Tailor-Made Dyes for Fluorescence Correlation ... - CiteSeerX

Biol. Chem., Vol. 382, pp.495 – 498, March 2001 · Copyright © by Walter de Gruyter · Berlin · New York

Short Communication

Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)

Peter Czerney1,*, Frank Lehmann1, Matthias Wenzel1, Volker Buschmann2, Anja Dietrich2 and Gerhard J. Mohr3 1

Dyomics GmbH, Bioinstrumentezentrum, Winzerlaer Str. 2a, D-07745 Jena, Germany 2 Physikalisch-Chemisches Institut, Universität Heidelberg, INF 253, D-69120 Heidelberg, Germany 3 Center for Chemical Sensors, ETH-Technopark, Technopark Str. 1, CH-8005 Zurich, Switzerland * Corresponding author

Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes. Key words: Diode lasers / Fluorescent labels / Life time/ Quantum yield.

Recently, there has been a revolution in the use of fluorescence in biotechnology, biomedical research, and clinical diagnostics. Powerful new fluorophores, which have been developed using techniques known to both organic chemistry and molecular biology, are combined with automated imaging workstations to define the content, activity, and dynamics of various materials in the life sciences. The excitation of fluorophores by powerful low price light sources like diode lasers enables detection in the ‘optical window’ of biological tissue where absorption and autofluorescence of the sample, as well as light scattering, are reduced to a minimum (Imasaka, 1999). One of the newest fluorescence technologies is fluorescence correlation spectroscopy (FCS) (Eigen and Rigler, 1994). FCS allows molecular interactions to be studied at the single-molecule level. A convenient way to investigate such binding events is to label one of the binding partners with a fluorescent tag and to irradiate the resulting host/guest complex with a light beam of very high photon flux, such as a focused laser source. Other methods using long wavelength fluorescence are fluorescence in situ hybridization (FISH), DNA sequencing and high-throughput screening (HTS) and several multiplex techniques (Gwynne and Page, 2000).

These innovative applications have led to a new dye chemistry. At present, the design of functional fluorophores is the most significant and enticing field in dye chemistry (Daehne et al., 1998). There are several requirements for an ideal fluorescent label in biotechnology. First, the fluorophore should be soluble and stable in water because most measurements are performed in aqueous solution. This requirement already excludes the great majority of commercially available organic fluorophores because many of them are not water-soluble, and others decompose in water. Second, labels should possess a reactive group for covalent coupling to targets. Third, molar absorbances of potential labels should be quite high, because only the photons that are absorbed can be emitted as fluorescent light again. Fourth, labels should exhibit a high emission quantum yield to reduce detection limits of tagged species. Other, more special demands include thermal and photostability as well as tunable charge of the labels to avoid undesired electrostatic attraction between label and target. Until now there has been no single label that fulfills all the requirements described above. Thus the search for optimized fluorophors for bioanalytical application is currently a challenge in chemistry (Czerney and Ehricht, 2000; Czerney and Grummt, 2000). We developed the formerly unknown hemicyanines DY-630 and DY-635 (see Figure 1) especially for use in FCS and at 633 nm excitation. The synthesis can be performed in three steps with high yields followed by activation of the dye to the NHS-ester (Dyomics, Jena, Germany; patent pending). The structural difference between the two dyes is the bridging of the aminoalkyl group to the neighboring phenyl ring in the benzopyran moiety of DY-635. This structure variation causes a slight red shift in light absorbance and is known to increase the fluorescence quantum yield due to rigidization of the chromophore and fixing the π-orbital of the amino nitrogen atom parallel to the π-orbitals of the aromatic system. The tert-butyl substituent in ortho-position to the heterocyclic oxygen reduces the tendency of the dyes to form aggregates in aqueous solution, which would reduce signal intensity and solubility. Furthermore, this group increases the chemical stability of the chromophore because it is space-filling and prevents nucleophilic agents from attacking the positively-charged 2-position of the benzopyran unit. Additionally the tert-butyl group increases the electron density in the 2-position and there-

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Fig. 1 635.

P. Czerney et al.

Structures of the Fluorescent Labels DY-630 and DY-

fore reduces the probability of a nucleophilic attack even more. The negatively-charged sulfonate group at the indole moiety increases the water solubility of the dyes and sets the total charge of the molecule to zero. This minimizes the influence of the label on the electrostatic properties of the tagged target molecule, which play an important role in separation processes like electrophoresis. The number behind ‘DY’ indicates approximately the absorption maximum in water. Both dyes exhibit high molar absorbances of more than 120 000 l mol-1 cm-1 in the long wavelength maximum and have fluorescence quantum yields of around 5% in water with a Stokes' shift of about 25 nm. A typical spectroscopic feature for this type of chromophore is the additional absorption maxi-

Table 1

mum near 500 nm, which allows an excitation by the argon ion laser (488 nm emission) widely used in medical diagnostics. The new dyes have been successfully tested on a ConfoCor 2 fluorescence correlation microscope (Carl Zeiss, Jena, Germany). A He/Ne-laser with an output power of 5 mW, attenuated to 30 percent, was used as excitation source. The sample volume had an elliptic shape with a length of approximately 1 µm and a diameter of around 400 nm. A HFT 633 beam splitter filter was used as dichroic mirror, and emission light passed an LP 650 long pass filter and was detected by an avalanche photo diode. To evaluate the results the ConfoCor software version 2.5 was used. The amplitudes of the correlation curves were corrected for transient non-fluorescent states. At a concentration of 10-8 M optimal values of 30 CPM (counts per molecule in kHz) for DY-635 and of 20 CPM for DY-630 were found under these conditions. These count rates are poor compared to the often used Cy5™ (Amersham Pharmacia Biotech), for which we determined a value of 90 CPM under the same conditions. The obtained results for the free dyes correlate well with the fluorescence lifetimes determined by single photon counting, yielding 0.4 ns and 0.2 ns for DY-635 and DY630, respectively. The diffusion time of the new dyes (molecular mass of DY-630 and DY-635: approx. 650 g mol-1) is approximately 50 µs. For details see Table 1. The dyes show a strong increase in fluorescence intensity and quantum yield, respectively, in the presence of proteins. Figure 2 demonstrates the rise of the fluorescence signal when bovine serum albumin (BSA) is added to an aqueous solution of DY-635 (as free acid). We observed, depending on the amount of BSA added, an up to eight-fold fluorescence intensity compared to aqueous solution of the free dye. Together with increasing quantum yield a bathochromic shift of both absorption and emission maxima occurs. The absorption maximum

Spectroscopic and FCS Data of DY-630 and DY-635. Absorption [nm]

Emission [nm]

FCS data

Dye

PBS

PBS

Concentration [l mol-1]

Counts/molecule [kHz]

Diffusion time [µs]

DY-630 DY-6301 DY-630-BSA2 DY-630-avidin2 DY-635 DY-6351 DY-635-BSA4

621 645 641 637 634 652 653

652 660 657 660 664 670 673

10-8 – 10-8 10-8 10-8 – 10-8

19.9 n.d. 148.2 100.8 31.0 n.d. 129.4

52 n.d. 280 265 47 n.d. 270

1

After addition of an excess of BSA. Dye/protein ratio 0.6. 3 Dye/protein ratio 0.5. 4 Dye/protein ratio 1.1.

2

New Dyes for FCS

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Fig. 2 Selected Absorption and Emission Spectra of DY-635 in Water (Dotted Line) and Noncovalently Attached to Bovine Serum Albumin (BSA, Solid Line). The excitation wavelength was 600 nm, spectra were recorded on a Perkin-Elmer LS50B fluorimeter.

Fig. 3 Fluorescence Decay of Free and BSA-Bound DY-635 in Water. Measurements were performed with a TC-SPC spectrometer 5000U (IBH, Glasgow, UK). Excitation by a 635 nm diode laser with a repetition rate of 1 MHz. Fluorescence was detected at 690 nm.

changes thereby from 635 nm up to 652 nm, while the emission maximum shifts from 664 nm to 670 nm. Oswald et al. (1999) described a similar behavior for squaraine dyes, but the Stokes shift of their compounds was much smaller. Due to the incorporated NHS-ester function covalent coupling of the labels to biomolecules can be easily achieved. In bicarbonate buffer solution of pH 9.0 the reactive dyes bind to amino-modified DNA or a protein by stirring at room temperature for a couple of minutes. Separation of the conjugate from unreacted or hydrolized dye can be readily achieved by gel permeation chromatography. The conjugates of DY-630 and DY-635 with BSA, avidin and an amino-modified DNA-18 mer [H2N-(C2H2)65’-TGT AAA ACG ACG CGG AGT-3’] exhibit a remarkable increase in fluorescence intensity together with a much

longer emission lifetime and a slight red shift in absorption and emission compared to the free dye in aqueous solution. Figure 3 illustrates the rise in emission lifetime in the case of the labeled albumin, which was measured using time correlated single photon counting (Birch and Imhof, 1991). For details concerning decay behavior of the albumin conjugate see Table 2. A monoexponential decay with an emission lifetime of 1.7 ns was found for the labeled oligomer. For details of the obtained results for all observed conjugates refer to Tables 1 and 2. Together with the increased fluorescence intensity and emission lifetime a rise in the count rate by a factor of 4 to 7 for FCS experiments with the protein conjugates under the same measurement conditions as the free dyes were observed. The diffusion time, which increased by a factor of 4, indicates that the high molecular weight conjugates were detected. The described change of the optical properties of the covalent and noncovalent conjugates can be explained as follows. After binding to the target some vibrational and rotational degrees of freedom of the chromophore

Table 2 in PBS.

Emission Life Times of DY-Labels and Its Conjugates

DY-630 DY-630-BSA DY-630-DNA DY-635 DY-635-BSA DY-635-DNA

Decay time [ns]

Fractional intensity

0.2 2.6 2.2 0.44 0.29 2.9 1.4 1.7

1.0 1.0 1.0 0.904 0.096 0.769 0.231 1.0

χ2

1.137 1.28

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decrease due to electrostatic interaction with charged compartments of the biomolecule as already observed by Patonay and colleagues with squaric acid derivatives (Sophianopoulos et al., 1997). Thus the nonradiative deactivation of the excited state becomes less probable and fluorescence emission increases, which can be observed both in lifetime and quantum yield. After attachment to biomolecules the spectroscopic properties of the presented labels improve significantly. The thereby occuring bathochromic shift of about 20 nm makes the dyes fully compatible with the fluorescent label Cy5 (Amersham Pharmacia Biotech), which has been commercially available for some years and which is the standard fluorophore for the red spectral region. Thus the emission of the DY-labels matches the filter systems for red fluorescence widely used in bioanalytics and medical diagnostics.

Acknowledgements The authors thank Dr. Volker Jüngel, Carl Zeiss Jena GmbH, for performing the FCS measurements, and the research group of Prof. Ulrich-Walter Grummt, Institute of Physical Chemistry, University of Jena, Germany, for the steady-state fluorescence measurements. P. C., F. L. and M. W. thank the German Federal Ministry of Economics and Technology for financial support within the FUTOUR program (reference no. 03FU 176 B).

References Birch, D.J.S., and Imhof, R.E. (1991). Time-Domain Fluorescence Spectroscopy Using Time Correlated Single Photon Counting. In: Topics in Fluorescence Spectroscopy, Vol. 1 Techniques, J.R. Lakowicz, ed. (New York, London: Plenum Press), pp. 1– 88. Czerney, P., and Ehricht R. (2000). New functional π-systems as chromophors for bio-diagnostics. Innovationen durch Sensorik (Dresden, Germany: w.e.b.-Universitäts-Verlag), pp. 21 – 24. Czerney, P., and Grummt, U.-W. (2000). Signals from the world of molecules. BioTec 12, 34 – 36. Daehne, S., Resch-Genger, U., and Wolfbeis, O.S. (1998). NearInfrared Dyes for High Technology Applications (Dordrecht, The Netherlands: Kluwer Academic Publishers). Eigen, M. and Rigler, R. (1994). Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl. Acad. Sci. USA 91, 5740 – 5747. Gwynne P., and Page, G. (2000). Laboratory and technology trends: fluorescence and labeling. Science 288, 1081– 1091. Imasaka, T. (1999). Diode lasers in analytical chemistry. Talanta 48, 305 – 320. Oswald, B., Duschl, J., Patsenker, L., Wolfbeis, O.S. and Terpetschnig, E. (1999). Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels. Bioconjug. Chem. 10, 925 – 31. Sophianopoulos, A.J., Lipowski, J., Narayanan, N. and Patonay, G. (1997). Association of near-infrared dyes with bovine serum albumin. Appl. Spectroscopy 51, 1511 – 1515. Received June 15, 2000; accepted January 9, 2001