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The following paper appeared in Proceedings of the fifty-third Annual Meeting, Microscopy Society of America; G. W. Bailey, M. H. Ellisman, R. A. Hennigar, and N. J. Zaluzec (Eds.). Jones and Begell, New York, NY, 1995, pp. 858-859 (1995).


James F. Hainfeld and Frederic R. Furuya*

Department of Biology, Brookhaven National Laboratory, Upton, NY 11973
*Nanoprobes, Inc., 25 East Loop Rd., Suite 124, Stony Brook, NY 11790-3355.

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 a-amino1. With some enzymes, catalytic activity after fixing is preserved2; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine3. It has also been reported that glutaraldehyde can polymerize in aqueous solution4. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates5. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

In order to explore the potential advantages and unique features of aldehyde linking, gold clusters were prepared with one or more aldehydes on their surface. A phosphine was first synthesized that contained a dihydroxy terminating group, tris [p-2,3-dihydroxypropylcarboxamido) phenyl] phosphine. This was used to form undecagold clusters, which were then oxidized with NaIO4 to produce a polyaldehyde gold cluster, having the formula Au11(P(C6H4)CONHCH2CHO)3)7, which was then purified by column chromatography. This showed high reactivity with Schiff's reagent for testing aldehydes, indicating multiple aldehydes per cluster.

The aldehyde-gold was reacted with BSA or Fab' fragments in varying ratios (5 gold clusters to 1 protein (5:1), or 1:1), along with 20 mM NaCNBH3 in 0.1 M HEPES pH 7.5 and incubated overnight. The unreacted aldehyde was blocked with 0.1 M glycine for 1 hr, and the reaction purified on a Superose-12 (Pharmacia) gel exclusion column in PBS to separate protein from unreacted gold, and to separate larger complexes, such as protein dimers or multimers. With a 5:1 mixing ratio, most of the protein was in the monomer peak with gold labeling; with a 1:1 mixing ratio, the monomer protein peak was largest, but smaller peaks corresponding to dimer, trimer, and aggregate protein were seen. In one case, the monomer peak had ~20 % gold labeling, and the dimer peak had a calculated labeling of 2.2 gold clusters per BSA.

Scanning transmission electron microscopy (STEM) of the chromatographic peaks showed respectively, protein monomers (Fig. 2), which had one Au11 attached, oligomers, or small aggregates of BSA-gold (Fig. 3). A similar preparation of aldehyde-gold using the 1.4 nm Nanogold cluster showed similar results (Fig. 4); Fig. 5 shows high multiple gold labeling of one or a few protein moleucules.

Aldehyde gold clusters therefore provide an interesting method of preparing conjugates which may also be of interest in producing gold clusters with multiple small molecules attached, since for example, the undecagold cluster discussed has 21 aldehyde groups around its surface. A monofunctional aldehyde cluster has also been synthesized, and should eliminate any aggregation or oligomer formation.


  1. B.F. Erlanger, Meth. Enzym. 70 (1980) 85.
  2. F.A. Quiocho and F.M. Richards, Biochemistry, 5 (1966) 4062.
  3. A.F.S.A. Habeeb and R. Hiramoto, Arch. Biochem. Biophys. 126 (1968) 16.
  4. M.A. Hayat, in Principles and Techniques of Electron Mocroscopy: Biological Applications, v.1, NY: Van Nostrand Reinhold Co. (1970) 79.
  5. J.C. Saccavini et al., in S.C. Srivastava, Ed., Radiolabeled monoclonal antibodies for imaging and therapy, NY: Plenum Press (1988) 239.
  6. The authors would like to thank N.I. Feng for biochemical assistance, J. Marecek for synthetic chemistry, and M. Simon and B. Lin for STEM microscopy. This work was partially supported by US Dept of Energy, OHER.

[Figure 1: Aldehyde Formation] (9k)

[Figures 2-5: Micrographs] (131k)

Fig. 1. Reaction scheme of coupling aldehyde gold to proteins; R = protein; Au = gold cluster.

Fig. 2. Darkfield STEM micrograph of unstained Fab' labeled with aldehyde Au11. Thin arrow points to Au11 cluster (bright spot); thick arrow to protein (grey mass). Full width, 64 nm.

Fig. 3. STEM micrograph of Fab' aggregate crosslinked with aldehyde Au11. Full width, 64 nm.

Fig. 4. STEM micrograph of unstained BSA labeled with aldehyde Au1.4nm. Full width, 64 nm.

Fig. 5. STEM micrograph of unstained BSA labeled with multiple Au1.4nm clusters. Full width, 64 nm.

© 1996 San Francisco Press. Used with permission.

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