Kong Xianglei, Huang L C Lora, Liau S-C Vivian, Han Chau-Chung, Chang Huan-Cheng
Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei, Taiwan 106.
Anal Chem. 2005 Jul 1;77(13):4273-7. doi: 10.1021/ac050213c.
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.
已开发出一种基于胺化金刚石纳米晶体的方案，可在一个微量离心管中对DNA寡核苷酸进行分离、浓缩、纯化和消化，用于基质辅助激光解吸/电离（MALDI）飞行时间（TOF）质谱分析。结果表明，使用金刚石纳米晶体作为固相萃取支持物，不仅能浓缩高度稀释溶液中的寡核苷酸，还能促进从严重污染溶液中分离寡核苷酸与蛋白质。酶促消化可在颗粒上进行，此外，消化产物可轻松从溶液中回收用于碱基测序。在此方法中，通过用聚（L-赖氨酸）（PL）非共价包覆羧化/氧化金刚石制备胺化金刚石纳米晶体（直径约100 nm），PL与DNA寡核苷酸形成稳定复合物。虽然复合物足够稳定，可承受用去离子水反复洗涤，但在高温下将金刚石加合物在氢氧化铵水溶液中孵育后，DNA分子可轻易洗脱。后续MALDI-TOF质谱分析无需预先分离PL或金刚石纳米晶体。
