Femtosecond Laser-Induced Crystallization of Amino Acid and Protein at the Air/Solution Interface
|關鍵字:||飛秒雷射;雷射誘發結晶化;甘胺酸;溶菌酶;氣液介面;光崩解;femtosecond laser;laser-induced crystallization;glycine;lysozyme;air/solution interface;optical breakdown|
Laser-induced crystallization is now becoming an indispensable technique to obtain molecular crystal. Particularly, femtosecond (fs) laser-induced crystallization was demonstrated in 2002 and is developing especially toward protein crystallization technique. When an intense fs laser pulse is focused into a supersaturated solution, multiphoton absorption of solvent occurs and induces mechanical stress to the surrounding area, which results in crystallization of solute. In this study, we explore how fs laser parameters such as pulse energy, repetition rate, and focal position affect crystallization and obtained crystals, and then demonstrate that solution surface irradiation of fs laser pulses can improve molecular crystallization probability drastically. Firstly, fs laser induced crystallization of glycine from the supersaturated solution is demonstrated and relations among laser parameters and crystallization probability, crystal morphology, and the polymorph are examined. Pulse energy and repetition rate dependences show that the frequency of cavitation bubble generation induced by multiphoton absorption of water mainly determines crystal morphology and number. Furthermore, significant increase of crystallization probability is also demonstrated by focusing fs pulses at the air/solution interface, whose mechanism may be ascribed to molecular adsorption at the interface. Based on the inference, we have succeeded in single glycine crystal formation induced by single fs pulse irradiation for the first time as far as we know. Secondly, we demonstrate fs laser induced crystallization of lysozyme as the most standard enzyme and protein in protein crystallography in the similar manner to glycine. It is revealed that irradiation at the air/solution interface of the solution droplet much improved the crystallization probability, compared with that of fs laser irradiation inside the solution and that of spontaneous crystallization. X-ray diffraction analysis of the obtained crystals clarified that crystal structure was the same under each crystallization condition. It is known that protein can be adsorbed and be localized at the solution surface leading to the denaturation. However, this result indicates that the salting-out effect can stabilize the structure even at the surface, which is contemporarily reported in elsewhere. The present result clearly shows that utilizing solution surface irradiation in the fs laser-induced crystallization sharpens the existent crystallization technique. Further spectroscopic dynamics study on the early stage of molecular nucleation occurring at the surface is promising to establish novel interface/surface molecular science from this crystallization methodology.
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