RAPID DETECTION AND QUANTITATION OF PATHOGEN-SPECIFIC BIOMARKERS USING NANOPOROUS DUAL- OR MULTI-LAYER SILICA FILMS

• US 20150260715A1
• Filed: 05/22/2015
• Published: 09/17/2015
• Est. Priority Date: 11/30/2012
• Status: Abandoned Application

• Current Assignee: Methodist Hospital
• Original Assignee: Houston Methodist Research Institute
• Inventors: HU, Ye, MA, Xin, WU, Hung-Jen, FAN, Jia, SUN, Tong
• Application Number:
  US14/720,715
• Publication Number:
  US 20150260715A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01N 2333/35 from Mycobacteriaceae (F)
• CPC Class Codes: G01N 2469/10 Detection of antigens from microorganism in sample from host
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/5695 Mycobacteria
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Improved methods for detecting active tuberculosis are disclosed. A method comprises enriching at least one M. tuberculosis-specific biomolecule from a sample by contacting the sample with a nanoporous film; and detecting the presence of the M. tuberculosis-specific biomolecule or fragment(s) thereof. The method may further comprise digesting the enriched M. tuberculosis-specific biomolecule with an enzyme to produce a digestion product comprising at least one fragment of the M. tuberculosis-specific biomolecule. Improved sensitivity and speed achieved.

Claims

1. A method of identifying at least one pathogen-specific peptide or polypeptide from a biological sample, comprising contacting the sample with a nanoporous dual- or multi-layer silica film;

and detecting the presence of the pathogen-specific peptide or polypeptide, or one or more proteolytic fragment(s) thereof.

2. The method of claim 1, wherein the pathogen-specific peptide or polypeptide is an M. tuberculosis-specific peptide or polypeptide.

3. The method of claim 2, wherein the M. tuberculosis-specific peptide or polypeptide comprises a contiguous amino acid sequence from an early secretory antigenic target protein (ESAT-6) or a culture filtrate protein 10 (CFP-10).

4. The method of claim 1, wherein the biological sample is obtained from a mammal.

5. The method of claim 1, wherein the biological sample comprises sputum, pleural effusion, cerebrospinal fluid, urine, serum, plasma, or whole blood.

6. The method of claim 1, wherein the at least one peptide or polypeptide within the biological sample is concentrated prior to contact with the nanoporous dual- or multi-layer silica film.

7. The method of claim 1, wherein the nanoporous dual- or multi-layer silica film comprises at least a first layer of silica film comprising a plurality of pores of substantially the same average diameter, into which the at least one pathogen-specific peptide or polypeptide is absorbed.

8. The method of claim 1, wherein the nanoporous dual- or multi-layer silica film comprises at least a first layer of silica film comprising a plurality of pores having an average diameter of about 3 to about 10 nm.

9. The method of claim 8, wherein the nanoporous dual- or multi-layer film comprises at least a first layer of silica film comprising a plurality of pores having an average diameter of about 6 to about 8 nm.

10. The method of claim 7, wherein the nanoporous dual- or multi-layer film comprises a second layer of silica film positioned upon the first layer, the second layer comprising a plurality of pores having an average diameter that is different from that of the pores of the first layer.

11. The method of claim 10, wherein the second layer of silica film contains a plurality of pores having a first average diameter that is larger than that of the plurality of pores in the first layer.

12. The method of claim 1, further comprising washing the nanoporous film after contacting the film with the biological sample.

13. The method of claim 1, further comprising digesting the sample containing the pathogen-specific peptide or polypeptide with a protease or a peptidase to produce one or more proteolytic fragment(s) of the pathogen-specific peptide or polypeptide.

14. The method of claim 13, wherein the protease is trypsin.

15. The method of claim 13, wherein proteolysis of the sample is performed on or within the nanoporous dual- or multi-layer silica film.

16. The method of claim 15, further comprising isolating the one or more proteolytic fragment(s) from the nanoporous dual- or multi-layer silica film with an elution buffer.

17. The method of claim 13, wherein the presence of the pathogen-specific peptide or polypeptide, or the one or more proteolytic fragment(s) thereof is detected by identifying at least one mass fingerprint of the peptide, the polypeptide, the proteolytic fragment(s), or a combination thereof, by mass spectrometry.

18. The method of claim 17, wherein the at least one mass fingerprint is detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

19. The method of claim 18, wherein the at least one mass fingerprint is identified at about 1895-1910 Da ([M+H]⁺) at about 2003-2005 Da ([M+H]⁺) at about 1900.9511 Da ([M+H]⁺) at about 1907.9246 Da ([M+H]⁺) at about 2003.9781 Da ([M+H]⁺) at about 1668.7170 Da ([M+H]⁺) at about 1593.7503 Da ([M+H]⁺) at about 1142.6276 Da ([M+H]⁺) at about 908.4584 Da ([M+H]⁺) or any combination thereof.

20. The method of claim 3, wherein the M. tuberculosis-specific peptide or polypeptide comprises an at least 8 contiguous amino acid sequence from any one of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, and SEQ ID NO;

7.

21. The method of claim 20, wherein the M. tuberculosis-specific peptide or polypeptide comprises an at least 12 contiguous amino acid sequence from any one of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, and SEQ ID NO;

6.

22. The method of claim 21, wherein the M. tuberculosis-specific peptide or polypeptide comprises an at least 14 contiguous amino acid sequence from any one of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, and SEQ ID NO;

5.

23. A method, comprising:

a) enriching at least one target protein or polypeptide from a sample by contacting the sample with a nanoporous dual- or multi-layer silica film under conditions to absorb the target protein or polypeptide to the film, and subsequently washing the nanoporous dual- or multi-layer silica film to remove extraneous material;

b) digesting the enriched target protein or polypeptide on the nanoporous dual- or multi-layer silica film to produce at least one digestion product comprising at least one proteolytic fragment thereof; and

c) detecting the presence of the at least one proteolytic fragment of the target protein or polypeptide.

24. The method of claim 23, wherein the target protein or polypeptide is specific to a pathogen associated with an infectious disease.

25. The method of claim 23, wherein the nanoporous dual- or multi-layer silica film comprises at least a first layer having a plurality of pores with an average pore diameter of about 3- to about 10-nm.

26. The method of claim 23, wherein the at least one target protein or polypeptide is an ESAT-6- or a CFP-10-specific protein or polypeptide.

27. The method of claim 23, wherein the detecting is performed using mass spectrometry.

28. The method of claim 24, wherein the infectious disease is tuberculosis.