1. NanoMed 2009 - Berlin Study of the micro- and nanostructured silicon for biosensing and medical applications Irina Kleps (irina.kleps@imt.ro) Mihaela Miu, Monica Simion, Teodora Ignat, Adina Bragaru, Florea Craciunoiu, Mihai Danila, National Institute for Research and Development in Microtechnology (IMT- Bucharest), Erou Iancu Nicolae 126 A, 72996, Bucharest, Romania

2. OUTLINE Motivation Micro- and nanostructured silicon preparation; PS as sensing element - Photoluminescence sensors; - Microarray substrates for protein detection; - DNA detection by Impedance spectroscopy; - SERS sensors; Nanostructured Si as carriers for controlled drug delivery Conclusions

3. Motivation - porous silicon (PS) is a low cost material; - PS has controllable pore size; - it has high surface area within a small volume (internal surface of 600 m 2 /cm 3 ); - convenient surface chemistry; - compatibility with conventional silicon microfabrication technologies; - PS is biocompatible [1, 2] and bioresorbable (nanometric size Si), with silicic acid releasing without toxic effects for the body [3]. [1] S. C. Bayliss, R. Heald, D. I. Fletcher, L. D. Buckberry, Adv. Mater., 11, 318 (1999). [2] A. Angelescu, I. Kleps, M. Miu, M. Simion, T. Neghina, A. Bragaru, S. Petrescu, C. Paduraru, A. Raducanu, N. Moldovan, Rev. Adv. Mater. Sci. 5 34-40 (2003). [3] Uracha Rungsardthong, Susan I. Anderson and Leigh T. Canham, Chiang Mai J. Sci. 2005; 32(3) : 487-494.

4. Micro- and nanostructured silicon preparation Porous silicon – PS - is obtained by electrochemical dissolution of silicon in HF-based solution Etching requires holes (electron injection) to break bonds. Resistivity ~ 10 6 Ω.cm similar to intrinsic Si. Schematic view of anodization cell Si + 4HF + (4-n)h + → SiF 4 + 4H + + ne - Dissolution chemistries A.M.M.T etching system for 4’’ Si wafers with programmable power supply and dedicated software for time-based current profiles.

5. PS morphology – SEM characterisation nano-PS (< 15 nm) meso-PS ( 50- 100 nm) macro-PS (ca. 1µm) Pore size / wire size Electrolyte type HF concentration Doping type and level Illumination

6. Experimental – PS fabricated by electrochemical etching:  p-type silicon (100) with 6 - 10  ·cm resistivity;  HF - C 2 H 5 OH electrolyte with 15, 18, 25% concentration;  current densities have been 15 mA/cm 2 and 25 mA/cm 2 No. Concetration HF (%) Current density (mA/cm 2 ) Time (min) Porosity (%) 12515658 22520660 325 662 41815665 51820670 615 680 71525688 The orange-red photoluminescence from the porous silicon is clearly visible when the wafer illuminated by UV light Photoluminescence spectra of PS / Si-p samples with different porosities (58% - 88%) PL peak position (nm) Porosity (%) PL maximum dependence with porosities The PL emission from PS is observable at wavelengths ranging from the ultraviolet to the infrared, the normalized spectra recorded for different experimental samples demonstrate the dependence on porosity. PL emission from PS the PL peaks for high porosity PS samples are centred around 650-720 nm, in visible range due to quantuum confinement and surface states effects; a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed;

7. thermal treatment at different temperatures between 300°C and 750°C Wavelength (nm) PL intensity (a.u.) Wavelength (nm) The PL peaks for high porosity PS samples are centred around 650-720 nm, in visible range due to quantuum confinement and surface states effects; a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed; the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a blueshift of PL peak when anodic oxidation was used; supplementary an improvement of PL intensity was observed. Additional treatments for PS optoelectronic properties stabilisation anodic oxidation

8. Porous silicon biosensor Chalenges: (i)integration of biological systems with PS: increasing of the immobilised biomolecule number on the surface; creation of stable covalent bonds; (ii)using micro/nanoscale materials for amplification of the detected signal (SERS, SEIR, SEF); (iii)using PS as receptor and transducer; (iv)integrating optics with low-power, portable devices PS is RECEPTOR for biological molecules selective for the analyte (DNA single strand, proteins, antibodies, enzymes) constitutes the molecular recognition element PS is DETECTOR (signal transducer) for biochemical interactions electrical, conductance, impedance, (electro)chemical Optical Luminescence Phosphorescence Internal Reflection Spectroscopy Reflectometric Interference Spectroscopy Ellipsometry Fluorescence—intensity, lifetime, polarization Fluorescence resonance energy transfer Absorbance Raman Scattering, SERS Surface plasmon resonance Interference 1.Fixed position arrays 2.Single Particle Encoding PS host matrix/support for immobilization of sensing biomolecule Techniques for immobilization range from physical adsorption to the replacement of hydride bonds with Si alkyls, and to antibody bonding at functionalized PSi surface with subsequent antibody-antigen interactions. The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte.

9. PS functionalisation Material in the pores changes the spectral response PL shift towards smaller energies after functionalisation SEM image of porous silicon surface before (a) and after (b) BSA deposition Affinity anchoring This method is commonly used to load proteins. -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface. Covalent binding To prepare the surface for the capture of BSA proteins, the device is first thermally oxidized at 800 0 C to form a silica-like internal surface. The sensor is then treated with 2% amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition. PCs can be designed to localize the electric field in the low refractive index region (e.g. air pores), which makes the sensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls. 2% 2% amino propyltrimethoxy silane 800 0 C

10. LN3 interconnected lines similar recording conditions were (488 nm frequency of excitation and 87 mW nominal power) the intensity of PL emission is three times larger in the case of semicircular microcavities leading to an important improvement of detection. Si substrate microstructuration and porosification LN2 pyramidal structures Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-circular cavities (left);

11. Si substrate microstructuration and porosification PL spectra recorded for three array of microcavities 0.3, 0.6 si 1 µm 1 µm 0.6 µm 0.3 µm The micropatterned substrate is an array of piramidal cavities which have to act similar to a collimating device and light emitted from sample traveling in detector direction to be reflected by the side walls improving the sensitivity of the biosensor

12. → gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer. → thiol molecules self assembled on AuNP, so that they can recognize a given protein. → In the absence of any protein, the photoluminescence of the PS is absorbed by the AuNP at a given wavelength. → when the protein specifically binds to the AuNP, the absorption due to the SPR is modified and the detected signal is enhanced. Elaboration of PS/Metallic plasmonic nanostructures The combination of the PS with Au-NP makes possible to design an optical biosensor where the light source is the silicon itself (photoluminescence) and the chemical transducer is the functionalized AuNP. Functionalised gold nanoparticles act as nano amplifiers Luminescence of the PS is modified by the SPR of the AuNPs A specific protein is recognised by the AuNP (specific absorption) Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their advantages in: -local scattering field enhancing -surface chemical modifications - biocompatibility - well established chemical synthesis process. The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent nanospheres are considered as the key and necessary conditions for local field enhancing.

13. Gold /PS X ray diffraction analyses: The metallic nanocrystallites orientation on nanostructurated Si substrates and the influence of additional anealing treatments – The Au (111)/nc-Si surface has a higher density of atoms comparatively with Au (100); this favours the attachment of a higher number of atoms and bio- molecules on the gold surface. Au / 38% PS Au / 60% PS For Au/PS (60%) nucleation begin on Au (111) planes more rapid than for Au/PS (38%) (i)in the as-deposited mesoporous films, the initial nucleation begin on Au (220) planes; (ii)after the thermal annealing at 500 0 C the crystallisation process on the (111) planes become dominant; and a (111) texture is obtained; (iii)the thermal annealing at 900 0 C induces an increase of the (111) crystallites, indicating a clear crystallisation on (111) planes In Au/ macroporous silicon, the Au (111) texture of crystallites became predominant Au / PS Experimental: The Au/PS/Si and Ag/PS/Si nanocomposites layers were thermal treated at 500 and 900 0 C in reducing atmosphere (H 2 and N 2 ). Au/macroporous silicon

14. Experimental AgNO 3 salt and AgNO 3 1% ethanolic solution were also deposited on the PS/Si substrate The Ag as-deposited films obtainrd from diluted solution of AgNO 3 are in an initial stage of crystallisation, with a high amorphous content In the case of Ag/PS samples, the annealing treatment at T=500 o C has no effect from the point of view of the microstructure analysis of the Ag films on PS (crystallization continue on (200) planes, the films have a higher crystalline content), while annealing at 900 o C produces a mixed (111) and (200) texture, with bigger grain sizes. Silver/Si nanocomposite layers Optical microscope image using a UV filter of Ag/PS

15. Experimental: macroporous silicon: p-type (100) Si, (5–10 Ω cm), 4% HF in DMF; 7.7 mA/cm2 current density; 100 nm gold layer was deposited by cathodic sputtering, 11-mercaptoundecanoic acid (11-MUA) was auto- assembled on all investigated substrates by their immersion in 2mM MUA in ethanol solution. SEM image of PVD-evaporated gold on macroPS substrate SEM image of macroPS layer Raman measurements on the Au/macroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate. Moreover the Au/macroporous Si is a suitable substrate for (bio)sensors, organic molecule conformation on the solid surface being achieved SERS spectra of 11- MUA adsorbed on different investigated substrates Nanostructured Au/Si substrate for organic molecule SERS detection In the normal resonance Raman the molecule interacts directly with the electromagnetic field associated with the traveling wave. In SERS this field is already modulated by the electron cloud oscillation in the metal and the molecule experience an enhanced field.

16. → Nanocrystalline Au(111)/PS substrates have important applications in biochemistry, especially in self-assembly of the thiol-end DNA molecule (SAM), such as HS-(CH2)6-5′-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3′. Immobilisation of a fluorescent oligonucleotide on Au/Ps (A – 20×, B – 40×; Nikon fluorescence microscope). The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip; FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample. NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production. TRYPSIN is a serine protease from the pancreas of vertebrates. Cleaves peptide bonds involving the amino groups of lysine or arginine. Au/PS enhance fluorescence PS as sensitive element for neurons in NutMix culture Main applications: immunologic biosensors; protein and DNA microarray technology

17. Investigation of the CHO cells immobilised in PS microreactor PS as sensitive element for CHO cells investigation Emission spectra recorded at 280 nm wavelength excitation PS has an intrinsic fluorescence spectrum, and the emission is modified – suffer a peak shift – after the PS treatment for cell growth and their process of fixing.

18. Microarray technology and immunologic sensors PS substrates advantages: low surface wetting (providing mild immobilisation conditions – a hydrophilic surface at the molecular level); small spots (increased immobilisation density over a spot, improved reaction kinetics, high density arraying); homogeneous probe molecule coverage (uniform fluorescence intensity over a spot, improved data quality); low fluorescence background (auto-fluorescence); biocompatibility (immobilisation and/or adsorption of biospecific binders with maintained affinity and selectivity, allowing protein digestion to be performed directly on the surface, allowing complex sample analysis such as blood and tissue lysates) The need to measure multiple parameters was solved by bundling several sensors together in order to multiplex them. Today, arrays with tens of thousands, and even hundreds of thousands of features are realised in the DNA/protein microarray technology. The microarrays use fluorescence signals as the transduction mechanism. Main requirements for microarray substrates inert and resistent to non-specific adsorption surface; the surface should contain functional groups for the facile immobilization of protein molecules of interest; bonding between a protein molecule and a solid surface should to be strong enough to retain the molecule on the surface, but also sufficiently non intrusive to have minimal effect on the 3D structure; the linking chemistry should control of protein orientation the local chemical environment favors the immobilized protein molecules to retain their native conformation;

19. PS and Au/PS as substrates for protein immobilisation and fluorescent detection in microarray technology Experimental: microarray technique for printing and characterization. -PS and Au/PS substrates were investigated for BSA immobilization - each sample was spotted with bovine serum albumin (BSA). - the spots contain Cy3 fluorophores, ranging from 2-9 to 1 fluors/µm2. - after 24 hours of incubation at 40C, they were laser scanned. - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions; - the fluorescence intensity remains almost constant after each washing run. Conclusion: Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array.

20. Electrical / Impedance detection Binding of DNA inside the PSi matrix induces a change in capacitance and conductance. Mott-Schottky plots for different concentration of DNA in electrolyte solution The capacitance of the Electrolyte/PS/Si has been measured as function of DC potential and the data recorded for different concentration of DNA in electrolyte solution are presented. The figure illustrate that the test structure capacitance plots suffer modifications by adding different concentrations of DNA in electrolyte solution: the flat band potential is shifted to lower values as DNA immobilization starts from 0.31V to 0.24V and 0.21V respectively. Experimental data Phosphate buffered saline- PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH. DNA 1 1 µM /50 ml PBS solution DNA 2 2 µM/50ml PBS solution

21. Bode (a, b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30° at higher frequency region, which tends to -20° as the concentration of DNA is increased; opposing, in lower frequencies domain, the maximum is initially around -70° and its value increases to -80° going concomitantly with a gradual shift to lower frequencies, proving that the interface phenomena became dominant. The corresponding impedance module plots fig. (b) show a similar behavior, with a more evident decrease of values in low frequencies domain. The Nyquist graphs presented in fig. (c) indicate that the data are not well separated above 200 Hz and clear distinct below, with a dominant interface capacitance.

22. Nanostructured Si as carriers for controlled drug delivery AIM: different methods for fabrication of PS based nanostructured microparticles as well as their loading with organic molecule or anorganic nanoparticles with therapeutic effect were experimented. Schematic representation of the p/n Si structure fabrication in view of porosification Optical image of a Si microparticle obtained by porosification using Si 3 N 4 layer as mask, followed by an ultrasonation process SEM images of nanostructured Si microparticles obtained by selective porosification: (a) n-epi/p+ Si process and (b) n-diffusion in p+Si process. Si p/n junctions selective porosification Si porosification using Si 3 N 4 mask Si porous multilayers

23. Nanostructured Si multilayers as carriers PS multilayers obtained on p+ Si Nanoporous Si obtained on p+ Si (50 nm pore size) Microparticles with nanoporous structure lower than 8 µm Current-potential-time diagram for 30 cicles (0.570 A: 10.0 sec and 3.000 A: 10.0 sec) Ball mill (1h) - The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied. - The dimensions of Si microparticles are given by the time of each step, and we have used a 10 sec. time interval for each porosification step Current-potential-time diagram for 150 cicles ( 0.554 A, 10 sec, 2.825 A, 4 sec) PS multilayers obtained on p+ Si

24. Loading of molecule of therapeutic interest Chondroitin sulfate (CS) (sulfated glycosaminoglycan -GAG) Lactoferrin (immunomodulatory compound- globular protein with antimicrobial activity) N-butyl-deoxynojirimycin (NB-DNJ) (an imino sugar that inhibits the growth of the CT-2A brain tumour) Ellipsometric parameters Delta and Psi for PS/Si, APTS/PS/Si and NB- DNJ/APTS/PS/Si Mouse melanoma B16 F10 cells proliferation on different APTS/PS devices: A- control cells; B- APTS/PS-CS; C-APTS/PS-Lf; D- APTS/PS-NB-DNJ;

25. Magnetite nanoparticles in PS carriers Gold, silver, and iron oxides were chemically or deposited by evaporation on porous silicon in order to assure biocompatibility, targeting, antimicrobial and terapeutic properties. SEM images of iron oxide nanoparticles on PS and EDAX analysis of the iron/iron oxide on the PS substrate. P S + F e 2 + / F e 3 + + N H 4 O H F e 3 O 4 / P S Fe/PS for drug delivery - Two steps (deposition – diffusion) process of iron deposited from corresponding salts was realized. - Nitrate Fe(NO3)3  9H2O - and sulfonate - Fe(SO4)  7H2O – were deposited on PS surface from saturated solutions. - Annealing temperatures: 1 h or in oven at 65 0 C or 90 0 C. - Iron drive-into the PS skeleton, was performed at high temperature, 6000C, in inert (Ar) or reducing (H2:N2) atmosphere Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

26. Gold nanoparticles on porous silicon carriers 5% mercaptopropyl trimetoxysilane MPTS SEM image of gold nanoparticles (7 nm) self-assembled on PS Optical microscope image using a UV filter of Ag/PS Silanization protocol for thiol groups attachement (Anal. Chem. 2001, 73, 2476-2483)

27. CONCLUSION 1.Medical diagnosis – Bionanodetection Biosensors Microarray technology 2. Medical therapy Carriers for controlled drug delivery The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area host material with the function of an optical transducer, which makes it an ideal material for biosensing applications. We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process, obtaining an effect like in a collimating device, where light emitted from sample travelling in detector direction is reflected by the side walls. PS substrate has been used for optimisation microarray imaging technique parameters. We have shown how electrochemical impedance spectroscopy (EIS), next to surface plasmon resonance (SPR), could become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the binding of charged molecules. Other challenging field of research presented herewith was related to nanostructured silicon particles which can combine optical with drug delivery properties. Micro/nanoporous silicon technology is very promising to develop novel devices and systems that have a biomedical impact

28. IMT-Nanotechnology Laboratory Team: Irina Kleps: Project coordinator Mihaela Miu: Impedance Spectroscopy Monica Simion : Microarray Technology Teodora Ignat: SERS substrates Florea Craciunoiu: Microparticle preparation Adina Bragaru: Porous silicon fabrication Mihai Danila: X-ray diffraction