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Singlet oxygen detection on a nanostructured porous silicon thin film via photonic luminescence enhancements Siti Nurul Aisyiyah Jenie, Sally E Plush, and Nicolas H. Voelcker Langmuir, Just Accepted Manuscript • Publication Date (Web): 16 Apr 2017 Downloaded from http://pubs.acs.org on April 16, 2017

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Singlet oxygen detection on a nanostructured porous silicon thin film via photonic luminescence enhancements S. N. Aisyiyah Jenie1,3, Sally E. Plush2 and Nicolas H. Voelcker1,4* 1

Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia

2

School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia

3

Research Centre for Chemistry, Indonesian Institute of Sciences (LIPI), Kawasan Pusat

Penelitian, Ilmu Pengetahuan dan Teknologi (Centre for Research, Science and TechnologyPUSPIPTEK), Serpong, Tangerang, Banten 15310, Indonesia 4

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia *Corresponding author: Prof. Nicolas H. Voelcker 381 Royal Parade, Parkville, Victoria 3052, Australia

Tel: +61 (0)3 99039230, Fax: +61 (0)3 9903 9581

email: [email protected] 1 ACS Paragon Plus Environment

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KEYWORDS. Singlet oxygen, porous silicon, microcavity, luminescence enhancement

ABSTRACT. Since reactive oxygen species are involved in a range of pathologies, developing analytical tools for this group of molecules opens new vistas for biomedical diagnostics. Herein, we fabricate a porous silicon microcavity (pSiMC) functionalized with the luminescent singlet oxygen (1O2) probe EuA ((Eu(III)-2,2',2''-(10-(2-((4-(2-((4-(2((anthracen-9-ylmethyl)amino)ethyl)-1H-1,2,3-triazol-1-yl)amino)-2-oxoethyl)-2-oxo-1,2dihydroquinolin-7-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) as proof-of-concept of an optical sensor for reactive oxygen species. We characterize each surface modification step of the pSiMC by means of FTIR and X-ray photoelectron spectroscopy as well as by determining the optical shifts of the resonance wavelength of the pSiMC. The luminescence signal upon detection of 1O2 on the EuA-modified pSiMC is enhanced ~2-fold compared to that of a single layer and detuned microcavity. The sensing performance of the EuA probe is improved significantly on the pSiMC compared to that in aqueous solution giving a limit of 1O2 detection of 3.7 x 10-8 M.

INTRODUCTION Reactive oxygen species (ROS) are involved in a diverse range of pathologies including ageing,1 cancer,2 inflammation,3 cardiovascular,4 autoimmune and neurodegenerative diseases.2b, 3-4 Singlet oxygen (1O2) is the major ROS involved in generating oxidative stress in cells and tissues

1, 3, 5

. Over the past decades, there has been significant progress in the

development of optical detection for

1

O2. These optical sensors mainly exploit the

phosphorescence property of the molecule at ~ 1270 nm. The quantum yield of this phosphorescence, however is disappointingly small (10-5 -10-8), resulting in a weak emission.6 Studies have been conducted to overcome this limitation by applying metal

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nanostructures for amplifying the phosphorescence detection. Gold nanoparticles, for example, which were tuned to have the appropriate localized surface plasmons have been reported to give a phosphorescence enhancement of the 1O2 up to a factor of several hundred.7 Using the same plasmonic principle, silver island films (SIF) have also found their applications in 1O2 phosphorescence enhancement. The near-IR phosphorescence of 1O2 was reported to be enhanced by a factor of 35.8 Although the indirect monitoring of 1O2 through the fluorescence or phosphorescence of a chemical probe on a nanostructured surface has not been thoroughly studied, sensors that apply this principle are very attractive. The emission occurs in the visible region, hence simple optical detectors can be employed. Moreover, the use of an organic molecule as the chemical probe may improve the quantum efficiency giving an excellent response as opposed to the weak 1O2 signal at 1270 nm. Probes containing an anthracene derivative, such as 9-[2(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX),9 [4’-(10-methyl9-anthryl)-2,2’:6’,2”-terpyridine-6,6”-diyl]bis(methylenenitrilo)tetrakis(acetate)-Eu(III) (MTTA-Eu(III))10 and its derivative (MTDTA-Eu(III))11 are initially non-luminescent. In the presence of 1O2, these probes become luminescent as a result of the formation of an endoperoxide in the 9,10 position. The quantum yield has been reported to be in the order of 0.5-0.9 in most cases.9, 12 In our previous work, we have established that a photonic structure based on a porous silicon microcavity (pSiMC) enhances the luminescence of a confined fluorophore.13 This is in agreement with previous reports, which confirmed that besides being sensitive to changes in the optical thickness of the spacer layer,14 pSiMCs are an excellent host-matrix for luminescent molecules since they both sharpen and amplify the emission.15 We have reported a comprehensive study on the preparation of pSiMC for the luminescent enhancement of a fluorophore. The pSiMC was prepared by controlled electrochemical etching in which the


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porosity, layer thickness and incidence angle towards the excitation light can be tuned precisely.13a The fluorophore applied in that study was a europium complex with weak emission characteristics due to the absence of a sensitizing antenna. The results showed that the emission of the complex was amplified more than 9-fold in the pSiMC when compared to a single layer. Biosensors can exploit this effect by monitoring target analytes or molecules via their direct emission or an additional luminescent label.16 As described in our previous research, we have developed a model biosensor which incorporated the use of an emissive europium complex consisting of a sensitizing carbostyril antenna.17 The proof-of-principle sensing experiment was conducted in buffer and wound fluid as the biological matrix. The model pSiMC platform was able to detect the europium labeled protein to a concentration as low as 150 nM. Herein, we report the development of a europium based sensor for the detection of 1O2; EuA. This probe, EuA, consists of a highly emissive europium complex with a carbostryil antenna to overcome the inherent low molar absorptivity of europium and an anthracene moiety as the 1O2 chemical trap. EuA was designed to recognize 1O2 and be attached into the pSiMC through the secondary amine moiety available in the molecule. The emission of the probe in response to exposure to 1O2 was studied.

EXPERIMENTAL Preparation of pSi Films The pSi films were prepared using boron doped, [100]-oriented silicon wafers (0.000550.001

Ωcm

resistivity,

475-525

µm

thickness,

Siltronix,

France)

which

were

electrochemically etched in a 1:1 mixture of 48% (v/v) hydrofluoric acid (HF) (Scharlau, Australia) and ethanol (EtOH) (100%, ChemSupply, Australia) in a custom built Teflon cell at 25°C using a source meter (Keithley 2425, USA) as the current source. The cathode used


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was a platinum mesh while aluminum foil was used to contact the silicon anode. The area of the exposed region of the silicon wafer was 1.767 cm2. Prior to sample preparation, the parasitic layer of the Si wafer was removed by etching the silicon wafers in a 1:1 HF/EtOH solution at 56.8 mA/cm2 for 30 s, followed by thorough rinsing of the layer with ethanol, drying with nitrogen (N2) gas and immersing the layer in 1 M sodium hydroxide (NaOH) (Sigma-Aldrich, Australia) for 2 min. Finally, the wafer was cleaned with deionized H2O and EtOH consecutively before being dried under N2 gas.18 The microcavity structures were fabricated by etching alternating layers of high and low refractive indices. The corresponding current densities for each of the refractive indices are shown in Table 1. The configuration of the microcavities were designed using the commercial program SCOUT that is based on the transfer matrix method19 and the best fit between the theoretical and experimental spectra were obtained as previously reported.13a, 1718

The angular dependence of the resonance wavelength was taken into consideration in

designing the pSiMC. In both microcavity samples, MC and detuned-MC, the first layer of the mirror was etched at 56.8 mA/cm2 (high porosity, H) while the second layer (low porosity, L) was etched at a current density of 5.7 mA/cm2. The anodization time for each of the samples followed that listed in Table 6.1. Our optimum design resulted in a pSiMC configuration of (HL)3-HHHH(LH)3, which consists of three periods of Bragg reflectors and four periods of H as the spacer layer. Freshly etched samples were washed thoroughly with EtOH and dried under N2 gas. In order to obtain the same film thickness as the pSiMC (~ 2 µm), pSi single layers were prepared by etching the silicon wafers at 56.8 mA/cm2 for 120 s, then washed thoroughly with EtOH and dried under a stream of N2 gas.


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Table 1. Etching conditions and pore parameters for the pSiMC and pSi single layers. Porosity, layer thickness and refractive index were determined by the transfer matrix method. Pore sizes were determined by means of scanning electron microscopy (SEM). pSi Sample

Current density (mA/cm2)

Etching time (s)

Porosity (%)

Layer thickness (nm)

Refractive index

Pore size (nm)

MC H Layer

56.8

5.4

86.2

130.5

1.3

65-100

L Layer

5.7

34.4

67.3

101.8

1.7

17-41

H Layer

56.8

6.7

86.2

150

1.3

65-100

L Layer

5.7

39.4

67.3

110.3

1.7

17-41

Single Layer

56.8

120

86.2

1980

1.3

65-100

Detuned-MC

The range of pore sizes for the H and L layers as well as the single layer were obtained through imaging of the samples by scanning electron microscopy (SEM). The pore sizes of the H and L layers were measured by preparing single layers at 56.8 mA/cm2 and 5.7 mA/cm2, respectively.

Modification of the pSi Films The modification of the samples followed Scheme 1. Freshly etched pSi samples were oxidized under ozone at room temperature for 1 h. The oxidized samples were then silanized with 5% (v/v) (3-Iodopropyl)trimethoxysilane (IPTMS) (Sigma-Aldrich, Australia) in dry toluene at 80 °C overnight. The iodoalkane-terminated surfaces were then exposed to 40 µl of 1 mg/ml of EuA in dimethylformamide (DMF) (Sigma-Aldrich, Australia) and H2O with volume ratio of 3:1. The synthesis and characterization of EuA followed the procedure published elsewhere.20 The modification of the silanized surface with EuA was conducted at 6 ACS Paragon Plus Environment

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65 °C overnight. The reaction vial was sealed with Teflon tape to avoid evaporation. The samples were washed with DI water thoroughly and dried under a stream of N2 gas.

Scheme 1. Surface modification of MC with EuA (MC-EuA). The silanization reaction was performed at 80 °C and 50 °C, and reaction times were varied from 15 min, 30 min, 1 h and 3 h for each reaction temperature. This optimization was conducted on pSi single layers etched at 56.8 mA/cm2 for 120 s. The coverage of EuA immobilized on the MC film was also optimized by exposing the silanized pSiMC samples to different concentrations of EuA ranging from 0.025, 0.05, 0.1, 0.25, 0.5, 1 to 4 mg/ml. The reaction was conducted at 65 °C overnight.

Characterization of pSi Films SEM images were obtained on a FEI QuantaTM 450 field emission gun environmental scanning electron microscope. The cross-sectional profiles of the pSiMC samples were measured with a 30 kV field emission source. Interferometric reflectance spectroscopy (IRS) measurements were conducted by applying white light from a tungsten lamp (Ocean Optics, USA), which was focused through a collimating lens onto the pSi surface at normal incidence. Light reflected from the surface was collected through the same optics and the distal end of the bifurcated fiber optic cable was connected to a CCD spectrometer (Ocean Optics S-2000). Reflectivity spectra were recorded over the wavelength range of 450-1000 nm. 7 ACS Paragon Plus Environment

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Fourier transform infrared (FT-IR) spectroscopy for p-type Si[100] wafers were recorded on a Bruker Vertex 70 Hyperion microscope coupled to a liquid N2 cooled MCT detector, in reflectance mode. Background spectra were acquired for clean unetched silicon wafers, at 3.5 cm-1 resolution, over the range of 500-3800 cm-1 with an accumulating average of 64 scans. The base line was corrected and normalized with OPUS 7.2 Spectroscopy software (Bruker). Luminescence spectroscopy measurements from the modified films with the luminescent probes were carried out on a LS55 fluorescence spectrometer (Perkin Elmer, USA). The samples were placed in a quartz cuvette with a customized holder and the emission was recorded over the range of 550 - 650 nm with an excitation wavelength of 348 nm at a 45° angle to the surface. The measurement was conducted in phosphorescence mode with a cycle time of 20 ms, delay time of 0.1 ms and a photomultiplier voltage set to 900 V.

Detection of Singlet Oxygen on the Modified pSi Film The detection of 1O2 via luminescence enhancement was conducted on the MC, detuned MC and single layer samples modified with EuA. The samples were immersed in 1 ml of solution containing 1 M of hydrogen peroxide (H2O2) (Sigma-Aldrich, Australia) and 10 mM of sodium hypochlorite (NaOCl) (Sigma-Aldrich, Australia) in 0.1 M phosphate buffered saline (PBS) (Merck, Australia) at pH 7.4. The sensing experiments were conducted at room temperature after which the samples were rinsed with PBS buffer and dried under a stream of N2 gas. Luminescence spectra were taken from the dried samples. The exposure time of the MC-EuA samples with H2O2/NaOCl/PBS solution was optimized. The time was varied from 2, 5, 10, 15, 30 to 60 min. The samples were rinsed with carbonate buffer, dried under a stream of N2 gas and the luminescence spectra were recorded. Following the optimization of the exposure time, the limit of detection of the MC-EuA samples for 1O2 detection was determined. The samples were immersed in a NaOCl/PBS


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solution with different concentrations of H2O2 ranging from 5 x 10-12 to 1 M for an optimized time at room temperature, rinsed with PBS buffer and dried under a stream of N2 gas. Luminescence spectra were obtained from the dried samples.

RESULTS AND DISCUSSION Preparation of pSi Films The pSiMC was designed according to the parameters detailed in Table 1. The refractive indices of the H and L layer were 1.3 and 1.7, respectively. The experimental reflectance spectra under normal illumination of the pSiMC were compared with the corresponding bestfit simulations obtained by applying the transfer matrix method (Figure 1). Excellent agreement between the experimentally observed and the simulated cavity modes was observed. The full width at half maximum (FWHM) was ~17 nm. The Q value, i.e. the amount of energy stored within the microcavity 13a, 21 was 40.

Figure 1. Reflectance spectrum of the freshly etched pSiMC sample (solid curve) compared to the calculated reflectance spectrum (dashed curve) at normal illumination. The SEM images in Figure 2 show the top view images of the L layer, H layer generated by applying a current density of 5.7 mA/cm2 and 56.8 mA/cm2, respectively, and the cross9 ACS Paragon Plus Environment

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sectional view of sample pSiMC. Pore sizes were in the range of 17 - 41 nm for the L layer and 65 - 100 nm for the H layer. Figure 3(c) shows that the microcavity is ~2 µm thick and features the desired (HL)3-HHHH-(LH)3 structure.

Figure 2. SEM micrographs of a) top-view L surface, etched at 5.7 mA/cm2, b) top view H surface, etched at 56.8 mA/cm2 and c) cross-sectional view of pSiMC.

Modification of pSi Films and their Characterization Freshly etched pSiMC films were modified for the covalent attachment of Probe EuA. Ozone oxidation was performed for the freshly etched samples to improve the surface stability and generate silanol groups. Silanization with 5% IPTMS in dry toluene was conducted at 80 °C for 30 min to ensure the anchoring of the iodosilane on the oxidized pSi surface (see Supporting Information).22 The samples were rinsed with dry toluene to remove the excess physisorbed silane. Rinsing with EtOH and acetone were avoided since those solvents might react with iodoalkane modified surface. The silanized samples were finally exposed to EuA overnight. The highly reactive iodo group on the silanized surface underwent nucleophilic displacement by the secondary amine moiety on EuA.23


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Figure 3. Overlays of FTIR reflectrance spectra for the surface modification steps on the pSi films according to Scheme 1. (1) fresh etched pSi, (2) modified pSi after oxidation, (3) after reaction iodosilane and (4) after nucleophilic substitution with EuA. Inset: Overlays of FTIR spectra of modified pSi films between wavenumbers 2800 and 3500 cm-1. The samples after each modification step were characterized by FTIR in reflectance mode. As shown in Figure 3, spectrum (1) exhibits bands at 916 and 2100 cm-1, assigned to the SiH2 scissor bending and the Si-Hx stretching mode, respectively. After ozone oxidation, the appearance of the specific band of the O-Si-O bending and stretching vibrations at 815 and 1008 cm-1, respectively, followed by the disappearance of the Si-Hx peak confirms the formation of the oxide layer on the silicon surface (Figure 3 - spectrum (2)). Silanization was confirmed by the appearance of the peaks at 1460, 2920 and 2892 cm-1 (Figure 3 – spectrum (c)), which were attributed to CH2 stretching vibrational modes of aliphatic C-H bonds of the iodosilane. The peak observed at 692 cm-1 is attributed to the C-I bond.24 Confirmation of iodosilane attachment came from XPS analysis which showed a distinct peak of the I3d (2.7 at.%) at 620 eV (Table S1). Finally, in spectrum (d) the C=O peak at 1616 cm-1 further confirms the immobilization of EuA on the silanized surface. We also observed the


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appearance of the broad peak around 3110-3410 cm-1, which corresponds to the coordinated water molecule (O-H) group in EuA. The optical shifts in the resonance of the pSiMC after each modification steps were also determined (Figure 4). After ozone oxidation of the freshly etched samples, a ~20 nm blueshift was observed. This effect is consistent with surface oxidation 18, 25 The formation of the organic layer by the iodosilane resulted in a clear redshift of ~38 nm confirming the increase in the effective refractive indices of the porous layer due to binding of an organic molecule. Finally, a redshift of ~9 nm was seen after immobilization of EuA on the silanized surface, which was expected on the basis of the increased refractive index of the porous layer. The optical quality of the freshly etched and functionalized pSiMC was preserved with a Qvalue maintained at ~40 for each reflectance spectrum. The final position of the resonance wavelength of the EuA-modified pSiMC (MC-EuA) was measured at 711 nm at normal incidence. At 45° incidence, the resonance wavelength blueshifted and overlapped with the emission of EuA at 614 nm.


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Figure 4. Reflectance spectra of pSiMC sample after the surface modification steps shown in Scheme 1 at normal incidence: freshly etched (blue), ozone oxidized (red), IPTMS silane modified (green) and EuA modified (purple).

Detection of Singlet Oxygen on pSi Films The detection of 1O2 by EuA in aqueous buffer at neutral pH, was explained in detail in our previous work.20 Briefly, at pH 7.4, probe EuA reacted with 1O2 (generated by reacting NaOCl and H2O2) through the anthracene moiety, followed by the recovery of the emission of the carbostyril antenna and the Eu(III) complex. This was indicated by a strong luminescence enhancement in the presence of 1O2. Hence, the luminescent probe shows a clear ‘off-on’ signal in the presence of 1O2. This detection principle is herein translated onto the pSiMC sensor. Sample MC-EuA was immersed in a 1 ml solution of NaOCl (10 mM) in 0.1 M PBS buffer. H2O2 (1 M) was added and the samples were immersed for 15 min. Figure 5a shows the emission spectra of the MC-EuA in its initial condition (dashed red curve) and in the presence of NaOCl (solid black curve) upon excitation at 348 nm. The figure also illustrates the 13 ACS Paragon Plus Environment

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emission spectrum of MC-EuA in the presence of 1O2 (solid red curve). In the presence of OCl- only, the EuA emission was quenched.26 Upon addition of H2O2 and formation of 1O2, the sample showed a 2.5-fold emission increase, consistent with the formation of an endoperoxide in the anthracene ring.26-27

Figure 5. (a) Emission spectra of MC-EuA in the presence of OCl- (solid black curve) and MC-EuA+1O2 (solid red curve) upon excitation at 348 nm. The dashed grey and red curve corresponds to the emission of the MC without EuA and MC-EuA, respectively. (b) Schematic of 1O2 sensing on MC-EuA surface. To illustrate the luminescence enhancement effect of the MC upon 1O2 sensing, the emissions were compared between the MC and other pSi structures. The structures included a pSi single layer and a detuned MC, which both have the same total thickness as the tuned pSiMC (~2 µm). As shown in Figure 6, the emission intensity at 614 nm (5D0→7F2 transition) using the tuned pSiMC was 2 and 1.8-fold higher than that using a single layer and a detuned pSiMC, respectively. This is in agreement with previous reports that spectral alignment of a MC or other aperiodic multilayers with the maximum emission of the emitting molecule enhances the emission intensity of the immobilized fluorophore.28 In contrast to our


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previous works,13b, 13c a narrowing of emission was not observed due to the larger bandwidth of the microcavity (~17 nm) compared to that of EuA (~10 nm).

Figure 6. Emission enhancement of the detection of 1O2 on MC-EuA versus other modified pSi structures upon excitation at 348 nm (Inset: emission intensities at 5D0→7F1 (585 nm) and 5

D0→7F2 (614 nm). Measurements were performed in triplicate (n=3). We determined spectral color purity feature of the Eu(III) complex on the pSi films using

the ratio of 5D0→7F2/5D0→7F1.13a, 17 This feature is regarded as an important parameter in studies related to the luminescence properties of lanthanides29 considering that these ions generate emissions at five different wavelengths. The 5D0→7F2/5D0→7F1 ratio of the immobilized EuA on the aligned microcavity increased more than 2-fold compared to the 5

D0→7F2/5D0→7F1 ratio of EuA in solution (Table 2), which further confirms that the pSi is a

suitable host for this probe. This is in agreement with previous studies that Si nanocrystallites may enhance the emission of Eu(III). 30


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Table 2. The ratios of luminescence intensity of 5D0→7F2/5D0→7F1 for EuA in solution and when covalently immobilized onto pSi structures upon detection of 1O2. 5

D0→7F2/5D0→7F1

50 µM of EuA in H2O

0.90

On pSi structures: MC

2.21

Single layer

1.74

Detuned MC

1.84

Towards a Singlet Oxygen Sensor In order to improve the sensitivity of the MC-EuA, we optimized both the surface modification steps, through changes in the concentration of EuA immobilized on MC, and the sensing of 1O2 by altering the detection time. The initial concentration of EuA used for immobilization on the pSiMC (Figures 3-5) was 1 mg/ml. Due to the bulky structure of EuA and the possibility that the probe may self-quench at high concentrations, the concentration of EuA used for reaction with the silanized surface was varied from 0.025, 0.05, 0.1, 0.250, 0.5, 1 and 4 mg/ml. The emissions from the MC-EuA with different concentrations of the probe, prior to NaOCl and H2O2 addition, were then recorded as shown in Figure 7a. The emission of MC-EuA reached its maximum when a concentration of 0.5 – 1 mg/ml of EuA was employed. The emission of the samples at concentrations below 0.5 mg/ml showed a significant decrease, which may be attributed to the lower amount of EuA attached on the surface. Interestingly, when we attempted immobilization at a high concentration of 4 mg/ml, we observed that the emission also decreased. This may be attributed to the self-quenching of


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the probe at higher surface density.31 Steric hindrance of the molecule when infiltrating through the multilayers does not apply here since the emission decreased only at higher concentration.

Figure 7. (a) Luminenscence intensity of MC-EuA immobilized with different concentrations of EuA in the absence of NaOCl and H2O2. (b) Luminescence intensity of MC-EuA (concentration of EuA applied was 1 mg/ml) at different reaction times with NaOCl and H2O2 (n=3). The optimized reaction conditions were then applied for the development of the 1O2 sensor. The detection of 1O2 was then conducted and the response time observed. The emission of the MC-EuA-EP samples at 614 nm was recorded 2, 5, 10, 15, 30 and 60 min after the addition of


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H2O2. The data in Figure 7b show that the highest intensity was obtained only 5 min after the addition of H2O2. At extended incubation times, the intensity however did not plateau but decreased. The observed decrease in intensity at longer incubation times could possibly be explained by the reaction of 1O2 with the carbostyril moiety of EuA over time, as has been observed with coumarin derivatives.32 Consequently, this leads to structural changes of the carbostyril moiety. As a result, the antenna can no longer sensitize the Eu(III) ion hence the luminescence decreases. The luminescence intensity at 614 nm of the MC-EuA as a function of H2O2 concentration in the NaOCl/PBS solution is illustrated in Figure 8. Different concentrations of H2O2 ranging from 5 x 10-12 to 1 M were applied. Linear correlation was observed within the concentration range of 5 x 10-8 to 1 M with an equation of y=0.7701x + 6.4017 (R2=0.9991). The sensing experiments were performed in triplicate. The limit of detection (LOD) was 3.7 x 10-8 M, calculated using the equation of yb+3Stdb, where yb is the average of the emission intensity measured for the blank control and Stdb is the associated standard deviation.


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Figure 8. Plot of normalized luminescence intensity of MC-EuA at 614 nm against the logarithmic H2O2 concentration added to NaOCl (10 mM) in PBS buffer (pH 7) (n =3). The broad working concentration range, covering eight orders of magnitude and the low LOD of 37 nM for our MC-EuA platform compares favorably with the literature. In Kiryu et al. 1O2 detection system, a dual charge integrating amplifier with two InGaAs/InP pin photodiodes was used to detect near infrared light and a working concentration range of 10100 µM of H2O2 was reported.33 Other singlet oxygen sensors rely on the phosphorescence enhancement of singlet oxygen using nanostructured materials, such as gold nanoparticles or silver island films.7b, 8 However, the detection limits were not reported. Hence, we believe that the MC-EuA is a promising detection platform for 1O2. In designing a sensor with potential for real life applications, the information of the physiological concentration of the analyte of interest is required. Numerous studies have been conducted to quantitatively determine the concentration of 1O2 in regards to cell death or necrosis, oxidative stress and photodynamic therapy (PDT).5c, 34 Recently, a comprehensive study by Zhu et al. has reported the in vivo threshold dosage of 1O2 for PDT using two types of photosensitizers, i.e. photofrin and the benzoporphyrin derivative monoacid ring A.34a The 19 ACS Paragon Plus Environment

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study identified that a threshold dose in the range of 0.56-0.72 mM of 1O2 produces necrosis. Another report suggested a much lower threshold of 1 µM of 1O2 in order to trigger apoptosis of cancerous cells.2b Given that our sensor has an LOD of 37 nM we believe that our sensing platform may find applications as a point-of-care (POC) sensor. Further development of the POC sensor would require modification of the rinsing and drying steps (i.e the use of microfluidic channels). Another possible development of the sensor is the use of the sensing platform as dipstick diagnostic device to detect singlet oxygen.

CONCLUSIONS We have demonstrated the detection of 1O2 on a pSiMC modified with EuA. The detection experiment was conducted by exploiting luminescence enhancement effects of the probe inside the pSiMC in the presence of 1O2. The covalent immobilization of EuA on the surface was achieved via nucleophilic substitution of the secondary amine of the probe with the iodo silane functionalized pSiMC. FTIR, XPS and the optical shift of the resonance wavelength of the microcavity were used to confirm the surface modification steps. The emission intensity measured upon 1O2 detection on pSi microcavities was ~ 2-fold higher than that on single layer or a detuned microcavity. This signal enhancement led to an excellent LOD of 3.7 x 108

M with a linear response between 5 x 10-8 to 1 M. These values are a significant

improvement to sensing performance of EuA in aqueous solution. The sensor demonstrated here is expected to find applications in POC diagnostics where singlet oxygen serves as a biomarker.

ACKNOWLEDGMENT This research was conducted and funded by the Australian Research Council Centre of Excellence

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CE140100036). SNAJ would like to thank the Australian Government for the Australia Award Scholarship and acknowledge funding from the Wound Management Innovation CRC (Australia).


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