Shrink-Induced Silica Structures for Far-field Fluorescence Enhancements

2 structures causes changes in the photophysical properties of the dye. Together, this results in both dramatic increases in the signal intensity and ﬂ uorescence enhancement factor. To understand the mechanisms of such enhancements, we characterize our SiO 2 structures, demonstrate ﬂ uorescence signal enhancements, investigate the effects of dye–silica interaction on absorption, emission


. Introduction
Fluorescence is widely used for chemical sensing, biomolecule detection, disease diagnostics, and various applications in biology.Ongoing challenges persist in improving detection sensitivity and the signal-to-noise ratio (SNR) of low-abundance target molecules.] These structures typically use expensive materials such as gold and silver, require extensive fabrication steps for precise control of homogeneous or periodic surface features, and necessitate specialized equipment (e.g.2-photon microscopy).Importantly, while large fl uorescence enhancements have been observed, such plasmon-coupled effects suffer from practical limitations including: localized near-fi eld effects with enhancements occurring only within nanometric lengths from the surface, heterogeneous enhancements with small areas of 'hot spots', and enhancements which are wavelength dependent.Such practical drawbacks impede progress in translating these large enhancements into solutions for deployable robust detection.
2][13] Studies of dye-encapsulated SiO 2 nanoparticles have suggested that these photophysical changes are dependent on internal architecture of the SiO 2 structures. [ 14 ]Immobilization of the fl uorescent dye within the SiO 2 core-shell is suggested to restrict molecular mobility, which can lead to reduced nonradiative relaxation and subsequently increased quantum yield. [ 15,16 ]] Leveraging these properties, as well as the mechanical stability and chemical versatility of SiO 2 , we develop a strategy to create a robust and scalable fl uorescence enhancing platform based on low-cost commodity shrink wrap fi lm.We have previously shown that we are able to achieve concentration of adsorbed molecules by leveraging the heat-induced retraction properties of polyolefi n (PO) sheets. [ 18 ]We have also shown that the deposition of a stiff, non-shrinkable material such as metal onto the PO fi lm results in buckling when the substrate is heated. [ 19 ]Here, we demonstrate that this strategy is extensible for SiO 2 .2][23][24] We predict that by covalently linking the biomolecules onto the shrinkable SiO 2 substrate: (1) the fl uorophores are brought closer in proximity to each other which results in signal concentration, (2) the SiO 2 structures create a highly porous surface that results in light scattering, and (3) the covalent attachment of fl uorophore within the SiO 2 structures causes changes in the photophysical properties of the dye.Together, this results in both dramatic increases in the signal intensity and fl uorescence enhancement factor.To understand the mechanisms of such enhancements, we characterize our SiO 2 structures, demonstrate fl uorescence signal enhancements, investigate the effects of dye-silica interaction on absorption, emission, and look into the detection sensitivity of the substrate.

. Preparation of PO-SiO 2 Substrate and Biomolecule Attachment
We leverage the stiffness mismatch between the thin SiO 2 layer and the PO fi lm to create SiO 2 micro-and nanostructures.The multi-scale substrate that results in enhanced fl uorescence signal is prepared following the procedure illustrated in Figure 1 .Briefl y, a home-made shadow mask was applied to a clean PO surface prior to sputter deposition of SiO 2 to form PO-SiO 2 substrates.The surfaces were chemically activated through O 2 plasma treatment and then functionalized with primary amine groups for further biomolecule attachment.The PO-SiO 2 surfaces were biotinylated and then hybridized with a STRITC (here on referred to as PO-SiO 2 -STRITC).Substrates were heated at T = 155 °C, which induces retraction of the substrate and causes the SiO 2 thin fi lm to buckle and crack.
Figure 2 a,b are top down SEM images of the shrunk PO-SiO 2 substrate.The SEM image illustrates the formation of a continuous population of heterogeneous surface structures.The cross section SEM image (Figure 2 c) demonstrates integration of the SiO 2 layer with the PO fi lm that occurs when the PO fi lm is heated above its glass transition temperature.Integration of the SiO 2 into the PO fi lm is also supported from energydispersive X-ray spectroscopy (EDS) data (Figure S1).The SiO 2 structures are patterned into distinct islands by applying a shadow mask prior to SiO 2 deposition (Figure 2 d).

. Optical Characterization
To examine the effects of the SiO 2 structures on the photophysical properties of the linked fl uorescent dye, optical properties of the reacted substrate and controls were measured.The UV-Vis absorption spectra of STRITC, glass-STRITC (STRITC bound to glass), and shrunk PO-SiO 2 -STRITC are shown in Figure 3 a.STRITC exhibits two absorption maxima at 520 and 549 nm attributed to the presence of dimeric and monomeric species reported to occur for rhodamine dyes at high concentrations (A 520/549 = 0.89). [ 25 ]Glass-STRITC also exhibits an absorption maximum at 549 nm.Covalent linkage of STRITC within the SiO 2 structures is not observed to cause spectral shifts in peak absorbance.The absorption spectra of the shrunk PO-SiO 2 -STRITC substrate shows higher optical density at higher energies, which fi ts well to Rayleigh scattering, suggesting that shrinking of the PO-SiO 2 substrate creates rough porous structures with small surface features that together result in light scattering. [ 26,27 ]he emission spectra are shown in Figure 3 b.The emission intensity maximum for the STRITC occurs at 575 nm.While covalent attachment of the STRITC on glass does not cause a change in the absorption wavelength, it is observed to cause a slight red shift of 3.0 nm for the emission wavelength for STRITC on the PO-SiO 2 substrate.This slight red shift in emission wavelength can be attributed to the change in molecular surrounding experienced by the STRITC dye since it is known that dye molecules are affected by microenvironment polarity. [ 28 ]However, the observed spectral shifts are insignificant and it can be suggested that confi nement of the dye molecules within the SiO 2 structures does not result in changes of the dye's electronic structure.

. Fluorescence Signal Enhancement on PO-SiO 2 Substrate
Fluorescence signal enhancement on PO-SiO 2 substrates were investigated using the model biotin-streptavidin binding  system.Substrates were prepared as described above and shrunk thermally.The fl uorescence images of the patterned substrates following shrinkage are shown in Figure 4 a.The 3D intensity distribution profi les (Figure 4 b) illustrate fl uorescence uniformity over the SiO 2 islands on the shrunk PO-SiO 2 -STRITC substrates and that enhanced fl uorescence signal is not localized to nanometric hotspots.The increase in fl uorescence signal is assessed and results are compared to that obtained on glass-STRITC and thermally shrunk PO (PO-STRITC).The average fl uorescence signal increase (SI) of the substrate is calculated to be the fl uorescence signal obtained after shrinking minus the background (here defi ned as the substrate without the presence of dye), over the fl uorescence signal before shrinking minus the background: Intens ity pos ts hr unk − Intens ity pos ts hr unk,bg Intens ity pr es hr unk − Intens ity pr es hr unk,bg (1)   As previously reported, the PO fi lm experiences a 77% reduction in each length upon heating, which results in a theoretical 20-fold consolidation of surface area. [ 18 ]The PO-STRITC experiences a fl uorescence SI of 14-fold (standard error (SE) = 0.57) due to concentration of the surface bound fl uorophores.This slight decrease in experimental value compared to the theoretical expectation is attributed to concentration quenching effect from bringing the fl uorescent molecules close in proximity.Interestingly, an approximate 50-fold (SE = 1.9) fl uorescence SI is observed on the shrunk PO-SiO 2 -STRITC substrates.Notably, the fl uorescence SI is accompanied with a signifi cantly increased SNR (defi ned as the ratio of the raw fl uorescence signal to the background signal) from 11:1 to 76:1.The increased fl uorescence intensity from shrinking the PO-SiO 2 -STRITC substrate exceeds that observed from just concentrating the fl uorescent molecules (as seen on the PO-STRITC substrate).This suggests that the additional increase of fl uorescence signal on the PO-SiO 2 -STRITC substrate is

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www.MaterialsViews.comwww.advopticalmat.dedetection (LOD) is defi ned to be the mean of the background plus three times the standard deviation of the background.The LOD is calculated to be 0.26 μ g mL −1 (SE = 0.026) on the heated glass surface.In contrast, the shrunk PO-SiO 2 substrate is able to yield a lower LOD of 11 ng mL −1 (SE = 0.0027).This proofof-concept demonstrates that the PO-SiO 2 substrate has higher detection sensitivity relative to planar glass surface.This ability to reach lower limits of detection suggests the possibility for applications in disease diagnostics and point-of-care testing.

. Conclusion
In this work, we have presented a rapid method to create SiO 2 micro-and nanostructures, demonstrated the ability of our structures to enhance fl uorescence signal of bound fl uorophores, and achieved lower limits of detection on the PO-SiO 2 substrates.We also investigated the photophysical properties of the dye on the SiO 2 structures.Fabrication of SiO 2 structures is simple and rapid, leading to signal enhancement within minutes.Structures are directly integrated on chip and defi ned regions of SiO 2 structures are easily established.The observed far-fi eld fl uorescence enhancements on our structures are robust and highly reproducible.
Due to the well-established conjugation chemistries and high biocompatibility of SiO 2 , our PO-SiO 2 substrate has applications in surface sensing technologies.Integration of our SiO 2 structures into microfl uidic devices for point-of-care applications is readily realized as similar structures have been demonstrated to be robustly integrated into the plastic used for microfl uidic chips. [ 30 ]

. Experimental Section
Fabrication of Functionalized PO-SiO 2 Substrate : PO fi lm (955-D, Sealed Air Corporation) was cleaned in isopropyl alcohol (IPA) and double deionized water (ddH 2 O) and dried with pressurized air.To due to presence of SiO 2 structures and not from bringing the fl uorophores into close proximity of each other.We also show on the glass surface that the application of heat does not cause signal amplifi cation or signifi cant signal degradation (SI , glass = 0.92, SE = 0.019) and that the SNR does not change considerably (from 5.7:1 to 5.2:1 following heating).Therefore the observed increase in fl uorescence intensity on the shrunk PO-STRITC and the shrunk PO-SiO 2 -STRITC cannot be attributed to changes in the fl uorophore due to heating.
For comparison between substrates, the average fl uorescence enhancement factors (EFs) are calculated.The fl uorescence EF is defi ned to be the fl uorescence intensity of substrate minus the background over the fl uorescence intensity of the comparison substrate minus its respective background: Compared to the glass-STRITC, the shrunk PO-SiO 2 -STRITC substrate has an average fl uorescence EF of 116 (SE = 9.7).We observe a higher fl uorescence signal on the fl at PO-SiO 2 -STRITC relative to glass-STRITC, and we attribute the higher signal to the increased surface area that forms during the sputter deposition of SiO 2 .Increased surface area subsequently allows for an increased number of binding sites for biomolecule attachment.To distinguish between concentrating surface biomolecules and additional effects from the SiO 2 structures, the EF of the shrunk PO-SiO 2 -STRITC substrate over the shrunk PO-STRITC is evaluated.Experimental results suggest that the SiO 2 structures contributes in an additional 5.0-fold enhancement (SE = 0.74) of fl uorescence signal on top of the concentrating effects.

. Binding Study with Alternate Dye
While fl uorescence enhancements that arise from plasmon resonances are highly dependent on nanostructure size and shape, [ 29 ] we show that increased fl uorescence signal and fl uorescence enhancement factors on our PO-SiO 2 substrates are not restricted to a particular wavelength or structure size.10 μ g mL −1 Cy2-conjugated streptavidin were spotted onto unshrunk biotinylated substrates as previously performed.Upon shrinking the substrate, a 39-fold (SE = 1.6) and 11-fold (SE = 1.2) increase in the fl uorescence signal is observed on the shrunk PO-SiO 2 substrate and the shrunk PO substrate, respectively.The increases in fl uorescence signal correspond to averaged enhancement factors of 106 (SE = 9.5) and 5.0 (SE = 0.27) for the shrunk PO-SiO 2 relative to the heated glass and shrunk PO substrate.An increase in SNR is also experienced on the shrunk PO-SiO 2 substrate from 13:1 to 29:1.

. Lower Limits of Detection
To evaluate detection sensitivity of the PO-SiO 2 substrates, a concentration curve of STRITC was performed on the PO-SiO 2 and glass substrate.We use the biotin-streptavidin hybridization since this system can be applied towards real immunoassays through DNA, protein, or aptamer linking.The results are plotted in Figure 5 .The fl uorescence signal corresponding to the limit of

Dense multiscale silica (SiO 2 )
micro-and nanostructures are fabricated on a pre-stressed polymer fi lm.This novel SiO 2 substrate serves as a robust platform to enhance the fl uorescence signal of bound biomolecules.Through a combination of surface concentration, light scattering, and changes in the photophysical properties of the confi ned dye molecules, dramatic fl uorescence signal enhancements (average = 116 times greater than on planar glass) and increased signal-to-noise ratio (76:1) are demonstrated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated streptavidin (STRITC) on SiO 2 structures.Enhanced detection sensitivity of STRITC over glass on the SiO 2 structures is achieved down to a detection limit of 11 ng mL −1 .Such signifi cant fl uorescence signal enhancements have importance in practical applications such disease diagnostics and surface sensing.

Figure 1 .
Figure 1.Schematic illustrating the fabrication of a fl uorescence enhancing PO-SiO 2 substrate.Clean PO is coated with a thin layer of SiO 2 through ion-beam sputter deposition, treated with O 2 plasma, and reacted with APTMS for the formation of reactive primary amines.The silanized surface was linked with biotin, reacted with STRITC, and shrunk.

Figure 2 .
Figure 2. SEM images of the PO-SiO 2 substrate.Top down SEM images of shrunk PO-SiO 2 substrate showing micro-and nanostructures at different magnifi cations (a,b) and a cross section SEM image of the PO-SiO 2 substrate (c).The PO fi lm can be patterned to form discrete SiO 2 islands (d).

Figure 4 .
Figure 4. Fluorescent images of the patterned substrates for heated glass, thermally shrunk PO, and thermally shrunk PO-SiO 2 bound with biotin and STRITC (a) and the corresponding 3D fl uorescence intensity profi les (b).

Figure 5 .
Figure 5.A plot of the fl uorescence intensity as a function of STRITC concentration on the shrunk PO-SiO 2 substrate and the glass surface.The inset graph is a zoom of the lower STRITC concentrations, background subtracted for each respective substrate.