteristic cross-sectional dimension of less than about 1000 nm and a length of at least about 10 nm, wherein said step of translocating comprises introducing the labeled macromolecules into separate fluidic nanochannel segments having a cross-sectional diameter of less than about 200 nm so as to at least substantially linearize at least a part of the labeled macromolecules; and

monitoring, through a viewing window capable of permitting optical inspection of at least a portion of the contents of the fluidic nanochannel segment, one or more signals related to the translocation of one or more regions of the labeled macromolecules through the nanochannel, wherein said step of monitoring comprises determining the distance between two said labels on the sample macromolecule and the distance between two said labels on the control macromolecule, respectively; and

correlating the distance between the labels to one or more characteristics of the macromolecules.

2. The method of claim 1, further comprising exposing at least one biological entity to at least one agent of interest, to a metabolite of the agent of interest, to a salt of the agent of interest, or any combination thereof.

3. The method of claim 2, further comprising isolating at least one macromolecule from the exposed biological entity.

4. The method of claim 2, wherein the exposing the at least one biological entity to at least one agent of interest, to a metabolite of the agent of interest, to a salt of the agent of interest, or any combination thereof, comprises injecting, treating, spraying, transfecting, digesting, immersing, flowing, applying, or any combination thereof.

5. The method of claim 2, wherein a biological entity comprises a living being, a cell, a biological molecule, a protein, or any combination thereof.

6. The method of claim 2, wherein the isolating at least one macromolecule from the exposed biological entity comprises extracting, lysing, purifying, pulling, manipulating, reacting, distilling, electrophoresing, or any combination thereof.

7. The method of claim 2, wherein the one or more macromolecules comprise proteins, single-stranded DNA, double-stranded DNA, RNA, small interfering RNA, or any combination thereof.

8. The method of claim 2, further comprising dividing the one or more macromolecules into two or more segments.

9. The method of claim 2, wherein the dividing comprises lasing, sonicating, chemically treating, physically manipulating, biologically treating, or any combination thereof.

10. The method of claim 1, further comprising binding a fluorescent label, a radioactive label, a magnetic label, or any combination thereof to one or more regions of the macromolecule.

11. The method of claim 1, wherein translocating comprises applying a fluid flow, a magnetic field, an electric field, a radioactive field, a mechanical force, an electroosmotic force, an electrophoretic force, an electrokinetic force, a temperature gradient, a pressure gradient, a surface property gradient, a capillary flow, or any combination thereof.

12. The method of claim 11, wherein the translocating comprises controllably moving at least a portion of the macromolecule into at least a portion of a fluidic nanochannel segment.

13. The method of claim 11, wherein the translocating comprises moving at least a portion of the macromolecule through at least a portion of a fluidic nanochannel segment at a controlled speed and a controlled direction.

14. The method of claim 1, wherein monitoring comprises recording, displaying, analyzing, plotting, or any combination thereof.

15. The method of claim 1, wherein the one or more signals comprise an optical signal, a radiative signal, a fluorescent signal, an electrical signal, a magnetic signal, a chemical signal, or any combination thereof.

16. The method of claim 1, wherein the signal is generated by an electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, an electrical charged transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic particle, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, an antigen, a hapten, an antibody, an antibody fragment, a lipid, or any combination thereof.

17. The method of claim 1, wherein the molecule is unlabeled and monitored by infrared spectroscopy, ultraviolet spectroscopy, or any combination thereof.

18. The method of claim 1, wherein the signal is generated by using one or more excitation sources to induce fluorescence, chemoluminescence, phosphorescence, bioluminescence, or any combination thereof.

19. The method of claim 18, wherein the excitation source is a laser, a visible light source, a source of infrared light, a source of ultraviolet light, or any combination thereof.

20. The method of claim 1, wherein correlating comprises determining the features of a distinct macromolecule or a portion thereof from a population of macromolecules by partial or full elongation of the macromolecule in a fluidic nanochannel segment.

21. The method of claim 1, wherein at least a portion of the macromolecule is stationary during the monitoring.

22. The method of claim 1, wherein at least a portion of the macromolecule is translocated within at least a portion of the fluidic nanochannel segment more than one time.

23. The method of claim 1, wherein the correlating comprises determining the length of at least a portion of the macromolecule.

24. The method of claim 1, wherein the correlating comprises determining the apparent partially elongated length of at least a portion of the macromolecule as confined within one or more fluidic nanochannel segments.

25. The method of claim 24, wherein the apparent partially elongated length is determined as the linear distance along the fluidic nanochannel segment within which a partially elongated macromolecule is confined.

26. The method of claim 1, wherein the correlating comprises determining the identity of one or more components of the macromolecule.

27. The method of claim 1, wherein the correlating comprises determining the sequence of one or more components of the macromolecule.

28. The method of claim 1, wherein the correlating comprises determining the presence of one or more modifications to at least a portion of the macromolecule.

29. The method of claim 1, wherein the correlating is performed by automated means, computerized means, mechanical means, manual means, or any combination thereof.

30. The method of claim 1, wherein the correlating comprises an algorithm for characterizing a duplex nucleic acid molecule based on observed signal modulations through the detection region of a nanochannel, wherein said algorithm is present on a computer readable medium.

31. The method of claim 1, wherein the one or more characteristics of the macromolecule comprise one or more target features present on at least a portion of the macromolecule.

32. The method of claim 31, wherein a target feature comprises an epigenetic factor.

33. The method of claim 32, wherein an epigenetic factor comprises one or more methylation patterns.

34. The method of claim 31, wherein a target feature comprises one or more genomic structures.

35. The method of claim 34, wherein a genomic structure comprises the position of one or more particular molecular sequences, single-nucleotide polymorphisms, haplotypes, repetitive elements, copy numbers polymorphisms, a change in one or more loci on a DNA molecule, open reading frames, introns, exons, regulatory elements, or any combination thereof.

36. The method of claim 31, wherein a target feature comprises a compound or drug binding site or complex, a DNA repairing or cleaving binding site or complex, a small interfering RNA or anti-sense nucleotides nucleotide binding sites or complex, transcription or regulatory factor binding site or complex, a restriction enzyme binding or cleaving site or complex, or any other genetically engineered or chemically modified binding site or complex, or any combination thereof.

37. The method of claim 1:

wherein the labeling of the macromolecules comprises contacting the control macromolecule with a first labeled probe (S1) of known length L1, wherein the control macromolecule comprises a control genomic sequence whose copy number is known and the first labeled probe is complementary to the sequence of the control genomic sequence, and contacting the sample macromolecule comprising a nucleotide sequence of interest with a second labeled probe of known length L2, wherein the second labeled probe is specific to the nucleotide sequence of interest; and

wherein the step of monitoring comprises detecting binding between the first labeled probe and the control genomic sequence and binding between the second labeled probe and the nucleotide sequence of interest, and ascertaining the total length of the hybridization signals that correspond to the first labeled probe (S1) and the second labeled probe (S2).

38. The method of claim 37, further comprising calculating the copy number of the nucleotide sequence of interest, wherein the copy number is calculated by calculating the ratios N1=S1/L1 and N2=S2/L2, wherein N1 corresponds to the copy number of the genomic control sequence and N2 corresponds to the copy number of the nucleotide sequence of interest.

39. The method of claim 37, wherein the copy number of the control sequence is an integer.

40. The method of claim 37, wherein a difference between N2 and N1 indicates an abnormality in the genome.

41. The method of claim 37, wherein the control genomic sequence comprises separate portions whose total length per genome is known, wherein the sequence of interest comprises separate portions whose length per normal gene is known, and wherein a significant difference between N2 and N1 indicates a genetic abnormality in the genome.

42. The method of claim 37, wherein the sequence of interest relates to a trisomy-linked chromosome, wherein the control genomic sequence is from a chromosome other than the trisomy-linked chromosome, and wherein a N2/N1 ratio of approximately 1.5 indicates a trisomic genotype.

43. The method of claim 37, wherein the nucleotide sequence of interest comprises a deletion of a portion of a genome.

44. The method of claim 37, wherein the nucleotide sequence of interest comprises a repeating sequence.

45. The method of claim 37, wherein the control genomic sequence and the nucleotide sequence of interest are identical for a given genome, and wherein one or more different genomes are analyzed within one or more fluidic nanochannel segments so as to determine the respective quantities of each genome present.

46. The method of claim 37, wherein the N2/N1 ratio has a statistical error of less than 20%.

47. The method of claim 37, wherein the control genomic sequence and nucleotide sequence of interest are from the same genome.

48. The method of claim 37, wherein the control genomic sequence is from the same chromosome as the nucleotide sequence of interest.

49. The method of claim 1, further comprising (a) introducing the labeled nucleotides into separate fluidic nanochannel segments having a cross-sectional diameters sufficient to at least substantially elongate the labeled nucleotides, (b) determining the distance between the labels on the sample nucleotide and the control nucleotide, respectively, and repeating steps (a) and (b) one or more times so as to further linearize the sample and control nucleotides and so as to obtain additional information regarding the distance between the labels on the control and sample nucleotides as the nucleotides elongate.

50. The method of claim 49, further comprising determining the length of the zone of interest on the sample nucleotide by comparing the distance between the labels on the control and sample nucleotides.

51. The method of claim 50, wherein a difference between the distance between the labels on the control and sample nucleotides indicates an abnormality in the zone of interest on the sample nucleotide.