Suppression of Bulk Fluorescence Noise by Combining Waveguide

Feb 9, 2017 - A high surface to bulk fluorescence ratio is very useful in bioimaging, sensing, sequencing, and physical chemistry characterization. We...
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Letter pubs.acs.org/journal/apchd5

Suppression of Bulk Fluorescence Noise by Combining WaveguideBased Near-Field Excitation and Collection Md. Mahmud-Ul-Hasan,*,†,‡ Pieter Neutens,‡ Rita Vos,‡ Liesbet Lagae,†,‡ and Pol Van Dorpe†,‡ †

Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200D, 3001 Leuven, Belgium imec, Kapeldreef 75, 3001 Leuven, Belgium



S Supporting Information *

ABSTRACT: A high surface to bulk fluorescence ratio is very useful in bioimaging, sensing, sequencing, and physical chemistry characterization. We used the evanescent field of a photonic waveguide for highly localized excitation and collection of molecular fluorescence. As both near-field excitation and collection are strongly distance dependent, we were able to increase the surface to bulk fluorescence ratio significantly. We have also experimentally investigated the combined excitation and collection efficiency as a function of the position of the molecule in the near field. Finally, we formulated and experimentally verified a general condition for the waveguide−molecule interaction length for maximum optical efficiency of the device. KEYWORDS: near-field excitation, near-field collection, waveguide sensor, bulk fluorescence, evanescent sensing fluorescence generated during the evanescent excitation can be suppressed by distance-dependent near-field coupling. Although the scheme of excitation and collection by the same waveguide has been reported in a couple of publications on fluorescence9 and Raman sensing,10 the focus has been mainly on using the photonic waveguides as a mean of miniaturization for on-chip sensing applications. Here, we explore the possibility of using waveguides beyond the miniaturization aspect and rather use them as solution to the fundamental problem of background fluorescence noise in sensitive biosensing or single-molecule analysis. The experimental setup is illustrated in Figure 1. An input grating coupler has been used to couple 637 nm excitation light to a single-mode silicon nitride (SiN) strip waveguide. The input light excites the molecules residing within the evanescent field of the waveguide mode in the active sensor area. The subsequent emission from the molecules couples back to the waveguide. The fluorescence signal copropagating with the excitation signal is then coupled out of the chip using an output grating coupler. The light is collected and collimated by an objective. The collimated light passes through an appropriate filter set to the detector. We have investigated the excitation and collection efficiency as a function of position of the molecules in the near field above the waveguide. We deposited different thicknesses of SiO2 spacer layers (10−400 nm) using a PECVD process at 350 °C on top of our pilot-line processed chip described in the Methods section. This spacer was used as a controllable separation between waveguide and solution. During this step, a

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luorescence is widely used as the transduction medium for biosensing and physical chemistry characterization applications.1 A better surface to bulk fluorescence ratio is very useful for all these applications. In fluorescence-based surface biosensors, the target analyte molecules are captured on the surface. The emission from the fluorescently labeled captured molecules is detected and quantified to obtain the concentration. An improvement in surface to bulk ratio can help to speed up the measurement time in most current techniques, such as ELISA, FISH, next-generation DNA sequencing, and others by avoiding the washing steps currently needed. These washing steps are used to reduce the bulk fluorescence noise by removing the unbound fluorescent molecules floating in the bulk solution. A better surface to bulk ratio also means a shorter effective length along the vertical direction of the observation volume. Limiting the observation volume is useful for singlemolecule fluorescence correlation spectroscopy to isolate a few molecules.2,3 It is also instrumental for cell membrane studies. The evanescent field generated by the total internal reflection at the solid/liquid interface has been a good tool for surface−bulk separation.4,5 The on-chip version for compact devices to generate an evanescent field has been the usage of the waveguide mode.6 However, this is still far from an ideal situation, as there is a big mismatch between the typically used evanescent field that extends from 80 to 200 nm above the surface into the bulk7 compared to the typical biosensing layer that ends within 10 nm.8 We propose to use a photonic waveguide not only to excite the molecule in the near field but also to collect the subsequent emission in the near-field using the same waveguide. This way, both the excitation and collection efficiency have an exponential dependency on the molecule−waveguide distance. Hence, a big fraction of the bulk © XXXX American Chemical Society

Received: December 20, 2016 Published: February 9, 2017 A

DOI: 10.1021/acsphotonics.6b01016 ACS Photonics XXXX, XXX, XXX−XXX

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between waveguide and solution. The collection efficiency also depends exponentially on the distance between the molecule and waveguide surface with a 1/e decay length of 61 nm. The multiplication of these two curves (the black line with triangles) is the expected combined efficiency when both the excitation and collection are done with the same waveguide. The combined efficiency decreases exponentially at a faster rate with a 1/e decay length of 30 nm. To verify this experimentally, a droplet containing the Atto-633 fluorescent dye with a 100 μM concentration was applied on top of the waveguide with varying oxide spacer thicknesses. The fluorescent dyes were excited by the evanescent field of the waveguide mode, and the subsequent fluorescence emission coupled to the waveguide was detected through an output grating coupler. The output fluorescence power for varying separations between solution and waveguide surface has been compared for the same excitation power. The results in Figure 2 (black squares) matches very well with the simulated combined curve (the black line with triangles). It is clear from the result that using the same waveguide for excitation and collection benefits from the double-exponential dependency compared to the conventional technique, where only the excitation has the exponential dependency on surface−molecule distance. This combined excitation and collection technique reduces the effective 1/e decay length by half. We have investigated the possible impact of the Purcell effect on our findings. The fluorescence quantum yield (Q) changes due to the modification of the radiative decay rate in the presence of the waveguide mode in the near field. We found a maximum 1.1 times increase in Q at the waveguide surface. However, in the context of this Letter, it is important to consider how the quantum yield changes in comparison to the collection efficiency 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 from the surface (data are shown in section 7 of Supporting Information). Hence, the effect of radiative rate modification can be ignored in the context of this Letter. In addition, we have designed an experiment to quantify the surface to bulk fluorescence ratio improvement in the near-field collection compared to the conventional far-field collectionbased approach. We have set up an experiment that combines a monolayer of fluorescent dye (Atto-633) on the waveguide surface with a solution containing a different dye (Atto-680) (Figure 3a). This allows disentangling the bulk and surface contribution to the fluorescence. The detailed description of the monolayer binding can be found in the Methods section and in the section 2 of Supporting Information. The contribution of surface-bound Atto-633 dye versus unbound bulk Atto-680 dye in the emission spectra collected in the far field and near field has been compared. Figure 3a illustrates the experimental settings. Both fluorophores are excited simultaneously by the evanescent tail of the guided mode by a 637 nm laser. The excitation light was coupled to the chip by an input grating coupler. The subsequent mixed emission from both fluorophores was collected by a commercial microscope objective. The far-field emission spectrum was collected from the top of the sensor region of the waveguide. The near-field emission collected by the waveguide was out-coupled from the chip by an output grating coupler. The same microscope objective was then used to collect the near-field coupled emission by translating the microscope (Nikon Ni-E) on top of

Figure 1. Schematic of experimental setup for simultaneous excitation and collection of fluorescence by the same waveguide. BP Filter, fluorescence band pass filter; BR Filter, band rejection filter to reject the excitation light.

reference silicon chip was always put in the deposition tool, and the actual thickness of the deposited oxide layer was determined using ellipsometry. 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 (simulation details in Supporting Information section 1). The P excitation efficiency, Eexc = Pevanes , is defined as the ratio total

between the available excitation power (Pevanes) in the evanescent field above a certain distance from the waveguide surface and the total power (Ptotal) in the waveguide mode. The red continuous line in Figure 2 shows the excitation efficiency

Figure 2. Excitation and coupling efficiency as a function of distance above the surface. FDTD simulated excitation efficiency (red line), collection efficiency (blue dashed), and their multiplication (black line with triangles). Measured fluorescence power using the same waveguide to excite and collect fluorescence (black squares).

as a function of separation between the waveguide surface and solution, determined by the oxide spacer thickness. The excitation efficiency decays exponentially as a function of separation, following the mode profile of the field intensity (E2). In this case this corresponds to a 1/e decay length of 58 nm. The blue dashed line shows the simulated collection efficiency (η), the fraction of the total fluorescence emission that couples back to the waveguide as a function of separation B

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Figure 3. (a) Schematic of the experiment. (b) Far-field collected spectra. Surface-bound Atto-633 + bulk Atto-680 (black line with solid circles); individual normalized spectra of surface-bound Atto-633 (blue line with open circles) and unbound bulk Atto-680 (red line). (c) Near-field collected spectra. Surface-bound Atto-633 + bulk Atto-680 (black line with solid circles); individual normalized spectra of surface-bound Atto-633 (blue line with open circles) and unbound bulk Atto-680 (red line).

the output grating coupler. The collected emission was directed through an appropriate filter set toward the detector of a commercial portable spectrometer (Ocean Optics, QE Pro). The input fiber was first aligned to the input grating with a 690 nm laser to make sure that the surface dyes are not bleached during the alignment. Once the fiber was properly aligned, a 3.5 μM bulk Atto-680 in DMSO was applied on top of the Atto-633 monolayer, and both dyes were excited simultaneously by switching the excitation laser to 637 nm. The collected spectra are shown as a black line with solid circles for the far field in Figure 3b and for the near field in Figure 3c. The individual contribution of surface-bound and bulk dye from the mixed emission spectra in Figure 3b and c can be extracted if their individual spectra are known in the far field and near field. The combined emission spectrum in either far field or near field is related to its individual spectra by I633 + 680(λ) = m1I633(λ) + m2I680(λ)

line for far-field collection in Figure 3b and for near-field collection in Figure 3c. The contribution of surface-bound Atto-633 (m1) and unbound bulk Atto-680 (m2) is extracted from the mixed emission data using eq 1. The m1 and m2 values on the righthand side of the equation are varied for the known experimental data of I633(λ) and I680(λ). These variation of m1 and m2 give the expectation curve for I633+680(λ) according to the equation. The error between this expectation curve and measured data for I633+680(λ) has been minimized using a built-in optimization tool in Matlab, where the genetic algorithm11 has been used to search for the best value for m1 and m2. The matched curves for near and far field are shown in section 3 of the Supporting Information along with the experimental curve. A bulk to surface ratio of 1.02 has been deduced from the near-field collected spectra (Figure 3c) compared to a 1.95 ratio from the far-field collected spectra (Figure 3b). We have experimentally found a bulk fluorescence suppression of 1.91 in the near-field collection compared to the conventional far-field collection following excitation in the near field. This value of suppression closely matches with our expected value of 2.0 calculated from the result on excitation and collection efficiency described in the previous part of this Letter (calculation in Supporting Information section 4). To confirm that the emission from the Atto-633 is coming purely from surface-bound dyes and the Atto-680 is purely from bulk dyes, we were able to bleach the Atto-633 signal, while leaving the Atto-680 signal unaffected after 50 s of continuous excitation (results are shown in Supporting Information section 5). In the previous two sections, we saw that this evanescent excitation and collection using the same waveguide approach can be useful to suppress bulk fluorescence for sensitive detection and physical chemistry characterization. It can also be very useful for techniques such as Raman, CARS, and SERS,

(1)

where m1 and m2 are the multiplication factors related to the contribution from surface-bound Atto-633 and unbound bulk Atto-680, respectively, in the mixed spectra. I633(λ) and I680(λ) are the normalized individual spectra of surface-bound Atto-633 and unbound bulk Atto-680. Two reference experiments were performed to find the normalized individual spectra in the near field and far field. The individual spectrum of surface-bound Atto-633 was obtained by repeating the same experiment as described before except in the last step only DMSO buffer was applied instead of Atto-680 + DMSO solution. The collected individual spectra of Atto-633 are shown by the blue line with open circles for far-field collection in Figure 3b and for nearfield collection in Figure 3c. The individual spectra of Atto-680 were also obtained by repeating the same experiment but without a monolayer of the Atto-633 dye on the surface. The collected individual spectra of Atto-680 are shown with a red C

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Figure 4. (a) Collected fluorescence emission (P) at the detector as a function of the sensor length (L). Experimentally collected fluorescence (black squares), expectation using the full eq 2 (red solid), and simplified eq 7 (blue dashed) of our derived analytical model. (b) Waveguide length where the coupled power increases as a function of the propagation decay constant due to intrinsic waveguide loss (α) and absorption by the analyte (β). Top axis shows the same relation as a function of the analyte concentration. The continuous line and dashed line show Lcritical using eq 5 and the simplified eq 6, respectively.

analyte molecules, respectively. L is the sensing length of the waveguide. The term P0 is given by

where the analyte molecules have a relatively small scattering or absorption cross section. For these applications, a relatively long solution−light interaction length is required to gather as much emission/ scattered signal as possible. Thanks to photonic nanotechnology, a very long waveguide can be routed in a small area, making it a promising technology for these plethora of applications. Intuition says that the longer the light−solution interaction length, the more molecules will be excited and the more response from molecules will be collected. However, in Figure 4a, it is shown that initially the output collected power increases as the length increases, but after a certain length, which we refer to as the critical coupling length (Lcritical), it decreases with an increase in length. This nonintuitive behavior originates from the competition between the increase in collected power from the increased number of contributing molecules and the decrease in collected power due to the increase in propagation loss when the light−solution interaction length is increased. In the next section, we will derive a general condition to find Lcritical for any sensing technique based on waveguide excitation and collection. Then the condition will be simplified further for specific techniques such as Raman where the scattering cross section is small or a fluorescence application where the concentration of analyte is relatively low. Although the derivation and experiment are performed for fluorescence, the final conclusion applies for any evanescent sensing technique based on excitation and collection by the same waveguide. The full derivation of our analytical model describing nearfield excitation and collection by a photonic waveguide can be found in section 6 of the Supporting Information. The collected fluorescence by a detector placed at the exit of the sensor is given by P = P0(e−αL − e−(α + β)L)

P0 =

I0fC(2h + w)tQση β

(3)

where I0 is the excitation intensity at the entrance of the sensor, f is the fraction of the power in the evanescence field relative to the mode power, and C is the analyte concentration. The parameter t is the virtual thickness of the analyte layer above the waveguide that is sufficient to produce an equivalent result as an infinitely thick layer. Q is the quantum yield of the analyte molecule, σ is the absorption cross section of molecule, and η is the fraction of the total emitted power from the molecule that couples back to the waveguide. By taking the derivative with respect to the length we can find Lcritical.

dP =0 dL Lcritical =

(4)

1 ⎛ α ⎞ ln⎜ ⎟ β ⎝α + β ⎠

(5)

When α ≫ β the condition simplifies to lim Lcritical =

β→ 0

1 α

lim P ∝ Le−αL

β→0

(6) (7)

This simplification can be applied only when the loss due to absorption by the analyte molecules is negligible compared to the intrinsic waveguide loss. That is the case for low concentration fluorescence sensing or applications such as Raman sensing, where the analyte has a relatively small scattering cross section. In Figure 4b, Lcritical has been plotted as a function of β/α using the full eq 5 (continuous line) and simplified eq 6 (dashed line) to find the limit where the

(2)

where α and β are the propagation decay constants due to intrinsic waveguide loss and loss due to absorption by the D

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excitation and collection scheme. We also have found that Lcritical is equal to the reciprocal of the propagation decay constant (α) for the special case of low concentration sensing applications. These findings will pave the way for implementing a plethora of applications such as single-molecule analysis, wash-free biosensing, membrane-based Raman, and CARS sensing on a chip where a reduction of bulk fluorescence has a huge importance.

simplification is valid. The result shows that the simplified equation can be applied when the propagation decay constant due to absorption (β) is at least 1 order of magnitude smaller than the intrinsic waveguide loss (α). The top axis of Figure 4b translates the β/α relation to fluorophore concentration for a typical value for the propagation decay constant due to intrinsic waveguide loss (α = 0.737; loss = 3.2 dB/cm) and a value for the decay constant (β) due to absorption by the solution for varying concentration (absorption cross section used for the calculations 4.9 × 10−16 cm2; Atto-633) of a typical fluorescent dye. The analyte concentration limit where the simplification is valid for a typical fluorescent dye and typical waveguide loss is on the order of micromolar concentration. The importance of this finding is that for low concentration sensing applications the length for efficient sensing depends only on the intrinsic waveguide propagation loss, not the analyte concentration. Figure 4a shows the experimental collected emission power (black squares) as a function of sensor length (L) for a 0.1 μM fluorescence dye concentration. The experimental data has been compared with the theoretical curve using eq 2 (red continuous) and simplified eq 7 (blue dashed). The experimental result matches very well with the derived analytical model. It also validates the simplified equation for low concentration sensing. Excitation and collection through integrated waveguides has a number of advantages. Essentially it combines the advantage of a limited sensing depth with a very long excitation and collection length. Due to the confinement of the optical mode in a single-mode waveguide, it can be used to excite the fluorescent molecules with a high power density over a very long length, much unlike conventional techniques such as a confocal fluorescence microscope, where the power density is also high, but the collection area is very small. It has already been shown to increase the collection by more than 104 times using this advantage.19 In comparison, plasmonic effects have shown a large fluorescence enhancement of nearly 103 times,17,18 with the caveat that this applies only for relatively low quantum yield dyes. Moreover, due to the limited sensing depth, it is only sensitive to molecules in the near field of the waveguide, enabling wash-free sensing. In comparison to our sensing depth of 30 nm, the sensing depth for LSPR, SPR, zero-modewaveguides, and nanoplasmon antennas ranges from 1 to 30 nm.12−16 Although these metal-assisted techniques have a relatively small sensing depth, the collection area is much smaller, thus much smaller signals in a single measurement, and the all-dielectric solution also avoids fluorescence quenching, which is common close to metal surfaces. In addition, the technique is completely CMOS compatible, ensuring scalability. Our approach, which uses both evanescent excitation and collection, reduces the sensing depth by a factor of 2 compared to the best available all-dielectric techniques. This improvement in separating bulk signal by reducing the sensing depth has an edge in real-time sensing, as it does not need the washing steps to remove the bulk fluorophores. To conclude, we showed that a waveguide-based near-field excitation and collection scheme performs better to separate surface and bulk fluorescence. We measured an increase in the surface to bulk fluorescence ratio by a factor of 1.91 compared to the state-of-the-art far-field collection based technique. Finally, we have formulated a general condition for the sensor length (Lcritical) to maximize device efficiency for any kind of sensing applications based on this proposed near-field



METHODS Waveguide. The bottom part of Figure 1 shows the crosssectional view of the sample along the waveguide length. We used a SiN strip waveguide (340 nm × 220 nm) fabricated in imec’s 200 mm CMOS pilot line using a CMOS-compatible low-temperature PECVD process optimized for low loss.20,21 The 220 nm thick waveguide layer is separated from the Si substrate by a 2.3 μm thick SiO2 bottom cladding. A 1 μm thick top cladding of SiO2 layer was applied to isolate the solution from the waveguide. The top cladding was removed selectively in the active areas, exposing waveguides with varying lengths to controllably investigate the interaction with the solution. Finally, a microfluidic channel was defined to contain the solution only in the relevant areas using photopatternable adhesive (PA-S500h, JSR Micro NV). Binding a Monolayer to the Waveguide Surface. A monolayer of Atto-633 was covalently bound to the waveguide surface by introducing azido groups (N3) at the surface using a vapor-phase silanization process followed by copper-catalyzed “click reaction”22 to form a bond between the terminal azido groups and alkyne-modified Atto-633 (details in Supporting Information section 2). After the click reaction, the samples were extensively rinsed to remove excess remaining dyes, and then the surface was submerged in diluted HCl solution to remove Cu precipitates to prevent quenching. Finally, the samples were submerged in DBCO-PEG blocking reagent solution to block possible remaining active azide sites on the surface in order to prevent any nonspecific binding to these sites. The samples to acquire the individual spectra of non-surfacebound Atto-680 in bulk solution were prepared by repeating the same procedure as described above except that the N3 groups at the surface were allowed to react with a strained alkyne-modified PEG chain introducing an antifouling layer that acts as a blocking layer and prevents nonspecific binding of Atto-680. The spectra were taken by applying Atto-680 dissolved in DMSO solution on top of the blocked waveguide surface.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsphotonics.6b01016. Additional details on FDTD analysis, surface functionalization, click chemistry, separating surface and bulk contribution by the Matlab optimization tool, calculation of bulk suppression, fluorescence bleaching, analytical model, and effect of Purcell enhancement (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] E

DOI: 10.1021/acsphotonics.6b01016 ACS Photonics XXXX, XXX, XXX−XXX

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ORCID

of a CMOS Compatible Biophotonics Platform Based on SiN Nanophotonic Waveguides. In CLEO: 2014; OSA: Washington, D.C., 2014; p JTh2A.31. (21) Subramanian, A. Z.; Neutens, P.; Dhakal, A.; Jansen, R.; Claes, T.; Rottenberg, X.; Peyskens, F.; Selvaraja, S.; Helin, P.; Bois, B.; Leyssens, K.; Severi, S.; Deshpande, P.; Baets, R.; Van Dorpe, P. LowLoss Singlemode PECVD Silicon Nitride Photonic Wire Waveguides for 532−900 Nm Wavelength Window Fabricated Within a CMOS Pilot Line. IEEE Photonics J. 2013, 5, 2202809. (22) Liang, L.; Astruc, D. The copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) “click” reaction and Its Applications. An Overview. Coord. Chem. Rev. 2011, 255, 2933−2945.

Md. Mahmud-Ul-Hasan: 0000-0002-0058-7433 Notes

The authors declare no competing financial interest.



REFERENCES

(1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Springer Science & Business Media, 2013. (2) Zander, C.; Keller, R. R.; Enderlein, J. Single Molecule Detection in Solution; Wiley Online Library, 2002; Vol. 43. (3) Wenger, J.; Rigneault, H. Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection. Int. J. Mol. Sci. 2010, 11, 206−221. (4) Lukosz, W.; Tiefenthaler, K. Directional Switching in Planar Waveguides Effected by Adsorption-Desorption Processes. IEEE Conf. Publ 1983, 227, 152−155. (5) Thompson, N. L.; Burghardt, T. P.; Axelrod, D. Measuring Surface Dynamics of Biomolecules by Total Internal Reflection Fluorescence with Photobleaching Recovery or Correlation Spectroscopy. Biophys. J. 1981, 33, 435. (6) Mukundan, H.; Anderson, A. S.; Grace, W. K.; Grace, K. M.; Hartman, N.; Martinez, J. S.; Swanson, B. I. Waveguide-Based Biosensors for Pathogen Detection. Sensors 2009, 9, 5783−5809. (7) Saleh, B. E. A.; Teich, M. C.; Saleh, B. E. Fundamentals of Photonics; Wiley: New York, 1991; Vol. 22. (8) Paribok, I. V.; Zhavnerko, G. K.; Agabekov, V. E.; Zmachinskaya, Y. A.; Yantsevich, A. V.; Usanov, S. A. Local Modification of the Silicon Surface with Protein Molecules. Russ. J. Gen. Chem. 2007, 77, 363− 366. (9) Rigo, E.; Aparicio, F. J.; Vanacharla, M. R.; Larcheri, S.; Guider, R.; Han, B.; Pucker, G.; Pavesi, L. Evanescent-Field Excitation and Collection Approach for Waveguide Based Photonic Luminescent Biosensors. Appl. Phys. B: Lasers Opt. 2014, 114, 537−544. (10) Dhakal, A.; Subramanian, A. Z.; Wuytens, P.; Peyskens, F.; Thomas, N.; Baets, R. L. Evanescent Excitation and Collection of Spontaneous Raman Spectra Using Silicon Nitride Nanophotonic Waveguides. Opt. Lett. 2014, 39, 4025−4028. (11) Holland, J. H. Genetic Algorithms. Sci. Am. 1992, 267, 66−72. (12) Chen, S.; Svedendahl, M.; Käll, M.; Gunnarsson, L.; Dmitriev, A. Ultrahigh Sensitivity Made Simple: Nanoplasmonic Label-Free Biosensing with an Extremely Low Limit-of-Detection for Bacterial and Cancer Diagnostics. Nanotechnology 2009, 20, 434015. (13) Estevez, M.-C.; Otte, M. A.; Sepulveda, B.; Lechuga, L. M. Trends and Challenges of Refractometric Nanoplasmonic Biosensors: A Review. Anal. Chim. Acta 2014, 806, 55−73. (14) Langer, J.; Novikov, S. M.; Liz-Marzán, L. M. Sensing Using Plasmonic Nanostructures and Nanoparticles. Nanotechnology 2015, 26, 322001. (15) Unser, S.; Bruzas, I.; He, J.; Sagle, L. Localized Surface Plasmon Resonance Biosensing: Current Challenges and Approaches. Sensors 2015, 15, 15684−15716. (16) Levene, M. J.; Korlach, J.; Turner, S. W.; Foquet, M.; Craighead, H. G.; Webb, W. W. Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations. Science 2003, 299, 682−686. (17) Khatua, S.; Paulo, P. M. R.; Yuan, H.; Gupta, A.; Zijlstra, P.; Orrit, M. Resonant Plasmonic Enhancement of Single-Molecule Fluorescence by Individual Gold Nanorods. ACS Nano 2014, 8, 4440−4449. (18) Kinkhabwala, A.; Yu, Z.; Fan, S.; Avlasevich, Y.; Müllen, K.; Moerner, W. E. Large Single-Molecule Fluorescence Enhancements Produced by a Bowtie Nanoantenna. Nat. Photonics 2009, 3, 654−657. (19) Dhakal, A.; Wuytens, P. C.; Peyskens, F.; Jans, K.; Thomas, N.; Baets, R. Nanophotonic Waveguide Enhanced Raman Spectroscopy of Biological Submonolayers. ACS Photonics 2016, 3, 2141−2149. (20) Neutens, P.; Claes, T.; Jansen, R.; Subramanian, A.; UlHasan, M.; Rochus, V.; Shirley, F. J.; Du bois, B.; Helin, P.; Severi, S.; Leyssens, K.; Dhakal, A.; Peyskens, F.; Selvaraja, S.; Deshpande, P.; Baets, R. G. F.; Lagae, L.; Rottenberg, X.; Dorpe, P. Van. Development F

DOI: 10.1021/acsphotonics.6b01016 ACS Photonics XXXX, XXX, XXX−XXX