Optimising the Enzyme Response of Porous Silicon Photonic Crystal via the Modular Design of Enzyme Sensitive Polymers

We describe the immobilization within the pores of a porous silicon photonic crystal of an enzyme degradable polymer network, for optical biosensing. A porous silicon (PSi) rugate filter is a one-dimensional photonic crystal with a high-reflectivity optical resonance that is sensitive to small changes in the refractive index of the pore space permeating though the structure. An enzymatically degradable polymer network was constructed by first “clicking” antifouling copolymer poly(oligo ethylene glycol-co-acrylic acid)-N3 to an alkyne functionalized PSi surface via copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) reaction. MMP-2 or MMP-9 specific cleavable peptide sequences, with diamine functional groups, was then added, using a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/Nhysroxysuccinimide (EDC/NHS) chemistry to react with the acrylic acid group. The polymer network was completed by further attachment of sacrificial polymer poly(hydroxyl ethyl acrylate-co-N-hydroxysuccinimide ester acrylate). X-ray photoelectron spectroscopy (XPS) and optical reflectivity measurement reveal successful modification of the PSi with the Page 1 of 29 Polymer Chemistry P o ly m er C h em is tr y A cc ep te d M an u sc ri p t


Introduction
There is a need for devices that can detect various biological species for the development of biotechnology and medical diagnostics.2][3] One class of the proteinases is the matrix metalloproteinases (MMPs) which are known to be released by cells as part of their normal tissue remodelling processes, such as embryonic development and cell migration. 45] Currently, the most common proteinase activity measurements are performed using fluorogenic or calorimetric methods with commercially available proteinase assay kits. 69][10] The working principle of most of these systems is based on fluorescent molecules that are connected to a quencher through peptide sequences.2] An example of such system is demonstrated in the work by Wosnick et al. 13 where they conjugated poly(phenylene ethynylene) with fluorescence activation upon treatment with trypsin.In another study by Chien et al., 14 polymer micelles were utilised to observe a colour change upon exposure to MMPs.A further example is shown by Tanaka et al. 14 where a digestion of biodegradable polymer by phosphatase enzymes, causing the release of fluorescein to measure enzymatic activity of cell lysate.However, all these approaches require the need of labels.[17] PSi can be fabricated by the etching of silicon which creates a nanostructured architecture with pores that are perpendicular to the surface. 16These pores can be continuously tuned in size during fabrication to create a one-dimensional photonic crystal that reflects light at welldefined wavelengths.In particular, a rugate filter structure with small full width half maximum (FWHM) have been used because their narrow reflectance peaks assist in producing sensitive devices. 189] In this paper the nanopores are used as a medium to immobilize a network of enzymatic degradable polymers.As a transducer, PSi translates enzymatic reactions that occur inside the pores into an optical response as a function of the change in refractive index (n) of the material inside the pores. 16,20 his is essential for the use of PSi as the biosensor, as any biological or synthetic materials that have a higher refractive index than air or water can be immobilized into pores of the PSi.For instance, organic materials such as peptides and biopolymers have a refractive index in the range of 1.4-1.5.As the materials leaves the pores, they are replaced by water (n water = 1.33) or air (n air = 1), shifting the reflectivity spectrum to lower wavelengths of light, which is referred to as a blue shift.1] The lack of specificity of the current 'state of the art' in the literature is due to the immobilization of more generic biomolecules such as zein 22 (digestible by pepsin which degrades food protein) and gelatin 20 (digestible by subtilisin; a non-specific proteinase and gelatinases).As a result, the current PSi biosensors are limited by the choice of biomolecules that can be immobilized inside the pores.In this present study, the aim was to develop a generic strategy to allow the detection of a specific proteinases (e.g.MMP-2 or MMP-9).Improvement of the specificity is achieved by employing a new modular approach to form enzyme sensitive materials inside the PSi pores.This is carried out by attaching a custom made polymer-peptide network that is highly tuneable where the peptide linkage provides the selectivity.This innovative strategy will expand the range of capabilities for PSi based optical biosensors to provide a universal platform for enzyme detection systems.
Although there are plenty of examples of polymerization on surfaces, [23][24] the modification of copolymer structures, or networks, anchored inside the pores of photonic crystals is still scarce in the literature.One example was shown by Voelcker and co-workers 25 where surface initiated polymerization of poly(N-isopropyl acrylamide) inside the pores of porous silicon was achieved through Atom Transfer Radical Polymerization (ATRP) where the initiator was immobilized through silane chemistry on oxidised silicon.However, this method is not suitable for our purpose as the oxide layer within the PSi is not sufficiently stable in aqueous solution, under the timescales of the enzymatic measurements, resulting in signal drift. 26One way to maintain the optical integrity of the PSi is through passivation of the silicon via hydrosilylation. 279][30] In this work, building on our surface chemistry, [28][29][30] the PSi was further modified with a synthetic polymer-peptide network.2][33] The enzymatically degradable polymeric network was immobilized within the pores of PSi by first "clicking" P1 to an alkyne modified PSi surface, followed by immobilization of a peptide with diamine functionalities, and then the attachment of P2.These surfaces were then tested for their performance as an optical biosensor.

Polymerization of P1 and P2
Cu(0)-mediated polymerization of copolymer Poly(oligo ethylene glycol methyl ether acrylate -stat -acrylic acid)-N 3 P1 .In a typical polymerization, oligo(ethylene glycol) methyl ether acrylate (OEGA 480 , 2.5 g, 5.21E-03 moles), tert-butyl acrylate (tBA, 0.4 g, 3.12E-03 moles), DMSO (4 mL), EBiB (64µL), Me 6 TREN(12 µL), and CuBr 2 (7 mg) were charged to a polymerization flask fitted with a rubber septum and the mixture was degassed with nitrogen purging for 30 min.A slight positive pressure of nitrogen was then applied and the pre-activated copper wire (0.5 cm) was carefully added under nitrogen blanket.The polymerization flask was then resealed and polymerized at room temperature for 24 h.A sample of the reaction was carefully removed for 1 H NMR and GPC analysis.The samples for 1 H NMR were simply diluted with CDCl 3 while samples for GPC were first diluted with THF and then passed over an aluminium oxide column to remove metal salts.
Conversion of polymerization was calculated via NMR.Both OEGA and tBA conversion was calculated by comparing the ratio between the intensity of acrylate bond (CH=CH 2 ) and methyl ether bond (O-CH 3 ) and tert-butyl bond (C-C 3 H 9 ) for OEGA and tBA respectively.
The conversion for copolymerization is calculated to be >99%.
The polymer was purified via dialysis to remove any small molecules impurities.Pure poly(OEGA-stat-tBA) (2 g) was then dissolved in DMF (4.5 ml) and mixed with NaN 3 (100 mg) and left to react overnight.The resulting polymer was purified again via dialysis against water and methanol.The presence of the azide group was verified using IR measurements.
Finally the polymer was dissolved in dichloromethane (DCM) and mixed with trifluoroacetic acid (TFA) for the deprotection of tert-butyl group to generate the acrylic acid group.The final polymer was isolated via dialysis and vacuum to remove any solvent residue and analysed via NMR and IR.

Cu(0)-mediated polymerization of Poly(hydroxyl ethyl acrylate-stat-N-hydroxysuccinimide
ester acrylate) P2.In a typical polymerization, hydroxyl ethyl acrylate (HEA, 5 g, 4.3E-02 moles) N-hydroxysuccinimide ester acrylate (NHSA, 190 mg, 1.1E-03 moles), DMSO (5 mL), EBiB (100 µL), Me 6 TREN(15 µL), and CuBr2 (15 mg) were charged to a polymerization flask fitted with a rubber septum and the mixture was degassed via nitrogen for 30 min.A slight positive pressure of nitrogen was then applied and the pre-activated copper wire (1 cm) was carefully added under a nitrogen blanket.The polymerization flask was then resealed and polymerized at room temperature for 24 h.A sample of the reaction was carefully removed for 1 H NMR and GPC analysis.The samples for 1 H NMR were simply diluted with CDCl 3 while samples for GPC were first diluted with THF then passed over an aluminium oxide column to remove metal salts.
Conversion of polymerization was calculated via NMR.Both OEGA and tBA conversion was calculated by comparing the ratio between the intensity of acrylate bond (CH=CH 2 ) and alkyl ester bond (O-CH 2 ) and succinimide bond (CH 2 -CH 2 ) for HEA and NHSA respectively.The conversion for copolymerization was calculated to be 100% for NHSA and 75% for HEA.

Porous Silicon Rugate Filter Formation
Rugate filters with 60 sinusoidally varying refractive index layers, a porosity variation from 54.5% to 57.5%, and an average pore size of ca.27 nm were prepared in a custom-made electrochemical cell with 35% hydrofluoric acid ethanolic solution as described previously. 18iefly, silicon wafers were cut into pieces (approximately 10 x 10 mm), cleaned in acetone and ethanol with sonication for 5 min each, and placed in the cell back-contacted with a polished steel electrode.A circular platinum electrode was immersed in the ethanolic HF solution above the wafer (Figure S1).A current density varying between 48 and 65 mA cm -2 was applied to the cell sinusoidally.After etching, the wafer was rinsed with ethanol and pentane and dried with argon.
The first fabrication step to construct the PSi-polymer structure is by forming an alkyne selfassembled monolayer to form surface 1, Scheme 1 by a thermal hydrosilylation method previously described in our group (supplementary information). 30It was then followed by the attachment of polymer P1 as explained below;
Reaction was carried out at room temperature and stopped, after 17 to 18 h.The prepared surface-bound [1,2,3]-triazole samples (surfaces 2, Scheme 1) were rinsed consecutively with copious amounts of ethanol and water and then analyzed.

Activation of carboxylic acid
The anti-fouling polymer modified samples were further modified to activate the carboxylic

Peptide immobilization
PSi modified sample with activated ester surface through EDC / NHS reaction were transferred to a sample flask containing the peptide sequences.Two custom prepared peptide sequences, VPLSLYSGK and SGKGPRQITAK, selectively cleavable by MMP-2 and MMP-9 respectively were used.Peptide immobilization was achieved by incubating the activated sample with 1 mg/mL peptide in 1X PBS for 3 h followed by rinsing with MilliQ™ water and ethanol.This resulted in surface 3 (Scheme 1).

Attachment of the sacrificial polymeric unit, P2
To a reaction vial, 80-100 mg of the synthesized polymer P2 was added to 1 mL of MilliQ™ water.To this reaction vial, the peptide-modified samples were added.The reaction was allowed to take place for a period of 3 h to form surface 4, Scheme 1 after which the samples were rinsed with copious amounts of MilliQ™ water.Optical reflectivity measurements were performed in water following the rinsing of polymerized samples.To ensure there was not any physical absorption of material after each modification step, the chips were rinsed with copious amounts of MilliQ™ water and ethanol.

Proteinase assays
For the proteinase assays, the surface modified samples were placed in a sample with proteinase enzymes in PBS and incubated at 37 °C during the entire duration of measurements.The samples were transferred to the stage and measured under wet conditions at specific designated time intervals.Optical reflectivity spectra were measured using a custom built optical setup (Figure S2).
Characterization Techniques NMR Spectroscopy. 1 H NMR spectra were recorded using a Bruker ACF300 (300 MHz) spectrometer employing CDCl 3 as solvent.Monomer conversions were determined via 1 H NMR spectroscopy,

X-ray Photoelectron spectrometer (XPS).
A Kratos Axis ULTRA XPS incorporating 165 mm hemispherical electron energy was used.The incident radiation was monochromatic A1 X-rays (1486.6 eV) at 225 W (15 kV, 15 ma).Survey (wide) scans were taken at analyzer pass energy of 160 eV and multiplex (narrow) higher resolution scans at 20 eV.Survey scans were carried out over 1200 eV binding energy with 1.0 eV steps and a dwell time of 100 ms.
Optical reflectivity measurement.Optical reflectivity spectra were measured in the visible and near-infrared at normal incidence using a custom-built optical arrangement.The setup incorporated a USB2000+ miniature fibre-optic spectrometer (Ocean Optics Inc.) and a fibrecoupled halogen light source (Mikropack GmbH, Germany) and had a spectral resolution of 1 nm and measurement spot size of ∼100 µm.Spectra were processed using custom spectroscopy software platform driven by LabVIEW (National Instruments, TA).

Scanning electron microscopy (SEM).
Scanning electron microscopy (SEM) images were taken using a Hitachi S900 SEM with a cold field emission source (4 kV).PSi samples were cleaved in the centre of the film and mounted on a brass sample base.

Results and Discussion
The overall construction of the PSi-polymer platform for proteinase detection is shown in Two polymers were synthesized for the purpose of this work (Scheme 2).Firstly, the antifouling polymer (P1) developed in this study required several characteristics, 1) an antifouling domain, 2) the ability to be functionalized by a peptide, and 3) have a functional moiety to allow conjugation to the alkyne terminated PSi surface.In order to achieve these properties, controlled radical polymerization was used for the synthesis of P1.Controlled radical polymerization has proven to be a powerful tool to create complex macromolecular structures with control over molecular weight, topography, functional groups, as well as end groups. 34There are various controlled radical polymerization methods such as Reversible Addition Fragmentation Transfer (RAFT) 35 , Atom Transfer Radical Polymerization (ATRP) 36 , Nitroxide Mediated Polymerization (NMP) 37 and recently Cu(0)-Mediated Radical Polymerization. 32,38 n this work, all polymer synthesis was performed via Cu(0)-mediated polymerization.This polymerization technique was selected because of the ease of the polymerization procedure and its excellent end group fidelity which has been demonstrated previously. 33,39 irstly, P1 was synthesized by statistical copolymerization of OEGA 480 and tBA to make poly(OEGA-stat-tBA)Br.As can be seen from Figure S3, the polymerization undergoes virtually full conversion (>99%) with excellent molecular weight control (PDI = 1.11, Figure S4).The molecular weight obtained from GPC (M n = 8 400 g mol -1 ) is in good agreement with the molecular weight estimated by NMR (M n = 7 500 g mol -1 ).The bromine end-group of the polymer was then modified with NaN 3 to give poly(OEGA-stat-tBA)N 3 followed by the deprotection of the tert-butyl group with trifluoroacetic acid (TFA) to form acrylic acid group resulting in poly(OEGA-stat-AA)N 3 .Deprotection of tert-butyl group to give an acidic group can be observed using NMR spectroscopy by tracking the disappearance of peaks at 1.85 ppm (Figure 1).Moreover, this modification can be easily followed by FT-IR measurements as shown in Figure 2 where azide peaks is easily identifiable at a bending vibration of 2120 cm -1 and -OH stretch from hydrogen bonds in carboxylic group is seen as a broad peak at 3200 -3600 cm -1 after deprotection of the tert-butyl group with acid treatment.The sacrificial polymer P2 poly(HEA-stat-NHSA) was also synthesized via Cu(0)-mediated polymerization.P2 is a sacrificial unit to leave the pores after cleavage of the proteinase selective peptide by the relevant MMP.Hence it is required to couple to free amine groups of the peptide sequence that are already immobilized onto P1.Therefore, P2 required functional groups that can react with amine groups such as N-hydroxysuccinimide ester (NHS) or pentafluorophenyl group (PFP).NHS group is chosen over PFP as the pentafluorophenol group which will be liberated through the reaction with amine that could be toxic.In order to synthesize P2, N-hydroxysuccinimide ester acrylate (NHSA) was copolymerized with hydroxyethyl acrylate (HEA).The reason for this choice is two-fold, 1) hydroxyethyl acrylate (HEA) is known to be a biocompatible material, and 2) the hydroxyethyl acrylate group has a different chemical signature compared with POEGA used in P1 where the greater amount of the O-C=O group can be detected by X-ray photoelectron spectroscopy (XPS).The polymerization of HEA and NHSA was followed by NMR, as shown in Figure S5.The conversion after 14 h reaction was found to be 100% of NHSA and 75% of HEA.The molecular weight of P2 was calculated from NMR (M n = 7 500 g mol -1 ) and GPC (M n = 7 200 g mol -1 ) with a narrow polydispersity index (PDI) = 1.13 (Figure S6).The molecular weight obtained from GPC is in good agreement with theoretical calculation and molecular weight obtained from NMR.It is important to note that polymerization of NHSA has been shown to be challenging by Shunmugan et al. 40 when using ATRP polymerization as well as RAFT polymerization as demonstrated by D'Agosto et al. 41 .In this work the copolymerization of hydroxyl ethyl acrylate (HEA) and N-hydroxysuccinimide ester acrylate was reasonably straight forward and can be useful for other applications.More importantly, the presence of NHSA group in the polymer is essential for further reaction with the amine group that is presence in the peptide sequences employed.The presence of this functional group can be clearly seen in proton NMR with peaks at 2.85 ppm which arise from the CH 2 -CH 2 bond of the N-hydroxysuccinimide group (Figure S5).The presence of Nhydroxysuccinimide group is also confirmed using FTIR measurements with two small peaks at 1780 and 1810 cm -1 are indicative of N-hydroxysuccinimide moiety (Figure 2).with two peaks at 1780 and 1810 cm -1 indicating the presence of the N-hydroxysuccinimide ester moiety in the copolymer.

PSi fabrication and modification
Porous silicon was made via anodic etching of p-type Si(100) in ethanolic HF.Rugate filter structure was chosen due its optical reflectivity character which has a narrow and sharp peak.  Figure 3A shows the cross sectional views of the entire 60-layer porous silicon (PSi) rugate filter structure with the pores perpendicular to each other.The rugate filter was formed after anodization using a sinusoidal current density (freshly etched surface).The properties of the porous structure such as porosity and pore sizes can be controlled via the anodization parameters.Figure 3B shows a top view of the porous structure with the average pore size of 20 nm.Surface modification was then performed to modify the surfaces of the pores to allow passivation of the surface from water penetration and thus protecting the photonic crystal entity from surface oxidation (i.e.formation of Si-O x from Si). 42 This step is essential as the optical reflectivity of the material is highly dependent on the chemistry of the pores. 26drosilylation with 1,8-nonadiyne (Scheme S1) was chosen as it provides a close packed self-assembled monolayer that protects the silicon from oxidation as well as possessing a

A B
remaining alkyne functionalities which provide the opportunity for further modification via copper catalyzed azide-alkyne cycloaddition (CuAAc) "click" chemistry. 30

Polymer Chemistry Accepted Manuscript
The success of the surface modification of the PSi was determined using optical reflectivity measurements and XPS.Firstly, hydrosilylation of the 1,8-nonadiyne on PSi (freshly etched surface 1, Scheme S1) resulted in a significant red shift of the reflectivity peaks as seen from Figure S7.Previous work by Guan et al. has shown that PSi microparticles passivated via thermal hydrosilylation maintain their optical integrity for up to 7 days in biological environment such as phosphate buffered saline (PBS), human blood, or Dulbecco's modified eagle's medium (DMEM) cell culture media. 43Thus, this surface is suitable for the proteinase assay which is the subject of this study."Clicking" of anchored polymer poly(OEGA-stat-AA)N 3 P1 on alkyne functional surface was conducted through CuAAc reaction and resulted in further red shift (surface 2, Figure 4).Peptide immobilization was then conducted via the activation of the carboxylic acid via EDC/NHS chemistry followed by addition of peptide sequence VPLSLYSGK peptide for MMP-2 proteinase specific cleavable linker and SGKGPRQITAK for MMP-9 proteinase specific linker.These peptide sequences were chosen based on previous work by Turk et al. 44 and Kridel et al. 45 which determined the cleavage sites for different MMPs proteinase (MMP-2 and MMP-9 respectively) and its kinetics.It was concluded that the linkages between serine(S)-leucine(L) and glutamine(Q)isoleucine(I) are the most prevalent linkage sites for MMP-2 and MMP-9 proteinase enzyme respectively. 44Both peptide sequences were designed with two free amine functional groups, one was located at the N-terminal of the peptide and the other from alysine (K) with a free amine on the side chain at the C-terminal.This diamine functionality allows statistical crosslinking with sacrificial polymer P2 poly(HEA-stat-NHSA).Any amine group from the peptide which does not react with -COOH of P1 during the EDC/NHS step may react with NHSA group of P2.Both the immobilization of the peptide (surface 2 3) and the sacrificial polymer P2 (surface 3 4) was easily followed by reflectivity measurements as shown in  For further characterization of surface modification process, chemical analysis was performed using x-ray photoelectron spectroscopy (XPS).XPS survey spectra (Figure S8) show the element at each step of modification.It can be seen from the survey spectra that the expected elements Si, C, O, and F were present in surface 1 but no N present.N peaks appeared with surfaces 2-4 which indicates successful addition of polymers and peptide onto the pores.Furthermore, Figure 5 shows narrow scans of C 1s and N 1s of each step of the modification procedures.Surface 1 shows a peak at a binding energy of 285 eV indicative of the carbon bound to carbon (C-C).This signal came from the functionalized alkyne layer.
Since there was no elemental nitrogen present on the surface, no nitrogen signal was detected as expected.After attachment of P1 (surface 2), there were new peaks observed at ~ 286.8 eV in the C 1s spectrum.This was indicative of oxygen bound to carbon (C-O) which represented the ethylene glycol group of P1.The high-resolution N 1s data (Figure 5b) showed two peaks centred at 400.58 eV and 403.48 eV with ratio of around 2:1, indicating the presence of chemically distinct nitrogen atoms consistent with the formation of a triazole moiety.This strongly suggests the fusion of azido polymer P1 to the alkyne modified surface. 30After immobilization of the peptide, there was a significant increase in the intensity of the N 1s peak especially at 400.38 eV which is attributed to various nitrogen based peptide bonds in the peptide (mainly amine and amide). 18The best fit to the experimental N 1s emission curve was obtained when this spectral region was deconvoluted into two functions having binding energies of 402.

Proteinase assay
The performance of the modified PSi-polymer construct as an optical biosensor was tested by conducting proteinase assays and measuring the optical shift in the reflectivity spectra of the proteinase incubated surfaces over time.The PSi-polymer chip was incubated with different proteinase enzyme as well as a control (PBS buffer solution).Example of the reflectivity measurement can be seen from Figure 6 and Fisgure S9.Two different proteinases, MMP-2 and MMP-9 were chosen to test the selectivity of the surface to different proteinases.Upon exposure to proteinase enzymes incubated at 37 °C, cleavage of the peptide bond takes place due to the proteolytic activity of the enzymes as depicted in Figure 7 (top).As the peptide is cleaved by the proteinases, the amino acid fragments and the sacrificial polymeric material P2 leave the pores bringing the reflectivity shift to the peaks corresponding to surface 2, changing the bulk refractive index of the crystal.This change in refractive index is measured as a "blue shift" in the reflectivity spectra of the PSi sensor.The optical detection of proteinase activity was accomplished in 100 nM concentration of proteinase enzyme.Previous work by Kilian et al. 20 has shown the potential of using modified PSi as an optical biosensor by immobilizing peptide and biopolymer gelatin. 18,20 n the work presented here, improvements have been made by increasing the total reflectivity shift and the specificity through the formation of the crosslinked polymer network for a given MMP. Figure 7 demonstrates the specificity of the chip towards specific proteinases as an optical response.It was observed when the chips with specific peptides were incubated with the complementary proteinase (for which the chip was made selective).In control samples with PBS solution negligible optical shift was observed (< 5 nm).More importantly, incubation of the modified PSi with a mismatch proteinase also resulted in negligible optical shift (< 5 nm).That is the polymer-VPLSLYSGK networks does not cleaved under exposure to MMP-9 proteinase and polymer-SGKGPRQITAK network does not cleaved under exposure to MMP-2 proteinase.The proteinase assay activity was done up to 5 hours as the the plot has approach a plateau after around 3-4 hours.This indicates that the enzyme has digest all the "digestible" material inside the pores.
These smart surface chips will have potential as a selective and sensitive optical biosensor. 17,20 owever, the integration of polymer networks have added specificity to the system making it more versatile.For example, one could simply change the peptide sequence tailored to such sequence cleavable by a given target proteinase.

Conclusion Page 25 of 29
Polymer Chemistry

Polymer Chemistry Accepted Manuscript
We have demonstrated a generic approach to optimize the sensing capability of porous silicon through modular polymer conjugation strategy where the surface of the PSi was first modified with an antifouling polymer, then an enzyme cleavable link was added which bridged the antifouling polymer and a second sacrificial polymer that was lost upon enzyme cleave of the peptide.Polymers required for this work were synthesized through Cu(0)mediated polymerization which is suitable to synthesize copolymers poly(OEGA-stat-AA)N 3 and poly(HEA-stat-NHSA) with good control.PSi substrates with specific response to MMP-2 and MMP-9 were prepared through grafting of the copolymers and peptide sequences.
Cleavage of the peptide-polymer network by the appropriate proteinase decreases the average refractive index of the photonic crystal resulting in a change in the reflectivity peak to lower wavelengths (blue-shift).The PSi-polymer constructs were shown to have selectivity towards different MMP enzymes.The approach could easily be tailored for different chemical/biochemical moieties, thus increasing the potential of such smart surfaces for biosensor applications.These structures could be easily expanded for other proteinases enzyme by simple changing the specific peptides sequences.

Table of Content Figure
The incorporation of versatile and tuneable polymer-peptide network into the pores of porous silicon photonic crystals improve the selectivity of porous silicon optical biosensor to detect certain types of matrix metalloproteinases enzyme.
-3-(3dimethylaminopropyl)carbodiimide (EDC, 100 mg, 0.05 mmol) and N-hydroxysuccinimide (NHS, 120 mg, 1.05 mmol) were dissolved in MilliQ™ water (5 mL).The acid-terminated polymer on the porous silicon surface was added to this solution and the uncapped flask was left in the dark at room temperature for 3 hours.The activated silicon samples were removed from the reaction mixture and washed with copious amounts of water before being further reacted.

Figure 1 .
Figure 1.NMR characterization of anchored polymer P1 synthesis before and after deprotection.The NMR spectra show expected peaks at 4.27 ppm and 1.85 ppm corresponding to the functional group of OEGA and tBA respectively.After deprotection, there is an obvious disappearance of peak at around 1.85.Note: the red cross denotes solvents peaks.

Figure 2 .
Figure 2. FT-IR spectra of (Top, A) synthesis and modification of anchored polymer P1.The

Figure 3 .
Figure 3. Scanning electron microscopy of (A, left) Cross section view of a 60-layer PSi

Figure 4 .
Figure 4. Typical reflectivity measurement of PSi during different stages of surface

Figure 4 .
Figure 4.The total red shift of the peaks was measured to be around 25-30 nm which

Figure 5 .
Figure 5. XPS narrow scans for the C 1s and N 1s regions for each of the surfaces 1-4 shown 38 eV and 400.38 eV.The ratios of N/C and N/Si were calculated which are increased from 0.004 to 0.014 and 0.015 to 0.057 respectively from surface 2 to 3, indicating more N as the peptide and polymer attached.Finally, the C 1s narrow scan of surface 4 shows a significant increase in peaks at 289.1 eV which can be attributed to ester bonds (O-C=O).This is an evidence for the attachment of poly(HEA-stat-NHSA) which has a high component of ester group (O-C=O) in the HEA bond.It is important to note that from the N 1s narrow scan there is attenuation of the peak at around 403 eV due to the fact that the triazole peaks are small component of the whole construct especially after another layer of polymer is bound.The composition of N compared to C and Si is also increased to 0.019 and 0.066 from 0.014 and 0.057 respectively.

Figure 6 .
Figure 6.Reflectivity spectrum of PSi chip with VPLSLYSGK cleavable polymer network after exposure to protease enzyme MMP-2 with the reflectivity peaks shifting towards lower wavelength ("blue shift") indicating materials leaving the pores.The peaks shifts because as the protease digests the organic material over time, the space that is left by the organic material (n ~ 1.45) is filled with water (n =1.33), which has lower refractive index than organic material.