Whispering Gallery Mode Biosensors in the Low-Q Limit

A. Weller, F. C. Liu, R. Dahint, M. Himmelhaus*

Angewandte Physikalische Chemie, Universität Heidelberg, INF 253, 69120 Heidelberg, Germany

SUPPORTING INFORMATION

Estimation of mass sensitivity and detection limit of the methods listed in Table 2:

1.  Surface acoustic waves: Literature values for the mass sensitivity of surface acoustic wave sensors range from 6 to 20pg/mm2, while the sensing area is typically 5mm2 [[1],[2],[3]]. Accordingly, the detection limit is 30 – 100 pg.

2.  Micro cantilever: For the micro cantilever, a detection limit of 5.5fg on a sensing area of 12µm2 has been demonstrated [[4]]. Thus, a mass sensitivity of 458pg/mm2 is obtained.

3.  High-Q WGM: Here, a mass sensitivity of 6pg/mm2 has been reported achieved with a silica bead of 400µm in diameter [[5]]. Therefore, the detection limit is 3.0pg.

4.  Low-Q WGM: In the present article, we show that for adsorption of polyelectrolyte (PE) multilayers onto the surface of a polystyrene bead with 2µm in diameter, the shift in the WGM mode positions is 0.038nm per Ångstrom layer thickness. This value was determined from a linear fit to the data of Figure4, where the layer thickness was taken from ref.[22]. The latter was determined in-situ, while our WGM measurements on the coated particles were performed in the dry state. We, thus, expect a certain shrinkage of the layers during the drying process, which goes in line with an increase in the refractive index of the film. The optical density of the layer, however, i.e. the product "thickness x refractive index", can be assumed to stay constant, since we deal with an aliphatic system, where density-dependent dipole-dipole interactions are small due to the high spatial confinement of the s bonds. Therefore, we used the parameters of fully hydrated PE films as given in ref.[22] for calibration of the optical shift to the layer thickness and the corresponding refractive index for PE coatings of polystyrene (PS) beads in aqueous suspension as available from ref.[20] in the Mie simulations. Accordingly, for calculation of mass sensitivity and detection limit, we also have to use the density of the fully hydrated PE of 0.81g/cm3 as given in ref.[22]. Assuming that a WGM shift of 0.1nm is still discernible, which is about 5% of the FWHM width of a TE mode (cf. Table1), we obtain a mass sensitivity of 213pg/mm2 and a detection limit of 2.7fg. Alternatively, we can take the surface coverage data for 3 PAH/PSS layers from Table1 of ref.[20] and correlate it directly to the corresponding wavelength shift as displayed in the inset of Figure4. This yields (2.4 x 10-9g/mm2)/1.8nm x 0.1nm = 133pg/mm2 in reasonable agreement with the value determined by means of the neutron reflectometry data.

5.  Surface plasmons: The mass sensitivity as given in the literature amounts to 10pg/mm2, typically achieved on a macroscopic surface area [[6]]. In principle, the maximum lateral resolution that can be achieved is limited only by the propagation length of the plasmons, which is typically in the range from few to some tens of microns. This yields a theoretical detection limit of 10pg/mm2 x (2µm)2 = 0.04fg. In practise however, it has been not possible so far to achieve such high resolution due to the poor signal-to-noise ratio. Currently, SPR imaging systems achieve a resolution of (25µm)2, yielding a proven detection limit of 6.3fg [[7]].

6.  Localized surfave plasmons (large area): For the discussion of the performance of localized surface plasmons of metal particles, we distinguish those systems probing a large ensemble from those probing individual nanoparticles. For ensemble measurements, probably the work of Schatz, van Duyne and coworkers on surface-immobilized silver nanoparticles reports the highest sensitivity so far [[8]]. They extrapolate the detection limit to 1500 hexadecanethiol (HDT) molecules adsorbed on a single nanoparticle (NP). The AFM images of Figure5 in their article show that they have about 14.5NP/µm2, while the total probe area of their transmission measurements is given as 5mm2. This transforms into a macroscopic detection limit of 1.1x1011 HDT molecules per mm2, which corresponds to 47pg/mm2 given the molar mass of the thiolate as 257.5g/mole. With a probe area of 5mm2, this yields a detection limit of 253pg. Certainly, the probe area can be reduced by improving the sensitivity of the detection system. However, single NP spectroscopy by means of SNOM as proposed by the authors is probably not in close reach of state-of-the-art instrumentation.

7.  Localized surface plasmons (single NP): Dark field microscopy has proven to be a feasible way of performing spectroscopy on single metal nanoparticles [[9]]. However, so far few work has been published that can be used to determine experimental mass sensitivity and detection limit of this approach. We therefore refer to the article of Raschke et al., in which the authors perform Mie simulations of single gold nanoparticles and calculate the resonance shift as a function of molecular adsorption [[10]]. They prove their calculations by adsorbing biomolecules on the particles and find reasonable agreement between prediction and experiment. However, there is no independent proof given on the coverage of a single NP and the article does not state all details of the experiment, for example, with respect to the built-up of material on the surface in the immediate environment of the particle. Therefore, we treat the results of the simulations as theoretical sensitivity and detection limit in the following.

In the calculation, a gold nanoparticle of 40 nm in diameter is first coated with a 3nm thick shell with a refractive index of n=1.5, then with an additional layer of 2nm of same index. For the first shell, the Mie simulations yield a shift in the resonance peak (located at 2.28eV) of -16meV, i.e. 3.84nm, and for the second shell additional 7.5meV, i.e. 1.82nm. For an estimate of mass sensitivity and detection limit we assume that the shell is packed with bovine serum albumine (BSA), which is a good assumption for the first shell (since biotinylated BSA was used in the experiment) and a reasonable one for the second (where streptavidin was used, i.e. a protein with similar optical density). For determination of the density of BSA we use the data on thickness and packing density of a monolayer as determined by Arnold et al. [[11]], dBSA=3.6nm, sBSA=2.9x1012cm-2.

Given a molecular weight of MBSA=66kDa, we obtain rBSA=0.883g/cm3, which yields a total mass of BSA of 15.4ag in the first, and 12.8ag in the second shell. Assuming that a spectral shift of 0.5nm is still discernible (which corresponds to about 1% of the FWHM width of 57.5nm for the spectrum shown in Figure2b of the article), we arrive at a mass sensitivity of 398pg/mm2 for the first and 530pg/mm2 for the second shell, respectively. While these values are in line with the results obtained for other microscopic detection methods, the detection limits are extremely low due to the small area of a single particle. Here, we obtain 2ag for the first, and 3.52ag for the second shell, respectively.

8. Composite systems: In the literature, a number of composite systems combining thin metal films and metal nanoparticles separated by a thin dielectric isolation layer have been proposed for biosensing [[12],[13]]. For an estimate of the performance of such layered structures, we chose the system reported by Takei and coworkers [13], since it seems to be well characterized. For adsorption of a monolayer of octadecanethiol (ODT, MW 286.56 g/mol) from ethanolic solution onto gold-coated PS beads of 110 nm diameter a spectral shift of 6nm is reported [[14]]. This result was achieved with a macroscopic fiber probe. Hong and Kao report for the same system a fluctuation of the absorption peak position of 0.08nm at a lateral resolution of 25µm2 [[15]]. Assuming a surface area of 21.4Ǻ2/thiolate as in the case of densely packed self-assembled monolayers of alkanethiols on gold [[16]], the surface mass density of the ODT film amounts to 2.2ng/mm2. This translates into a mass sensitivity of the composite system of 55pg/mm2 assuming that a spectral shift of 0.15nm is still discernible. To determine the detection limit, we take into account that the nanoparticles cover about 54% of the underlying substrate [[17]] and calculate the total surface area of the nanoparticles located within the detection spot of (25µm)2 to finally obtain a value of 37fg.

[1]* Corresponding author. Present Address: Fujirebio, Inc., Hachioji-shi 192-031, Japan

Email:

. W. Welsch, C. Klein, M. von Schickfus, S. Hunklinger, Anal. Chem. 68, 2000-2004 (1996)

[2]. F. Josse, F. Bender, R. W. Cernosek, K. Zinszer, Anal. Chem. 73, 5937-5944 (2001)

[3]. D. W. Branch, S. M. Brozik, Biosens. Bioelectr. 19, 849-859 (2004)

[4]. N. V. Lavrik, P. G. Datskos, Appl. Phys. Lett. 82, 2697-2699 (2003)

[5]. F. Vollmer, S. Arnold, D. Braun, I. Teraoka, A. Libchaber, Biophys. J 85, 1974-1979 (2003)

[6]. U. Jönsson, L. Fägerstam, B. Ivarsson, B. Johnsson, R. Karlsson, K. Lundh, S. Läfas, B. Persson, H. Roos, I. Rönnberg, S. Sjölander, E. Stenberg, R. Stahlberg, C. Urbaniczky, H. Ostlin, and M. Malmqvist, Biotechniques 11, 620-623 (1991)

[7]. J. M. Brockman, B. P. Nelson, R. M. Corn, Annu. Rev. Phys. Chem. 51, 41-63 (2000)

[8]. M. Duval Malinsky, K. L. Kelly, G. C. Schatz, R. P. van Duyne, J. Am. Chem. Soc. 123, 1471-1482 (2001)

[9]. J. Yguerabide, E. E. Yguerabide, Anal. Biochem. 262, 157, 1998; C. Sönnichsen, T. Franzl, T. Wilk, G. von Plessen, J. Feldmann, O. Wilson, P. Mulvaney, Phys. Rev. Lett. 88, 077402 (2002)

[10]. G. Raschke, S. Kowarik, T. Franzl, C. Sönnichsen, T. A. Klar, J. Feldmann, A. Nichtl, K. Kürzinger, Nano Lett. 3, 935-938 (2003)

[11]. S. Arnold, M. Koshsima, I. Teraoka, S. Holler, F. Vollmer, Opt. Lett. 28, 272 – 274 (2003)

[12]. G. Bauer, F. Pittner, T. Schalkhammer, Mikrochim. Acta 131, 107 (1999)

[13]. M. Himmelhaus, H. Takei, Sens. Actuators B, 63, 24-30 (2000)

[14]. R. Dahint, E. Trileva, H. Acunman, U. Konrad, M. Zimmer, V. Stadler, M. Himmelhaus, Biosens. Bioelectron. 22, 3174-3181 (2007)

[15]. X. Hong, F.-J. Kao, Appl. Opt. 43, 2868-2873 (2004)

[16]. L. H. Dubois, B. R. Zegarski, R. G. Nuzzo, J. Chem. Phys. 98, 678-688 (1993)

[17]. M. Himmelhaus, H. Takei, Phys. Chem. Chem. Phys. 4, 496-506 (2002)