Hermanson G. Bioconjugate Techniques, Second Edition

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480 9. Fluorescent Probes

electrons to coordinate with the lanthanide metal ion. The result is a protected lanthanide that

cannot be quenched easily by water or other matrix components. Complexed with europium,

this label can be excited at 340 nm and provide sharp emission peaks at wavelengths of 589, 599,

618 (maximum emission), 623, 651, 689, 697, and 702 nm. The largest peaks in the  600 nm

range can optimally excite Cy5 dyes, such as the hydrophilic DyLight 649 (Section 8, this

chapter) and create an effective TR-FRET system for homogeneous assay design.

The TMT chelator containing an amine-reactive isothiocyanate group can be used to label

antibodies, proteins, or other molecules using the following protocol ( Figure 9.52 ).

Protocol

1. In a fume hood, dissolve the TMT chelator in a 1:1 mixture of DMF:DMSO at a concen-

tration of 10 mg/ml.

2. Dissolve a protein to be labeled in 0.1 M sodium bicarbonate, pH 9.0, at a concentration

of 1–10 mg/ml.

3. With mixing, add a quantity of the TMT chelator to the protein solution to provide a

5- to 10-fold molar excess of reagent. Optimization may have to be done to determine

the best ratio of chelator to protein for the intended application.

4. React at 4 ° C overnight with gentle mixing.

5. Purify the labeled protein by gel ﬁ ltration or dialysis.

As in the structure of the TMT chelator group, pyridine derivatives long have been known

to be enhancers of lanthanide luminescence (Thomas et al., 1978). One such compound,

Figure 9.52 The isothiocyanate group of a TMT europium chelate can be used to label amine-containing pro-

teins and other molecules to result in an isothiourea bond.

9. Lanthanide Chelates for Time-Resolved Fluorescence 481

482 9. Fluorescent Probes

dipicolinic acid (DPA), contains two carboxylates on both sides of a pyridine core, which pro-

vides three coordination sites per molecule to complex with lanthanides. In solution, three DPA

molecules can coordinate with one lanthanide ion to fully surround the metal and protect it from

quenching by water or other molecules. Each DPA pyridine group can absorb energy in the UV

region and transfer it to the central lanthanide, which results in strong emission at characteristic

wavelengths.

Lamture and Wensel (1995) synthesized a unique modiﬁ cation of the basic DPA structure

to make the chelator bifunctional and thus able to react with functional groups on other mol-

ecules. This reactive DPA reagent, 4-(iodoacetamido)-2,6-dimethylpyridine dicarboxylate, con-

tains an iodoacetyl group on the C-4 of the pyridine ring, thus making it particularly reactive

with thiol groups and amines at higher pH. This derivative was used to create a large poly-

meric-chelating structure from poly-

L-lysine that contained 50–100 DPA units along its length.

Loading this complex with terbium(III) ions resulted in highly intense luminescence. The

remaining amines on poly- L-lysine ﬁ nally were succinylated to create carboxylates for coupling

to proteins via carbodiimide conjugation ( Figures 9.53 and 9.54 ).

Another antenna group that is particularly effective for lanthanide luminescence is carbosty-

ril derivatives (7-amino-4-methyl-2(1H)-quinolinone), which can be attached to many chelating

Figure 9.53 DPA derivatives have been used as potent enhancers of lanthanide luminescence. Three DPA

groups can coordinate with a terbium ion. The iodoacetate derivative of DPA has been used to label covalently

molecules for lanthanide luminescence.

Figure 9.54 The iodoacetamide derivative of DPA has been used to create a chelating polymer of lanthanide

metals using poly-

L -lysine as the backbone.

9. Lanthanide Chelates for Time-Resolved Fluorescence 483

484 9. Fluorescent Probes

compounds via amide bond formation with the 7-amino group to a carboxylate ( Figure 9.55 ).

A carbostyril-DTPA derivative has intense luminescence for use in TR-FRET-based assays for

drug discovery. The 7-aminoquinolinone structure is a common choice for designing ﬂ uores-

cent lanthanide chelates (Soini and Lövgren, 1987; Sammes and Yahioglu, 1996; Selvin, 2002,

2003). The synthesis of various carbostyril derivatives is described in Ge and Selvin (2004).

The 7-aminoquinolinone group ﬁ rst is put onto DTPA by reaction with one of the chelator ’s

anhydride rings. Linking the chelator to proteins or other molecules is done through the anhy-

dride on the opposite end by forming an amide-linked spacer arm. In some cases, sulfonate

groups or other hydrophilic components on the carbostyril structure can provide increased

hydrophilicity for modifying biomolecules.

When used with europium or terbium ions, a carbostyril-based lanthanide chelate can be

excited at 340 nm and provide sharp characteristic emission bands for transfer of energy to

the appropriate acceptor ﬂ uor. Similar to the TMT chelator described previously, luminescence

from terbium FRET signals well with Cy3 dyes and luminescence from europium can be used

with APC or Cy5 dyes. Other ﬂ uorescent dyes that have similar excitation and emission ranges

to these also can be used as acceptors in TR-FRET assays. For instance, terbium chelates can

Figure 9.55 A carbostyril–DTPA europium chelator is a strong enhancer of lanthanide luminescence. The

chemical structure and three-dimensional structure of the chelator are shown.

be used with ﬂ uorescein and tetramethylrhodamine dyes, if the appropriate ﬁ lters are used for

measuring emission.

The potential for creating an expressed protein time-resolved luminescence system was real-

ized in the development of a novel lanthanide-chelating complex derived from a metal binding

peptide sequence. This sequence, called a lanthanide binding tag (LBT), can be used as a fusion

tag in recombinant protein expression (Franz et al., 2003; Nitz et al., 2003; Wöhnert et al. ,

2003; Lim and Franklin, 2004; Goda et al., 2007). To create this luminescent tag, a peptide

sequence of about 15–20 amino acids representing the basic structural domain of a calcium

binding loop from calmodulin was mutated to coordinate tightly a terbium ion. Luminescence

occurs through excitation in the UV of a nearby tryptophan residue with energy transfer to the

chelated lanthanide. Recombinant mutagenesis of the original calcium binding domain resulted

in optimizing the dissociation constant for terbium to within the nanomolar range, thus the

metal ion is tightly associated with the peptide tag (Martin et al., 2005). Recombinant proteins

containing this small fusion tag can be detected using time-resolved ﬂ uorescence techniques.

Such in vivo expression of a luminescent lanthanide tag can be used to study protein interac-

tions using LRET or track individual proteins within cells (Sculimbrene and Imperiali, 2006).

10. Quantum Dot Nanocrystals

Properties of Quantum Dots

Quantum dots (QDs) are nanoparticles typically made of a semiconductor metal alloy arranged

in a spherical crystalline core and capped with a shell consisting of a second metal alloy com-

position ( Figure 9.56 ). The size of this raw core/shell construct is usually less than 10 nm in

diameter or about the same sizes as many globular protein molecules. Upon exposure to light at

the appropriate wavelength range, QDs are able to absorb a photon of energy, which results in

Figure 9.56 The structure of a typical QD nanocrystal includes a semiconductor alloy core surrounded by a

shell consisting of a different alloy structure. Early QD compositions involved the use of CdSe core with a ZnS

shell, but many different alloy compositions have been used and are possible.

10. Quantum Dot Nanocrystals 485

486 9. Fluorescent Probes

the excitation of an electron within the core. The excited electron is conﬁ ned within the nanoc-

rystal, because its core diameter is less than the exciton Bohr radius, which thus leads to quan-

tum conﬁ nement. The shell structure aids in this conﬁ nement and prevents the electron from

tunneling out of the core and escaping into the outer medium or undergoing non-radiative

deactivation. The size and shape of a QD governs the discrete energy levels that the excited-

state electron can attain within it, thus dots can be tuned to have desired electronic properties

by careful adjustment of core diameter and composition. Upon return of the electron to its

ground state, a radiative QD emits a photon of light, the wavelength of which is dependent on

the alloy material type and its diameter (Alivisatos, 1996) ( Figure 9.57 ).

As a result of their unique optical and electronic properties, particularly their ability to ﬂ uo-

resce at discrete wavelengths directly proportional to their sizes and material compositions, QDs

have found use in many ﬁ elds, including electronics, biology, medicine, and even cosmetics.

The ﬁ rst attempts to modify their surface characteristics to make them water-soluble and bio-

compatible eventually led to their use as ﬂ uorescent labels for biomolecules in many applica-

tions (Rogach et al. , 1996; Bruchez et al. , 1998; Chan and Nie, 1998).

QDs have a number of advantages over other ﬂ uorescent molecules such as organic dyes,

including: (1) resistance to photobleaching, which allows them to be imaged over long peri-

ods without loss of ﬂ uorescence; (2) narrow, nearly symmetrical emission peaks with no red-

shift tail typical of organic ﬂ uors, thus creating a bright ﬂ uorescence signal at characteristic

wavelengths; (3) a broad absorbance band, which increases almost exponentially toward

shorter wavelengths with extremely high extinction coefﬁ cients (10

–10

 1

); (4) the

ability to excite at a single wavelength an entire family of QDs having different emission char-

acteristics, thus providing multiplexed assay capability; (5) the capacity to design QDs with

emission characteristics ranging from the low visible wavelengths to well within the IR region;

and (6) the potential for relatively high QY of ﬂ uorescence (0.65–0.95 for CdSe).

The emission properties of QDs can be adjusted based upon core diameter and nanoparticle

composition. Nanoparticle diameters typically are carefully controlled during manufacture to

be between 2 and 10 nm. In addition, the band gap energy or energy of ﬂ uorescence emission

is inversely proportional to the diameter of the QD particle. Thus, the smaller the particle, the

Figure 9.57 The size of a QD directly affects its emission wavelength. Careful control of nanocrystal diameter

during the manufacturing process can result in discrete QD populations having emission properties ranging

from the blue to the red region of the visible spectrum.

more blue-shifted is its emission and the larger the QD, the more red-shifted is its emission

bands. QDs also have an intrinsic color to their solutions that corresponds to the size of the

particles and their ﬂ uorescence emission characteristics. However, to create a single particle

population with a tight ﬂ uorescence emission pattern, the diameter of the particles must be

controlled to well within a nanometer. The emission peak width is directly proportional to the

size distribution of a particle population. This makes manufacturing reproducible QDs a con-

stant challenge for most suppliers that rely on size to control ﬂ uorescence properties.

However, as opposed to the difﬁ culty of tuning emission properties by particle diameter,

QD alloy composition instead may be adjusted independent of size to control the wavelength

of emission for a given particle population. In a QD having a concentration gradient com-

position, the concentration of an alloy of a ﬁ rst semiconductor gradually increases from the

core to the surface of the particle, while the concentration of a second semiconductor gradu-

ally decreases from the core to the surface (Nie and Bailey, 2007). A third semiconductor type

also may be added to ﬁ ne-tune further the emission properties. By careful adjustment of these

semiconductor concentration gradients, QD populations can be made having discrete emission

properties without changing the particle size. Therefore, tuning QD spectral characteristics can

be done using a single particle size and by making selective changes to the alloy composition.

This avoids the difﬁ culties in manufacturing particles of uniform size, because all particle pop-

ulations can have the same size, but only vary in their relative semiconductor gradient concen-

trations to attain particles having discrete ﬂ uorescence character.

The material types making up the core of a QD also affect the range of emission wave-

lengths that can be attained. For common material types, the ranges of emission wavelengths

that can be achieved by adjustment of particle diameter or composition are: CdSe   470–

660 nm, CdTe   520–750 nm; InP  620–720 nm; PbS  900 nm; and PbSe  1,000 nm.

QDs have been made using a number of techniques. A common method to make bulk

quantities of particles involves doing colloidal suspension synthesis in organic solvent with

nucleation of semiconductor metals under high-temperature conditions (Murray et al., 1993;

Hines and Guyot-Sionnest, 1996; Dabbousi et al., 1997). In one such process, a solvent such

as octadecene is stirred at constant rate and heated to  300 ° C at which point solutions con-

taining the semiconductor metals are injected. The metals at ﬁ rst decompose under high heat

and then recombine to form alloys consisting of nanoparticle seeds, which grow to create the

QDs. The reaction time determines the size of the nanoparticles and thus their spectral proper-

ties. Detergent molecules often are added to coat the resulting nanoparticles and prevent their

aggregation during nucleation. Originally, the solvent and detergent molecule used for mak-

ing QDs was TOPO (tri- n-octylphosphine oxide), which ends up coating the particles with the

phosphine component interacting with the semiconductor surface and the alkyl chains pointing

out into the organic solution ( Figure 9.58 ). Other additives, such as stearic or oleic acid, func-

tion similarly. The raw particles thus prepared are hydrophobic and not dispersible in aqueous

solution.

To use QDs in biological applications, the particles must be rendered biocompatible by

coating with a hydrophilic layer that masks the surface, thus preventing aggregation and

nonspeciﬁ c binding. This is not a trivial problem, as the successful commercialization of QDs

for biomolecule labeling took at least 5 years from the time the ﬁ rst two papers appeared in

Science describing water-soluble particles for bioconjugation (Bruchez et al., 1998; Chan and

Nie, 1998). The fact is, these early particles were not very soluble in aqueous environments

and tended to clump together or bind nonspeciﬁ cally with biomolecules.

10. Quantum Dot Nanocrystals 487

488 9. Fluorescent Probes

Figure 9.58 QDs made using the TOPO process typically have a layer of these molecules associated with their

outer surface. The TOPO groups must be displaced and replaced by water-soluble groups to provide biocom-

patibility for bioconjugation purposes.

The initial modiﬁ cations done to covalently link molecules to QDs need to displace

the TOPO or detergent coating on the raw nanocrystal surface with a new organic deriva-

tive imparting water solubility. The ﬁ rst attempts at making biocompatible QDs all involved

the use of simple monothioacids, such as thioacetic acid, which can link to the shell through

thiol dative bonding and provide a terminal carboxylate for further conjugation ( Figure 9.59 ).

However, monothiol linkers easily can oxidize back off QDs and leave behind surface gaps,

which become hydrophobic sites for particle clumping and nonspeciﬁ c binding to biomol-

ecules. A better approach is to use a dithiol compound, which forms two dative bonds per

linker on the QD surface. This makes the linkage to the QD resistant to oxidation and prevents

nonspeciﬁ c surface gaps from forming. One such dithiol compound that is particularly use-

ful is dihydrolipoic acid (DHLA), which contains a carboxylate group on the other end. QDs

modiﬁ ed with DHLA subsequently can be modiﬁ ed with polyethylene glycol (PEG) groups to

provide increased hydrophilicity of the surface or directly linked to proteins via electrostatic

interactions or through an EDC-mediated reaction (Mattoussi et al., 2000; Uyeda et al., 2003;

Medintz et al. , 2004; Anikeeva et al. , 2006; Clapp et al. , 2006).

The negative charge character of DHLA-modiﬁ ed QDs has been used to link noncovalently

positively charged proteins, such as avidin (Goldman et al., 2002a) or a recombinant protein

containing a positively charged fusion peptide. In this regard, the highly positive leucine zipper

Figure 9.59 One of the ﬁ rst methods of preparing water-soluble QDs was to use thioacetic acid modiﬁ cation of

the nanocrystal surface. This resulted in a negative charge on the surface of each dot that provides like charge

repulsion of particles suspended in aqueous solution. The carboxylate group also could be used for conjugation

with amine-containing molecules.

10. Quantum Dot Nanocrystals 489