Whispering Gallery Mode Biosensor

Transcription

1 Whispering Gallery Mode Biosensor Frank Vollmer*, Stephen Arnold #, Iwao Teraoka #, Albert Libchaber* *Rockefeller University, 1230 York Ave Box 155, New York, New York 10021, USA; # Polytechnic University, 6 Metrotech, Brooklyn, New York 11201, USA; We have developed a novel, fiber-optic biosensor which uses whispering gallery modes (WGMs) in dielectric microspheres for biomolecular detection. The WGMs are excited by evanescent coupling to an etch-eroded single mode optical fiber. WGM resonance positions are detected as Lorentzian dips in the intensity of the fiber-transmitted light. The polarization of molecules interacting with the evanescent field of the WGM on the microsphere surface leads to a red shift of a given WGM resonance wavelength. Chemical modification of the sensor surface allows specific detection of proteins, DNA and (nano-)particles such as viruses and bacteria. Introduction. Dielectric microspheres are ideal optical cavities because of their ability to confine light in a small volume over a long time period (high-q, up to ~10 9, Fig.1, Collot, 1993). For practical applications, the WGMs of microparticles are excited by evanescent coupling to a substrate such as eroded or tapered fibers, fiber half-couplers or prisms. Potential applications of such waveguide-sphere systems incluce low-threshold lasers, high-spectral purity filters for DWDM (dense wavelength division multiplexing), and integrated components for optical systems. WGMs are dependent on changes in refractive index, morphology and temperature and it has been suggested to use the evanescent field to probe the environment near the microsphere surface (Serpenguezel, 1995). Such fiber-optic microsphere sensors could be used in a variety Fig.1 of ways. Cavity ringdown measurements are applied in atmospheric sensing (Thompson, 2002), absorption by gases such as carbon dioxide and monoxide can be used in sensitve gas detectors (Rosenberger, 2001) and changes in the transmission characteristics of a microsphere-fiber system are predicted useful for biomolecular detection (Boyd, 2002). We are able to show that the perturbation of a WGM by polarizable molecules such as proteins and DNA leads to a sensitive increase of a given WGM resonance wavelength (Vollmer, 2002; Vollmer, 2003). A theory has been developed which relates the red shift of the wavelength to the surface density and polarizability of microsphere-bound or adsorbed molecules (Arnold, 2003; Teraoka 2003). In our novel resonant device, the light revisits an analyte molecule many thousand times (high-q) thus increasing the detection limit by orders of magnitude as compared existing single-pass sensors. Indeed, we are able to show a

2 sensitivity greater as compared to most commercial available surface plasmon detectors. Experimental Setup.The WGMs of a micronsized silica sphere are excited by evanescent coupling to an etch-eroded, single mode optical fiber (Fig.2a). A smf-28 fiber is etched in 25% hydrofluoric acid to a final diameter of 4 µm (fiber core diameter is 6.6 µm). Microspheres are fabricated by melting the tip of a piece of smf-28 fiber in a hot butane/nitrous oxide flame. Surface tension forms a spheroidal object. This sphere-on-a-stem is held in the sample cell which is mounted on a xyz stage and is moved towards the eroded part of the fiber. Coupling occurs upon mechanical contact (Fig.2b). Fig.2 spectrum. Amplitudes are different since coupling may vary %. Q-factors are on the order of 2 x Coupling of the spheroid to the fiber can lead to different resonant light orbits. The following fluorescent images (Fig.3) of orbits are taken using a 635 nm laser to excite WGMs of spheroids immersed in a fluorophore (Cy5) solution: Fig.3 The sample cell is built using two glass plates separated by two 4 mm rubber spacers on each end (Fig.2a). Experiments are done in solution: surface tension holds the liquid inbetween the glass plates. A tunable distributed feedback laser (~1340 nm wavelength) is coupled into one fiber end. The lasing wavelength is tuned by modulating the laser diode current with a sawtooth shaped function. The tuning coefficient has been determined as 0.01 nm/ma. The intensity transmitted through the fiber sphere system is recorded at the far end of the fiber using an InGaAs photodetector (Fig.2c). WGMs are identified as Lorentzian-dips in the The usual position of coupling the spheroid to the fiber is depicted in the upper left image. In this case the WGM orbit resembles that inside a corresponding sphere. To demonstrate the effect of adsorption of a typical protein (bovine serum albumin, BSA) to the sphere, the surface was chemically modified with an amino-silane agent. The so positively charged aminoilanated surface of the

3 sphere readily adsorbes the negatively charged protein. A labview program tracks the position of a resonant dip with a parabolic minimum fit. After injection of the protein BSA into the sample cell (filled with phosphate buffer), we observe an overall positive shift of the resonance position (Fig.4). This red shift of a given WGM resonance wavelength is entirely due to the adsorption of a monolayer of BSA protein. The initial negative wavelength shift is due to thermal contraction of the silica sphere as revealed by the temperature trace measured with a thermocouple proximal to the microsphere. Fig.4 with α... excess polarizability of BSA, σ...surface density of adsorbed BSA molecules, R...microsphere radius, ε...dielectric constant of the vacuum, the sphere and the buffer solution, respectively. Using this theory we predict from our wavelength shift of nm a surface density of 1.7 x adsorbed BSA molecules/cm 2. We were able to experimentally confirm the 1/ R size dependence using spheres of different sizes for our adsorption measurements (Fig.5). From the slope of this plot we are able to predict the smallest diameter of the BSA molecule as 3.6 nm, which compares well with crystallographic data (Arnold, 2003). Fig.5 δω/ω /R(µm -1 ) A first order perturbation theory (Arnold, 2003) predicts the fractional shift in wavelength ω/ω as: ω αexσ p ω ε ( ε ε )R rm 0 rs By coupling two microspheres (A and B, Fig.6a) to a common optical fiber we were able to show that each microsphere can be unambiguously identified by its own resonance wavelength (Fig.6b). To demonstrate a multiplexed measurement we modified each microsphere with its own DNA molecules. Sphere A was modified with a nucleic acid molecule (oligonucleotide) 11 bases in length. Sphere B was modified with an oligonucleotide which differed in only one of the 11 bases. Hybridization to the target oligonucleotide which is complementary to the one immobilized on sphere A should result in a much larger wavelength shift than hybridization to the mismatched sequence on sphere B which we could confirm experimentally (Fig.6c). Using the difference signal A-B we are able to discriminate the single nucleotide mismatch with a signal-to-noise of 54 (Fig.6d).

4 Fig.6a A B Modes in Microspheres by Protein Adsorption. Optics Letters 28, (2003). Boyd, R.W., Heebner, J.W. Sensitive disk resonator photonic biosensor. Applied Optics 40, (2002) Fig.6b Collot et.al. Very high Q whispering-gallery mode resonances observed on silica microspheres. Europhysics Letters 23, (1993) Rosenberger, A.T., Rezac, J.P. Whispering-gallery-mode evanescent-wave microsensor for trace-gas detection. Proc. SPIE 4265, (2001). Serpengüzel, A., Arnold, S., Griffel, G. Enhanced Coupling to Microsphere Resonances with optical fibers, Opt. Lett. 20, (1995). Teraoka, I., Arnold, S., Vollmer, F. Perturbation Approach to Resonance Shift of Whispering Gallery Modes in a Dielectric Microsphere as a Probe of a Surrounding Medium. in press Journal of the Optical Society B (2003). Fig.6c Thompson, J.E., Smith, B.W., Winefordner, J.D. Monitoring atmpspheric particulate matter through cavity ringdown spectroscopy. Anal. Chem., 74, (2002). Vollmer, F., Braun, D., Libchaber, A., Khoshsima, M., Teraoka, I., Arnold, S. Protein detection by optical shift of a resonant microcavity. Applied Physics Letters 80, (2002). Fig.6d Vollmer, F., Arnold, S., Braun, D., Teraoka, I., Libchaber, A. Multiplexed DNA Quantification by Spectroscopic Shift of Two Microsphree Cavities. Accepted Biophysical Journal (2003). References. Arnold S., Khoshsima, M., Teraoka, I., Holler, S., Vollmer, F. Shift of Whispering Gallery

5 Arnold S., Khoshsima, M., Teraoka, I., Holler, S., Vollmer, F. Shift of Whispering Gallery Modes in Microspheres by Protein Adsorption. Optics Letters 28, (2003). Boyd, R.W., Heebner, J.W. Sensitive disk reso nator photonic biosensor. Applied Optics 40, (2002) Collot et.al. Very high Q whispering-gallery mode resonances observed on silica microspheres. Europhysics Letters 23, (1993) Rosenberger, A.T., Rezac, J.P. Whispering-gal lery-mode evanescent-wave microsensor for trace-gas detection. Proc. SPIE 4265, (2001). Serpengüzel, A., Arnold, S., Griffel, G. Enhanced Coupling to Microsphere Resonances with optical fibers, Opt. Lett. 20, (1995). Teraoka, I., Arnold, S., Vollmer, F. Perturbation Approach to Resonance Shift of Whispering Gallery Modes in a Dielectric Microsphere as a Probe of a Surrounding Medium. in press Jour nal of the Optical Society B (2003). Thompson, J.E., Smith, B.W., Winefordner, J.D. Monitoring atmpspheric particulate matter through cavity ringdown spectroscopy. Anal. Chem., 74, (2002). Vollmer, F., Braun, D., Libchaber, A., Khoshsima, M., Teraoka, I., Arnold, S. Protein detection by optical shift of a resonant microcavity. Applied Physics Letters 80, (2002). Vollmer, F., Arnold, S., Braun, D., Teraoka, I., Libchaber, A. Multiplexed DNA Quantification by Spectroscopic Shift of Two Microsphree Cavities. Accepted Biophysical Journal (2003).