Label-Free Detection of Single Protein Molecules Using Deep UV

dues in Ecβ Gal protein as well as fluorescence correla- ..... Time-resolved fluorescence decay of Ecβ Gal in sodium phosphate solution excited at 2...
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Anal. Chem. 2006, 78, 2732-2737

Label-Free Detection of Single Protein Molecules Using Deep UV Fluorescence Lifetime Microscopy Qiang Li and Stefan Seeger*

Physikalisch-Chemisches Institut der Universita¨t Zu¨rich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland

We present the detection of single β-galactosidase molecules from Escherichia coli (Ecβ Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecβ Gal protein as well as fluorescence correlation spectroscopy of Ecβ Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence. Laser-induced fluorescence detection of single fluorescent molecules represents the ultimate level of sensitivity for fluorescence-based assays in analytical chemistry, molecular biology, biotechnology, and medical diagnostics. The most common method depends on the use of fluorescent tags, which can be excited by one-photon excitation in the visible range. So far, only a few reports have been published about ultrasensitive detection of dyes, biomolecules, and cells in the wavelength region of near UV to deep UV.1-4 Brand et al.1 investigated single Coumarin120 dye detection by confocal fluorescence microscopy with onephoton excitation (OPE) in the near UV (350 nm) as well as twophoton excitation at 700 nm. Schneckenburger et al.3 recorded autofluorescence lifetime imaging of cultivated cells using novel picosecond laser diode with 375-nm wavelength. Recently we demonstrated single UV dye molecule detection in the deep UV region after one-photon excitation at 266 nm.4 The fluorescence intensity and fluorescence lifetime images of individual 2,2′′′dimethy-p-quaterphenyl (BMQ) dye molecules have been obtained with a pulsed UV laser. This UV laser-based fluorescence lifetime imaging microscopy is suited for intrinsic fluorescence detection of biological macromolecules after UV excitation as well. The autofluorescence of biological samples results from the intrinsic fluorescence of proteins, coenzymes, and other components of body fluids. In proteins, the three aromatic amino acid * To whom correspondence should be addressed: Fax: +41-44-6356813. E-mail: [email protected]. (1) Brand, L.; Eggeling, C.; Zander, C.; Drexhage, K. H.; Seidel, C. A. M. J. Phys. Chem. A 1997, 101, 4313-4321. (2) Wennmalm, S.; Blom, H.; Wallerman, L.; Rigler, R. Biol. Chem. 2001, 382, 393-397. (3) Schneckenburger, H.; Wagner, M.; Weber, P.; Strauss, W. S. L.; Sailer, R. J. Fluoresc. 2004, 14, 649-654. (4) Li, Q.; Ruckstuhl, T.; Seeger, S. J. Phys. Chem. B 2004, 108, 8324-8329.

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residuesstryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe)smay contribute to their intrinsic fluorescence when excited in the ultraviolet region of 260-280 nm.5 Usually, single protein molecule detection relies on the use of a large variety of highly efficient fluorescent labels in the visible spectral range. One advantage of fluorescent labeling is the extremely high sensitivity, which enables the investigation in some instances down to the single-molecule level. But the addition of dye tags is timeconsuming, may change the environment of the molecule under investigation, and may produce unknown conformational effects. In such situations, the possibility of using the native fluorescence of a molecule is a considerable improvement. In recent years, autofluorescence has been used increasingly for diagnosis of cells and tissues for tumor detection.6-11 Autofluorescence-based diagnosis has significant advantages with respect to exogenous fluorescence-based techniques. It can be applied, in principle, to all kinds of tumors, whereas the latter ones can be used only for those tumors and photosensitizers for which selective uptake occurs. Recently, the intrinsic fluorescence emission of biomolecules has been investigated in some research groups by means of OPE12-20 and two- or three-photon excitation.21-23 The autofluorescence of single proteins such as blue (BFP), cyan (CFP), (5) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum Press: New York, 1999. (6) Gschwend, M. H.; Ruedel, R.; Strauss, W. S. L.; Sailer, R.; Brinkmeier, H.; Schneckenburger, H. Cell. Mol. Biol. 2001, 47, OL95-OL104. (7) Horvath, K. A.; Schomacker, K. T.; Lee, C. C.; Cohn, L. H. J. Thorac. Cardiovasc. Surg. 1994, 107, 220-225. (8) Anidjar, M. J. Biomed. Opt. 1996, 1, 335-341. (9) Croce, A. C.; Spano, A.; Locatelli, D.; Barni, S.; Sciola, L.; Bottiroli, G. Photochem. Photobiol. 1999, 69, 364-374. (10) Colasanti, A.; Kisslinger, A.; Fabbrocini, G.; Liuzzi, R.; Quarto, M.; Riccio, P.; Roberti, G.; Villani, F. Lasers Surg. Med. 2000, 26, 441-448. (11) Rigacci, L.; Alterini, R.; Bernabei, P. A.; Ferrini, P. R.; Agati, G.; Fusi, F.; Monici, M. Photochem. Photobiol. 2000, 71, 737-742. (12) Pierce, D. W.; Hom-Booher, N.; Vale, R. D. Nature 1997, 388, 338. (13) Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, W. E. Nature 1997, 388, 355-358. (14) Iwane, A. H.; Funatsu, T.; Harada, Y.; Tokunaga, M.; Ohara, O.; Morimoto, S.; Yanagida, T. FEBS Lett. 1997, 407, 235-238. (15) Sako, Y.; Minoghchi, S.; Yanagida, T. Nat. Cell Biol. 2000, 2, 168-172. (16) Harms, G. S.; Cognet, L.; Lommerse, P. H. M.; Blab, G. A.; Kahr, H.; Gamsjager, R.; Spaink, H. P.; Soldatov, N. M.; Romanin, C.; Schmidt, T. Biophys. J. 2001, 81, 2639-2646. (17) Harms, G. S.; Cognet, L.; Lommerse, P. H. M.; Blab, G. A.; Schmidt, T. Biophys. J. 2001, 80, 2396-2408. (18) Iino, R.; Koyama, I.; Kusumi, A. Biophys. J. 2001, 80, 2667-2677. (19) Garcia-Parajo, M. F.; Koopman, M.; van Dijk, E. M.; Subramaniam, V.; van Hulst, N. F. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14392-14397. (20) Sanchez-Mosteiro, G.; Koopman, M.; van Dijk, E. M. H. P.; Hernando, J.; van Hulst, N. F.; Garcia-Parajo, M. F. ChemPhysChem 2004, 5, 1782-1785. (21) Gryczynski, I.; Malak, H.; Lakowicz, J. R.; Cheung, H. C.; Robinson, J.; Umeda, P. K. Biophys. J. 1996, 71, 3448-3453. 10.1021/ac052166u CCC: $33.50

© 2006 American Chemical Society Published on Web 03/15/2006

Figure 1. Experimental setup for deep UV laser-based fluorescence lifetime microscopy system.

green (GFP), yellow (YFP), and red fluorescent protein (DsRed) have been investigated by OPE when excited in the visible range.12-20 Lippitz et al.23 reported results on two-photon excitation microscopy of hemocyanin and avidin-coated spheres by using intrinsic Trp emission. They suggested that under favorable conditions Trp-containing biomolecules can be investigated at the single-molecule level without introducing artificial dye labels. Until now, there is no report on single-molecule detection (SMD) of Trp or proteins containing Trp by means of their intrinsic fluorescence emission after one-photon excitation. The major problem is the limited photostability of Trp molecules. β-Galactosidase from Escherichia coli (Ecβ Gal) containing 156 Trp residues is a good candidate for UV OPE investigations. β-Galactosidase is an enzyme exhibiting broad substrate specificity; it is wide distributed in microorganisms, animals, and plants.24 The enzyme is a tetramer of four identical subunits with a monomer molecular mass of 116 352. Within each subunit, the 1023-amino acid polypeptide chain folds into five compact sequential domains, plus an extended segment of ∼50 residues at N-terminus. Each of the four active sites in the tetramer is formed by elements from two different subunits. Ecβ Gal catalyzed the hydrolysis of wide variety of β-galactosides, including natural substrates such as lactose and artificial substrates such as o-nitrophenyl-β-galactoside.24 Recently, many research groups have investigated the ultrasensitive detection of Ecβ Gal by means of bioluminescent assay and capillary electrophoresis laser-induced fluorescence detection based on the catalytic activity of Ecβ Gal.25-29 The limits of detection for Ecβ Gal has found from few (22) Maiti, S.; Shear, J. B.; Williams, R. M.; Zipfel, W. R.; Webb, W. W. Science 1997, 275, 530-532. (23) Lippitz, M.; Erker, W.; Decker, H.; Van Holde, K. E.; Basche, T. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2772-2777. (24) Jacobson, R. H.; Zhang, X. J.; DuBose, R. F.; Matthews, B. W. Nature 1994, 369, 761-766. (25) Rondelez, Y.; Tresset, G.; Tabata, K. V.; Arata, H.; Fujita, H.; Takeuchi, S.; Noji, H. Nat. Biotechnol. 2005, 23, 361-365. (26) Eggertson, M. J.; Craig, D. B. Biomed. Chromatogr. 1999, 13, 516-519.

thousand molecules down to single molecules. An individual Ecβ Gal molecule can catalyze the formation many product molecules in a short period, and the instrument can detect a very few of such product molecules. However, it must be noted that the Ecβ Gal itself was not detected but products formed by the activity of the single Ecβ Gal. In this paper, we present our results of time-resolved fluorescence identification of single tryptophan-containing proteins by means of UV fluorescence lifetime microscopy. We studied native fluorescence decay and photon bursts of single Ecβ Gal molecules in aqueous solution by a time-correlated single-photon counting method. The average number of Ecβ Gal protein molecules in the detection volume obtained from fluorescence correlation spectroscopy (FCS) measurements proves that we indeed observe single-molecule events. We are able to demonstrate the ultrasensitive identification of single biological macromolecules by ultraviolet native fluorescence detection. EXPERIMENTAL SECTION UV Confocal Fluorescence Lifetime Microscopy. The experimental setup for UV confocal fluorescence lifetime microscopy is shown in Figure 1. It consists of a 266-nm UV mode-locked diode-pumped picosecond laser (GE-100-XHP-FHG, Time-Bandwidth Products Inc.). The laser system provides pulses with a duration of less than 10 ps and with a repetition rate of 40 MHz; maximum output power is 30 mW. The polarized laser beam was split 50/50 by a beam splitter (Laser Components GmbH) sending 50% into a high-speed photodiode module (PHD-400, Becker & Hickl GmbH, Berlin, Germany) which is used as deriving the synchronization signal for triggering of the time-correlated singlephoton counting module. The second beam passed an excitation filter (254WB25, Omega Optical) and is directed into the quartz (27) Craig, D. B.; Dovichi, N. J. Can. J. Chem. 1998, 76, 623-626. (28) Craig, D. B. Recent Res. Dev. Anal. Biochem. 2002, 2, 229-238. (29) Craig, D.; Arriaga, E. A.; Banks, P.; Zhang, Y.; Renborg, A.; Palcic, M. M.; Dovichi, N. J. Anal. Biochem. 1995, 226, 147-153.

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microscope objective (40×, NA ) 0.80, Partec GmbH, Mu¨nster, Germany) by a dichroic beam splitter (290DCLP, Omega Optical). The laser power was adjusted by inserting different neutral density filters (Melles Griot). Surface scans were performed by moving the sample with a motorized x,y-translation stage (Ma¨rzha¨user, Wetzlar, Germany). The fluorescence light was collected by the same objective and transmitted through the dichroic mirror. An achromatic lens (LAU-25-200, OFR Inc., 200-mm focal distance) focuses the light onto a pinhole. After the pinhole, the fluorescence emission is detected by a high-speed photomultiplier tube (PMT) detector head (PMH-100-6, Becker & Hickl GmbH). Two emission band-pass filters (330WB60, Omega Optical), one positioned directly after the lens, the other directly in front of the detector, discriminate fluorescence against scattered light. The signal pulses of the PMT was fed into a time-correlated single-photon counting (TCSPC) PC interface card (SPC-630, Becker & Hickl GmbH) to acquire time-resolved data. The time-correlated single-photon counting was performed in the reversed mode; that is, the signal of the PMT was used to start the clock of the time-to-amplitude converter, and the reference signal of the laser from high-speed photodiode was used as stop signal. Software written in C++ was developed for synchronization of scanning motion with the data acquisition. The instrument response function (IRF) was measured by replacing the sample with a scattering dispersion of colloidal silicon dioxide particles in water (particle size 11 nm) and then recording the Rayleigh scattering of the excitation light without two emission filters. With this setup, an IRF of 240 ps (fwhm) was measured. The fluorescence decay time constants were obtained by deconvoluting the instrument response function. Ecβ Gal Preparation. The pure β-galactosidase from E. coli was purchased from Sigma. All experiments were done at room temperature. The buffer used was 50 mM sodium phosphate solution (pH 7.0). To adjust the protein concentration we used the UV/visible/NIR spectroscopy Lambda 900 (Perkin-Elmer). Steady-state fluorescence spectra were measured in standard quartz cuvettes with a Luminescence spectrometer LS 50B (Perkin-Elmer). Time-resolved data of bulk solutions were measured at concentrations of 1 × 10-8 mol/L. Prior to use, the quartz cover slides (SPI Supplies, 170-µm thickness) were cleaned for 60 min in CHCl3 in an ultrasonic bath followed by washing with double-distilled water and then dried in nitrogen flow. Safety Considerations. Exposure to the UV radiation of the laser is harmful to eyes and skin. Wearing laser protection glasses for deep UV light is absolutely recommended. RESULTS AND DISCUSSION The β-galactosidase from E. coli. (Ecβ Gal) is a large homotetrameric enzyme with molecular mass of 465 412 Da. Each subunit contains 39 tryptophan residues. The amino acids tyrosine and phenylalanine also show intrinsic fluorescence when excited with deep UV radiation. However, the intrinsic fluorescence of Tyr is ∼100 times weaker than that of Trp due to a low extinction coefficient, while fluorescence emission of Phe is negligible because of the low extinction coefficient and low quantum efficiency. Additionally, the transmission of our emission filter set for the fluorescence of Tyr and Phe is substantially lower due to the blue-shifted emission maximum compared to Trp. Also, due to energy transfer from proximal Phe and Tyr to Trp in the same protein, the intrinsic fluorescence emission of Ecβ Gal protein is 2734 Analytical Chemistry, Vol. 78, No. 8, April 15, 2006

Figure 2. (a) Absorption and fluorescence emission spectra of Ecβ Gal in sodium phosphate solution (pH 7.0). Fluorescence emission was excited at 266 nm with 1 × 10-8 mol/L solution of Ecβ Gal. (b) Time-resolved fluorescence decay of Ecβ Gal in sodium phosphate solution excited at 266 nm together with the IRF. Corresponding fit revealed three-exponential fluorescence decay with mean lifetime of 3.81 ns. The weighted residuals of the fit are also shown in the lower panel.

dominated by Trp residues.5 The UV absorption and fluorescence emission spectra of Ecβ Gal in sodium phosphate solution are shown in Figure 2a. Maximum absorption of Ecβ Gal is at 282 nm with an extinction coefficient of 2.26 × 105 L mol-1 cm-1; the fluorescence quantum yield of Ecβ Gal is 0.18.30 The fluorescence emission spectrum of Ecβ Gal shows a maximum at 340 nm, compared to N-acetyl-L-tryptophamide, which has the emission maximum of 352 nm; the position of emission maximum of Ecβ Gal is blue-shifted, which indicates some of tryptophanyl residues are embedded in the buried or unrelaxed microenvironment. Figure 2b shows the fluorescence decay of Ecβ Gal in buffer solution together with IRF. The decay parameters were determined by least-squares deconvolution using multiexponential models, and their quality was judged by the reduced χ2 value and the randomness of the weighted residuals (Table 1). The Ecβ Gal shows a three-exponential decay with mean lifetime of 3.81 ns. The mean lifetime of protein is longer than those exhibited in aqueous tryptophan solutions, indicating that the tryptophanyl residues are shielded from the solvent. The structural and functional aspects of Ecβ Gal have been investigated by frequency domain measurement of the intrinsic (30) Lakowicz, J. R.; Shen, Y.; D’Auria, S.; Malicka, J.; Fang, J.; Gryczynski, Z.; Gryczynski, I. Anal. Biochem. 2002, 301, 261-277.

Table 1. Intrinsic Fluorescence Intensity Decay of β-Galactosidase from E. coli. (Ecβ Gal) in Sodium Phosphate Solution (pH 7.0) temp (°C) Bismuto et al.31 15 15 D’Auria et al.32 10 40 60 this work room temp

〈τ〉

R1

τ1

R2

τ2

R3

τ3

χ2

3.82 3.85 3.7 3.6 3.2 3.81

0.853 0.641 0.03 0.07 0.06 0.05

4.174 4.543 9.2 7.3 6.9 8.07

0.147 0.357 0.54 0.48 0.45 0.51

1.780 2.637 3.2 2.9 2.6 2.85

0.002 0.43 0.45 0.49 0.44

0.010 0.66 0.59 0.59 0.20

6.7 6.1 2.3 1.8 2.5 2.3

fluorescence decays of tryptophanyl residues.31,32 To obtain more conformational dynamics information of Ecβ Gal, the emission decay has been analyzed in terms of sum of few discrete exponential components. Bismuto et al.31 found that the fluorescence decays of Ecβ Gal can be described by either two- or threeexponential decays model with χ2 value of 6.7 and 6.1 at pH 7.0 and 15 °C. They cannot distinguish both models due to slightly different χ2 values. On the other hand, D’Auria et al.32 found that the tryptophan emission decays were well described by a threeexponential decays model in temperature range of 10-60 °C. Table 1 shows the multiexponential analysis results of Ecβ Gal obtained by our time-domain measurements using TCSPC techniques. The fluorescence decay of Ecβ Gal exhibits a threeexponential model with lifetime at 8.07, 2.85, and 0.20 ns, respectively. The results of three lifetime components are in agreement with the frequency-domain data reported by D’Auria,32 while the lifetime of each component is slightly different from that observed by frequency-domain measurement. These results also suggest that tryptophan residues located in different microenvironments and confirm the conclusion obtained by D’Auria. In confocal fluorescence microscopy, the probe volume is effectively an elongated cylinder, and its radius is determined by optical diffraction and its length by spherical aberration.33 The diffraction limited focus has a lateral diameter of 1.0 µm at half the intensity maximum (fwhm) deduced from the spots originating from single BMQ molecule images4 and extends ∼3 µm (fwhm). The cylindrical probe volume is thus estimated to be 5 × 10-15 L. This probe volume is expected to contain an average of only one analytical molecule in a 3 × 10-10 mol/L solution. In more dilute solutions, the detection events are increasingly dominated by single molecules because the probability for two or more molecules in the probe volume becomes negligibly small. To demonstrate the high sensitivity of the instrument, we measured the intensity fluctuations caused by single molecules traveling through the detection volume. The measurement was carried out at a fixed spot having one drop of Ecβ Gal solution on the quartz glass surface. Figure 3 shows typical signal tracks for the blank and for the concentration of 2 × 10-11 mol/L Ecβ Gal solution. At a concentration of 2 × 10-11 mol/L, on average 0.06 Ecβ Gal molecule are present in the probe volume. Distinct fluorescence (31) Bismuto, E.; Nucci, R.; Rossi, M.; Irace, G. Proteins: Struct., Funct., Genet. 1999, 35, 163-172. (32) D’Auria, S.; Di Cesare, N.; Gryczynski, I.; Rossi, M.; Lakowicz, J. R. J. Biochem. 2001, 130, 13-18. (33) Rigler, R.; Mets, U.; Widengren, J.; Kask, P. Eur. Biophys. J. 1993, 22, 169175.

Figure 3. Fluorescence signal observed from (a) sodium phosphate buffer solution and (b) 2 × 10-11 mol/L Ecβ Gal in buffer solution. Data acquisition was performed at a speed of 1000 data points/s (1ms integration time).

signals generating from single molecules are observed in our experiment. On the basis of background signal, we calculated a signal-to-noise (S/N) ratio of 3 at the most intense bursts. To our knowledge, the detection of individual protein molecules with intrinsic amino acid fluorescence by one-photon excitation at UV region in ambient conditions has not been demonstrated before. To get more evidence of single-molecule detection, we measured events of the fluorescence bursts at different concentrations of Ecβ Gal solutions. Figure 4 shows fluctuating fluorescence signals observed from (a) 1 × 10-9, (b) 5 × 10-10, (c) 5 × 10-11, (d) 2 × 10-11, (e) 1 × 10-11, and (f) 5 × 10-12 mol/L Ecβ Gal solution. At these concentrations, the average number of Ecβ Gal molecules in the detection volume is 3, 1.5, 0.15, 0.06, 0.03, and 0.015, respectively. Figure 5a shows the dependence of the baseline of fluorescence signal on difference concentrations of Ecβ Gal solutions. We found that the baseline of observed fluorescence burst rates and the maximum fluorescence burst rates increase at concentrations of Ecβ Gal solutions higher than 5 × 10-10 mol/ L. The increase in the baseline and maximum fluorescence burst rates implies that the detection events are dominated by more than one molecule because the probability for zero molecule in the probe volume becomes small by Poisson statistics. However, no concentration dependence of the baseline and the maximum fluorescence burst rates at concentrations of 5 × 10-11 mol/L and less is observed. The number of events for observing single molecules increases with increasing concentration of Ecβ Gal solution (Figure 5b). We found less events of fluorescence bursts at lower concentration and need to wait significantly longer to observe the single molecules traversing the focus volume (Figure 4f), which indicates that the probability of one Ecβ Gal molecule Analytical Chemistry, Vol. 78, No. 8, April 15, 2006

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Figure 5. (a) Dependence of fluorescence signal baseline on difference concentrations of Ecβ Gal solutions. (b) Number of fluorescence bursts vs Ecβ Gal concentration when the signal is determined by counting single molecules as described in the text.

Figure 4. Fluorescence photon bursts observed from (a) 1 × 10-9, (b) 5 × 10-10, (c) 5 × 10-11, (d) 2 × 10-11, (e) 1 × 10-11, and (f) 5 × 10-12 mol/L Ecβ Gal solution and (g) sodium phosphate buffer solution. Data acquisition was performed at a speed of 1000 data points/s (1-ms integration time).

occupying the detection volume is small at low concentration. The results demonstrate that the observed fluorescence signals are single-molecule events at concentrations less than 5 × 10-11 mol/ L. The S/N ratio observed for Ecβ Gal molecules at an integration time of 1ms is lower than those of the frequently used fluorescent dye. The fluorescence burst size obtained from single Ecβ Gal molecules is determined by such photophysical properties as the absorption cross section, the fluorescence quantum yield, the fluorescence lifetime, and triplet quantum yield as well as photostability. The low S/N ratio for SMD of Ecβ Gal molecules is mainly due to limited photostability of Ecβ Gal. Lippitz et al.23 have studied the ratio of the fluorescence quantum efficiency to the photobleaching quantum efficiency for Trp residues in hemocyanin (Hc) and avidin-coated spheres. They found that the average number of emission photons per Trp is about 5 and 180 for hemocyanin and avidin-coated spheres before bleaching, respectively. Taking into account the detection efficiency and the Trp content of hemocyanin (148 residues) and avidin-coated spheres (340 residues), out of one Hc molecule and latex sphere 4 and 380 photon counts can be detected before photobleaching, respectively. The fluorescence quantum efficiency of Ecβ Gal is ∼3 times higher than Hc molecule;23,30 if we assume Ecβ Gal has a photobleaching quantum efficiency similar to that of Hc, the 2736 Analytical Chemistry, Vol. 78, No. 8, April 15, 2006

Figure 6. Time-dependent fluorescence intensity of 2 × 10-9 mol/L Ecβ Gal with high irradiation energy at stationary solution. The average laser power was 2 mW. The inset shows residuals of doubleexponential decay fit to the data.

detector should acquire more photon counts in an Ecβ Gal experiment than that in Hc. However, the limited S/N ratio for SMD of Ecβ Gal molecules indicates some of the Trp residues photobleach during a 1-ms transit time across the probe volume. To prove the photobleaching behavior of Ecβ Gal, we measured the dependence of the fluorescence intensity on irradiation time with high laser power at stationary solution. Figure 6 shows typical data for 2 × 10-9 mol/L Ecβ Gal solution; the time dependence of the fluorescence intensity displays a prompt decrease and a more gradual decrease above background fluorescence intensity. We believe that the prompt fluorescence decrease reflects the fast photobleaching process of Ecβ Gal molecules in probe volume.

After the equilibrium of the number of incoming new molecules and exiting bleached molecules, the fluorescence intensity gradual decreases because the total fluorescent Ecβ Gal molecules in the sample are decreasing. This kinetic scheme can be described by two-exponential decay; the inset in Figure 6 shows weighted residuals of this fitting. Further, we used FCS as a method to measure the statistical average of the number of protein molecules in the detection volume for an aqueous Ecβ Gal solution. The fluctuations of the fluorescence count flow F(t) are described in terms of the normalized intensity autocorrelation function G(tc) defined as34

G(tc) )

〈F(t)F(t + tc)〉 〈F(t)〉2 1+

)

(

(1 - IB/I)2

x8N

)(

)

1 1 (1) 1 + 4Dtc/ω02 1 + 4Dtc/z02

Here N is the average number of molecules in the detection volume, D is the translational diffusion coefficient, and τD ) ω02/ 4D is the characteristic time for diffusion. ω0 and z0 are 1/e2 radius for the radial direction and the axial direction of the input laser beam, respectively. IB is the background signal intensity, and I is the total signal intensity. A typical normalized fluorescence correlation function of 5 × 10-11 mol/L Ecβ Gal solution is shown in Figure 7. By fitting this curve with the autocorrelation function (1), using a diffusion coefficient D ) 3.12 × 10-7 cm2/s for Ecβ Gal solution35 yields an average number of molecules N of 0.2 ( 0.1. This result is in good agreement with the calculated value of 0.15 obtained from the detection volume and the concentration of Ecβ Gal solution. For this average molecule number, there is only a small probability to find more than one molecule in the detection volume. Hence, these data show that we detect singlemolecule events in the experiments described. CONCLUSION We have demonstrated single tryptophan-containing proteins detection in the deep UV region after one-photon excitation for the first time. This was achieved by observing intrinsic fluorescence of tryptophan residues by means of deep UV laser-based (34) Thompson, N. L. In Topics in Fluorescence Spectroscopy: Techniques; Lakowicz, J. R., Ed.; Plenum Press: New York, 1991; Vol. 1, pp 337-374. (35) Sober, H. A. CRC handbook of biochemistry: selected data for molecular biology, 2nd ed.; The Chemical Rubber Co.: Cleveland, OH, 1970.

Figure 7. Normalized fluorescence correlation function G(tc) for 5 × 10-11 mol/L Ecβ Gal solution: recorded data (full circles) and fitted curve using eq 1 (solid line). The following parameters were obtained with background intensity of 4.3 kHz and a signal intensity of 5.4 kHz: baseline 1.001, N ) 0.2, τD ) 1.9 ms, ω0 ) 0.5 µm, and z0/ω0 ) 12.

fluorescence lifetime microscopy using the technique of TCSPC. The bursts of autofluorescence photons from Ecβ Gal protein and a statistical analysis using fluorescence correlation spectroscopy of Ecβ Gal supply the evidence to detect single protein molecules. The lifetime distribution data further deliver information of the microenvironment of the Trp residues in the protein. Although the S/N ratio is weak due to the strong photobleaching of tryptophan compared to the S/N ratio delivered by fluorescent dyes, the signal is significant. This work opens the direct labelfree detection of proteins. Further improvement of the optics, in particular the microscope objective, will allow us to increase the S/N ratio and will give access to a broader range of proteins containing less number of Trp residues. ACKNOWLEDGMENT This work was supported by the Swiss National Science Foundation.

Received for review December 8, 2005. Accepted February 21, 2006. AC052166U

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