Hermanson G. Bioconjugate Techniques, Second Edition

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580 13. Silane Coupling Agents

Figure 13.10 The isocyanate-containing silane coupling agent can be used to couple hydroxyl-containing mol-

ecules to inorganic surfaces. The reactions should be done in dry organic solvent to prevent hydrolysis of the

reactive group.

Silva et al. (2005) used ICPTES to create novel chitosan-siloxane hybrid polymers by cou-

pling the isocyanate groups to the functional groups of the carbohydrate and forming a sil-

ica polymer using the triethoxysilane backbone. Boev et al. (2005) used this functional silane

coupling agent to prepare CdS ﬂ uorescent nanoparticles in a urea-silicate matrix. ICPTES and

APTS have been have been used in combination to create organically modiﬁ ed silica xerogels

through carboxylic acid solvolysis, which formed hybrid materials that have luminescent prop-

erties (Fu et al ., 2006).

The following protocol is based on the method of FitzGerald et al. (2005), who used the

technology to develop aluminum oxide-based biochips for high-throughput analysis.

Protocol

1. In a fume hood, dissolve ICPTES in anhydrous toluene at a concentration of 5 percent

(v/v) and also containing 1 percent DIPEA base.

2. Clean and prepare an inorganic substrate to remove unreactive metal salts and to create

⎯OH sites for coupling the silane compound. For glass, this can be done using acid or

base washes. Formation of ⎯OH groups on glass typically is done using piranha solu-

tion, which is 25 percent sulfuric acid and 15 percent hydrogen peroxide. For clean-

ing aluminum oxide sheets, use an alkaline detergent solution and apply sonication for

1 hour, then wash with water.

3. Treat the inorganic substrate with the ICPTES solution by immersion and react at 50°C

overnight with mixing.

4. Wash the derivatized substrate with toluene at least twice and then rinse a ﬁ nal time with

acetone.

5. Dry the modiﬁ ed substrate at 120°C for 30 minutes in an explosion-proof oven.

Many other functional silane coupling agents are available from commercial suppliers,

including hydroxyl, aldehyde, acrylate and methacrylate, and anhydride compounds. Substrate

modiﬁ cation procedures similar to those discussed above can be used with these reagents to

link a biomolecule to an inorganic surface or particle.

2. Functional Silane Compounds 581

582

The use of particles in biological assays and other applications dates back many decades

(Singer and Plotz, 1956). Assays and detection systems made by coupling afﬁ nity ligands to parti-

cles of nanometer or micrometer diameter have been used in diagnostic tests and numerous other

research procedures. Latex microspheres and gold nanoparticles perhaps were the earliest exam-

ples of solid phase spheres used for these purposes. With the recent explosion in nanotechnology,

the relevant particle size has shrunk by 2–3 orders of magnitude and the applications for particles

have dramatically expanded. Nanoparticles and microparticles are used in agglutination tests and

assays, particle capture ELISA methods, lateral ﬂ ow tests, solid phase assays, scintillation proxim-

ity assays, polymerase chain reaction (PCR) tests, superparamagnetic-based assays and magnetic

separation systems, biosensors, as enhancers of Raman spectral signals, in light scattering assays,

and as ﬂ uorescent labels or stains for detecting biological molecules.

1. Particle Types

The types of particles used in biological applications are extremely varied. Most often, they are

nonporous in nature and spherical in shape. However, the material science revolution in nanote-

chnology has provided particle types and compositions of almost limitless shape and size, includ-

ing spherical, amorphous, or aggregate particles, as well as elaborate geometric shapes like rods,

tubes, cubes, triangles, and cones. In addition, new symmetrical organic constructs have emerged

in the nanometer range that include fullerenes (e.g., buckyballs), carbon nanotubes, and den-

drimers, which are highly deﬁ ned synthetic structures used as bioconjugation scaffolds in various

applications (these specialized nanoparticles are discussed in Chapter 7; Chapter 9, Section 10;

and Chapter 15).

The chemical composition of particles can be just as varied as their shape. Commercial par-

ticles can consist of polymers or copolymers, inorganic constructs, metals and semiconductors,

superparamagnetic composites, biodegradable constructs, and synthetic dendrimers and dendrons.

Often, both the composition of a particle and its shape govern its suitability for a particular

purpose. For instance, composite particles containing superparamagnetic iron oxide typically are

used for small-scale afﬁ nity separations, especially for cell separations followed by ﬂ ow cytom-

etry analysis or ﬂ uorescence-activated cell sorting (FACS). Core–shell semiconductor particles, by

Microparticles and Nanoparticles

contrast, are the basis for quantum dot nanoparticle labels, which provide intensely ﬂ uorescent

detection reagents.

The original polymeric latex particles still are widely used for separation and detection.

Polymers provide a matrix that can be swollen for embedding other molecules in their core,

such as organic dyes or ﬂ uorescent molecules. Even nanoparticle quantum dots can be incorpo-

rated into larger latex particles to form highly ﬂ uorescent composite microparticles.

Such ﬂ uorescent or dyed latex particles are useful in multiplexed detection systems using

suspension arrays (e.g., Luminex technology; Armstrong et al., 2000). In this application, dyes

are incorporated within the beads having a range of different spectral properties to create a

series of different colored particles. In addition, changing the amount of dye molecules within

the beads or blending two or more dyes in a single particle population can form a gradient of

different color compositions. Particle subpopulations of a particular color or emission wave-

length and intensity then can be used to identify the type of target being measured in a suspen-

sion assay. Mixing together in a single solution such particle subpopulations having different

colors permits multiple targets to be assayed simultaneously, wherein each particle type is iden-

tiﬁ ed by its color and correlated to the analyte being targeted. Flow-cytometry-based instru-

ments then are used to detect and measure the particle color and assay result.

Dyed particles also are commonly used in diagnostic lateral ﬂ ow tests (like the common

home pregnancy test), as the colors can be seen with the eye without the need for special detec-

tors. In this type of assay, antibodies or antigens are coupled to the dyed particles and a sample

solution applied to the test strip carries them along within a membrane. The particles then are

captured at points in the membrane that represent either a control or a positive sample result.

Large numbers of color particles docking at these points within the membrane create the visual

lines associated with these disposable tests.

Polymeric particles can be constructed from a number of different monomers or copolymer

combinations. Some of the more common ones include polystyrene (traditional “ latex ” par-

ticles), poly(styrene/divinylbenzene) copolymers, poly(styrene/acrylate) copolymers, polymeth-

ylmethacrylate (PMMA), poly(hydroxyethyl methacrylate) (pHEMA), poly(vinyltoluene),

poly(styrene/butadiene) copolymers, and poly(styrene/vinyltoluene) copolymers. In addition, by

mixing into the polymerization reaction combinations of functional monomers, one can create

reactive or functional groups on the particle surface for subsequent coupling to afﬁ nity ligands.

One example of this is a poly(styrene/acrylate) copolymer particle, which creates carboxylate

groups within the polymer structure, the number of which is dependent on the ratio of mono-

mers used in the polymerization process.

Many of the common polymer particle types present a surface with hydrophobic charac-

ter, such as the polyaromatic styrene-containing ones. These in particular are better used with

modiﬁ cations on the particle surface to add charge or hydrophilicity, which masks the underly-

ing polymer core. Some constructs of this type provide a grafted hydrophilic surface to limit the

degree of nonspeciﬁ c binding and form a more biocompatible particle, which won ’t denature

or bind protein through unwanted hydrophobic interactions. Some polymer particles are made

from hydrophilic monomers and display better biocompatibility. HEMA is very hydrophilic

due to an abundance of hydroxyl groups, and its properties are extremely biocompatible with-

out further surface derivatization.

Inorganic particles are used extensively in various bioapplications, too. Gold nanoparticles

long have been used as detection labels for immunohistochemical (IHC) staining and lateral

ﬂ ow diagnostic testing. These dark, dense particles provide single particle detection capability

1. Particle Types 583

584 14. Microparticles and Nanoparticles

for sensitive staining techniques, especially in microscopy applications. In addition, the ease at

which clumps of gold particles can be seen visually makes their use in diagnostic strip tests a

very popular alternative to dyed latex particles (see Chapter 24 for a thorough review of gold

conjugation methods).

Invariably, the use of particles in bioapplications involves the attachment of afﬁ nity capture

ligands to their surface, either by passive adsorption or covalent coupling. The coupling of an

afﬁ nity ligand to such particles creates the ability to bind selectively biological targets in com-

plex sample mixtures. The afﬁ nity particle complexes thus can be used to separate and isolate

proteins or other biomolecules or to detect speciﬁ cally the presence of these targets in cells, tis-

sue sections, lysates, or other complex biological samples.

The reactions used for coupling afﬁ nity ligands to nanoparticles or microparticles basically

are the same as those used for bioconjugation of molecules or for immobilization of ligands onto

surfaces or chromatography supports. However, with particles, size can be a major factor in how

a reaction is performed and in its resultant reaction kinetics. Since particle types can vary from the

low nanometer diameter to the micron size, there are dramatic differences in how such particles

behave in solution and how the density of reactive groups or functional groups affects reactions.

2. Particle Characteristics and Stability

Particle size, surface composition, and density directly affect how a particle behaves in suspen-

sion. This in turn affects coupling protocols, especially in the handling and washing techniques

used for particles during the conjugation process. Larger particles of micron size generally will

settle over time just in normal gravity. As particle size decreases, however, a point is reached

that a true colloidal suspension may occur, wherein the particles won ’t separate no matter how

long they sit in suspension. This typically happens when particle size gets to about 100 nm,

and Brownian motion causes water molecules to collide with particles with high enough force-

to-mass ratios to prevent them from settling under gravity. Many dense particles of less than

100 nm, such as silica, still can be separated from solution using a bench-top centrifuge, except

as particles approach the size of biological macromolecules, or around 10 nm, at which point

an ultracentrifuge would be required for separation. For a comparison of particle sizes and

how they contrast to biomolecules (see Figures 14.1 and 14.2 ).

The forces acting on particles in suspension mainly can be explained by the Derjaguin,

Landau, Verwey, and Overbeek (DLVO) theory, which describes the attractive van der Waals

forces and repulsive electrostatic forces affecting their stability (Derjaguin and Landau, 1941;

Verwey and Overbeek, 1948). A corollary to this theory indicates that for hydrophobic sur-

faces, adding carboxylates can result in a stabilization of particles in solution and a preven-

tion of aggregation through charge repulsion. Of course, this is true provided the pH of the

solution is maintained above about pH 5, so that the carboxylates are not protonated, and

their negative charge character is maintained. Often, without the presence of surface charge

to create signiﬁ cant repulsive effects, many particles of commercial interest would aggregate

and fall out of suspension rather quickly due to hydrophobic surface interactions and van der

Waals forces (electrostatic attractions). The smaller the particle, the more signiﬁ cant the van

der Waals attractive forces may become.

By contrast, relatively hydrophilic particles like those made of pHEMA may maintain col-

loidal stability even at small size due to the “repulsive” effects of a water of hydration layer,

which forms around each particle in aqueous solution and is energetically difﬁ cult to remove

or penetrate ( Figure 14.3 ). A closely bound water layer apparently is the result of hydrogen

bonding and dipole interactions with polar surface groups, such as the hydroxyls on pHEMA

particles. Many such hydrophilic particles can display stability at high salt concentrations, and

even if DLVO theory may predict aggregation (Molina-Bolívar et al. , 1998).

The degree and type of surface charge can dramatically affect particle behavior in suspension.

Latex particles that have a very low density of charged groups, for instance, may still present

hydrophobic polymer surface areas large enough to cause instability. The type of charged groups

on the particle surface directly can affect the magnitude of charge repulsion. In particular, the acti-

vation of carboxylate particles using an EDC/sulfo-NHS two-step reaction (Chapter 3, Section 1.2)

will temporarily replace the negatively charged carboxylates with negatively charged sulfonates.

The sulfonate groups on the sulfo-NHS ester intermediates create a stronger negative charge on

the particle surface than the original carboxylates. In some cases, the increase in negative charge

Figure 14.1 Particles commonly used in biological applications can range in size over three orders of magni-

tude, from as small as macromolecules ( 10 nm) to approximately the diameter of cells (10 m). The diameter

of a particle population dramatically can affect its behavior in solution.

2. Particle Characteristics and Stability 585

586 14. Microparticles and Nanoparticles

Figure 14.2

Comparison of the relative size of a 10 nm particle and the diameter of a typical protein, human

serum albumin, at about 7 nm.

repulsion can result in an inability to pellet the particles by centrifugation after the activation

step, even if the particles could be separated by centrifugation before activation. This was true for

40 nm carboxylated silica nanoparticles in our hands (unpublished observations).

The charge repulsion effects between particles can be severely affected by the buffer and salt

composition of the solution they are suspended in. Charges can be eliminated or neutralized by

ionizable groups being protonated or unprotonated or by the concentration of ions in solution.

For instance, lowering the pH of an aqueous solution below the p K

of the surface carboxy-

lates will result in them being protonated. With most particles, especially hydrophobic ones, this

will cause particle aggregation due to loss of surface negative charge. Similarly, a high salt con-

centration can effectively mask the charge character of a carboxylated particle by having too

many positively charged ions associated with the surface charges. Most particle types that are

stable in suspension due to like charge repulsion can be made to aggregate if the pH is changed

or the buffer or salt concentration is too high. Part of the challenge of successfully working with

small particles is to maintain optimal solution characteristics to keep the particles dispersed

throughout the conjugation process. This includes all activation, coupling, and washing steps

that are used to conjugate an afﬁ nity ligand and subsequently use it in its intended applica-

tion. Therefore, for each particle type being used to conjugate a ligand, one should ﬁ rst become

knowledgeable of its physical and solution characteristics before doing any coupling reactions.

Another aspect of particle handling that should be worked out before actually coupling

an afﬁ nity ligand is the best procedure to wash the particles and remove unreacted ligands or

by-products of a coupling reaction. With small quantities of particles, most often, this can be

done by centrifugation or if working with very small nanoparticles, by gel ﬁ ltration or size

exclusion chromatography. Membrane ﬁ ltration also is another option, especially for larger

micron-sized particles. If the solution volume and quantity of particles is large enough, then

tangential ﬂ ow ﬁ ltration is a viable alternative, too.

Although many particle types in theory can be separated from solution by centrifugation,

some particles may become irreversibly aggregated upon pelletting and should not be centri-

fuged under any circumstances. The relative hydrophobicity and degree of charge on a par-

ticle’s surface directly affects their tendency to aggregate, and this property to a large extent

governs whether the particle population can be resuspended successfully after centrifugation.

In particular, particles that contain higher densities of like surface charge, which functions to

repulse and keep individual particles away from one another, typically can be resuspended suc-

cessfully after centrifugation. To aid in re-suspension, centrifuged particles should be subjected

to sonication, either by using a bath sonicator or a sonic probe to disrupt the pellet. Vortex

mixing usually should be avoided, as it is not very effective at re-dispersing small particles pel-

letted by centrifugation. Before using centrifugation or any other separation technique, it is

best to contact the manufacturer to see if they offer any guidelines for handling the particles.

Figure 14.3 Small particles often are stabilized in aqueous solution by like charge repulsion, which prevents

particle aggregation and precipitation.

2. Particle Characteristics and Stability 587

588 14. Microparticles and Nanoparticles

3. Particle Concentration

If individual particles in suspension are considered the equivalent of discrete molecules, then

the molar concentration of a given particle suspension can be calculated based on the known

particle diameter, density, and the mass of particles present. This allows particles to be treated

similarly to other biomolecules with respect to determining concentration for conjugation pur-

poses. However, there are important differences that should be realized when working with

particles as opposed to working with soluble macromolecules, like proteins. Since common

commercial particles can vary in size from the molecular range (approximating the size of an

antibody or 10 nm diameter) to a scale 1,000 times larger (or approaching the size of a cell

at 10 m), a change in diameter affects the concentration of particles as well as the effective

concentration of surface functional groups present in suspension.

In general, as particle size decreases, the molar concentration of particles in a constant vol-

ume of solution increases (for a given mass of particles). For instance, a 1 mg quantity of 1  m

latex microspheres represents far fewer particles than a 1 mg amount of 50 nm nanoparticles.

Thus, the effective molar concentration of nanoparticles in solution will be much greater than

the concentration of the same mass of microparticles (if both are suspended in the same vol-

ume of solution). In addition, as particle size decreases, the ratio of a particle ’s surface area to

mass increases. This means that the total surface area available for conjugation on the nano-

particles is much greater than the total surface area present on the microparticles. If both par-

ticles contain the same functional groups on their surfaces for coupling afﬁ nity ligands (i.e.,

carboxylates), then the effective concentration of these groups in solution for the nanoparticles

is much greater than the concentration of the same groups in a given solution for the micropar-

ticles (assuming both have about the same surface density or “parking area ” of the carboxylate

functional groups).

Thus, most conjugation reactions done with nanoparticles should take into account a poten-

tially greater reactivity than the same reactions done using microparticles, due to the higher

effective concentration of functional groups present in solution for the nanoparticles. As parti-

cle size decreases and particle concentrations increase, the available surface area increases, and

the effective concentration of reactive groups increases along with it. All of these factors must

be considered when optimizing conjugation reactions to particles. This means that an optimal

protocol for coupling proteins to 1 m carboxylated microspheres most likely will have to be

re-optimized for use with carboxylated nanoparticles.

The following sections discuss many of the major particle types and provide bioconjugation

options for the coupling of ligands to the surface of functionalized particles. Some additional

nanoparticle constructs, including gold particles, dendrimers, carbon nanotubes, Buckyballs

and fullerenes, and quantum dots are discussed more fully elsewhere (see Chapter 7; Chapter 9,

Section 10; Chapter 15; and Chapter 24).

4. Polymeric Microspheres and Nanospheres

Perhaps the most common particle type used for bioapplications is the polymeric microsphere

or nanosphere, which consists basically of a spherical, nonporous, “hard” particle made up

of long, entwined linear or crosslinked polymers. Creation of these particles typically involves

an emulsion polymerization process that uses vinyl monomers, sometimes in the presence of

divinyl crosslinking monomers. Larger microparticles usually are built from successive polym-

erization steps through growth of much smaller nanoparticle seeds. The investigation of parti-

cle surfaces by high-resolution electron microscopy often can reveal the presence of numerous

entangled, amorphous nanoparticles making up the morphology of much larger nanospheres

or microspheres. Instead of being smooth spheres, the surface of most polymeric particles actu-

ally is torturous and made up of many craters, pits, and crevasses, which may appear feature-

less under lower resolution imaging.

Some of the most common forms of polymeric particles consist of polystyrene or copolymers

of styrene, like styrene/divinylbenzene, styrene/butadiene, styrene/acrylate, or styrene/vinyltolu-

ene. Other common polymer supports include PMMA, polyvinyltoluene, and pHEMA and the

copolymer, poly(ethylene glycol dimethacrylate/2-hydroxyethylmetacrylate) [poly(EGDMA/

HEMA)] (Ayhan et al., 2002) ( Figure 14.4 ). The types of monomers used to form the polymers

ultimately govern the ﬁ nal particle characteristics. If the monomers are hydrophobic, such as

those that form most of the styrene-based particles, then the resultant particle surfaces will

be hydrophobic, as well. The inclusion of hydrophilic monomers, including charged groups

and polar constituents, will create more hydrophilic character on the surface. In addition, since

many particles are polymerized in the presence of detergents, particles typically have detergent

molecules non-covalently adsorbed on their surfaces, unless they have been purposely removed

by extensive cleaning after polymerization.

Polymeric particles traditionally have been called “ latex ” beads or spheres, probably from

the classic deﬁ nition of an “emulsion of rubber or plastic globules in water ”. However, due to

Figure 14.4 Some of the most common particles consist of these polymers.

4. Polymeric Microspheres and Nanospheres 589