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Detection of trophic factor activated signaling molecules in cells by a compact fiber-optic sensor
Biosensors and Bioelectronics 20 (2004) 345–349
Detection of trophic factor activated signaling molecules in cells by a compact fiber-optic sensor Rakesh Kapoor a,∗ , Navjot Kaur a , Emmanuel T. Nishanth a , Stanley W. Halvorsen b , Earl J. Bergey a , Paras N. Prasad a a
Institute for Lasers, Photonics and Biophotonics, University at Buffalo, State University of New York, 458 NSC, Buffalo, NY 14260, USA b Department of Pharmacology and Toxicology, University at Buffalo, State University of New York, Buffalo, NY 14214, USA Received 11 December 2003; received in revised form 2 February 2004; accepted 4 February 2004 Available online 16 March 2004
Abstract This paper describes a highly sensitive method to detect trophic factor activated signaling molecules in cells using a compact fiber optic biosensor. The method is demonstrated by quantitative detection of phosphorylation of signal transducers and activators of transcription 3 (STAT3) in neuroblastoma cells. A single fiber-optic probe based on total internal reflection fluorescence sensing system is used. A 405 nm diode laser is used for evanescent wave excitation of immobilized labelled analyte on the probe surface. A compact charged coupled device (CCD) based spectrometer is used for recording the fluorescence signal. The method is two orders of magnitude more sensitive than the Western blotting technique. © 2004 Elsevier B.V. All rights reserved. Keywords: Fiber-optic; TIRF; STAT; Biosensor; Fluorescence; Evanescent wave; Diode laser
1. Introduction In recent years considerable research effort has been devoted to the development of fiber-optic biosensors because of their potential sensitivity, detection speed and adaptability to a wide variety of assay conditions (Mehrvar et al., 2000; Prasad, 2003). Fiber-optic biosensors are analytical tools in which a fiber-optic device serves as a transduction element. The usual aim is to produce a signal that is proportional to the concentration of a chemical or biochemical to which the biological element reacts. The bioactive compound could be an enzyme, an antibody, or a nucleic acid. The signal generated in such a sensor is the result of a specific interaction between the analyte and the immobilized bioactive compound. The signal measured by the fiber-optic system, upon analyte binding, can originate from surface changes in the absorbance, scattering or fluorescence. The total internal reflection fluorescence (TIRF), or fluorescence using evanescent-wave excitation is especially well suited for measuring the concentration of fluorescent ∗ Corresponding author. Tel.: +1-716-645-6800x2221; fax: +1-716-645-6945. E-mail address: [email protected] (R. Kapoor).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.02.004
molecules attached to an analyte (Hirschfeld and Block, 1984; Andrade et al., 1985; Shirver-Lake et al., 1995; Zhou et al., 1997). In this technique an analyte has to be selectively immobilized on the surface of the fiber probe. Several strategies have been used for immobilization on fiber-optic biosensors. The sandwich assay technique is one such strategy (Wyatt et al., 1992) in which one antibody is immobilized on the surface of the waveguide, and a second antibody labelled with a fluorescent dye is added to the bulk solution. In the absence of the antigen, the second antibody remains in solution and little fluorescence is observed. However upon addition of antigen, a “molecular sandwich” is formed on the waveguide and the labelled second antibody is within the evanescent-wave excitation volume. Evanescent-wave excites the labelled antibody and produces the characteristic fluorescence of the dye molecule attached to the antibody. The fluorescence signal from these labelled antibodies is proportional to the number of bound antigens. In this paper we have demonstrated that, based on sandwich assay binding and TIRF detection, a single fiber-optic probe can be used for detection of trophic factor activated signaling molecules in cells. Phosphorylation of specific amino acids is critical for the regulation of protein activity involved in many cellular processes. The method has been demonstrated by
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detecting the fraction of phosphorylated signal transducers and activators of transcription 3 (STAT3) in neuroblastoma cells. This quantitative method is rapid and sensitive. Ciliary neurotrophic factor (CNTF) induces phosphorylation of STAT3 on tyrosine residues. After stimulation, activated STATs dimerize and translocate to the nucleus and interact with specific DNA response elements. This regulates the expression of targeted genes (Darnell, 1997; Stahl et al., 1994), required for proper growth, maintenance and development of cells and tissues (Leonard and Oshea, 1998). In addition, STAT3 plays an important role in regulating both innate and acquired immunity. The constitutive activation of STAT3 is associated with cancer (Bromberg et al., 1999).
2. Materials All solvents and chemical were either of analytical grade or chemically pure. Aminopropyltriethoxysilane (APTS) was obtained from Sigma (Milwaukee, WI, USA). The immobilization reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) was obtained from Pierce (Rockford, IL, USA). Monoclonal anti-STAT3 antibody was obtained from Transduction Laboratories (Lexington, KY, USA) and polyclonal anti-phospho STAT3 (Tyr705) antibody was obtained from Cell Signaling Tech. (Beverly, MA, USA). Alexa fluor 430 dye was obtained from Molecular Probes (Eugene, OR, USA) and Centrispin-10 columns were from Princeton Separations (Adelphia, NJ, USA). 2.1. Cell culture and whole cell extraction BE (2)-C human neuroblastoma cells were grown in a 1:1 mixture of Ham’s F12 and Eagle’s minimal essential medium supplemented with 10% (v/v) fetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin (Kaur et al., 2002). Before treatment, cells were placed in serum free media for 2 h. Whole cell extracts were prepared either before or after CNTF stimulation (1 nM, 30 min). Cells were rinsed with phosphate buffered saline (PBS) thrice and extracted in lysis buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl2 , 0.2 mM EDTA, 420 mM NaCl, 0.2 mM PMSF, 1 mM DTT, 100 M Na3 VO4 , and 20% glycerol) by keeping on ice for 30 min (Bhattacharya and Schindler, 2003) then freeze-thawed to insure complete cell lysis. Lysates were centrifuged at 14 000 × g at 4 ◦ C and supernatants were collected. 2.2. Antibody-dye conjugation Anti-phospho-STAT3 (PY-STAT3) antibody (1.1 g/ml) and anti-STAT3 (1.2 g/ml) antibodies were conjugated with optimum concentration of Alexa Flour 430 dye. The reaction mixture was incubated for 2 h at room temperature with constant shaking. The labelled-antibody conjugates
were separated from free dye using gel filtration columns (Centrispin-10). 2.3. Western blotting Cell extract proteins were separated on 7.5% sodium dodecyl sulphate polyacrylamide gels before transfer to polyvinylidene difluoride membrane (ImmobilonP, Millipore). Proteins on immunoblots were probed with antiphospho-STAT3 antibodies as described previously (Kaur et al., 2002) using Amersham enhanced chemiluminescencePlus (ECL-Plus). Exposed films were scanned with an Epson 636 Professional Series scanner. 3. Probe preparation The probe was a 10 cm long 600 m core multimode optical fiber (Ocean Optics Inc.). Protective polymer (2.5 cm) surrounding the cladding of the fiber was removed from one end by heating. The fiber was then decontaminated by sonicating it in a soap solution (DeContam from ESPI). This was followed by sonicating the fiber in a solution of de-ionized water to get rid of any carbon soot on the surface of the fiber. The probe was then transferred to a solution of 10% hydrofluoric acid for the appropriate duration of time to etch the cladding. To immobilize antibodies tips were cleaned and neutralized in ethanol, and then the etched part of the fiber was silanized for 3 h with a 2% solution of APTS. After silanization, about 1 cm of the tip of the etched part of fiber was exposed to 400 l of 0.5 mM NHS-ASA in the dark for 1 h. Then the fiber was washed and rinsed with PBS (pH 7.2) to remove any unbound NHS-ASA from the fiber. This procedure minimized any non-specific binding of foreign molecules to the surface of the fiber, which would result in background fluorescence. The treated fiber tips were then placed in the anti-STAT3 antibody solution at a concentration of 2.5 g/ml for about 10–15 min and exposed to long wave UV light in order to initiate the photo activation of the cross-linker. This leads to the immobilization of anti-STAT3 antibody onto the probe surface. Anti-STAT3 has the same binding affinity for both the non-activated STAT3 protein and the activated STAT3 (PY-STAT3) protein. 4. Instrumentation A typical diagram of our setup is shown in Fig. 1. A 405 nm laser diode (Nichia, Japan) is used for excitation. A dichroic band pass filter (405 nm, band width 10 nm) was placed in front of the diode laser to block any red tail emission. The diode light passes through a dichroic beam combiner/splitter. The beam combiner/splitter is a long pass filter with high reflectivity for the excitation wavelength at 405 nm and high transmission at wavelengths longer than 480 nm. We used Alexa 430 as a fluorescence label with emission peak at 530 nm. Laser output is a collimated beam and a
R. Kapoor et al. / Biosensors and Bioelectronics 20 (2004) 345–349
347
Fig. 1. A schematic diagram of the experimental setup: ND, neutral density; BS, beam splitter; BF, bandpass filter; and LD, laser diode.
short focal length lens is used to focus the laser beam into a 600 m core fiber. The fiber-probe was connected to this fiber with the help of a detachable SMA connector (Thorlabs Inc., NJ, USA). This arrangement helped us in changing the probe fiber for different experiments. The analyte fluorescence couples back into the probe fiber and gets transmitted into the collection fiber of a miniature charged coupled device (CCD) based fiber-optic spectrometer (Ocean Optics, model HR2000). The signal from the spectrometer is coupled to a computer (IBM). All the spectra were collected with the help of this computer. The auto fluorescence generated in the transporting fibers can drastically reduce the signal to background ratio, therefore the choice of fiber is an important task. After testing several fibers it was found that a 600 m core fiber model ZDF VIS/NIR from Ocean Optics Inc. gave negligible auto fluorescence at the 405 nm excitation wavelength. For better coupling efficiency, the core diameter of the collection fiber was also chosen to be 600 m.
Fig. 2. Signals from two probes prepared in identical conditions. Both the probes were placed for an hour in Alexa 430 labelled anti-STAT3 antibody solution. Sharp peaks are mercury lines from leaked stray room light.
signal obtained from the extract of non-activated cells was much smaller than that obtained from the extract of activated cells. This confirms the ability of our technique to discriminate between the activated and non-activated cells. To quantitate the phosphorylation of STAT3 protein, the ratio of concentration of PY-STAT3 to that of total STAT3 (activated plus non-activated) was determined. A fiber probe with immobilized anti-STAT3 antibody on its surface was placed in protein extracts from activated cells. To saturate the probe we placed it in the solution for 3 h. Under saturating conditions the ratio of PY-STAT3 molecules to STAT3 molecules, attached to the immobilized antibodies on the probe surface, will correspond to their ratio in cell extract solution. After rinsing with PBS-Tween-20 the probe was kept in Alexa 430 labelled anti-PY-STAT3 solution for an
5. Results and discussions First, we compared the reproducibility of our probes. Two probes were prepared under identical conditions and were placed in for 3 h in whole cell protein extracts from stimulated cells. The probes were then rinsed with PBS containing 0.05% Tween-20 and placed for 1 h in a solution of Alexa 430 labelled antibodies, specific to phosphorylated STAT3 (anti-PY-STAT3). The fluorescence signals recorded from these probes are shown in Fig. 2. It can be seen that the signals obtained from the both the probes are nearly identical. Thus results obtained from the probes prepared under identical conditions were reproducible. Next the signals obtained from activated and non-activated cell extracts were compared using two probes prepared under identical conditions. The first probe was placed in extract solution from cells activated with 1 nM CNTF and the second in extract solution from non-activated cells, in each case for 3 h. After rinsing with PBS-Tween-20, both the probes were placed in Alexa 430 labelled anti-PY-STAT3 solution for 1 h. The spectra recorded from the probes are shown in Fig. 3. The
Fig. 3. Fluorescence spectrum from two probes. The higher amplitude signal was obtained from the probe kept in extract solution from activated cells while the lower amplitude signal is from the probe kept in extract solution from non-activated cells.
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hour. It was found that 1 h was sufficient to saturate the signal. After rinsing the probe with PBS-Tween-20, the fluorescence spectrum was recorded. The amplitude of this spectrum is proportional to the total PY-STAT3 concentration. Next the same probe is placed in Alexa 430 labelled second antibody anti-STAT3 solution for an hour. After rinsing the probe with PBS-Tween-20, the fluorescence spectrum was recorded again. The amplitude of this spectra is proportional to total concentration of STAT3 proten (activated plus non-activated). Since the same optical probe was used, the spectral recording conditions for both the signals were identical. Under identical conditions, if SPY is the signal corresponding to the PY-STAT3 protein and ST is the signal corresponding to the total STAT3 protein (activated plus non-activated), the ratio of the concentrations will be given as
Table 1 Measurement of PY-STAT3 fraction determined in different dilutions of whole cell protein extract (the cells were activated with 1nM concentration of CNTF)
CPY SPY = , CT [SPY + (DA /DN )(ST − SPY )]
Fig. 5. Western blotting technique: (A) unstimulated cells; (B) undiluted stimulated cell extract; (C) 1:5 times diluted stimulated cell extract; (D) 1:25 times diluted stimulated cell extract; (E) 1:625 times diluted stimulated cell extract.
(1)
where DA and DN are dye/antibody ratio for labelled anti-PY-STAT3and anti-STAT3, respectively. The dye/ antibody ratio of the labelled-antibody solutions was determined by measuring the absorbance at 430 and 280 nm using the following formula: [dye] 203 000 × [A430 ] = , [antibody] 16 000 × [(A280 − (0.28 × A430 )]
(2)
where the A430 and A280 represent the absorbance at wavelengths of 430 and 280 nm, respectively. The molar excitation coefficients of typical antibody and Alexa 430 are 203 000 and 16 000 cm−1 M−1 , respectively and 0.28 is the correction factor to account for absorption of the dye at 280 nm. In our experiment the ratio DA /DN was 1.05. Eq. (2) is valid if the signal corresponding to PY-STAT3 and STAT3 are recorded either with two identical fiber-probes or with same probe. Both the spectra recorded with our
Relative dilution
PY-STAT3 factor (%)
1 1:5 1:25 1:625
38 35 37 33
Average
(36 ± 2)
probe are shown in Fig. 4. The signal showing higher amplitude in Fig. 4 is proportional to the total STAT3 protein (activated plus non-activated) concentration. To estimate the fraction of PY-STAT3, all spectra were corrected for background, and the signal amplitude was computed from the area under the corrected spectral curve. The fraction of PY-STAT3 protein in the cell extract was obtained by using Eq. (1). The PY-STAT3 fraction in extract solution obtained from cells activated with 1 nM of CNTF, was found to be 38%. To further test the sensitivity of our method, the experiment was repeated to determine the PY-STAT3 fraction in three different dilutions (1:5, 1:25, and 1:625) of the extract solution obtained from cells activated with 1 nM of CNTF. We could detect signals for all the dilutions and the estimated fraction of activated STAT3 was unchanged with increasing dilution of cell extract solution (Table 1). The experiment was also repeated with 1:3125 dilution but we could not detect any signal above the background. For comparison purposes we determined PY-STAT3 signal for different dilutions of the cell extract using Western blotting (Fig. 5). We could detect signal corresponding only to the 1:5 dilution, but no signal was detected for 1:25 and 1:625 dilutions. This demonstrates that a single fiber optic probe can be used to measure tyrosine phosphorylation of STAT3 in neuroblastoma cells, with sensitivity orders of magnitude better than that provided by by a state-of-the-art Western blotting method.
6. Conclusion
Fig. 4. Fluorescence signal corresponding to the concentrations of PY-STAT3 and total STAT3 (activated + non-activated) in the extract solution from activated cells.
A rapid and sensitive quantitative detection of trophic factor activated signaling molecules in cells has been developed using a TIRF based fiber-optic method. The method is demonstrated by determining the fraction of tyrosine
R. Kapoor et al. / Biosensors and Bioelectronics 20 (2004) 345–349
phosphorylation of STAT3 protein induced in neuroblastoma cells. The complete assay takes only 2–4 h with the fiber-optic probe method, a significant improvement over the 2–3 days required by Western blotting technique. It is also demonstrated that the proposed fiber optic method is two orders of magnitude more sensitive than Western blotting. This technique can also be adapted for the quantitative detection of activation of other signaling proteins such as ERK1/2 (extracellular signal regulated protein kinase 1/2), p38/MAPK (p38/mitogen-activated protein kinase), JNK/SAPK1 (Jun N-terminal kinase/stress-activated protein kinase) and AKT/PI3K (AKT/phosphatidylinositol 3-kinase), using highly specific antibodies to activated proteins that are commercially available. Acknowledgements We thank Regeneron Pharmaceuticals (Terrytown, NY) for recombinant human CNTF. This work was supported by Universal Technology Corporation through contract #02-S470-020-C1, Infotonics through contract #Q550000 and Enhanced Center for Advanced Technology through subcontract #55415-00-01A. References Andrade, J.D., VanWagenen, R.A., Gregonis, D.E., Newby, K., Lin, J.N., 1985. Remote fiber-optic biosensors based on evanescent-excited fluo-
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roimmunoassay: concept and progress. IEEE Trans. Electron Devices ED-32 (7), 1175–1179. Bhattacharya, S., Schindler, C., 2003. Regulation of Stat3 nuclear export. J. Clin. Invest. 111 (4), 553–559. Bromberg, J.F., Wrzeszezynsta, M.H., Devgan, G., Zhoo, Y., Pestell, R.G., Albanese, C., Darnel, J.E., 1999. Stat3 as an oncogene. Cell 98, 295– 303. Darnell, J.E., 1997. STATs and gene regulation. Science 277, 1630– 1635. Hirschfeld, T.E., Block, M.J., 1984. Fluorescent immunoassay employing optical fiber in capillary tube, US Patent No. 4447546. Kaur, N., Wohlhueter, A.L., Halvorsen, S.W., 2002. Activation and inactivation of signal transducers and activators of transcription by ciliary neurotrophic factor in neuroblastoma cells. Cell. Signaling 14, 419– 429. Leonard, W.J., Oshea, J.J., 1998. Jaks and STATs-biological implications. Ann. Rev. Immunol. 16, 293–322. Mehrvar, M., Bis, C., Scharer, J.M., Young, M.M., Luong, J.H., 2000. Fiber-optic biosensors—trends and advances. Anal. Sci. 16, 677–692. Prasad, P.N., 2003. Introduction to Biophotonics. Wiley, New York, pp. 327–334. Shriver-Lake, L.C., Golden, J.P., Patonay, G., Narayanan, N., Ligler, F.S., 1995. Use of three longer-wavelength fluorophores with the fiber-optic biosensor. Sens. Actuators B 29, 25–30. Stahl, N., Boulton, T.G., Farruggella, T., Ip, N.Y., Davis, S., Witthuhn, B.A., Quelle, F.W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J.N., Yancopoulos, G.D., 1994. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science 263 (5143), 92–95. Wyatt, G.M., Lee, H.A., Morgan, M.R.A., 1992. Immunoassays for Food-Poisoning Bacteria and Bacterial Toxins. Chapman & Hall, London, pp. 29–67. Zhou, C., Pivarnik, P., Auger, S., Rand, A., Letcher, S., 1997. A compact fiber-optic immunosensor for Salmonella based on evanescent wave excitation. Sens. Actuators B 42, 169–175.
Detection of trophic factor activated signaling molecules in cells by a compact fiber-optic sensor Rakesh Kapoor a,∗ , Navjot Kaur a , Emmanuel T. Nishanth a , Stanley W. Halvorsen b , Earl J. Bergey a , Paras N. Prasad a a
Institute for Lasers, Photonics and Biophotonics, University at Buffalo, State University of New York, 458 NSC, Buffalo, NY 14260, USA b Department of Pharmacology and Toxicology, University at Buffalo, State University of New York, Buffalo, NY 14214, USA Received 11 December 2003; received in revised form 2 February 2004; accepted 4 February 2004 Available online 16 March 2004
Abstract This paper describes a highly sensitive method to detect trophic factor activated signaling molecules in cells using a compact fiber optic biosensor. The method is demonstrated by quantitative detection of phosphorylation of signal transducers and activators of transcription 3 (STAT3) in neuroblastoma cells. A single fiber-optic probe based on total internal reflection fluorescence sensing system is used. A 405 nm diode laser is used for evanescent wave excitation of immobilized labelled analyte on the probe surface. A compact charged coupled device (CCD) based spectrometer is used for recording the fluorescence signal. The method is two orders of magnitude more sensitive than the Western blotting technique. © 2004 Elsevier B.V. All rights reserved. Keywords: Fiber-optic; TIRF; STAT; Biosensor; Fluorescence; Evanescent wave; Diode laser
1. Introduction In recent years considerable research effort has been devoted to the development of fiber-optic biosensors because of their potential sensitivity, detection speed and adaptability to a wide variety of assay conditions (Mehrvar et al., 2000; Prasad, 2003). Fiber-optic biosensors are analytical tools in which a fiber-optic device serves as a transduction element. The usual aim is to produce a signal that is proportional to the concentration of a chemical or biochemical to which the biological element reacts. The bioactive compound could be an enzyme, an antibody, or a nucleic acid. The signal generated in such a sensor is the result of a specific interaction between the analyte and the immobilized bioactive compound. The signal measured by the fiber-optic system, upon analyte binding, can originate from surface changes in the absorbance, scattering or fluorescence. The total internal reflection fluorescence (TIRF), or fluorescence using evanescent-wave excitation is especially well suited for measuring the concentration of fluorescent ∗ Corresponding author. Tel.: +1-716-645-6800x2221; fax: +1-716-645-6945. E-mail address: [email protected] (R. Kapoor).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.02.004
molecules attached to an analyte (Hirschfeld and Block, 1984; Andrade et al., 1985; Shirver-Lake et al., 1995; Zhou et al., 1997). In this technique an analyte has to be selectively immobilized on the surface of the fiber probe. Several strategies have been used for immobilization on fiber-optic biosensors. The sandwich assay technique is one such strategy (Wyatt et al., 1992) in which one antibody is immobilized on the surface of the waveguide, and a second antibody labelled with a fluorescent dye is added to the bulk solution. In the absence of the antigen, the second antibody remains in solution and little fluorescence is observed. However upon addition of antigen, a “molecular sandwich” is formed on the waveguide and the labelled second antibody is within the evanescent-wave excitation volume. Evanescent-wave excites the labelled antibody and produces the characteristic fluorescence of the dye molecule attached to the antibody. The fluorescence signal from these labelled antibodies is proportional to the number of bound antigens. In this paper we have demonstrated that, based on sandwich assay binding and TIRF detection, a single fiber-optic probe can be used for detection of trophic factor activated signaling molecules in cells. Phosphorylation of specific amino acids is critical for the regulation of protein activity involved in many cellular processes. The method has been demonstrated by
346
R. Kapoor et al. / Biosensors and Bioelectronics 20 (2004) 345–349
detecting the fraction of phosphorylated signal transducers and activators of transcription 3 (STAT3) in neuroblastoma cells. This quantitative method is rapid and sensitive. Ciliary neurotrophic factor (CNTF) induces phosphorylation of STAT3 on tyrosine residues. After stimulation, activated STATs dimerize and translocate to the nucleus and interact with specific DNA response elements. This regulates the expression of targeted genes (Darnell, 1997; Stahl et al., 1994), required for proper growth, maintenance and development of cells and tissues (Leonard and Oshea, 1998). In addition, STAT3 plays an important role in regulating both innate and acquired immunity. The constitutive activation of STAT3 is associated with cancer (Bromberg et al., 1999).
2. Materials All solvents and chemical were either of analytical grade or chemically pure. Aminopropyltriethoxysilane (APTS) was obtained from Sigma (Milwaukee, WI, USA). The immobilization reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) was obtained from Pierce (Rockford, IL, USA). Monoclonal anti-STAT3 antibody was obtained from Transduction Laboratories (Lexington, KY, USA) and polyclonal anti-phospho STAT3 (Tyr705) antibody was obtained from Cell Signaling Tech. (Beverly, MA, USA). Alexa fluor 430 dye was obtained from Molecular Probes (Eugene, OR, USA) and Centrispin-10 columns were from Princeton Separations (Adelphia, NJ, USA). 2.1. Cell culture and whole cell extraction BE (2)-C human neuroblastoma cells were grown in a 1:1 mixture of Ham’s F12 and Eagle’s minimal essential medium supplemented with 10% (v/v) fetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin (Kaur et al., 2002). Before treatment, cells were placed in serum free media for 2 h. Whole cell extracts were prepared either before or after CNTF stimulation (1 nM, 30 min). Cells were rinsed with phosphate buffered saline (PBS) thrice and extracted in lysis buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl2 , 0.2 mM EDTA, 420 mM NaCl, 0.2 mM PMSF, 1 mM DTT, 100 M Na3 VO4 , and 20% glycerol) by keeping on ice for 30 min (Bhattacharya and Schindler, 2003) then freeze-thawed to insure complete cell lysis. Lysates were centrifuged at 14 000 × g at 4 ◦ C and supernatants were collected. 2.2. Antibody-dye conjugation Anti-phospho-STAT3 (PY-STAT3) antibody (1.1 g/ml) and anti-STAT3 (1.2 g/ml) antibodies were conjugated with optimum concentration of Alexa Flour 430 dye. The reaction mixture was incubated for 2 h at room temperature with constant shaking. The labelled-antibody conjugates
were separated from free dye using gel filtration columns (Centrispin-10). 2.3. Western blotting Cell extract proteins were separated on 7.5% sodium dodecyl sulphate polyacrylamide gels before transfer to polyvinylidene difluoride membrane (ImmobilonP, Millipore). Proteins on immunoblots were probed with antiphospho-STAT3 antibodies as described previously (Kaur et al., 2002) using Amersham enhanced chemiluminescencePlus (ECL-Plus). Exposed films were scanned with an Epson 636 Professional Series scanner. 3. Probe preparation The probe was a 10 cm long 600 m core multimode optical fiber (Ocean Optics Inc.). Protective polymer (2.5 cm) surrounding the cladding of the fiber was removed from one end by heating. The fiber was then decontaminated by sonicating it in a soap solution (DeContam from ESPI). This was followed by sonicating the fiber in a solution of de-ionized water to get rid of any carbon soot on the surface of the fiber. The probe was then transferred to a solution of 10% hydrofluoric acid for the appropriate duration of time to etch the cladding. To immobilize antibodies tips were cleaned and neutralized in ethanol, and then the etched part of the fiber was silanized for 3 h with a 2% solution of APTS. After silanization, about 1 cm of the tip of the etched part of fiber was exposed to 400 l of 0.5 mM NHS-ASA in the dark for 1 h. Then the fiber was washed and rinsed with PBS (pH 7.2) to remove any unbound NHS-ASA from the fiber. This procedure minimized any non-specific binding of foreign molecules to the surface of the fiber, which would result in background fluorescence. The treated fiber tips were then placed in the anti-STAT3 antibody solution at a concentration of 2.5 g/ml for about 10–15 min and exposed to long wave UV light in order to initiate the photo activation of the cross-linker. This leads to the immobilization of anti-STAT3 antibody onto the probe surface. Anti-STAT3 has the same binding affinity for both the non-activated STAT3 protein and the activated STAT3 (PY-STAT3) protein. 4. Instrumentation A typical diagram of our setup is shown in Fig. 1. A 405 nm laser diode (Nichia, Japan) is used for excitation. A dichroic band pass filter (405 nm, band width 10 nm) was placed in front of the diode laser to block any red tail emission. The diode light passes through a dichroic beam combiner/splitter. The beam combiner/splitter is a long pass filter with high reflectivity for the excitation wavelength at 405 nm and high transmission at wavelengths longer than 480 nm. We used Alexa 430 as a fluorescence label with emission peak at 530 nm. Laser output is a collimated beam and a
R. Kapoor et al. / Biosensors and Bioelectronics 20 (2004) 345–349
347
Fig. 1. A schematic diagram of the experimental setup: ND, neutral density; BS, beam splitter; BF, bandpass filter; and LD, laser diode.
short focal length lens is used to focus the laser beam into a 600 m core fiber. The fiber-probe was connected to this fiber with the help of a detachable SMA connector (Thorlabs Inc., NJ, USA). This arrangement helped us in changing the probe fiber for different experiments. The analyte fluorescence couples back into the probe fiber and gets transmitted into the collection fiber of a miniature charged coupled device (CCD) based fiber-optic spectrometer (Ocean Optics, model HR2000). The signal from the spectrometer is coupled to a computer (IBM). All the spectra were collected with the help of this computer. The auto fluorescence generated in the transporting fibers can drastically reduce the signal to background ratio, therefore the choice of fiber is an important task. After testing several fibers it was found that a 600 m core fiber model ZDF VIS/NIR from Ocean Optics Inc. gave negligible auto fluorescence at the 405 nm excitation wavelength. For better coupling efficiency, the core diameter of the collection fiber was also chosen to be 600 m.
Fig. 2. Signals from two probes prepared in identical conditions. Both the probes were placed for an hour in Alexa 430 labelled anti-STAT3 antibody solution. Sharp peaks are mercury lines from leaked stray room light.
signal obtained from the extract of non-activated cells was much smaller than that obtained from the extract of activated cells. This confirms the ability of our technique to discriminate between the activated and non-activated cells. To quantitate the phosphorylation of STAT3 protein, the ratio of concentration of PY-STAT3 to that of total STAT3 (activated plus non-activated) was determined. A fiber probe with immobilized anti-STAT3 antibody on its surface was placed in protein extracts from activated cells. To saturate the probe we placed it in the solution for 3 h. Under saturating conditions the ratio of PY-STAT3 molecules to STAT3 molecules, attached to the immobilized antibodies on the probe surface, will correspond to their ratio in cell extract solution. After rinsing with PBS-Tween-20 the probe was kept in Alexa 430 labelled anti-PY-STAT3 solution for an
5. Results and discussions First, we compared the reproducibility of our probes. Two probes were prepared under identical conditions and were placed in for 3 h in whole cell protein extracts from stimulated cells. The probes were then rinsed with PBS containing 0.05% Tween-20 and placed for 1 h in a solution of Alexa 430 labelled antibodies, specific to phosphorylated STAT3 (anti-PY-STAT3). The fluorescence signals recorded from these probes are shown in Fig. 2. It can be seen that the signals obtained from the both the probes are nearly identical. Thus results obtained from the probes prepared under identical conditions were reproducible. Next the signals obtained from activated and non-activated cell extracts were compared using two probes prepared under identical conditions. The first probe was placed in extract solution from cells activated with 1 nM CNTF and the second in extract solution from non-activated cells, in each case for 3 h. After rinsing with PBS-Tween-20, both the probes were placed in Alexa 430 labelled anti-PY-STAT3 solution for 1 h. The spectra recorded from the probes are shown in Fig. 3. The
Fig. 3. Fluorescence spectrum from two probes. The higher amplitude signal was obtained from the probe kept in extract solution from activated cells while the lower amplitude signal is from the probe kept in extract solution from non-activated cells.
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hour. It was found that 1 h was sufficient to saturate the signal. After rinsing the probe with PBS-Tween-20, the fluorescence spectrum was recorded. The amplitude of this spectrum is proportional to the total PY-STAT3 concentration. Next the same probe is placed in Alexa 430 labelled second antibody anti-STAT3 solution for an hour. After rinsing the probe with PBS-Tween-20, the fluorescence spectrum was recorded again. The amplitude of this spectra is proportional to total concentration of STAT3 proten (activated plus non-activated). Since the same optical probe was used, the spectral recording conditions for both the signals were identical. Under identical conditions, if SPY is the signal corresponding to the PY-STAT3 protein and ST is the signal corresponding to the total STAT3 protein (activated plus non-activated), the ratio of the concentrations will be given as
Table 1 Measurement of PY-STAT3 fraction determined in different dilutions of whole cell protein extract (the cells were activated with 1nM concentration of CNTF)
CPY SPY = , CT [SPY + (DA /DN )(ST − SPY )]
Fig. 5. Western blotting technique: (A) unstimulated cells; (B) undiluted stimulated cell extract; (C) 1:5 times diluted stimulated cell extract; (D) 1:25 times diluted stimulated cell extract; (E) 1:625 times diluted stimulated cell extract.
(1)
where DA and DN are dye/antibody ratio for labelled anti-PY-STAT3and anti-STAT3, respectively. The dye/ antibody ratio of the labelled-antibody solutions was determined by measuring the absorbance at 430 and 280 nm using the following formula: [dye] 203 000 × [A430 ] = , [antibody] 16 000 × [(A280 − (0.28 × A430 )]
(2)
where the A430 and A280 represent the absorbance at wavelengths of 430 and 280 nm, respectively. The molar excitation coefficients of typical antibody and Alexa 430 are 203 000 and 16 000 cm−1 M−1 , respectively and 0.28 is the correction factor to account for absorption of the dye at 280 nm. In our experiment the ratio DA /DN was 1.05. Eq. (2) is valid if the signal corresponding to PY-STAT3 and STAT3 are recorded either with two identical fiber-probes or with same probe. Both the spectra recorded with our
Relative dilution
PY-STAT3 factor (%)
1 1:5 1:25 1:625
38 35 37 33
Average
(36 ± 2)
probe are shown in Fig. 4. The signal showing higher amplitude in Fig. 4 is proportional to the total STAT3 protein (activated plus non-activated) concentration. To estimate the fraction of PY-STAT3, all spectra were corrected for background, and the signal amplitude was computed from the area under the corrected spectral curve. The fraction of PY-STAT3 protein in the cell extract was obtained by using Eq. (1). The PY-STAT3 fraction in extract solution obtained from cells activated with 1 nM of CNTF, was found to be 38%. To further test the sensitivity of our method, the experiment was repeated to determine the PY-STAT3 fraction in three different dilutions (1:5, 1:25, and 1:625) of the extract solution obtained from cells activated with 1 nM of CNTF. We could detect signals for all the dilutions and the estimated fraction of activated STAT3 was unchanged with increasing dilution of cell extract solution (Table 1). The experiment was also repeated with 1:3125 dilution but we could not detect any signal above the background. For comparison purposes we determined PY-STAT3 signal for different dilutions of the cell extract using Western blotting (Fig. 5). We could detect signal corresponding only to the 1:5 dilution, but no signal was detected for 1:25 and 1:625 dilutions. This demonstrates that a single fiber optic probe can be used to measure tyrosine phosphorylation of STAT3 in neuroblastoma cells, with sensitivity orders of magnitude better than that provided by by a state-of-the-art Western blotting method.
6. Conclusion
Fig. 4. Fluorescence signal corresponding to the concentrations of PY-STAT3 and total STAT3 (activated + non-activated) in the extract solution from activated cells.
A rapid and sensitive quantitative detection of trophic factor activated signaling molecules in cells has been developed using a TIRF based fiber-optic method. The method is demonstrated by determining the fraction of tyrosine
R. Kapoor et al. / Biosensors and Bioelectronics 20 (2004) 345–349
phosphorylation of STAT3 protein induced in neuroblastoma cells. The complete assay takes only 2–4 h with the fiber-optic probe method, a significant improvement over the 2–3 days required by Western blotting technique. It is also demonstrated that the proposed fiber optic method is two orders of magnitude more sensitive than Western blotting. This technique can also be adapted for the quantitative detection of activation of other signaling proteins such as ERK1/2 (extracellular signal regulated protein kinase 1/2), p38/MAPK (p38/mitogen-activated protein kinase), JNK/SAPK1 (Jun N-terminal kinase/stress-activated protein kinase) and AKT/PI3K (AKT/phosphatidylinositol 3-kinase), using highly specific antibodies to activated proteins that are commercially available. Acknowledgements We thank Regeneron Pharmaceuticals (Terrytown, NY) for recombinant human CNTF. This work was supported by Universal Technology Corporation through contract #02-S470-020-C1, Infotonics through contract #Q550000 and Enhanced Center for Advanced Technology through subcontract #55415-00-01A. References Andrade, J.D., VanWagenen, R.A., Gregonis, D.E., Newby, K., Lin, J.N., 1985. Remote fiber-optic biosensors based on evanescent-excited fluo-
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roimmunoassay: concept and progress. IEEE Trans. Electron Devices ED-32 (7), 1175–1179. Bhattacharya, S., Schindler, C., 2003. Regulation of Stat3 nuclear export. J. Clin. Invest. 111 (4), 553–559. Bromberg, J.F., Wrzeszezynsta, M.H., Devgan, G., Zhoo, Y., Pestell, R.G., Albanese, C., Darnel, J.E., 1999. Stat3 as an oncogene. Cell 98, 295– 303. Darnell, J.E., 1997. STATs and gene regulation. Science 277, 1630– 1635. Hirschfeld, T.E., Block, M.J., 1984. Fluorescent immunoassay employing optical fiber in capillary tube, US Patent No. 4447546. Kaur, N., Wohlhueter, A.L., Halvorsen, S.W., 2002. Activation and inactivation of signal transducers and activators of transcription by ciliary neurotrophic factor in neuroblastoma cells. Cell. Signaling 14, 419– 429. Leonard, W.J., Oshea, J.J., 1998. Jaks and STATs-biological implications. Ann. Rev. Immunol. 16, 293–322. Mehrvar, M., Bis, C., Scharer, J.M., Young, M.M., Luong, J.H., 2000. Fiber-optic biosensors—trends and advances. Anal. Sci. 16, 677–692. Prasad, P.N., 2003. Introduction to Biophotonics. Wiley, New York, pp. 327–334. Shriver-Lake, L.C., Golden, J.P., Patonay, G., Narayanan, N., Ligler, F.S., 1995. Use of three longer-wavelength fluorophores with the fiber-optic biosensor. Sens. Actuators B 29, 25–30. Stahl, N., Boulton, T.G., Farruggella, T., Ip, N.Y., Davis, S., Witthuhn, B.A., Quelle, F.W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J.N., Yancopoulos, G.D., 1994. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science 263 (5143), 92–95. Wyatt, G.M., Lee, H.A., Morgan, M.R.A., 1992. Immunoassays for Food-Poisoning Bacteria and Bacterial Toxins. Chapman & Hall, London, pp. 29–67. Zhou, C., Pivarnik, P., Auger, S., Rand, A., Letcher, S., 1997. A compact fiber-optic immunosensor for Salmonella based on evanescent wave excitation. Sens. Actuators B 42, 169–175.