NMR systems and methods for the rapid detection of analytes

• US 8,409,807 B2
• Filed: 02/01/2012
• Issued: 04/02/2013
• Est. Priority Date: 10/22/2010
• Status: Active Grant

• Current Assignee: T2 Biosystems, Inc
• Original Assignee: T2 Biosystems, Inc
• Inventors: Neely, Lori Anne, Audeh, Mark John, Blanco, Matthew, Chepin, James Franklin, Demas, Vasiliki, Dhanda, Rahul K., Lowery, Thomas Jay Jr.
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US13/363,916
• Publication Number:
  US 20120164644A1
• Time in Patent Office: 426 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation
• CPC Class Codes: C12Q 2565/633 NMR
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 24/08 by using nuclear magnetic resonance G01N24/12 takes precedence
• CPC Class Codes: G01N 27/745 for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01R 33/302 Miniaturized sample handling arrangements for sampling small quantities, e.g. flow-through microfluidic NMR chips
• CPC Class Codes: G01R 33/448 Relaxometry, i.e. quantification of relaxation times or spin density G01R33/50 takes precedence
• CPC Class Codes: G01R 33/465 applied to biological material, e.g. in vitro testing
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

This invention features systems and methods for the detection of analytes, and their use in the treatment and diagnosis of disease.

Claims

1. A method for detecting the presence of a Candida species in a whole blood sample, the method comprising:

(a) providing an extract produced by lysing the red blood cells in a whole blood sample from a subject, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet prior to resuspending the pellet and optionally repeating the centrifuging, discarding, and resuspending steps;

(b) lysing cells in the extract to form a lysate;

(c) amplifying nucleic acids in the lysate to form an amplified lysate solution comprising from 40% (w/w) to 95% (w/w) of a Candida species target nucleic acid and from 5% (w/w) to 60% (w/w) of nontarget nucleic acid;

(d) following step (c), adding to the amplified lysate solution from 1×

10⁶ to 1×

10¹³ magnetic particles per milliliter of the amplified lysate solution to form a mixture, wherein the magnetic particles have a mean diameter of from 700 nm to 950 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of a target nucleic acid or a multivalent binding agent, wherein said magnetic particles have a T₂ relaxivity per particle of from 1×

10⁹ to 1×

10¹² mM<sup>−

1</sup>s<sup>−

1</sup>;

(e) providing the mixture in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the mixture to a bias magnetic field created using one or more magnets and an RF pulse sequence;

(f) exposing the mixture to a bias magnetic field and an RF pulse sequence;

(g) following step (f), measuring the signal from the detection tube;

(h) on the basis of the result of step (g), detecting the target nucleic acid, wherein step (g) is carried out without any prior purification of the amplified lysate solution; and

(i) on the basis of the result of step (h), determining whether the Candida species was present in the sample.

2. The method of claim 1, wherein said whole blood sample is from 0.05 to 4.0 mL.

3. The method of claim 1, wherein the method is capable of detecting a Candida species concentration of at least 3 cells/mL in the whole blood sample.

4. The method of claim 1, wherein the magnetic particles are substantially monodisperse.

5. The method of claim 1, wherein step (g) comprises measuring the T₂ relaxation response of the mixture, and wherein increasing agglomeration in the mixture produces an increase in the observed T₂ relaxation time of the mixture.

6. The method of claim 1, wherein the amplifying of step (c) comprises amplifying a nucleic acid to be detected in the presence of a forward primer and a reverse primer, each of which is universal to multiple Candida species to form a solution comprising a Candida amplicon;

and

said magnetic particles of step (d) have a first probe and a second probe conjugated to their surface, the first probe operative to bind to a first segment of the target nucleic acid and the second probe operative to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid.

7. The method of claim 6, wherein the forward primer comprises the oligonucleotide sequence 5′

-GGC ATG CCT GTT TGA GCG TC-3′

(SEQ ID NO;

     1).

8. The method of claim 6, wherein the reverse primer comprises the oligonucleotide sequence 5′

-GCT TAT TGA TAT GCT TAA GTT CAG CGG GT-3′

(SEQ ID NO;

     2).

9. The method of claim 6, wherein the Candida species is Candida albicans, and wherein the first probe comprises the oligonucleotide sequence:

10. The method of claim 6, wherein the Candida species is Candida krusei, and wherein the first probe and the second probe comprise an oligonucleotide sequence selected from:

11. The method of claim 6, wherein the Candida species is Candida glabrata, and wherein the first probe comprises the oligonucleotide sequence:

12. The method of claim 6, wherein the Candida species is Candida parapsilosis or Candida tropicalis, and wherein the first probe and the second probe comprise an oligonucleotide sequence selected from:

13. The method of claim 1, wherein steps (a) through (i) are completed within 3 hours.

14. The method of claim 6, wherein the magnetic particles comprise two populations, a first population bearing the first probe on its surface, and the second population bearing the second probe on its surface.

15. The method claim 1, wherein said magnetic particles comprise one or more populations having a first probe and a second probe conjugated to their surface, the first probe operative to bind to a first segment of the Candida amplicon and the second probe operative to bind to a second segment of the Candida amplicon, wherein the magnetic particles form aggregates in the presence of the Candida amplicon.

16. The method of claim 1, wherein said magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and a multivalent binding agent comprising a first probe and a second probe, the first probe operative to bind to said first binding moiety and the Candida amplicon, the second probe operative to bind to a second binding moiety and the Candida amplicon, and the binding moieties and multivalent binding agent operative to alter an aggregation of the magnetic particles in the presence of the Candida amplicon.

17. The method of claim 1, wherein the amplified lysate solution of step (c) comprises whole blood proteins and non-target oligonucleotides.

18. The method of claim 1, wherein said magnetic particles have a mean particle diameter between 700 and 900 nm.

19. The method of claim 1, wherein said magnetic particles have a mean particle diameter of between 700 and 850 nm.