Nanostructured porous silicon in imaging and biosensing

Giugno 6 - 10, 2022 – Ischia (NA) Nanostructured Porous Silicon in Imaging and Biosensing Giuseppe Barillaro Information Engineering Dpt University of Pisa [email protected] Outline •The University of Pisa •Photonicand plasmonicencoded microlens for flurescence microscopy using a smartphone •Bioresorbable chemical sensors for in-vivo tracking of analytes of clinical iterest Pisa-Tuscany-Italy The University of Pisa The Barillaro’s group @ the University of Pisa www.iet.unipi.it/g.barillaro [email protected] Advanced microand nano-structuring of materials (silicon, metal, polymers, composites) for application electronics, photonics, (bio)sensing and (nano)medicine Outline •The University of Pisa •Photonicand plasmonicencoded microlens for flurescence microscopy using a smartphone •Bioresorbable chemical sensors for in-vivo tracking of analytes of clinical iterest 1 cm + _ 1 cm The mass of silicon dissolved for unit time (and depth) depends on the etching current density Nanostructured porous silicon M.J. Sailor, Porous Silicon In Practice, Wiley (2012) Mimicking (trying to) Nature in lab 10 nm Over the latest 50 years, micro and nanotechnologies enabled the integration on a smartphone of a computational power 1.000.000 times larger that the Apollo’s mission computers Google image What we have done so far 31 Figures Figure 1. Preparation and characterization of PDMS lenses on nanostructured porous silicon. (a) Sketch of PDMS lens preparation on nanostructured PSi: 1etching of a PSi templating layer; 2drop of a prescribed mass of PDMS pre-polymer on PSi and subsequent thermal curing; and, 3free-standing PDMS lens obtained by peeling the lens off the PSi chip. (b) Picture of a PDMS lens as-formed on the PSi chip (left) and after peeling it off the PSi chip (right). (c) Main geometrical and optical characteristics of PDMS lenses for different porosity values (and, in turn, etching current density values) of the PSi templating layer, for constant PSi Photonic-encoded lenses for smartphone fluorescence microscopy S. Mariani, et al., Adv. Funct. Mater. (2020). Implantable and bioresorbable chemical sensor systems for in-vivo and continuous tracking of chemotherapeutic drugs D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programm e under grant agreement No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M . Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu implant monitor resorb From ex-vivo to in-vivo chemical sensors that degrade in in safe byproducts An EIC PathFinder Open Project funded with about 3 MEUR by the EC (2022-2025) Biofunctionalization of PSi with charged polyelectrolytes 1 oxidized PSi Biotin 2 PAH caoting 3 PMAA-biotin S. Mariani et al., Nat. Commun. 9(1), 5256 (2018) 15 µm silicon PSi air 2 4 Strept detection 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 IAW-IAW 0 (a.u.) Concentration (nM) 0 2 4 6 8 10 0.00 0.05 0.10 LbL functionalization Covalent functionalization IAW-IAW 0 (a.u.) Concentration (nM) noise floor LoD∼100 fM Figure 3 globally-neutral streptavidin in acetate negatively-charged streptavidin in HEPES 1 2 3 LbL biofunctionalization target binding repulsive unspecific removal 1 2 3 bare PAH/PMAA LbL coating unspecific binding repulsive unspecific removal 0 20 40 60 80 -125 -100 -75 -50 -25 0 HEPES Acetate Acetate EOT - EOT 0 [nm] Time [min] acetate HEPES acetate a) C on t i n uo u s Be f ore P B S A f t e r P B S B ef o re H E P ES Aft e r H E P E S -80 -70 -60 -50 -40 -30 -20 -10 0 10 Acetate EOT - EOT0 [nm] Acetate b) c) e) 0 20 40 60 80 100 120 140 160 180 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 IAW-IAW 0 (a.u.) Time (minutes) f) 2 strept 3 HEPES 1 acetate acetate a c et at e d) 0 50 100 150 200 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 IAW-IAW 0 (a.u.) Time (minutes) 2 strept acetate acetate 1 3 HEPES acetate g) Streptavidin Pepsin BSA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 IAW-IAW 0 (a.u.) pH = 7.4 > pIstrept, pIBSA, pIpepsin streptavidin 0 20 40 60 80 100 120 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 IAW-IAW0 (a.u.) Time (minutes) 2 strept 3 HEPES 1acetate acetate streptavidin 83 nM in saliva g) 83 830 8300 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 IAW-IAW0 (a.u.) Streptavidin (nM) Saliva 1:10 83 nM 830 nM 8300 nM 0 100 200 300 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 IAW-IAW0 (a.u.) Time (minutes) f) streptavidin spiked in saliva Performance of PSi biosensors in raw saliva The chemical composition of saliva consists of 99.5% water and 0.5% (5000 ppm) of solid components (0.1% electrolytes, 0.3% proteins, 0.1% small molecules) S. Mariani et al., Nat. Commun. 9(1), 5256 (2018). 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 Bioresorbable chemical sensor for pH sensing in vivo 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 M. Corsi, A. Paghi, et al., Advanced Science (2022) 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 18 Figure S1. Images of as-prepared nPSi scaffold, oxidation to nPSiO2, and transfer-printing on PLGA foil. a) Top-view SEM image (magnification 250000×) of a nPSi scaffold with porosity of ~77 %. Scale bar is 400 nm. Inset shows the histogram of the size distribution of pores with average diameter of 35 nm. b) Cross-section SEM image (magnification 35000×) of the nPSi scaffold in a). Scale bar is 1 µm. Inset shows a magnification (250,000×) of the bottom part of the scaffold that allows to better appreciate the columnar morphology of the pores. Scale bar is 400 nm. c) Picture of a 5-µm-thick nPSi membrane lifted-off from the native bulk silicon. d) Picture of the nPSi membrane in c) oxidized to nPSiO2 at 1000 °C for 5 min. e) Picture of nPSiO2 membrane in d) after transferprinting on a PLGA foil. Figure S2. Photoluminescence spectrum of a nPSiO2 membrane coated with PAH:Rh and PMAA:Rh polyelectrolytes. a) Sketch of a nPSiO2 scaffold on PLGA foil coated with Rh-labelled polyelectrolytes via LbL technique. Rh excitation peak at 520 nm/emission peak at 580 nm. b) Photoluminescence spectrum of a nPSiO2 scaffold on PLGA foil coated with (PAH:Rh/PMAA:Rh)2+(PAH:Rh)1. 18 Figure S1. Images of as-prepared nPSi scaffold, oxidation to nPSiO2, and transfer-printing on PLGA foil. a) Top-view SEM image (magnification 250000×) of a nPSi scaffold with porosity of ~77 %. Scale bar is 400 nm. Inset shows the histogram of the size distribution of pores with average diameter of 35 nm. b) Cross-section SEM image (magnification 35000×) of the nPSi scaffold in a). Scale bar is 1 µm. Inset shows a magnification (250,000×) of the bottom part of the scaffold that allows to better appreciate the columnar morphology of the pores. Scale bar is 400 nm. c) Picture of a 5-µm-thick nPSi membrane lifted-off from the native bulk silicon. d) Picture of the nPSi membrane in c) oxidized to nPSiO2 at 1000 °C for 5 min. e) Picture of nPSiO2 membrane in d) after transferprinting on a PLGA foil. Figure S2. Photoluminescence spectrum of a nPSiO2 membrane coated with PAH:Rh and PMAA:Rh polyelectrolytes. a) Sketch of a nPSiO2 scaffold on PLGA foil coated with Rh-labelled polyelectrolytes via LbL technique. Rh excitation peak at 520 nm/emission peak at 580 nm. b) Photoluminescence spectrum of a nPSiO2 scaffold on PLGA foil coated with (PAH:Rh/PMAA:Rh)2+(PAH:Rh)1. 18 Figure S1. Images of as-prepared nPSi scaffold, oxidation to nPSiO2, and transfer-printing on PLGA foil. a) Top-view SEM image (magnification 250000×) of a nPSi scaffold with porosity of ~77 %. Scale bar is 400 nm. Inset shows the histogram of the size distribution of pores with average diameter of 35 nm. b) Cross-section SEM image (magnification 35000×) of the nPSi scaffold in a). Scale bar is 1 µm. Inset shows a magnification (250,000×) of the bottom part of the scaffold that allows to better appreciate the columnar morphology of the pores. Scale bar is 400 nm. c) Picture of a 5-µm-thick nPSi membrane lifted-off from the native bulk silicon. d) Picture of the nPSi membrane in c) oxidized to nPSiO2 at 1000 °C for 5 min. e) Picture of nPSiO2 membrane in d) after transferprinting on a PLGA foil. Figure S2. Photoluminescence spectrum of a nPSiO2 membrane coated with PAH:Rh and PMAA:Rh polyelectrolytes. a) Sketch of a nPSiO2 scaffold on PLGA foil coated with Rh-labelled polyelectrolytes via LbL technique. Rh excitation peak at 520 nm/emission peak at 580 nm. b) Photoluminescence spectrum of a nPSiO2 scaffold on PLGA foil coated with (PAH:Rh/PMAA:Rh)2+(PAH:Rh)1. Nanostructured porous scaffolds boost fluorescence intensity 11 1 Figure 2. Assessment of the bioresorbable pH sensor in vitro. a) Photoluminescence spectra of 2 the bioresorbable pH sensor measured in the pH range 4 – 7.5 in PBS at 37 °C. b) Calibration curve 3 (photoluminescence intensity at 580 nm vs. pH value) of the bioresorbable pH sensor measured 4 over the pH range 4 – 7.5 in PBS at 37 °C (n=3 pH cycles). c) Real-time measurement of the 5 photoluminescence intensity of the pH sensor at different pH values from 7.5 to 4, and back, in PBS 6 at 37 °C. pH variation in the physiological range 6.5 – 7.5 were of 0.25 points. d) Calibration curve 7 of the pH sensors in different configurations, namely, LbL-coated nPSiO2 on native silicon chip 8 (red) and on a PLGA film (blue), LbL-coated nPSiO2 with barrier layer on a PLGA film (green) 9 (n=3 pH cycles). e) Sensitivity values of the sensors in d). (n=3 samples, 3 full cycles per sample). 10 f) Photoluminescence intensity changes between pH 4 and 7.5 measured on the pH sensor in 11 solution and between artificial skin flaps in two different configurations. (n=3 samples per 12 architecture, 3 full cycles per sample). Two-tailed t-student test (significance level < 0.01) 13 confirmed that data were statistically equivalent. g). Calibration curve (photoluminescence intensity 14 at 580 nm vs. pH value) of the bioresorbable pH sensor measured over the pH range 4 – 7.5 in ISF 15 at 37 °C (n=3 pH cycles). Inset show the comparison of sensitivity values of the pH sensor in PBS 16 and ISF (n=3 samples, 3 full cycles per sample). h) Sketch of the setup used to perform pH sensing 17 with a compact and cheap light source-photodiode pair. i) Calibration curve (photodiode response 18 vs. pH value) of the bioresorbable pH sensor in the range of pH 4-7.5 measured with the setup in a) 19 (n=3 samples, 3 full cycles per sample). Data are presented as mean (± s.d). 20 21 In-vitro pH sensing 11 1 Figure 2. Assessment of the bioresorbable pH sensor in vitro. a) Photoluminescence spectra of 2 the bioresorbable pH sensor measured in the pH range 4 – 7.5 in PBS at 37 °C. b) Calibration curve 3 (photoluminescence intensity at 580 nm vs. pH value) of the bioresorbable pH sensor measured 4 over the pH range 4 – 7.5 in PBS at 37 °C (n=3 pH cycles). c) Real-time measurement of the 5 photoluminescence intensity of the pH sensor at different pH values from 7.5 to 4, and back, in PBS 6 at 37 °C. pH variation in the physiological range 6.5 – 7.5 were of 0.25 points. d) Calibration curve 7 of the pH sensors in different configurations, namely, LbL-coated nPSiO2 on native silicon chip 8 (red) and on a PLGA film (blue), LbL-coated nPSiO2 with barrier layer on a PLGA film (green) 9 (n=3 pH cycles). e) Sensitivity values of the sensors in d). (n=3 samples, 3 full cycles per sample). 10 f) Photoluminescence intensity changes between pH 4 and 7.5 measured on the pH sensor in 11 solution and between artificial skin flaps in two different configurations. (n=3 samples per 12 architecture, 3 full cycles per sample). Two-tailed t-student test (significance level < 0.01) 13 confirmed that data were statistically equivalent. g). Calibration curve (photoluminescence intensity 14 at 580 nm vs. pH value) of the bioresorbable pH sensor measured over the pH range 4 – 7.5 in ISF 15 at 37 °C (n=3 pH cycles). Inset show the comparison of sensitivity values of the pH sensor in PBS 16 and ISF (n=3 samples, 3 full cycles per sample). h) Sketch of the setup used to perform pH sensing 17 with a compact and cheap light source-photodiode pair. i) Calibration curve (photodiode response 18 vs. pH value) of the bioresorbable pH sensor in the range of pH 4-7.5 measured with the setup in a) 19 (n=3 samples, 3 full cycles per sample). Data are presented as mean (± s.d). 20 21 M. Corsi, A. Paghi, et al., Advanced Science (2022) 14 220 h (Figure 3g-i). The PL intensity decrease can be ascribed to the degradation, chemical and/or 1 mechanical, of the fluorescent polymer multilayer, given that the silica scaffold dissolved in about 2 50 hours. The polymer brush architecture of the fluorescent multilayer after dissolution of the silica 3 scaffold might impact significantly on its degradation (Figure S14). Remarkably, full sensor 4 degradation occurred on the same timescale as the sensor operation, that is in about 100 hours. By 5 best fitting the PL reduction trend with a linear model, degradation rates of about -0.9 and -0.8 % h6 1 were consistently achieved for pH sensors on native silicon in a flow cell and on PLGA foil under 7 artificial skin, respectively. The polymer multilayer degradation was further confirmed by both 8 optical/fluorescence microscopy. No macroscopic polymer residues or residual photoluminescence 9 were found in the solution collected at the output of the flow cell or in the skin around the sensor 10 location at the end of the experiment, indicating a full degradation of the polymer and, in turn, of 11 the sensor occurred, apart from the PLGA foil that is known to degrade at a slower rate2. 12 13 14 220 h (Figure 3g-i). The PL intensity decrease can be ascribed to the degradation, chemical and/or 1 mechanical, of the fluorescent polymer multilayer, given that the silica scaffold dissolved in about 2 50 hours. The polymer brush architecture of the fluorescent multilayer after dissolution of the silica 3 scaffold might impact significantly on its degradation (Figure S14). Remarkably, full sensor 4 degradation occurred on the same timescale as the sensor operation, that is in about 100 hours. By 5 best fitting the PL reduction trend with a linear model, degradation rates of about -0.9 and -0.8 % h6 1 were consistently achieved for pH sensors on native silicon in a flow cell and on PLGA foil under 7 artificial skin, respectively. The polymer multilayer degradation was further confirmed by both 8 optical/fluorescence microscopy. No macroscopic polymer residues or residual photoluminescence 9 were found in the solution collected at the output of the flow cell or in the skin around the sensor 10 location at the end of the experiment, indicating a full degradation of the polymer and, in turn, of 11 the sensor occurred, apart from the PLGA foil that is known to degrade at a slower rate2. 12 13 Ex-vivo pH sensing and sensor degradation M. Corsi, A. Paghi, et al., Advanced Science (2022) In-vivo pH sensing vs. biocompatibility/dissolution 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 M. Corsi, A. Paghi, et al., Advanced Science (2022) Acknoledgments TEM analysis on PS_PL_p_23 (60s) Fabrizio Toia 23 August 2013 Collaborators: - Prof. M. Sailor, University of California, San Diego - Dr. L. Dahne, Surflay Nanotec, Berlin - Prof. D. Giuliani, University of Modena and Reggio Emilia - Prof. G. Di Giuseppe, University of Pisa D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programm e under grant agreem ent No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M. Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu Open position available for PhD and postdocs @[email protected] Thank You D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programm e under grant agreement No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M . Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu