Hermanson G. Bioconjugate Techniques, Second Edition

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620 14. Microparticles and Nanoparticles

alkaline conditions above pH 8.0. Surface treatment with organosilane derivatives can stabilize

particles to hydrolysis, but long-term exposure to highly alkaline environments still should be

avoided. It is also recommended that at least 0.05 M NaCl be maintained to increase stability

of the particles in aqueous environments. The best stability for silica is obtained at neutral or

acidic pH conditions containing a low concentration of salt.

5.1. Fluorescent Silica Particles

Silica particles have been exploited in virtually every assay or detection strategy that polymer

particles have been used in for bioapplication purposes. Recently, ﬂ uorescent dye-doped sil-

ica nanoparticles have been developed by a number of groups that have similar ﬂ uorescence

characteristics to quantum dot nanocrystals (Chapter 9, Section 10). Fluorescent silica nano-

particles can be synthesized less expensively than quantum dots due to the fact that the silica

particles incorporate standard organic dyes (Ow et al., 2005; Wang et al., 2006) and are not

dependent on making reproducible populations of semiconductor particles with precise diam-

eters to tune emission wavelengths.

The preparation of ﬂ uorescent silica particles can be done using a number of strate-

gies. Santra et al. (2001) describe a water-in-oil emulsion using detergent-mediated reverse

micelle formation and controlled hydrolysis of TEOS to create mono-disperse silica nanopar-

ticles (see also Arriagada and Osseo-Asare, 1995). Adding the water-soluble ﬂ uorescent dye,

tris(2,2-bipyridyl) dichlororuthenium (II) hexahydrate (Ru(II)bpy

2

) to this emulsion

resulted in dye molecules being entrapped within the silica particle structure as it formed

(Figure 14.23 ) (also see Chapter 28, Section 4.2 for additional properties of Ru(II)bpy

2

Subsequent functionalization of the surface with silane derivatives can be done to facilitate lig-

and immobilization.

These Ru(II)bpy

2

ﬂ uorescent silica nanoparticles were used to detect single bacterial

cells using antibodies conjugated to the surface after functionalization with trimethoxysilyl-

propyldiethylenetriamine followed by succinylation to create carboxylates. Speciﬁ c antibody

molecules against E. coli O157 then were coupled to this modiﬁ ed ﬂ uorescent particle using

the carbodiimide method with EDC and NHS (Zhao et al. , 2004).

Another type of ﬂ uorescent silica particle was formed from silica bubbles created on the

surface of gold nanoparticles (Liz-Marzan et al., 1996; Makarova et al., 1999). Fluorescein

isothiocyanate (FITC) was adsorbed onto the gold nanoparticle surface and then reacted with

3-aminopropyltrimethoxy silane. The isothiocyanate groups on the adsorbed dye molecules

coupled to the amine groups on the silane as it polymerized on the gold surface, effectively

forming a silica shell around the gold particle. The gold core then was dissolved by reaction

with cyanide ions to leave behind the silica nanobubbles ﬁ lled with water, which also left the

ﬂ uorescent molecules attached on the inner surface ( Figure 14.24 ).

Fluorescent silica nanoparticles, called FloDots, were created by Yao et al. (2006) by two

synthetic routes. Hydrophilic particles were produced using a reverse micro-emulsion process,

wherein detergent micelles formed in a water-in-oil system form discrete nanodroplets in which

the silica particles are formed. The addition of water-soluble ﬂ uorescent dyes resulted in the

entrapment of dye molecules in the silica nanoparticle. In an alternative method, dye molecules

were entrapped in silica using the Stöber process, which typically results in hydrophobic par-

ticles. Either process resulted in luminescent particles that then can be surface modiﬁ ed with

Figure 14.23 Silica nanoparticles containing ﬂ uorescent dye molecules can be prepared using a reverse micelle

suspension process: (a) The water-in-oil emulsion is formed with the aqueous phase droplets containing TEOS

and dye molecules in detergent. (b) The ﬁ nal particles contain entrapped dye within the silica particle matrix,

creating highly ﬂ uorescent particles.

5. Silica Particles 621

622 14. Microparticles and Nanoparticles

Figure 14.24 Fluorescent silica nanobubbles have been created using gold nanoparticle seeds that initially are

coated by adsorption with a ﬂ uorescent dye. The particles then are capped by a layer of silica by polymerizing

TEOS and entrapping the dye molecules within it. Finally, the gold core is dissolved by reaction with cyanide,

leaving behind hollow ﬂ uorescent silica nanobubbles.

functional silanes to contain appropriate functionalities for coupling to afﬁ nity ligands or bio-

molecules. Ru(II)bpy

2

or standard organic ﬂ uorescent dyes can be incorporated into such sil-

ica particles with high efﬁ ciency. In one example, a 70 nm particle containing Ru(II)bpy

2

was

found to be equal in ﬂ uorescence intensity to 39 quantum dots having an emission at 605 nm

(Yao et al., 2006). Another silica nanoparticle prepared using a rhodamine dye contained about

1,290 molecules of the dye entrapped within each particle, which produced intensely ﬂ uores-

cent labels.

In a different method of producing dye-doped silica particles, van Blaaderen and Vrij (1992)

and Verhaegh and van Blaaderen (1994) developed a method to covalently link amine-reactive

dyes to APTS and then polymerize the resultant conjugate with TEOS in mixtures of ammonia,

water, and ethanol to form ﬂ uorescent “ organosilica ” spheres. The dye molecules could be dis-

persed throughout the entire particle, contained in the core, or within discrete spherical regions

within the particles. Either hydrophilic or hydrophobic particles could be created, depending

on the surface treatment used subsequent to particle formation. Fluorescent particles consisting

of either ﬂ uorescein or rhodamine dyes made by this method were shown to be susceptible to

photobleaching similar to the organic dyes in solution.

The following protocol is based on the creation of ﬂ uorescent silica core/shell particles using

the method of van Blaaderen and Vrij (1992).

Protocol

Formation of dye-silica core particles:

1. React APTS (11.5 mg) with FITC (10.6 mg) in anhydrous ethanol (1 ml) with mixing for

12 hours (protect from light). The use of anhydrous conditions will prevent the hydroly-

sis of the APTS alkoxy groups, which would cause premature condensation.

2. Prepare a solution consisting of 75 ml of ethanol containing 8.5 ml of ammonia.

3. To the stirring ethanol/ammonia solution, add 3.3 ml of TEOS along with the completed

reaction solution from step 1, which is now the conjugate of ﬂ uorescein and APTS linked

through the amino group on the alkyl silane.

4. The reaction is allowed to continue for 24 hours with slow stirring.

5. Wash the resultant core particles twice with the ethanol/ammonia solution using

centrifugation.

Formation of the silica shell:

1. Dilute the particles to 1.2 l using a 10:1 mixture of ethanol:ammonia. Add 28 ml of

TEOS to the particle suspension and mix to dissolve.

2. React for 24 hours with slow mixing.

3. Wash the particles with the ethanol/ammonia solution several times using centrifuga-

tion. Finally, wash the particles into the desired solution (ethanolic or aqueous) without

ammonia present to prevent silica hydrolysis upon storage.

Ow et al. (2005) developed an improved method of incorporating ﬂ uorescent molecules into

silica particles using a modiﬁ ed Stöber synthesis, which resulted in both enhanced ﬂ uorescence

and photostability of the encapsulated dyes. In this two-stage procedure, reactive organic dyes

5. Silica Particles 623

624 14. Microparticles and Nanoparticles

ﬁ rst are conjugated to a silane derivative and condensed to form a polysiloxane-dye-rich nano-

particle core structure. No TEOS is added at this point. These dyed core nanoparticles then are

used as seed for the addition of silica sol–gel monomers to condense around the core and cre-

ate a silica network (shell) around the dye-rich core ( Figure 14.25 ). Nyffenegger et al. (1993)

used a similar process to form ﬂ uorescein particles by ﬁ rst coupling FITC to 3-aminopropylt-

rimethoxy silane to form a thiourea derivative, and then condensing the dye silane derivative

with tetramethoxy silane to form the dyed silica particles.

Figure 14.25 The preparation of highly controlled ﬂ uorescent silica nanoparticles can be done by ﬁ rst polymer-

izing APTS that has been covalently modiﬁ ed with an amine-reactive dye to form ﬂ uorescent core particles. The

core then is capped by a shell of silica by polymerization of TEOS. The shell layer can be further derivatized

with silane coupling agents to provide functional groups for conjugation.

The method developed by Ow et al. permits control over the size of the particles and allows

the incorporation of virtually any organic dye into silica, provided it can be ﬁ rst conjugated

to a silane derivative to form the core. Fluorescent particles made by this procedure may be

made as monodisperse populations having diameters from less than 10 nm to over 1 m sized

spheres, with a high degree of precision. The core size also can be varied by changing the con-

centration of the dye-silane derivative during the condensation process. The procedure for

making these particles is similar to that described above using the method of van Blaaderen

and Vrij (1992), but without the addition of TEOS for creation of the dye-silane core. Exact

procedures are given in Wiesner et al. (2006).

In the preparation of 15 nm core–shell ﬂ uorescent silica particles, Ow et al. (2004) reported

that the naked core (2.2 nm) alone produced a ﬂ uorescence intensity of less than the free dye

in solution, presumably due to dye quenching. However, upon addition of the outer silica shell

around the core, the brightness of the particles increased to 30 times that of the free dye (using

tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)). They speculate that shell may pro-

tect the core from solvent effects, as evidenced by a lack of spectral shift upon changing the

solvent in which the particles are suspended.

The enhanced photophysical properties of these ﬂ uorescent core–shell silica nanoparticles

make them potentially as useful as semiconductor quantum dots for bioconjugation purposes.

In this case, standard, commercially available, organic dyes can be incorporated into the sil-

ica nanoparticles to provide a range of emission properties as diverse as those available using

the dyes alone. In addition, once encapsulated in the particles, the dyes display much better

photostability and increased ﬂ uorescence (brightness) compared to the free dyes in solution.

A major advantage of silica-based particles is that they are known to have greater biocom-

patibility than quantum dots in that they are nontoxic, hydrophilic, and can be conjugated

to proteins and other targeting molecules with relative ease. The ability to add any desired

ﬂ uorescence characteristic to such particles simply by choosing the appropriate organic dye or

metal chelate luminescent molecule makes dye-doped silica nanoparticles especially useful for

bioapplications.

5.2. Silane Functionalization of Silica Particles

Surface functionalization of silica particles or ﬂ uorescent silica particles typically is done using

functional alkyl silanes. The process may be used to add a reactive group to the surface of the

particles for spontaneous coupling to biomolecules or it may be used to add the appropriate

nucleophilic group to the surface, such as an amine or a carboxylate. Silane modiﬁ cation chem-

istry is discussed in more detail in Chapter 13.

The following protocol for modiﬁ cation of silica nanoparticles is based on the method of

Zhao et al. (2004), which describes the addition of amine functionalities using trimethoxysilyl-

propyldiethylenetriamine. Other functional silane modiﬁ cations may be done similarly.

Protocol

1. Add 32 mg of silica nanoparticles (ﬂ uorescent or plain) to 20 ml of 1 mM acetic acid con-

taining 1 percent trimethoxysilyl-propyldiethylenetriamine with stirring. Other concen-

trations of silane derivatives used for particle modiﬁ cation typically range from 1 to 5

5. Silica Particles 625

626 14. Microparticles and Nanoparticles

percent (w/w). Optimization of this concentration may have to be done for a particular

silica particle size and type.

2. React with mixing for 30 minutes at room temperature.

3. Wash the amine-derivatized particles at least 3 times with water using centrifugation to

remove excess reactants.

4. Store the particles in a suitable buffer at neutral or slightly acidic pH containing a preservative.

The amine-modiﬁ ed silica particles may be used to couple with carboxylate-containing

ligands using a carbodiimide reaction. Similar coupling protocols may be used as that

previously described for amine-containing polymer particles (Section 4, this chapter). The

amine-particles also may be further derivatized by reaction with succinic anhydride to

create carboxylated particles for coupling to proteins or other amine-containing ligands.

5. To prepare the succinylated carboxylate derivative of the amine particles, wash the parti-

cles with water and then into DMF.

6. Suspend the amine-particles in DMF containing 10 percent succinic anhydride.

7. React for 6 hours under nitrogen gas with mixing.

8. Wash the carboxylated particles at least 3 times with DMF by centrifugation. Resuspend

in water and wash 3 times with water to remove DMF. Store the particles in water or a

suitable buffer at neutral or slightly acidic pH.

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins,

using a carbodiimide reaction with EDC. A similar protocol to that previously described for cou-

pling to carboxylate polymer particles may be used. The following protocol is based on the method

of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli O157.

Protocol

1. Suspend the carboxylated silica particles from step 8, above, in 10 ml of 0.1 M MES, pH 6.8.

2. With mixing, add 500 mg of EDC and 500 mg of NHS (or sulfo-NHS).

3. React for 25 minutes at room temperature with stirring.

4. Quickly wash the activated particles with water using centrifugation to remove excess

reactants. Resuspend the washed particles in 10 ml of 0.1 M sodium phosphate, pH 7.3.

5. Add a quantity of protein or antibody to the activated particles representing a 1–10  excess

of protein over the calculated monolayer for the type of particles used. For coupling a limit-

ing amount of antibody to the activated ﬂ uorescent particles, something that may be desir-

able when using expensive monoclonals, the reaction should be carried out in very dilute

particle suspension (i.e., 0.1 mg/ml) to prevent aggregation of particles due to the possibility

of one antibody reacting with more than one particle. Zhao et al. (2004) used an antibody

concentration of just 5 g/ml to create successfully anti-bacterial ﬂ uorescent particles.

6. React for 2–4 hours at room temperature.

Add a blocking agent, such as a non-relevant protein (e.g., BSA) to a ﬁ nal concentration of

1 percent to mask any nonspeciﬁ c binding sites and to couple with any remaining reactive

groups on the silica particle surface. This is important especially if a limiting amount of

antibody was initially reacted with the particles in step 5. React for 30 minutes to 1 hour

at room temperature.

8. Wash the particles several times with PBS, pH 7.2, to remove excess protein and reaction

by-products. Store products in neutral or slightly acidic buffer containing a preservative.

627

1. Buckyballs and Fullerenes

1.1. Properties of Fullerenes

Carbon is an incredible element that is able to form structures having highly diverse properties

depending on its bonding patterns and three-dimensional organization. Natural allotropes of

carbon include diamond, graphite, amorphous carbon, and several other known forms ( Figure

15.1 ). Depending on the bond structure and atomic orientation that carbon takes on within the

structure of an allotrope, the resultant characteristics can range from the hardest known abra-

sive mineral, diamond, to the extremely soft, graphite, which is used as a lubricant.

In 1985, the story of carbon allotropes took a dramatic turn with the discovery of C

which resulted in a new type of carbon structure, called the fullerenes (Kroto et al., 1985).

This discovery earned the 1996 Nobel Prize in chemistry for Harold Kroto, Robert Curl, and

Buckyballs, Fullerenes, and Carbon Nanotubes

Figure 15.1 Three major allotropes of carbon (l to r): diamond, lonsdaleite, and graphite.

628 15. Buckyballs, Fullerenes, and Carbon Nanotubes

Richard Smalley. Buckminsterfullerene (named after Buckminster Fuller for his geodesic dome

architectural design) is a spherical cage of carbon having 60 atoms forming a truncated icosa-

hedron of average diameter 0.72 nm, which contains 12 pentagons and 20 hexagons of bonded

carbon ( Figure 15.2 ). The shape is exactly the same as a modern soccer ball, with the pentagon

and hexagon conﬁ gurations clearly outlined on its surface.

There now are known to be a whole family of caged carbon structures having various num-

bers of carbon atoms, including C

, and the huge C

540

. The name

“ fullerene” has replaced the unwieldy, “Buckminsterfullerene” used to describe this general

spheroid structure of carbon, although they still are referred to as “Buckyballs”.

Of all the fullerene forms, the nearly spherical properties of C

have attracted the greatest

attention, especially in the ﬁ eld of bioconjugation. In addition to its physical properties, C

fullerenes have unique photo-optical and electro-chemical properties, which make them useful

as carriers for biomedical research applications. For instance, upon exposure to light C

will

generate singlet oxygen, which can be used in vivo to cleave biological molecules, particularly

DNA and RNA. Studies indicate that irradiation of C

in solution can be used to destroy virus

contamination (Kasermann and Kempf, 1997). Solutions of Buckminsterfullerene are a deep

purple color, whereas other sizes of fullerenes display a variety of other colors.

Figure 15.2 The structure of a C

fullerene, also called a Buckyball.

Fullerene C

also functions efﬁ ciently as an antioxidant, actually being better than other

lipid-soluble antioxidants at scavenging reactive oxygen species (ROS) (Wang et al., 1999).

Water-soluble derivatives of C

, such as a poly-hydroxyl form, are able to function in the same

respect in aqueous environments.

Pure fullerenes are insoluble in aqueous environments and only sparingly soluble in many

organic solvents. The greatest solubility is found in 1,2,4-trichlorobenzene (20 mg/ml), carbon

disulﬁ de (12 mg/ml), toluene (3.2 mg/ml), and benzene (1.8 mg/ml) (Wikipedia.org). Solubility

calculations have been performed on C

in 75 different organic solvents (Sivaraman et al., 2001).

1.2. Modiﬁ cation of Fullerenes

Many chemical derivatization methods have been developed to afford fullerene solubility in

particular environments and to provide functional handles for bioconjugation (Bosi et al .,

2003). The combination of adding polar groups and reactive functionalities to fullerenes, such

as  COOH,  NH

, and  OH groups, provides water solubility and bioconjugation targets.

Examples of these modiﬁ cations include the method of Brettreich and Hirsch (1998) to add

multiple carboxylates in a dendritic fashion and Wang et al. (1999) who added multiple pairs

of carboxylates to the surface carbons. In addition, Cusan et al. (2002) developed a C

–PEG

dendrimer-based diamine derivative using a substituted fulleropyrrolidine modiﬁ cation linked

to the surface. Polymer carriers also have been used to provide water solubility and sites of

attachment. Cyclodextrins have been found to be excellent carriers of C

by holding the fuller-

ene within its hydrophobic core (Andersson et al., 1992; Braun, 1997; Samal and Geckeler,

2000; Filippone et al ., 2002).

In many methods for derivatization of C

, the initial modiﬁ cation is based on the reac-

tion at a 6,6 ring junction on the fullerene with an azomethine ylide to form the 1,3-dipolar

cycloaddition product, a fulleropyrrolidine (Prato et al., 1996). The reaction is done overnight

with heating to reﬂ ux in organic solvent. Typical reactants that combine with C

in this reac-

tion include an N-glycine derivative (with a constituent off the -amino group) and an alde-

hyde derivative, which gives the fulleropyrrolidine compound according to Figure 15.3 . By

judicious choice of the right starting materials, the process provides a range of derivative pos-

sibilities to employ fullerenes in various bioconjugate applications. If the reactants are added

in large excess over the concentration of the fullerene, then up to 9 such pyrrolidine groups

can be introduced per C

molecule. The number of modiﬁ cations actually ends up being a bell

shaped curve from 5 to 9 pyrrolidine derivatives, with a peak at 7 modiﬁ cations (Prato and

Maggini, 1998). By controlling the length of the reaction, a mono-substituted product can be

obtained in 40–50 percent yields.

In addition, the use of appropriate hydrophilic constituents on the aldehyde or glycine reac-

tants can result in excellent water solubility of the C

derivative. Two such modiﬁ cation arms

can be added simultaneously to the pyrrolidine ring, thus providing a functional group for fur-

ther conjugation and a hydrophilic arm for increased water solubility. PEG derivatives have

been formed in this manner, which create highly soluble fullerene derivatives.

The following procedure adapted from Prato et al. (1996) is an example of how glycine and

formaldehyde derivatives may be used to create fullerene modiﬁ cations for subsequent biocon-

jugation purposes.

1. Buckyballs and Fullerenes 629