Suppression of bulk fluorescence noise by combining near-field excitation and collection

Transcription

1 Supporting information for: Suppression of bulk fluorescence noise by combining near-field excitation and collection Md. Mahmud-Ul-Hasan 1,2, Pieter Neutens 2, Rita Vos 2, Liesbet Lagae 1,2, Pol Van Dorpe 1,2 1. KU Leuven, Department of Physics and Astronomy, Celestijnenlaan 200D, 3001 Leuven, Belgium. 2. imec, Kapeldreef 75, 3001 Leuven, Belgium 1. FDTD analysis A commercial-grade simulator (Lumerical Solutions, Inc) based on the Finite-Difference Time-Domain (FDTD) method was used to simulate the excitation and collection efficiencies. The values used in the FDTD simulation for different parameters are given below- Waveguide Material Ref. Index = 1.93 Waveguide Width = 340 nm Waveguide Height = 220 nm Substrate Material = Si Cladding Material = SiO 2 Solution Material Ref. Index = 1.33 (water) Bottom Cladding Height = 2.3 μm Top Cladding Height = varied according to the experimental condition Excitation Efficiency The excitation efficiency is defined as the ratio between the available excitation power in the evanescent field above a certain distance from the waveguide surface and the total power in the waveguide mode. To find the excitation efficiency for different separation between waveguide surface and solution, the upper cladding thickness was varied from 0 nm to 400 nm with a step of 10 nm. A fundamental TE mode was injected to the waveguide at 637 nm excitation wavelength. A frequency domain electric field profile monitor was placed to record the field profile at different co-ordinates in the plane orthogonal to the light propagation direction. The excitation efficiency at different position in near-field was then calculated from the electric field values by post-processing in Matlab.

2 Collection Efficiency Collection Efficiency, the fraction of the total fluorescence emission that couples back to the waveguide as a function of separation between waveguide surface and solution, was calculated by placing the electrical dipole source above the waveguide surface. To find the collection efficiency for a specific separation between molecule and waveguide surface, the upper cladding thickness was set to the separation value. To simulate the real experimental situation, dipoles were placed at 10 different heights away from the cladding surface. This simulates the bulk molecules separated from the waveguide surface. The simulations were always done for 3 different axis polarized dipole sources at each position. The average collection efficiency value from those 3 different polarization simulations is assumed to be collection efficiency from a molecule residing at that position. The same procedure has been repeated for 41 times to find the collection efficiencies for different separation values from 0 nm to 400 nm with a step of 10 nm.

3 2. Surface functionalization and Click Chemistry The monolayer of Atto-633 was bound to the waveguide surface using azido-terminated organo-silanization process and copper catalyzed click chemistry to form a bond between terminal azide (N N + -N - ) group and Alkyne (C C) modified Atto The silanized surface was submerged for 1h for click chemistry in a mixture solution of 140 µm Alkyne (C C) modified atto mm Tris (benzyltriazolylmethyl) amine in DMSO + 2 mm copper catalyst in DMSO mm Sodium-L-ascorbate in H2O. 2. A cleaning procedure was followed to get rid of any dyes that did not bind to the surface. 3. The surface was then submerged in HCl : H 2 O (1:100) solution for 15 minutes to get rid of any Cu left on the surface that might quench the fluorophores during experiment. 4. Finally, the samples were submerged in 600 µm DBCO-PEG blocking reagent solution for 1 hour to prevent any non-specific binding to the active azide site on the surface where atto-633 might not bind in the last step.

4 3. Finding surface and bulk contribution using Matlab optimization tool Figure 1 Near field collected spectra. Experimental value for bound Atto-633+ bulk Atto-680 (black curve). Matched curve obtained from the summation of separate near field spectra of Atto-633 and Atto-680 (red curve). Multiplication factors for best match were found by using built-in optimization tool of Matlab Figure 2 Far field collected spectra. Experimental value for bound Atto-633+ bulk Atto-680 (black curve). Matched curve obtained from the summation of separate near field spectra of Atto-633 and Atto-680 (red curve). Multiplication factors for best match were found by using built-in optimization tool of Matlab

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6 4. Calculating estimated bulk suppression Number of molecules / length = Number of molecules in to length away from the surface = Bulk fluorescence in Far-field collection from to, = = = (1) Where, is the fluorescence generated by a molecule at surface. is the exponential decay constant in the excitation efficiency curve (fig. 2). Following the same procedure one can find the bulk fluorescence in near-field collection compared to the a fluorophore at surface = 2 (2) Comparing equation (1) and equation (2), a suppression of 2 in bulk fluorescence with respect to the surface fluorescence in near field collection is expected in near field collection.

7 5. Bleaching of the surface dye Figure 3: near field collected spectra of surface bound Atto-633+unbound bulk Atto-680. Black curve with closed circle is the integrated spectra over 0-50 second. Red curve with open circle is the integrated spectra over second.

8 6. Waveguide based evanescent excitation and coupling Figure 4 Number of contributing molecules in fluorescence emission present in a virtual box shown in Figure 4 (above) with infinitely small volume with a length 2, width of and thickness of around the waveguide, = 2 (3) = concentration of the fluorescent molecules = waveguide height = waveguide width = effective 1/ decay length of the combined efficiency. This is the virtual thickness of the analyte layer above the waveguide which is sufficient to produce an equivalent result as infinitely thick layer. Average excitation intensity in evanescent field at position = = (4) Where, = fraction of total mode power available in the evanescent field above the surface. = average excitation intensity in the waveguide mode = (5) Where α and β are the propagation decay constants due to intrinsic waveguide loss and loss due to absorption by the analyte molecules respectively. is the excitation intensity at the entrance of the sensor. Combining equation (4) and (5) = (6) Power absorbed in to length along the propagation direction, Where σ is the absorption cross-section of the fluorophore = (7)

9 Emitted fluorescence power, Where is the quantum yield of the fluorophore using equation (7) and (8) we can say, = (8) = (9) Emitted power coupled back to the waveguide, Where, is the collection efficiency Using equation (3), (6) and (9) we get = = 2 (10) Emission power at detector, for the total sensor length of = = 2 (11) Where, = (12) = 2 (13) The collected fluorescence by a detector placed at the exit of the sensor with a length, Where, = = 1 = = 2 = (14) = 2 (15)

10 7. Effect of purcell enhancement We found a maximum 1.1 times increase in Quantum Yield at the waveguide surface due to the Purcell effect. However, in the context of this letter, it is important to consider how the Quantum Yield changes (black line with rectangle in fig. 5) in comparison to the collection efficiency (red line with closed circle in fig. 5) as a function of the distance between the molecule and waveguide surface. We found a negligible variation of quantum yield compared to the variation of collection efficiency as a function of distance away from the surface. Hence, the effect of radiative rate modification can be ignored in the context of this letter. Figure 5: Quantum Yield (black line with closed rectangle), Collection Efficiency (red line with closed circle), multiplication of Quantum Yield and Collection Efficiency (blue line with open circle) as a function of distance above the waveguide surface. All the values have been normalized to the respective values on the surface.