Nanoporous substrates for the analytical methods
Nanoporous materials can be used to enrich samples for subsequent analysis of substances contained in the sample. The method is shown to enrich the yield of species in the low molecular weight proteome, allowing detection of small peptides in the low nanomolar range.
1. A method of fractionating or separating very low molecular weight components from a fluid sample, said method comprising the steps of:
(a) providing a fluid sample comprising a first component consisting of molecules having a molecular weight no higher than 20 kDa and a second component having a molecular weight higher than 20 kDa;
(b) providing a substrate comprising a nanoporous material, wherein the nanoporous material comprises pores with a defined specific pore size that allows the entry of molecules of the first component and retains substantially all molecules of the first component and excludes substantially all molecules of the second component;
(c) exposing the substrate to the sample for a first period of time sufficient for the first component to penetrate a nanoporous surface of the substrate and enter into the pores of the nanoporous material while the second component does not penetrate the nanoporous material;
(d) washing the substrate for a second period of time sufficient to displace the second component from the substrate without removing the first component from pores of the nanoporous material;
(e) exposing the substrate to a solvent to extract the first component from the pores substrate; and
(f) analyzing a fraction of the first component that penetrated and entered into the nanoporous material.
2. The method of claim 1 , wherein the fluid sample is a biological fluid selected from the group consisting of blood serum, blood plasma, blood, urine, seminal fluid, seminal plasma, pleural fluid, ascites, nipple aspirate, feces or saliva.
3. The method of claim 1 , wherein the first component comprises peptides, antigens, antibodies, proteins, protein fragments, RNA and/or DNA.
4. The method of claim 1 , wherein the nanoporous material is a nanoporous silicon.
5. The method of claim 1 , wherein the nanoporous material is a nanoporous oxide material.
6. The method of claim 5 , wherein the nanoporous oxide material is a nanoporous silica.
7. The method of claim 1 , wherein the surface of the nanoporous material is modified to promote binding or adsorption of the first component thereto.
8. The method of claim 7 , wherein the nanoporous material has an electrically charged surface.
9. The method of claim 7 , wherein the nanoporous material has a surface modified with functional groups.
10. The method of claim 1 , wherein the substrate is a film, a wafer, a particle or a microchip.
11. The method of claim 1 , wherein the substrate is fabricated by a top-down technique.
12. The method of claim 1 , wherein the substrate is fabricated by a top-down technique selected from photolithography, electron beam lithography, X-ray lithography, deep UV lithography and nanoprint lithography.
13. The method of claim 1 , further comprising the step of extracting the first component from the nanoporous material.
14. The method of claim 1 , further comprising the step of washing the nanoporous material subsequent to the step of exposing the nanoporous material to the sample.
15. The method of claim 1 , wherein the nanoporous material directly adsorbs the first component by means of silanol groups present on the silica-based nanoporous material.
16. The method of claim 1 , further comprising the step of analyzing the first component.
17. The method of claim 16 , wherein the first component is analyzed by mass spectrometry, gel electrophoresis, chromatography, bioassay or a combination thereof.
18. The method of claim 17 , wherein the mass spectrometry is MALDI-TOF mass spectrometry, LC/MS mass spectrometry, ESI-MS mass spectrometry, tandem mass spectrometry or SELDI mass spectrometry.
19. The method of claim 1 , wherein the first component consists of molecules having a molecular weight no higher than 15 kDa.
20. The method of claim 1 , wherein the first component consists of molecules having a molecular weight no higher than 10 kDa.
21. The method of claim 1 , wherein the first component consists of molecules having a molecular weight no higher than 5 kDa.
22. The method of claim 1 , wherein the first component is present in the sample at a concentration no higher than 1000 ng/ml.
23. The method of claim 1 , wherein the first component is present in the sample at a concentration no higher than 200 ng/ml.
24. The method of claim 1 , wherein the first component is present in the sample at a concentration no higher than 100 ng/ml.
25. The method of claim 1 , wherein the first component is present in the sample at a concentration no higher than 20 ng/ml.
26. The method of claim 1 , wherein the first component is present in the sample at a concentration no higher than 10 ng/ml.
27. The method of claim 7 , wherein the nanoporous material has a surface modified with metals.
28. The method of claim 1 , wherein the substrate has a surface constructed with several nanoporous regions distributed over an inert, non-porous, non-adsorbent silicon chip surface.
29. The method of claim 28 , wherein each nanoporous region has a different molecular-weight specificity.
30. The method of claim 1 wherein the nanoporous material has a pore size distribution centered at 2-20 nm.
31. The method of claim 1 wherein the nanoporous material has a pore size distribution centered at 2-10 nm.