Hermanson G. Bioconjugate Techniques, Second Edition

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490 9. Fluorescent Probes

peptide has been used as a fusion tag (Goldman et al., 2002b) as well as a penta-histidine pep-

tide tag (Medintz et al., 2003). Combinations of positively charged fusion proteins and avidin

also have been used to control the resultant biotin binding density on a DHLA-QD surface for

use in live cell imaging (Jaiswal et al. , 2004).

One major advantage of DHLA modiﬁ cation is that the diameter of the QD remains as

small as possible, while still creating a water-soluble particle. QDs of 10 nm diameter have

been created using this process and were successfully used to image intracellular proteins

(Jaiswal et al. , 2003).

Another type of simple surface modiﬁ cation involves the noncovalent coating of the QDs

with detergents or lipids. The hydrophobic tails of these molecules bind to the QD particle,

while the hydrophilic portions interact with the aqueous phase and render the dots dispers-

ible. Still other surface modiﬁ cation schemes use polymeric coatings containing multiple bind-

ing points to the QD, thus eliminating the possibility for leaching. Coatings containing PEG

spacers also can be used to create a highly hydrophilic layer on top of the semiconductor sur-

face. All of these modiﬁ cation strategies provide QDs that are water-soluble (or dispersible and

stable in suspension) and that contain functional groups for covalent attachment of proteins or

other afﬁ nity molecules.

Masking the surface of semiconductor QD particles also can be done by adding another

inorganic layer to the outer shell alloy structure. This layer can take the form of a silica coating

formed by the reaction of a silane derivative with the shell (Darbandi and Nann, 2005). For

instance, a pure silica surface can be created by controlled polymerization of the raw nanoc-

rystals with tetraethyl orthosilicate (TEOS), which forms a siliceous sphere with silanol groups

on the outer surface. Another organosilane derivative that is appropriate for use with metal-

lic particles is mercaptopropyl-tris-hydroxy-silane. The thiol groups on the silane compounds

datively bind to the surface while the hydroxy-silane groups polymerize to form a new silica

coating. The resultant silanol-containing surface then can be functionalized using other orga-

nosilane compounds containing functional groups or reactive groups for further conjugation

with biomolecules (see Chapter 13 for organosilane reagents and protocols). The only disad-

vantage of this approach is the increasingly greater particle diameter that results from building

successive layers on the initial QD core, which may inhibit their use for probing within cells or

tissues ( Figure 9.60 ).

Water-soluble QDs now are available from a number of manufacturers (Invitrogen, Evident

Technologies, and Crystalplex). Each supplier uses their own proprietary methods of surface

paciﬁ cation to create biocompatible particles. Even coated QD clusters are available that

contain hundreds of particles bound together in a polymer matrix (Crystalplex). These form

intensely bright labels for biomolecules, because the nanocrystals do not quench when clus-

tered together at high density.

Most QD surfaces for biological applications contain negatively charged carboxylates for con-

jugation with amine-containing molecules via a carbodiimide reaction with EDC and (sulfo)NHS

(Chapter 3, Section 1). The negative charges on the QD surface prevent particle aggregation

through like charge repulsion. An alternative method of creating water dispersible dots is to

form a hydrophilic coating that carries along with it a layer of hydration consisting of hydrogen-

bonded water molecules. This often is done using hydroxylic polymers or PEG modiﬁ cations.

This too prevents aggregation due to the high energy needed to remove the bound water layer.

QDs have been used successfully in many biological applications, which exploit their best

properties of brightness, photostability, and multiplex capability. There are many publications

Figure 9.60 Many different thiol-containing linkers can be used to prepare water-soluble QDs. The mono-

thiol compounds suffer from the deﬁ ciency of being easily oxidized or displaced off the surface, thus creating

holes for potential nonspeciﬁ c binding. The dithiol linkers are superior in this regard, as they form highly stable

dative bonds with the semiconductor metal surface that do not get displaced. The PEG-based linkers are espe-

cially effective at creating a biocompatible surface for conjugation with biomolecules.

10. Quantum Dot Nanocrystals 491

492 9. Fluorescent Probes

that use QDs for in cell or whole organism-based imaging, including tracking of targets within

cells (Dahan et al., 2003), gene localization within chromosomes (Xiao and Barker, 2004),

embryo developmental monitoring (Dubertret et al., 2002), tumor imaging in vivo (Gao et al. ,

2004), and multiplexed imaging and assays (Medintz et al., 2003; Wu et al., 2003), including

FRET signaling (Han et al., 2001). For a review on the use of QDs for cancer imaging and

treatment, see Vashist et al. (2006).

However, QD labels still are not without potential problems. Although the raw dots typi-

cally are less than 10 nm in diameter, the addition of thick surface layers for biocompatibil-

ity and conjugation can increase the hydrodynamic radius considerably. Most particles with

polymer coatings are in the 20–50 nm range in diameter, which often limits their use for cell-

based detection due to their inability to easily penetrate cells and diffuse freely to intracellu-

lar targets. Most cell imaging applications with this type of QD involve cell-surface staining

or transport within cells by endocytosis, which limits particle access to other areas within the

cell. In addition, nonspeciﬁ c binding still plagues some QD probes when used with complex

biological samples.

Another potential deﬁ ciency of QDs is the toxic nature of their metallic composition.

Most particles contain at least one known toxic metal (e.g., cadmium) or contain alloys with

unknown toxilogical properties. Cadmium-based QDs exposed to UV light for long periods

release cadmium ions, which are highly toxic to cells (Derfus et al., 2004). The initial proposal

that QDs would be ideal as ﬂ uorescent probes for in vivo diagnostic imaging may not be fully

realized due to the potential for heavy metal toxicity in humans. Even the use of QDs for in

vitro research purposes must be done with care, as the solutions should be regarded as haz-

ardous waste and disposed of according to standards for handling heavy metal contaminated

solutions.

Another potential difﬁ culty with using QDs relates to their special spectral characteristics.

Excitation of QDs optimally occurs in the low region of the spectrum, typically below 400 nm,

while emission usually is measured in regions that can be hundreds of nanometers away from

the excitation wavelength. Unfortunately, many instruments for imaging or ﬂ uorimetry still

don’t contain lasers and ﬁ lter sets that exactly match QD excitation and emission patterns.

Most instruments in use today initially were designed for organic ﬂ uors with matched ﬁ lter sets

for such common dye derivatives as ﬂ uorescein, rhodamine, and the cyanine dyes. Using QDs

with these instruments may mean exciting at a non-optimal, higher wavelength than is recom-

mended to obtain full brightness.

However, the most important issue with QD ﬂ uorescence is their tendency to blink or to

be completely dark and not ﬂ uoresce at all. Individual QDs can undergo an on/off cycle that

results in a dark period of no light emission after a photon has been emitted (Nirmal et al. ,

1996; Efros and Rosen, 1997). This blinking can be observed on the order of milliseconds and

can be problematic if imaging at the single dot resolution or if using QDs for ﬂ ow cytometry

purposes is important. When imaging a larger population of QDs, the problem of blinking will

be overcome by the fact that at any given moment many of the particles will be emitting light

and thus contributing to the overall signal. Blinking will lower the apparent QY for the com-

bined QD population, but it won ’t eliminate signal entirely. In addition, certain solution addi-

tives used at relatively high concentration (i.e., 100 mM DTT or 2-mercaptoethanol) may serve

to limit the blinking phenomena (Hohng and Ha, 2004).

A more severe issue with QDs, however, is the problem of dark or nonradiant dots in aque-

ous solution. Yao et al. (2005) documented that a signiﬁ cant fraction of commercially available

QD particles in a population can be entirely dark. The nonradiant properties probably are due

to defects in their nanocrystalline structure that occurred during manufacture. The percentage

of dark dots varies for each sample, but they can represent 44–47 percent of all dots in a popu-

lation. Thus, the use of QD conjugates for biomolecule imaging may mean that nearly half of

all particles in an assay do not contribute to the resultant ﬂ uorescence signal.

Conjugation to QDs

Many antibodies, proteins, and other targeting or afﬁ nity ligands have been conjugated to QDs

for biological applications. Antibodies to tumor markers have been used to image cancer cells in

vivo (Tada et al., 2007), ﬂ uoroimmunoassays have been developed using antibody-conjugated

QDs (Goldman et al., 2005), peptide–QD conjugates have been made to target proteins in vivo

(Ness et al., 2003), antibody–QD conjugates containing tumor toxic agents have been designed

to image and kill tumor cells (Gao et al., 2004), and sugar–QD conjugates have been made to

detect carbohydrate binding proteins (Babu et al. , 2007).

The conjugation of proteins and other molecules to QDs involves standard coupling reactions

with the added caveat related to the potential difﬁ culties of working with particles. Many of the

coupling strategies described in Chapter 14 for dealing with nanoparticles and microparticles

are valid for use with QDs, but it is best when using commercially available particles to pay

close attention to the manufacturer ’s suggested protocols.

When designing a protein–QD conjugate, it is also important to consider the optimal

number of proteins to be coupled per particle. In some applications, a low ratio of protein-to-

QD may result in the highest signal for ﬂ uorescence detection. This often is the case for anti-

body–QD conjugates where two to three antibodies coupled per particle are sufﬁ cient to target

antigens. However, other applications may require a high density of protein on the QD surface.

For instance, as a result of investigations into the interaction of CD8 with HLA A2 complexes,

it was found to be important to create multiple contacts between the HLA molecules and the

CD8 molecules on cell-surface membranes. Therefore, up to 12 HLA A2 complexes per QD

were coupled (the maximal possible to ﬁ t on the particle surface) to assure the greatest interac-

tion potential with cells (Anikeeva et al., 2006). Additionally, when preparing (strept)avidin–

QD conjugates, it may be desirable to maximize the biotin binding ability of the complex to

interact with as many biotinylated molecules as possible. Commercial streptavidin–QD conju-

gates typically have at least 5–10 proteins coupled per particle. Since each QD conjugate will

have its own unique use, the conjugation process should be optimized to perform best in its

intended application.

As is the case with most nanoparticles, buffer and salt compatibility should be taken into

account when working with QDs. Small charged particles maintain colloidal stability by like

charge repulsion, which prevents aggregation by van der Waals or hydrophobic attraction. Any

salt or buffer constituents that neutralize or eliminate the surface charge on the QDs will cause

clumping or precipitation. Some buffer additives also will cause loss of ﬂ uorescence intensity

and should be avoided in concentrations above a certain level. Consult the supplier ’s guidelines

for buffer suggestions to maintain particle stability.

The following methods are based on those cited in the literature or in company instruction

manuals for coupling molecules to ﬂ uorescent nanoparticles. The coupling of unique proteins

or other molecules to QD surfaces may need further optimization of reactant ratios as well as

time and temperature to obtain the best conjugates.

10. Quantum Dot Nanocrystals 493

494 9. Fluorescent Probes

Conjugation of Proteins to QDs Using EDC

Carbodiimide coupling to carboxylate-containing QDs usually involves the use of EDC in a single-

step or two-step process to form an amide bond. If a one-step reaction is done, the QD is activated

with EDC in the presence of an amine-containing molecule, such as a protein. Many protocols use

this method, but it can result in protein polymerization in addition to coupling, because proteins

contain both carboxylates and amines. A two-step protocol results in better control of the reaction

(Figure 9.61 ). In the ﬁ rst step, EDC is used in the presence of sulfo-NHS to activate the carboxy-

lates on the particles to intermediate sulfo-NHS esters. After a quick separation step to remove

excess reactants, the activated QDs are added to the protein solution to be coupled. This then

results in amide bond formation without polymerization of the protein in solution. See Chapter 3,

Section 1 and Chapter 14, Section 1 for additional information on this process.

The following protocol describes the coupling of amine-containing proteins to carboxylated

QDs using a single-step EDC reaction, as recommended by several manufacturers.

Figure 9.61 QDs containing carboxylate groups can be coupled to amine-containing proteins or other mol-

ecules using the EDC/sulfo-NHS reaction to form amide bond linkages. The intermediate sulfo-NHS ester is

negatively charged and will help maintain particle stability due to like charge repulsion between particles.

Protocol

1. Prepare a carboxylated QD solution in 10 mM sodium borate, pH 7.4 (reaction buffer),

at a concentration of 1 M. The supplier of QDs usually will provide the reagent concen-

tration as a molar quantity, which treats each particle as though it was a single molecule.

A typical QD solution as obtained from a manufacturer may be about 8 M starting

concentration.

2. Dissolve the protein to be conjugated to the QD in reaction buffer at a concentration of

1–10 mg/ml.

3. Add a quantity of the protein solution to the QD solution with mixing to obtain the

desired molar excess of protein over the concentration of nanoparticles. Using a 1- to 20-

fold molar excess typically works well, but optimization should be done to determine the

best ratio for a particular application.

4. Prepare a solution of EDC in water at a concentration of 10 mg/ml. Immediately add

57 l of this solution to the protein/QD solution. Mix well.

5. React for 2 hours at room temperature with gentle mixing.

6. Filter the solution through a 0.2 m ﬁ lter (low protein binding type) to remove any pre-

cipitated protein or particles.

7. Separate excess protein from the protein–QD conjugate by use of ultraﬁ ltration spin col-

umns using an exclusion limit appropriate for allowing passage of the unconjugated pro-

tein, but retention of the protein–QD conjugate. For proteins of molecular weight below

100 kD, a membrane of this cutoff will work well.

QD nanoparticles containing carboxylate groups also may be reacted in a two-step EDC/

sulfo-NHS reaction to couple proteins and other molecules containing both amines and car-

boxylates. This type of reaction is designed to remove excess EDC activating agent before addi-

tion of protein, so protein polymerization cannot occur.

Protocol

1. Prepare a QD solution in 25 mM PIPES buffer, pH 7.0 (reaction buffer), at a concentra-

tion of 100  g/ml.

2. Prepare a protein solution in reaction buffer at a concentration of 1 mg/ml.

3. Prepare a solution of 20 mM EDC, 50 mM sulfo-NHS in water immediately before use.

4. To each ml of QD solution, add 50 l of the EDC/sulfo-NHS stock solution. Maintain

the pH at 7.0 by the addition of base, if necessary. Small volume reactions may be con-

trolled using a pH stat.

5. React for 10 minutes at room temperature with gentle mixing.

6. Add 1.4 l of 2-mercaptoethanol to each ml of the reaction mixture to quench excess

EDC. Sonicate the QD solution several times to maintain particle dispersion.

7. Add 5  l of the protein solution to each 100  g quantity of activated QDs.

8. React for 60 minutes at room temperature with mixing.

9. Remove excess reactants and block remaining sulfo-NHS ester sites by dialysis against

50 mM Tris, pH 7.4. Use a membrane cutoff appropriate to allow passage of the protein

being coupled, but retention of the protein–QD conjugate.

10. Quantum Dot Nanocrystals 495

496 9. Fluorescent Probes

Conjugation to QDs Using Sulfo-SMCC

Sulfo-SMCC is a heterobifunctional crosslinking agent containing an amine-reactive NHS ester

on one end and a thiol-reactive maleimide group on the other end (Chapter 5, Section 1.3).

Amine-containing QDs may be activated with sulfo-SMCC to contain sulfhydryl-reactive male-

imides for conjugation with thiol-containing proteins or other molecules ( Figure 9.62 ). The fol-

lowing protocol illustrates this process.

Protocol

1. Prepare a 200 l solution of amine-containing QDs at a concentration of 2.5 M in

50 mM sodium phosphate, pH 7.4 (reaction buffer). This represents 0.5 nmole of QD in

200 l buffer.

2. Add 1 mg of sulfo-SMCC to the QD solution with mixing to dissolve the crosslinker.

3. React for 60 minutes at room temperature with mixing.

4. Remove excess crosslinker and reaction by-products by gel ﬁ ltration on a desalting resin.

The QD fraction is identiﬁ ed by its characteristic ﬂ uorescence. This operation should be

Figure 9.62 Sulfo-SMCC can be used to conjugate amine-containing QDs with thiol-containing proteins or

other molecules using a two-step coupling procedure.

done quickly to limit the degree of maleimide hydrolysis. The resulting particles contain

reactive maleimide groups for coupling to a thiol-containing protein or other molecule.

5. Prepare a protein or antibody containing an available thiol group in reaction buffer at

a concentration of 1–10 mg/ml. Add a quantity of this protein solution to the puriﬁ ed

nanoparticle suspension to attain the desired molar excess of protein over the concentra-

tion of activated QDs. Typically, a 1- to 20-fold molar excess of protein over the QD

concentration works well. The optimal amount of protein to be added should be deter-

mined experimentally by considering the best performance of the ﬂ uorescent conjugate in

its intended application. Tada et al. (2007) created a monoclonal anti-HER2 antibody–

QD conjugate at a level of three antibodies per nanoparticle to target tumors in mice.

Creating thiol groups on proteins or peptides may be done from disulﬁ des by reduction.

Alternatively, a thiolation reagent may be used to add thiols to the protein surface for

coupling (see the protocols in Chapter 1, Section 4.1).

6. React with mixing for 2 hours at room temperature. At the completion of the reaction,

cysteine may be added at 50 mM to block excess maleimide-reactive sites.

7. Purify the QD conjugate using gel ﬁ ltration or ultraﬁ ltration using a micro-spin device.

The use of a molecular weight cutoff for gel ﬁ ltration that will accommodate both the

conjugate and the not-coupled protein is appropriate (i.e., Superdex-200 resin). For

ultraﬁ ltration, use a membrane cutoff that will retain the conjugate, but permit the not-

coupled protein to pass through.

10. Quantum Dot Nanocrystals 497

498

Monoclonal antibodies provide extremely high antigen speciﬁ city that can be useful as can-

cer targeting and therapeutic reagents in vivo (Waldmann, 1991). Radiolabeled monoclonals

are currently undergoing developmental clinical trials for their use in the diagnosis or treatment

of cancer. The antigen-binding speciﬁ city of the radioimmunoconjugate provides the targeting

capability to localize in tumor sites, while the associated radiolabel provides cytotoxic proper-

ties or detectability for imaging applications (Schlom, 1986).

Iodine-131 was among the ﬁ rst radioactive isotopes used for radioimmunoconjugate prepa-

ration (Order, 1982; Regoeczi, 1984). Since the earliest studies on the efﬁ cacy of radiotherapy,

additional isotopes have been employed, such as iodine-125, bismuth-212, yttrium-90, yttrium-88,

technetium-99 m, copper-67, rhenium-188, rhenium-186, galium-66, galium-67, indium-111,

indium-114 m, indium-115, and boron-10.

There are several methods commonly used to label monoclonal antibodies with radionuclides.

In a direct labeling process, a radioactive atom is attached to functional groups on the antibody

without the use of an intervening chemical spacer. For instance, radioiodination can be done

through modiﬁ cation of tyrosine side chains using established techniques and reagents (Chapter 12).

Another direct method uses indigenous sulfhydryl groups or those formed through disulﬁ de

reduction to couple covalently certain metal nuclides (Holmberg and Meurling, 1993; Ranadive

et al., 1993). Thiolation reagents also can be used in this regard to create the requisite  SH groups

by modiﬁ cation of other protein functional sites (Joiris et al., 1991) (Chapter 1, Section 4.1).

Indirect methods of protein labeling with radiolabels utilize organic compounds able to

chelate metal ions in a coordination complex. Bifunctional chelating agents (BCAs), as they

are called, contain a reactive group for coupling to proteins or other molecules and a strong

metal-chelating group for complexing certain radioactive metals. Their extensive use with

monoclonal antibodies that are able to target speciﬁ c cellular antigens has resulted in impor-

tant radiopharmaceutical applications for the diagnosis and treatment of cancer (Wessels and

Rogus, 1984; Meares, 1986; Otsuka and Welch, 1987; Hnatowich, 1990; Liu and Wu, 1991;

Subramanian and Meares, 1991). The BCAs may be loaded with the radioactive metal before

or after their conjugation with a monoclonal antibody (Frytak et al., 1993). If they are loaded

with radionuclides prior to modifying the antibody, the BCA–metal pair is called a preformed

complex (Kasina et al ., 1991).

The following sections describe the major methods and BCAs used to create

radioimmunoconjugates.

Bifunctional Chelating Agents and

Radioimmunoconjugates

1 . D T P A

DTPA is diethylenetriaminepentaacetic anhydride, a BCA containing two amine-reactive anhy-

dride groups. The compound reacts with N-terminal and -amine groups of proteins to form

amide linkages. The anhydride rings open to create multivalent, metal-chelating arms able to

bind tightly metals in a coordination complex ( Figure 10.1 ) (Hnatowich et al., 1982). Metal-

chelate bonds are created through dative interactions with the unshared pair of electrons on

each oxygen and nitrogen atom on DTPA, thus creating the potential for eight coordination

sites with metal ions (from 3 nitrogens and 5 oxygens).

Optimal reaction conditions for antibody modiﬁ cation with DTPA are neutral to slightly alka-

line pH environments containing no extraneous amines. A pH of 7–8 may be used with buffering

provided by phosphate or bicarbonate buffers at 0.1 M. Since two anhydride groups are present

on each DTPA molecule there is potential for creating crosslinks between two amine-containing

molecules. Conjugation of antibody through DTPA crosslinks may be a major reason some immu-

noglobulins lose antigen-binding activity after modiﬁ cation (Lanteigne and Hnatowich, 1984).

Optimization of the amount of protein present and the quantity of DTPA added to the reaction

may have to be done to avoid this type of crosslinking and polymerization.

Figure 10.1 DTPA reacts with amine-containing molecules via ring opening of its anhydride groups to create

amide bond linkages. The potential also exists for both anhydride groups to react and cause crosslinking of

modiﬁ ed molecules, which is undesirable.

1. DTPA 499