ABSTRACT
A protocol based on aminated diamond nanocrystals has been developed to isolate, concentrate, purify, and digest DNA oligonucleotides in one microcentrifuge tube for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. It is shown that use of diamond nanocrystals as a solid-phase extraction support not only permits concentration of oligonucleotides in highly diluted solutions but also facilitates separation of oligonucleotides from proteins in heavily contaminated solutions. Enzymatic digestions can be conducted on particle, and additionally, the digests can be easily recovered from the solution for base sequencing. In this method, the aminated diamond nanocrystals ( approximately 100 nm in diameter) were prepared by noncovalent coating of carboxylated/oxidized diamonds with poly(L-lysines) (PL), which form stable complexes with DNA oligonucleotides. While the complexes are sufficiently stable to sustain repeated washing with deionized water, the DNA molecules can be readily eluted after incubation of the diamond adducts in aqueous ammonium hydroxide at elevated temperatures. No preseparation of PL or diamond nanocrystals is required for subsequent MALDI-TOF mass analysis.
Subject(s)
DNA/analysis , Diamond/chemistry , Nanoparticles/chemistry , Oligonucleotides/analysis , Polylysine/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Adsorption , Biotinylation , DNA/chemistry , Oligonucleotides/chemistryABSTRACT
Methods have been developed to immobilize proteins onto the surfaces ofnanodiamonds with an average size of 5 +/- 1 nm. The immobilization started with carboxylation/oxidization of diamonds with strong acids, followed by coating the surfaces with poly-L-lysine (PL) for covalent attachment of proteins using heterobifunctional cross-linkers. The feasibility of this approach is proven with fluorescent labeling of the PL-coated diamonds by Alexa Fluor 488 and subsequent detection of the emission using a confocal fluorescence microscope. Immobilization of proteins onto the surfaces is also demonstrated with yeast cytochrome c, which possesses a free SH group for linkage and a characteristic Soret absorption band for observation.
