2 . 1 Gold nanoparticles:
Metal nanoparticles have attracted much attention in the past few years due to their potential for use as device elements in nanoelectronic and optical application Successful fabrication f useful devices depends on the ability to prepare ordered asemblies of nanoparticles in a rapid and inexpensive manner.
The synthesis of nanoparticles with desired size/shape has, therefore, enormous importance, especially in the emerging field of nanotechnology . However, it is a fact that the reproducible preparation of stable particles with controlled prechosen size is a very difficult task using the popular colloid-chemical approach. This, in fact, demands development of a new general method as well as improvement over the existing ones. Among the known colloidal nanoparticles systems, gold is one of the most widely studied ones. And a few systematic approaches already exist to obtain particles of prechosen size via its controlled formation［5］. For example, Frens (1973) proposed a method where reducing/stabilizing agent, citrate to gold ratiowas varied. This results in particles with a broad size range (diameters between 10 and 150 nm). But for more than 30 nm diameter particles the monodispersity was observed to be poor.
Leff et al. (1995) synthesized gold particles with diameters ranging from 1.5 to 20 nm by varying the Au(III) ion to stabilizer thiol molar ratio. This approach requires long time, 12 h or so to allow the reaction products to attain equilibrium.
Another methodology, known since 1906, is ‘seed’- or ‘germ’-mediated growth which promises to obtain particles of desired size ［6］. Here, appropriate amounts of precursor ions are reduced over the preformed ‘seed’ or ‘germ’, i.e.,
small particles by suitable reducing agents. The reducing agent used in the second stage of ‘seed’-mediated growth is generally a weaker one, viz., H2NOH, ascorbate ion. They should reduce only the precursor ions which are adsorbed onto the‘seed’ surface without creating any new nucleation center. The final size of the particles would depend on the size of the ‘seed’ and the amount of the precursor ions to be reduced on them (Schmid, 1992). Therefore, in principle, the smaller is the starting seed, the lower will be the desired size limit of the particles. It allows preparing particles over a broad size range. Smaller
particles are generally produced by using stronger reducing agents, viz., NaBH4, phosphorus, tetrakis (hydroxymethyl) phosphonium chloride, etc., or radiolytic method. Furthermore, nature of the particle stabilizer, solvent, reaction condition, viz., pH, temperature, etc., plays crucial role in determining the final size of the particles. Recently, a radiolytic and a chemical size control with improved monodispersity via seed-mediated growth of colloidal gold nanoparticles were reported by Henglein and Meisel and Natan’s group (Brown & Natan, 1998;
Brown et al., 2000), respectively. They have followed iterative growth method, i.e., particles were grown in the immediately previous step were used as seeds in the next growth step. In the present study, we examine the applicability of a combination of photochemical approach with a non-iterative growth method to develop a simple and fast technique of size control. Here, particles of various size ranging from average diameter 5 to 20 nm could be obtained within few minutes by UV irradiation at room temperature (28◦C) in presence of air.
Furthermore, larger size particles (diameter 25–110 nm) were produced directly from the original seed particles by varying the ratio of original [seed] to [Au(III)]
and using ascorbic acid as reductant. It should be noted that ascorbate ion is frequently used as reducing agent for reduction of Au(III) or Ag(I) ions. Goia
and Matijevic (Goia et al., 1999) recently used isoascorbic acid at various pH conditions to synthesize relatively large spherical gold particles (ranging in modal diameter from 80 nm to 5m) directly from Au(III) ions.
［6-15］
Fig. 2.1 Absorption spectra of colloids obtained at various reaction temperatures.
Fig. 2.2 SEM image of the gold nanoparticles synthesized by microwave heating. Gold nanoparticle preparation conditions: 10mL of aqueous solution containing
3.5mM citrate and 1mM HAuCl4. Microwave irradiation operation conditions:
600W of applied energy, reaction temperature controlled at 125_C with temperature increase speed at 80_C/min and holding time for 15 min.
Fig. 2.3 Absorption spectra of colloids obtained at various citrate concentrations.
The vertical arrow indicates the order intensity at 520nm with different citrate concentrations.
2 . 2 Water-based gold nanoparticle synthesis
2 . 2 .1 Advantages:
– Water is a good solvent for a number of metal ions as well as a variety of capping molecules. The synthesis involves preparation of an aqueous gold salt solution followed by reduction of the metal ions in a single step21. It is therefore considerably simpler than the multi-step Brust protocol.
– No additional stabilization against aggregation of the gold nanoparticles is required – surface bound ions citrate ions, chloroaurate ions, etc.) stabilize the nanoparticles electrostatically in solution.
– Electrostatic layer-by-layer assembly involving, for example, oppositely charged polyelectrolytes/surfactants nd nanoparticles may be readily ccomplished on suitably functionalized surfaces.
– Nanoparticle shape control can be easily effected by using suitable micelles (arising due to spontaneous assembly of suitable surfactants in water) as templates.
– Perhaps the biggest advantage of a water-based synthesis procedure is that bioconjugation of the gold nanoparticles with DNA19, enzymes46, etc. may be easily accomplished. ［15-21］
2 . 2 . 2 Disadvantages:
– Ionic interactions limit the concentration of gold nanoparticles in the aqueous phase to very dilute levels, a ig drawback in biological labeling of the nanoparticles.
– Control over the particle size and monodispersity in a particular reduction protocol is not very good.
– The gold nanoparticles do not spontaneously assemble into a close-packed hexagonal arrangement on solvent evaporation.
– The gold nanoparticles are not easily separated from solution in the form of a powder that would be readily re-dispersible in water after storage.
2 . 3 Solution-based synthesis of gold nanoparticles
2 .3 .1 Advantages:
– High degree of control may be exercised over the gold nanoparticle size, monodispersity29 and chemical nature of the nanoparticle surface (via capping with terminally functionalized thiols)
– High concentrations of the gold nanoparticles in solution may be easily prepared.
– Functionalized gold nanoparticles may be stored as a powder without sintering of the particles.
– The nanoparticles spontaneously assemble into closepacked, hexagonal monolayers upon solvent evaporation. The collective properties of the nanoparticle assembly may be controlled by varying the interparticle separation via capping with different chain length alkanethiols.
2. 3. 2 Disadvantages:
– The procedure is a multi-step one involving independent phase transfer of the gold ions followed by their reduction and capping.
– While close-packed monolayers of the gold nanoparticles may be deposited by solvent evaporation, there is little control over the process of assembly.
Furthermore, superlattices of the gold nanoparticles cannot be readily deposited, in contrast with the layer-by-layer assembly that is possible for electrostatically stabilized gold nanoparticles in water.
– Formation of bioconjugates with gold nanoparticles is not possible in an organic environment. It is clear that both methods for the synthesis of gold nanoparticles have characteristic advantages. Depending on the particular application of the nanoparticles, the ideal condition would be to somehow marry the two methods and thus maximize their advantages. This may be conveniently done by effecting a phase transfer of gold nanoparticles synthesized in one medium (water/organic solvent) to the second medium (organic solvent/water). In addition to maximizing the benefits accruing from a combination of the two syntheses methods, the ability to move nanoparticles across liquid interfaces into environments of specific physicochemical properties to probe, for example, variation in the optical properties of the nanoparticle solution49 is an attractive feature of phase transfer protocols. In the remaining part of this article, I discuss some of the methods developed to carry out the phase transfer of gold nanoparticles in both directions.
Figure 2.4 Five sols of colloidal gold prepared in water and in mixtures of butyl acetate and CS2.
2 . 4 Immunoglobulin G
Two intact IgG molecules with genetic hinge deletions have been analyzed by X-ray diffraction. Both of these myeloma proteins, Dob and Mcg, were conformationally constrained as a consequence of missing hinge polypeptides.
The structure of another, Kol, was a normal immunoglobulin with an intact hinge, but the Fc was crystallographically disordered and could not be visualized . In all cases the antibodies exhibited global 2-fold symmetry for the visible components.
Recently, the structure of an intact IgG2a was determined which had no overall symmetry (Harris et al., 1992, 1997). That anti-canine lymphoma Mab contained two independent, local dyads, one relating Fab constant domains and the second relating heavy chains in the Fc. The two dyads were obliquely oriented and non-intersecting.
The intact murine IgG1K (Mab 61.1.3), described here, is speci®c for the anti-epileptic drug phenobarbital. The crystals were monoclinic space group P21 with one entire molecule as the asymmetric unit. No crystallographic obligation existed for the Mab to possess exact global symmetry, suggesting that the IgG1 might exhibit a considerable degree of segmental ¯exibility, as The residue numbering convention for the entire
paper is that of Kabat et al. (1991).
Atomic coordinates are deposited with the Brookhaven Protein Data Bank using this same numbering system. Abbreviations used: IgG, immunoglobulin G; Mab, monoclonal antibody; CDR, complementarity determining region; NCS,
non-crystallographic symmetry. J. Mol. Biol. (1998) Academic Press Limited for the IgG2a. Based on ¯uorescent depolarization studies, IgG1 is known, however, to be the most rigid of the four mouse subclasses ［20-28］
Figure 2.5 Structure of an intact IgG1K for phenobarbital. Light chain1 is in pink, heavy chain1 in magenta , light chain2 in yellow, and heavy chain2 in orange. In (a) the IgG1 is viewed perpendicular to the approximate dyad axis relating Fab segments. The 2-fold rotation is near exact, but Fabs are translated by 9 AÊ with respect to one
another along the rotation axis. In (b) the IgG1 is viewed perpendicular to the pseudo 2-fold axis relating heavy chains of the Fc segment. The angle between the Fab and Fc dyads is 107_, thus the long axis of the Fc is dislocated and roughly at a right angle to the plane of the Fabs. Hinge angle differences are evident here as well. The Fc segment of the IgG1 lies in the crystallographic xz plane, with its dyad approximately along the face diagonal.
2 . 5 Enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) has been shown to be a highly sensitive technique capable of detecting antibodies to a wide variety of antigens (4-6, 8-10, 13, 18). This technique has the following advantages: the enzyme conjugates and the substrate reaction products are stable for long
periods of time, results can be read visually or on a spectrophotometer, and ELISA lacks the potential radiation hazards ofradioimmunoassay. In this report an ELISA technique is used to measure relative amounts of isotypically specific antibodies against the lipopolysaccharide (LPS) extracted from a hybrid of Shigella flexneri and Escherichia coli (Shigella X16). The ELISA technique used in this study is a modification of our earlier procedure which did not directly relate the absorbance units from the substrate reaction to specific antibody against Shigella X16 LPS (X16 LPS) in gravimetric terms (13, 18).
Data from the present study (i) establish sensitivity and specificity of the technique, (ii) assess parameters affecting the ELISA system, and (iii) compare this ELISA to passive hemagglutination and quantitative precipitation by using immunoglobulin G (IgG) fractions from rabbit antisera to X16 LPS. ［29-34］