Hermanson G. Bioconjugate Techniques, Second Edition

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470 9. Fluorescent Probes

efﬁ ciency. Avoid amine-containing buffers such as Tris or imidazole. Also, avoid the addi-

tion of thiol reducing agents or glycerol, as these also will react with the NHS ester. If the

protein concentration is below 1 mg/ml, the molar excess of dye added to the reaction

will have to be greatly increased to maintain yield of the reaction.

2. In a fume hood, dissolve the NHS ester-cyanine dye in DMF at a concentration of

10 mM. Protect all dye solutions from light.

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

desired molar excess of dye over protein. For instance, for an antibody dissolved in

buffer at 10 mg/ml, the addition of 66 l of dye solution will give a 10-fold molar excess

of dye over protein.

4. React for 1 hour at room temperature with gentle mixing.

5. Purify the labeled protein from excess dye and reaction by-products using dialysis or gel

ﬁ ltration on a desalting resin.

Thiol-reactive Cyanine Dyes

Cyanine dyes that have a thiol-reactive group typically are maleimide derivatives. Maleimide groups

react with sulfhydryls under neutral pH conditions to form a thioether linkage ( Figure 9.47 ).

Since thiols are present in proteins in limited amounts, dye labeling through these groups often

will result in modiﬁ cations that occur only at discrete locations on the molecule. Labeling thiol

groups also may be done using peptides that have been synthesized with terminal cysteine resi-

dues or using oligonucleotides containing a 5  thiol group.

The pH of a maleimide conjugation reaction should be in the range of 6.5–7.5 to assure

speciﬁ city toward thiol groups. Higher pH conditions will begin to result in cross-reactions

with amines through a Michael addition process. Coupling to proteins can be done through

disulﬁ de reduction or through use of a thiolation reagent (Chapter 1, Section 4.1). Reduction of

disulﬁ des may be done using immobilized TCEP (Thermo Fisher), which effectively forms free

thiols on proteins and peptides while not contaminating a reaction with the reducing agent.

The following protocol is based on the methods recommended by Thermo Fisher for use of

the cyanine dye DyLight 649. Antibody reduction is based on the methods of Sun et al. (2005),

which results in partially reduced bispeciﬁ c immunoglobulin containing available thiols in the

hinge region for labeling.

Protocol

Partial Reduction of Antibody IgG

1. Dissolve an antibody in 50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, pH

7.2 (reaction buffer), at a concentration of 1–10 mg/ml.

2. Dissolve TCEP in reaction buffer at a concentration of 10 mM and readjust the pH if

necessary.

3. Add a quantity of the TCEP solution to the antibody solution with mixing to achieve a

2.75 M excess of the reducing agent over the amount of antibody present.

4. Incubate for 2 hours at 37 °C.

The reduced antibody can be used immediately to label with a maleimide-based cyanine dye

using the following protocol.

Labeling of Reduced Antibody with Thiol-Reactive Cyanine Dye

5. In a fume hood, dissolve the maleimide-cyanine dye in DMF at a concentration of

10 mM. Protect all dye solutions from light.

6. With mixing, add a quantity of the dye solution to the antibody solution to provide

the desired molar excess of dye. For instance, for an antibody dissolved in buffer at

Figure 9.47 A maleimide-containing cyanine dye can be used to label thiol-containing molecules to form

thioether bonds.

8. Cyanine Dye Derivatives 471

472 9. Fluorescent Probes

10 mg/ml, the addition of 66 l of dye solution will give a 10-fold molar excess of dye

over protein.

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

8. Purify the labeled protein from excess dye and reaction by-products using dialysis or gel

ﬁ ltration on a desalting resin. Protect the labeled protein from light to avoid photob-

leaching the dye and losing ﬂ uorescence intensity.

Carbonyl-reactive Cyanine Dyes

Cyanine-type dyes containing hydrazide functional groups may be used to label molecules

containing carbonyl groups, especially aldehydes ( Figure 9.48 ). Aldehydes may be created on

carbohydrates, sugars, and glycans by periodate oxidation, which cleaves the carbon–carbon

bonds between diols to form reactive formyl groups. Thus, glycoproteins and other glyco-

conjugates can be labeled speciﬁ cally with a cyanine ﬂ uorescent dye only through carbohydrate

components, which often avoids binding sites or active centers. Hydrazide-containing cyanine

dyes also may be used to detect glycoproteins in cells, tissues, gels, or on Western blots after

periodate oxidation. In this regard, bands containing glycoproteins can be detected in a sample

separately from the rest of the non-glycosylated protein pool (Thermo Fisher).

Glycan molecules that have been released from a protein by enzymatic means also can be

labeled at their reducing ends by a hydrazide-containing cyanine dye. This reaction results in

a single ﬂ uorescent label on the C-1 carbon of the inner most sugar of the glycan tree. The

labeled glycan can be tracked through separation steps or detected for its speciﬁ c interactions

with carbohydrate binding proteins (see Chapter 1, Section 4.6).

Hydrazide-based cyanine dyes are reactive with common formaldehyde ﬁ xatives for cell and

tissue studies. This enables these dyes to function as general stains for protein-rich areas within

cells, and they get crosslinked into place by the formaldehyde reaction process.

The following protocol relates to the labeling of glycoproteins with hydrazide-cyanine

dyes. Similar methods can be used to detect glycoproteins in other applications, such as within

electrophoresis gels or on blots. For methods related to the labeling of glycans at their reducing

ends with hydrazide reagents, see Chapter 11, Section 6.

Protocol

1. Dissolve a glycoprotein to be labeled in 50 mM sodium phosphate, pH 7 (reaction

buffer), at a concentration of 1–10 mg/ml. For sialic acid modiﬁ cation, place the sample

in ice to cool to near 0 °C.

2. Dissolve sodium meta-periodate in reaction buffer at a concentration of 10 mg/ml

(0.046 M). Protect from light. To obtain approximately a 1 mM concentration of sodium

periodate in the reaction solution (suitable for oxidizing only sialic acid residues), add

21.8l of this stock solution to each ml of the glycoprotein solution to be oxidized.

Maintain the solution on ice. For general oxidation of carbohydrates other than just

sialic acid, add 218 l of the stock solution to obtain an approximate ﬁ nal concentration

of 10 mM periodate in the reaction. Use room temperature conditions for general carbo-

hydrate oxidation. Wrap the vial containing the reaction solution with aluminum foil to

protect from light. The use of an amber vial also is suitable for this purpose.

3. React for 15–30 minutes at room temperature.

4. Quench the reaction by immediate gel ﬁ ltration on a desalting column. If a dextran-based

resin is used for the chromatography, the support itself will react with sodium periodate

to quench excess reagent. Alternatively, N-acetylmethionine may be added to quench the

reaction, because the thioether of the methionine side chain will react with periodate to

form sulfoxide or sulfone products (Geoghegan and Stroh, 1992). In addition, sodium

Figure 9.48 A cyanine dye containing a hydrazide group can be used to label glycans at their reducing end or

other reducing sugars, forming a hydrazone linkage. Glycoproteins also can be labeled after periodate oxidation

to form aldehyde groups.

8. Cyanine Dye Derivatives 473

474 9. Fluorescent Probes

sulﬁ te (Na

) was used by Stolowitz et al. (2001) to quench the periodate oxidation of

horseradish peroxidase (HRP) in solution. To quench the reaction with cellular samples,

wash the cells with buffer to remove remaining traces of periodate.

5. In a fume hood, dissolve the hydrazide-cyanine dye in DMF at a concentration of 10 mM.

Protect all dye solutions from light.

6. With mixing, add a quantity of the dye solution to the oxidized antibody solution to pro-

vide the desired molar excess of dye. For instance, for an antibody dissolved in buffer at

10 mg/ml, the addition of 66 l of dye solution will give a 10-fold molar excess of dye over

protein.

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

8. Purify the labeled protein from excess dye using dialysis or gel ﬁ ltration on a desalting

resin. Protect the labeled protein from light to avoid photobleaching the dye and losing

ﬂ uorescence intensity.

9. Lanthanide Chelates for Time-resolved Fluorescence

Lanthanide metals are sometimes called rare earths and are located in period 6 of the periodic

table of elements with atomic numbers 57–71, beginning with lanthanum (although there is

some discrepancy over exactly where they start and end). Due to their unique electronic

properties, lanthanide ions display luminescent properties with very sharp emission peaks.

Unfortunately, the metal ions alone have extremely low extinction coefﬁ cients of no more than

about 1–10 M

 1

, which makes them poorly ﬂ uorescent. The dim nature of lanthanide

luminescence can be overcome by associating the metal ion with a chromophore group that

can act as a light antenna. Lanthanide metals can form nine coordination bonds with ligands,

creating three-faced centered trigonal prism structures. Coordinated in this way in an organic-

chelate structure that also contains a strongly absorbing chromophore nearby, the dimness of

lanthanide metal luminescence can be transformed into an extraordinarily bright complex. The

appropriate antenna group is one that can absorb energy and transfer it to the lanthanide metal,

which then emits light at speciﬁ c wavelengths, depending on what lanthanide element is in

the coordination complex. For a review of chelating compounds for lanthanide luminescence,

see Arnaud and Georges (2003). Some examples of antenna-chelate structures are shown in

Figure 9.49 ( Figure 9.50 ).

Fluorescent labels built from lanthanide-chelating groups of this type are extremely useful in

biological applications due to their large Stoke ’s shift (nearly 290 nm), no overlap between exci-

tation and emission peaks, and very long ﬂ uorescent lifetimes. When bound by a chelating group

that completely envelops and interacts with most of the lanthanide ’s nine possible coordination

sites, the metal also is protected from any solvent quenching effects, which may reduce lumi-

nescence (Pietraszkiewicz et al., 1993). If the antenna-chelating group also contains a reactive

group, then the lanthanide chelate complex can be covalently linked as a label on targeting mol-

ecules or afﬁ nity ligands. The result is one of the most intensely ﬂ uorescent probes available for

biological detection applications.

The ﬂ uorescence lifetimes of lanthanide chelates are among the longest of all ﬂ uorescent

compounds. Whereas most organic ﬂ uors or inorganic nanocrystals have lifetimes measured in

the nanosecond or picosecond range, lanthanide chelates typically emit light for microseconds

to milliseconds. The advantage of this long-lived emission is that time-gated measurements can

Figure 9.49 Examples of antenna-chelator compounds that have been used for lanthanide luminescence.

9. Lanthanide Chelates for Time-resolved Fluorescence 475

476 9. Fluorescent Probes

be taken, which detect lanthanide luminescence only long after emission from other organic

ﬂ uors has decayed to zero. In complex biological samples, often there are many organic compo-

nents that have ﬂ uorescent properties, especially in the lower visible or UV regions of the spec-

trum. When using lanthanide chelates as ﬂ uorescent probes and labels, any indigenous sample

ﬂ uorescence can be eliminated before the chelate emission is measured. This “time-resolved”

luminescence technique virtually abolishes background ﬂ uorescence and makes detection and

assays using lanthanide chelates among the most sensitive ﬂ uorescence measurements possi-

ble. For reviews of time-resolved ﬂ uorescence using lanthanide chelates in biological assays, see

Soini and Kojola (1983); Soini et al. (1990); Diamandis and Christopoulos (1990); Hemmila

(1985); Diamandis (1993); and Hemmila (1998).

Different lanthanide metals also produce different emission spectrums and different intensi-

ties of luminescence at their emission maximums. Therefore, the relative sensitivity of time-

resolved ﬂ uorescence also is dependent on the particular lanthanide element complexed in the

chelate. The most popular metals along with the order of brightness for lanthanide chelate

ﬂ uorescence are europium(III)  terbium(III)  samarium(III)  dysprosium(III). For instance,

Huhtinen et al. (2005) found that lanthanide chelate nanoparticles used in the detection of

human prostate antigen produced relative signals for detection using europium, terbium,

samarium, and dysprosium of approximately 1.0:0.67:0.16:0.01, respectively. The emission

Figure 9.50 The principle of enhanced lanthanide luminescence using antenna-chelating compounds. The

organic dye antenna group absorbs energy and transfers it to the lanthanide metal ion coordinated within the

chelate structure. The best chelating groups fully coordinate the metal and protect it from the quenching effects

of water. The lanthanide atom then emits light at a longer wavelength, which is characteristic of the lanthanide

metal. Lanthanide chelates also contain a reactive group that can be used to attach covalently the label to tar-

geting molecules.

wavelengths for each of these lanthanides also are different. Terbium has a main peak of lumi-

nescence in range of 545 nm, dysprosium at about 575 nm, europium at about 615 nm, and

samarium at approximately 645 nm (although there are other emission bands for all of these

lanthanide metals as well).

In assays, europium chelates usually produce the greatest sensitivity in an assay, followed

closely by terbium chelates. Another advantage of europium is that it has the longest ﬂ uores-

cent lifetime of all the lanthanides, making it the best choice for time-resolved applications. For

these reasons, most applications of lanthanide ﬂ uorescence use europium or terbium chelates

to modify biological molecules, as they provide the brightest conjugates achievable when using

this technology.

Another advantage of lanthanide chelates is their lack of self-quenching effects. Since the

excitation and emission peaks don ’t overlap for a given lanthanide metal, the chelates don ’t

quench each other if modiﬁ ed at high density on other molecules or immobilized close together

on surfaces or particles. Therefore, creating complexes having multiple lanthanide-chelating

groups is a strategy that can dramatically increase ﬂ uorescence. This property differs from

organic ﬂ uorescent reagents, as dyes usually start to quench if as few as 6–8 ﬂ uorescent labels

modify a single protein. By contrast, it is possible to form polymer or particle labels contain-

ing dozens, hundreds, or even thousands of lanthanide-chelating groups, which are capable

of increasing the ﬂ uorescent signal in assays equal to the sum of the total number of groups

present. Using this approach, Huhtinen et al. (2005) created nanoparticle labels containing

hundreds of ﬂ uorescent lanthanides. Similarly, Scorilas et al. (2000) created a polyvinylamine

polymer chains with multiple europium chelates and also added biotin labels to form huge

complexes with streptavidin in solution. Such reagents increase dramatically the lanthanide

luminescence signal in immunoassays or other detection applications beyond that possible with

standard organic ﬂ uors.

Lanthanide chelates also can be used in FRET applications with other ﬂ uorescent probes

and labels ( Figure 9.51 ). In this application, the time-resolved (TR) nature of lanthanide lumi-

nescent measurements can be combined with the ability to tune the emission characteristics

through energy transfer to an organic ﬂ uor (Comley, 2006). TR-FRET, as it is called, is a pow-

erful method to develop rapid assays with low background ﬂ uorescence and high sensitivity,

which can equal the detection capability of enzyme assays (Selvin, 2000).

FRET signaling is limited by the distance requirements for energy transfer between ﬂ uores-

cent molecules. The donor molecule must be constrained within the immediate molecular vicin-

ity of the acceptor ﬂ uorescent molecule. FRET systems can be described in terms of the Forster

radius, which is the distance at which energy transfer efﬁ ciency is 50 percent (Forster, 1948;

Lakowicz, 1999). For energy transfer between organic ﬂ uors, the Forster radius is usually no

more than about 15–20 Å. This means that for FRET signaling to occur with high retention of

ﬂ uorescence yield, the donor and acceptor molecules must be in extreme proximity, held much

closer than the radius of the average globular protein.

Lanthanide chelates have distinct advantages in FRET systems, because their Forster radius

can be on the order of 80–100 Å. This means that two biological molecules coming together in

solution, one labeled with a donor lanthanide chelate and the other labeled with an acceptor

organic ﬂ uor, usually are positioned close enough to undergo efﬁ cient energy transfer. Thus,

lanthanide TR-FRET assays can be developed that are completely homogeneous in nature;

taking advantage of both the long lifetime of ﬂ uorescence and the efﬁ cient energy transfer

characteristics of a long Forster radius. For instance, an antibody labeled with a europium

9. Lanthanide Chelates for Time-Resolved Fluorescence 477

478 9. Fluorescent Probes

chelate can be used along with another antibody labeled with an organic ﬂ uorescent label able

to absorb energy from the lanthanide. If both antibodies recognize different epitopes on a tar-

get antigen, then a solution phase assay can be designed wherein the FRET signal is observed

only if both antibodies are bound to the antigen. The excess labeled antibodies in solution

still will be free to diffuse throughout the sample and thus not be held in enough proximity to

undergo energy transfer. The result is low background interference, even though excess ﬂ uor is

not washed away.

To make a luminescence resonance energy transfer (LRET) system work, the proper choice

of acceptor ﬂ uor must be made for the lanthanide chelate. The acceptor must have an excita-

tion band corresponding to or overlapping an emission band of the lanthanide label, so that

excitation energy may be transferred. Although energy transfer is never 100 percent efﬁ cient as

would be evident by a complete disappearance of the lanthanide emission band, the efﬁ ciency

can be high. To maximize the yield of ﬂ uorescence transfer, the acceptor also should be an efﬁ -

cient absorber with a high extinction coefﬁ cient and have bright ﬂ uorescent properties (high

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an

organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an anti-

body labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in

solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if

they are held within close enough proximity to allow efﬁ cient FRET to occur. Under these conditions, excitation

of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not

bound to a target.

QY) to assure good signal in the ﬁ nal assay application. In this regard, one of the best acceptor

molecules for lanthanide luminescence is the phycobiliprotein, APC. APC often is paired with

europium chelates to create LRET assays. The protein ’s multiple ﬂ uorescent bilin groups

enhance its ability to receive energy transfer from the luminescence of the donor europium. In

an europium–APC system, excitation in the UV range with subsequent energy transfer results

in emission in the red region of the spectrum, giving an effective Stoke ’s shift of over 300 nm.

Combine the FRET signal with a time-gated measurement and the result is a highly sensitive

assay with virtually no interference from other biological or organic molecule ﬂ uorescence.

Organic ﬂ uorescent dyes with the appropriate spectral properties also can be paired with

lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye

Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate.

The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies

for developing speciﬁ c analyte assays. In addition, if using a terbium chelate as the donor, then

a Cy3 ﬂ uorescent dye can be used in assays as the acceptor.

Antenna-chelate molecules for lanthanide luminescence must have a reactive group to allow

conjugation with targeting molecules to be useful for TR-FRET in biological applications.

Some of the reactive groups that have been designed for polyaminocarboxylate chelates include

the amine-reactive isothiocyanate derivatives (Li and Selvin, 1997), thiol-reactive maleimide

groups, and pyridyl disulﬁ de groups (Chen and Selvin, 1999).

Two of the more common chelate structures are built from an EDTA or DTPA backbone

(Chapter 10, Section 1). Depending on the method of antenna group addition, the DTPA-

chelate structure can have a maximum of eight coordination groups consisting of carboxylates

and amines to hold the lanthanide atom. An EDTA-chelating group can have up to six coor-

dination groups per chelate molecule. The remaining coordination sites are taken up by water

molecules in aqueous solution, which potentially can quench luminescence, because they are

effective acceptors of excitation energy from the lanthanide. Both EDTA- and DTPA-chelating

groups with antenna molecules do perform well in luminescent assays. However, it is better

to have a chelating group that can coordinate with all or a majority of the nine coordination

bonds on a lanthanide metal ion. This avoids the potential for quenching effects from other

chelators in solution or from water itself. In this regard, reagents built from a DTPA-chelating

group potentially are better than those based on EDTA.

In some cases, the initial lanthanide-chelating group may not have an antenna group built

into its structure. One of the early commercial applications of europium luminescence in a TR-

FRET-based assay system used an isothiocyanatophenyl-EDTA or an isothiocyanatophenyl-

DTPA derivative to modify proteins (Delﬁ a system from Wallac). This system uses an

isothiocyanate chelate group to carry the lanthanide and attach it to antibodies and other mol-

ecules, but it doesn ’t have an antenna group. In an assay, this system requires the use of a

secondary ﬂ uorescence enhancer to create the lanthanide ﬂ uorescence signal. This secondary

reagent typically is  -naphthoyltriﬂ uoroacetone, which when added in excess at the end of an

assay extracts the lanthanide metal from the nonﬂ uorescing chelate and forms another soluble

chelate that is highly ﬂ uorescent.

More advanced designs incorporate efﬁ cient antenna groups directly into the chelating

structure of the label. For instance, the terpyridine- bis(methylenamine) tetraacetic acid (TMT)

chelator of europium developed by Saha et al. (1993) has multiple pyridine rings and two imino-

diacetic acid groups on each side to create a chelator having nine coordination sites. The pyrid-

ine groups provide the antenna structure and also supply nitrogen atoms with unshared pairs of

9. Lanthanide Chelates for Time-Resolved Fluorescence 479