Labeling and Purification of Alcalase and Fluorescent Nanocrystals Synthesized in Water Phase†

doi:10.7503/cjcu20140803

Abstract

Abstract:

CdTe nanocrystals were synthesized in aqueous solution and capped with amercaptopropionic acid, which were used as a kind of fluorescent probe to label Alcalase. The fluorescence spectra showed that CdTe nanocrystals were accompanied by a narrow size distribution and high stability. The fluorescence intensity decreased 8.36% after 30 d at room temperature. Transmission electron microscopy(TEM) imaging revealed that CdTe nanocrystal’s diameter was about 3 nm, and X-ray diffraction(XRD) pattern showed it with zinc blende structure. After being conjugated with Alcalase using 1-ethyl-3-(3-dimethyl aminopropye) carbodii-mide hydroclloride(EDC) and N-hydroxysulfosucinimide sodium salt(NHSS) methods, the CdTe-Alcalase conjugates were separated and purified by ultra-filter-centrifugation, a Sephadex G-100 column and sodium dedecye sulfate(SDS)-polyacrylamide gel electrophoresis. Fluorescent spectral analysis showed that CdTe-Alcalase conjugates also presented good optical stability compared with CdTe nanocrystals. The enzyme activity was also relatively high at 61.06 U/μg. The fluorescent emission of the CdTe-Alcalase conjugates was visualized with a fluorescence microscope, which would be suitable for tracing the enzyme reaction in the future.

Key words: Fluorescent nanocrystal, Alcalase, Labeling, Purification

CLC Number:

O629
R318.08

TrendMD:

ZHU Jinming, SUN Zhuo, FU Yao, GUO Yi, XU Li. Labeling and Purification of Alcalase and Fluorescent Nanocrystals Synthesized in Water Phase^†[J]. Chem. J. Chinese Universities, 2015, 36(3): 511.

Figures/Tables 5

Fig.1 TEM image of CdTe nanocrystals The circle indicate nanocrystals.

Fig.2 Schematic illustration of MPA functionalized CdTe nanocrystals conjugated with Alcalase

Fig.3 Column purification of CdTe nanocrystals and Alcalase (A) CdTe nanorystals; (B) Alcalase; (C) simple mixture of CdTe nanocrystals and Alcalase; (D) EDC and NHSS mixture; (E) reactive mixture after coupling without ultra-filter-centrifugation; (F) reactive mixture after coupling and ultra-filter-centrifugation.

Fig.4 Image of SDS-Polyacrylamide gel electrophoresis Line 1. CdTe-Alcalase conjugates; Line 2. Alcalase; Line 3. Marker.

Fig.5 Fluorescence microscope images of CdTe nanocrystals(A) and CdTe-Alcalase conjugates(B)

References 21

[1]	Resch-Genger U., Grabolle M., Cavaliere-Jaricot S., Nitschke R., Nann T., Nat. Methods,2008, 5(9), 763—775
[2]	Zhang J., Campbell R. E., Ting A. Y., Tsien R. Y., Nat. Rev. Mol. Cell Biol., 2002, 3(12), 906—918
[3]	Wu J., Liu S., Shen X. Y., Yang N. Y., Liu Y., Tsuji I., Yamaura T., Li J., Li X. M., Chem. Res. Chinese Universities,2013, 29(5), 911—916
[4]	Xiao Y., Li S., Huang H. M., Zhang Y., Xue Z. Z., Tang X. Y., He X. X., Wang K. M., Chem. Res. Chinese Universities,2013, 29(5), 845—849
[5]	Michalet X., Pinaud F. F., Bentolila1 L. A., Tsay J. M., Doose S., Li J. J., Sundaresan G., Wu A. M., Gambhir S. S., Weiss S., Science,2005, 307(5709), 538—544
[6]	Guo Y., Li C. R., Shen H. B., Zhang X. Z., Li L. S., Yu Q., Xu L., Chem. Res. Chinese Universities,2011, 27(5), 832—835
[7]	Wang Y. L., Liu S. Y., Mo F. P., Pan H. Q., Chem. J. Chinese Universities,2013, 34(1), 45—49
	(王益林, 刘声燕, 莫凤萍, 潘华桥. 高等学校化学学报, 2013, 34(1), 45—49)
[8]	Zhang H., Wang D. Y., Yang B., Mohwald H., J. Am. Chem. Soc., 2006, 128(31), 10171—10180
[9]	Warner J. H., Hoshino A., Yamamoto K., Tilley R. D., Angew. Chem. Int. Ed., 2005, 44(29), 4550—4554
[10]	Hines D. A., Kamat P. V., J. Phys. Chem. C,2013, 117(27), 14418—14426
[11]	Finetti C., Colombo M., Prosperi D., Alessio G., Morasso C., Sola L., Chiari M., Chem. Commun., 2014, 50, 240—242
[12]	Yuan J., Gaponik N., Eychmuller A., Anal. Chem., 2012, 84(11), 5047—5052
[13]	Lin J. H., Tseng W. L., Biosens. Bioelectron., 2013, 41, 379—385
[14]	Iyer A., Chandra A., Swaminathan R., BBA-Gen. Subjects,2014, 1840(9), 2935—2943
[15]	Hua X. F., Liu T. C., Cao Y. C., Liu B., Wang H. Q., Wang J. H., Huang Z. L., Zhao Y. D., Anal. Bioanal. Chem., 2006, 386(6), 1665—1671
[16]	Shahidi F., Han X. Q., Synowiecki J., Food Chem., 1995, 53(3), 285—293
[17]	Rogach A. L., Franzl T., Klar T. A., Feldmann J., Gaponik N., Lesnyak V., Shavel A., Eychmüller A., Rakovich Y. P., Donegan J. F., J. Phys. Chem. C,2007, 111(40), 14628—14637
[18]	Rogach A. L., Katsikas L., Kornowski A., Su D. S., Eychmuller A., Weller H., Ber. Bunsenges. Phys. Chem., 1996, 100(11), 1772—1778
[19]	Zhang H., Zhou Z., Yang B., Gao M. Y., J. Phys. Chem.B,2003, 107(1), 8—13
[20]	Gutiérrez-Lazos C. D., Ortega-López M., Pérez-Guzmán M. A., Espinoza-Rivas A. M., Solís-Pomar F., Ortega-Amaya R., Silva-Vidaurri L. G., Castro-Pena V. C., Pérez-Tijerina E., Beilstein J. Nanotechnol., 2014, 5, 881—886
[21]	Meinardi F., Colombo A., Velizhanin K. A., Simonutti R., Lorenzon M., Beverina L., Viswanatha R., Klimov V. I., Brovelli S., Nat. Photonics,2014, 8(5) 392—399