Submicron machining and biomolecule immobilization on porous silicon by electron beam
© Imbraguglio et al.; licensee Springer. 2012
Received: 30 April 2012
Accepted: 13 September 2012
Published: 25 September 2012
Three-dimensional submicrometric structures and biomolecular patterns have been fabricated on a porous silicon film by an electron beam-based functionalization method. The immobilized proteins act as a passivation layer against material corrosion in aqueous solutions. The effects' dependence on the main parameters of the process (i.e., the electron beam dose, the biomolecule concentration, and the incubation time) has been demonstrated.
KeywordsPorous silicon Electron beam Lithography Micromachining Biomolecules 87.85.Va
Electron beam lithography (EBL) is known to be a pattern-designing technique of integrated electrical circuits, with writing resolution even down to tens of nanometers, which allows the fabrication of innovative and advanced devices for nanotechnological applications [1, 2]. In EBL procedures, electron-sensitive polymeric resists are usually spun on top of samples' surfaces prior to irradiation in a scanning electron microscope (SEM). Depending on the nature of the resist, the irradiated geometry or its specular negative is used as a mask for subsequent material etching or deposition steps in order to obtain structures with nanometer-scale features. However, the EBL, when directly used to desorb chemical species from a surface, can be also exploited as a local functionalization method  to create molecular modified or biopatterns without employing any resist. This has been the case, for instance, of porous silicon (PS) surface-based biosensors .
PS is a silicon nanosponge produced by electrochemical etching of crystalline silicon in a hydrofluoric (HF) acid-containing solution. It is one of the most investigated structures in nanomaterials science due to some fascinating properties which can be harnessed in many applied research fields . Morphological parameters of PS matrices, i.e., pore sizes, porosity (voids-to-total volume ratio), and thickness, can be easily controlled just by varying the experimental electrochemical conditions (such as current density, HF concentration, or etching duration), allowing the use of PS for size-selective filtration or separation processes, drug delivery, or sensing applications.
The huge specific surface area (in the order of 200 m2/cm3) of PS is hydride-terminated in as-etched samples [6, 7], and a well-established modification chemistry has been developed in the past years to selectively bind large amounts of different target analytes within the pores . In so far as biological species are concerned, the immobilization of proteins , enzymes , and antibodies  on PS surfaces can be achieved by different techniques. Among the ones which allow submicrometric definitions, the EBL has already proved its capability in designing chemical and biopatterns solely on selected and very small region of PS [3, 4], opening the possibility of future development of highly-sensitive nanobiosensors and multiplexed analysis based on this material. From this perspective, PS is a very convenient substrate for EBL processes because of its porous structure. Indeed, due to the lower quantity of Si atoms with respect to an equal volume of bulk Si, PS behaves as a low-density material and exhibits a reduced proximity effect , which is the main limitation factor in writing resolution with standard EBL-based methods. Moreover, the absence of the polymeric resist during irradiation further contains the phenomenon because the only cause of overexposure of PS irradiated regions stems from secondary and backscattered electrons just coming from the underlying PS/Si substrate. This means that more defined and small patterned features can be, in principle, obtained by direct, i.e., not resist-mediated, irradiation of PS. Therefore, by combining EBL nanostructuring capabilities with those of PS whose pore dimensions can be varied from the macro- to the microscale (<2 nm), sophisticated optical nanostructures could, in principle, be fabricated, such as three-dimensional (3D) photonic crystals, waveguides, or optical gratings.
As far as the application of the direct EBL method on PS is concerned, some previous works have already demonstrated its capability in material structuring [12, 13] as well as the feasibility to fabricate a reliable biosensor . Different kinds of geometries had been patterned on PS surfaces, and the control of feature dimensions is possible by tuning the process parameters. Furthermore, proteins which were bound on PS surfaces by such a resistless EBL technique had been shown to retain their full functionality after the process and can act as bioreceptors for molecular and biomolecular analytes. We report on recent studies and advances on both sides, i.e., the (submicron) machining and the immobilization of biomolecules on PS. The obtained results let us discover a new and intriguing property of the interaction between biomolecular species and a solid-state nanomaterial which, to the best of our knowledge, has never been observed before.
PS single-layer films have been anodically etched from highly boron-doped single-crystal Si wafers <100> (resistivity ranging from 0.008 to 0.012 Ω cm) in a 1:2 solution of aqueous 50% HF/ethanol in volume; the electrochemical etching procedure is described in several papers [3–5]. Typically, a double-step etching/stop loop (consisting of 0.2-sec etching at a current density of 400 mA/cm2 followed by a 10-sec stop etching time) was repeated, the number of times depending on the desired film thickness, using a Keithley 2400 SourceMeter (Keithley Instruments Inc., Cleveland, OH, USA). PS samples with thicknesses of 6.5 and 9 μm were fabricated, with pore dimensions comprised in the mesoscale regime (approximately 20 to 50 nm) and porosity of about 80%, as confirmed by profilometric and SEM measurements. Anyway, no substantial differences related to the different thickness values of the samples or due to decreasing the current density to 300 mA/cm2 were observed in the experimental results.
After dipping in an HF-based solution in order to remove the native silicon oxide layer, PS samples were introduced into the vacuum chamber of a FEI Quanta 3D (FEI Co., Hillsboro, OR, USA) field emission gun (FEG) SEM equipped with a nano pattern generator system (NPGS) 9.0 from J.C. Nabity Lithography Systems (Bozeman, MT, USA). Simple rectangular geometries, formed by line series 0.8-μm wide and 30-μm long (spaced by a distance of 0.7 μm), were written by NPGS on the PS sample surfaces, applying a 20-kV accelerating voltage to the electron beam. The electronic dose (i.e., the number of electrons per area units) range explored in this work has been varied from 40 up to 200 mC/cm2.
In order to allow as much consistent comparison between samples as possible, identical patterns were written on specular portions of the same PS chip, which were then divided after irradiation and immediately immersed in buffered solutions. Depending on the test performed, pure 1 X phosphate buffered saline (PBS) or bovine serum albumin (BSA) protein-containing solutions were prepared. PBS tablets and BSA lyophilized powders from Sigma-Aldrich (St. Louis, MO, USA) have been dissolved in purified water provided by a Millipore Elix 3 purification system (Millipore Co., Billerica, MA, USA). The pH of pure PBS solutions has been checked each time by a CyberScan pH/Ion 510 meter (Eutech Instruments, Vernon Hills, IL, USA) to be equal to a nominal value of 7.4. Four different BSA concentrations (5, 1, 0.5, and 0.1 μM) were obtained by multiple dilutions from a 15-μM mother solution of BSA in PBS buffer. The irradiated PS samples were incubated for times ranging from 5 to 120 min, after which they were profusely rinsed first in PBS, then in deionized water and finally dried with nitrogen gas.
Fourier transform infrared (FTIR) reflectance spectra have been acquired using a Nicolet Nexus FTIR spectrophotometer (Thermo Scientific, Logan, UT, USA) coupled with a Nicolet Continuum FTIR microscope equipped with a MCT detector. The spectra were collected both in the irradiated area and just a few micrometers outside by registering a total of 512 interferograms for each spectrum with a resolution of 4 cm−1. Finally, the samples were observed with a FEI Inspect F FEG SEM in a tilted (60°) secondary electron configuration.
Results and discussion
It is well known that high-surface-area nanomaterials such as PS undergo rapid oxidation and even dissolution when exposed to ambient air conditions or immersed in aqueous solutions because of their poor stability . These kinds of processes can be locally further stressed and speeded up by using different techniques, such as laser writing [15, 16] or contact atomic force microscopy-based methods . From this point of view, EBL can potentially be used to remove material from a solid substrate only in submicrometer-wide regions due to the high focusing power of the electron beam. Among other different approaches, EBL can be thought of as a good compromise in so far as writing speed and resolution are concerned. As previously mentioned, the interaction between PS and the electron beam has already been studied in some former works [3, 4, 12, 13]. Rocchia et al.  first demonstrated that the electron irradiation provokes hydrogen desorption from the native SiHx bonds of PS surfaces, leading to high reactive electron beam-activated PS (EBAPS) regions which easily oxidize in ambient air. Such a strong reactivity is most likely due to the formation of dangling bonds and defects as a consequence of the hydrogen depletion caused by the electron bombardment. Nevertheless, in the reported previous cases, even if the written geometries were clearly distinguishable immediately after irradiation by the electron microscope, optical, profilometric, and SEM investigations did not reveal any substantial structuring of the PS along the z-axis (perpendicular to the PS surface). Anyway, very tiny z-depth profiles were measured only after removing the electro-oxidized PS areas in acid or alkaline solutions, as the difference step between the top non-exposed PS surface and the bottom crystalline Si one after EBAPS removal. The amplitude of such thicknesses had been studied as a function of different oxidizing conditions (air, water, H2O2, incubation time) as well as energies and electronic doses provided by the electron beam. The results showed that the stronger the oxidizing procedure is, the thicker is the depth measured by profilometry, and so the final vertical structuring of PS. Besides, monotonic increases in the thickness were observed by augmenting the electronic dose and the accelerating voltage of the electron beam. In the latter case, a 25-kV saturation value was found, beyond which the electrons lose their energy also through the Si bulk underlying the PS substrate, and the effect no longer depends on the electron energy. The maximum depth value (≈180 nm) was obtained with a 12-μm thick sample irradiated at 25 kV at the maximum electronic dose (120 mC/cm2) . It has to be pointed out that the reported thickness values were detected only after dipping EBAPS samples in HF or KOH etchants. This is a quite aggressive etching procedure which may cause, in some case, the removal of PS non-exposed areas too due to the low degree of selectivity of such methods. Obviously, as far as micro- or nano-machining of PS is concerned, easiness and precise reproducibility in fabrication of machined structures are two important requirements.
In the present case, the conservation of the Fabri-Pérot interference pattern uniformity has been used as a roundabout optical method to exclude that the preservation of the PS matrix, observed in Figure 2, was due to just a superficial passivation acted by the immobilized proteins. BSA biomolecules are small enough to penetrate inside the nanopores of EBAPS samples as well as water molecules, whose number is, however, several orders of magnitude larger in a few micromolar concentrated BSA buffer solution. It is very unlikely that BSA molecules could directly bind to the activated Si sites through a covalent bond because of, firstly, the highest probability which water molecules have to saturate with EBAPS dangling bonds. Secondly, it had been demonstrated in few cases that the biomolecular functionality of the immobilized target is retained after the process , and such a kind of attachment would rather denature the biomolecule of interest. On the other hand, the strong affinity of BSA for Si dioxide is well established , and it is therefore plausible that oxygen atoms could act also as a linker for the immobilization of BSA proteins to the EBAPS pore walls. During the incubation time of the EBAPS samples with the BSA solution, two fast reactions probably occurred one after the other: the enhanced oxidation of the irradiated areas and the consequent selective attachment of BSA molecules to these regions. As soon as the reaction proceeds, the constrained space available within the nanopores favors the packaging of the biomolecules, thus creating a sort of scaffold which prevents material corrosion. In this way, a soft matrix as that formed by the BSA proteins is able to protect and sustain a nanostructured solid one.
We found that electron beam irradiation on PS has no noticeable effects on its morphological structure as long as, after EBL writing, samples were left in air or just a few minutes in buffered solutions. On the other hand, if the irradiated PS samples were dipped for incubation times greater than 20 min in pure PBS (or very low BSA concentrated) solutions, the irradiated strips appear, after drying, as well-defined submicrometric vertical structures embedded into the porous matrix, suggesting that a heavy EBL-controlled erosion of the nanomaterial can be accomplished in these conditions. A valuable option to common 3D micro- and nano-machining techniques of PS has thus been proposed.
In addition, submicrometric bio-PS composite patterns have been successfully fabricated by the same technique, and a nanoscale biomolecular passivation effect has also been observed. We are confident to transfer the acquired knowledge to the immobilization of other and more useful nano and biomolecular targets (i.e., conductive biomolecules or functionalized metallic nanoparticles), which could be suitable for applications in different emerging research fields, such as molecular and bioelectronics or surface-enhanced Raman spectroscopy.
This work has been performed at Nano Facility Piemonte, INRiM, a laboratory supported by Compagnia di San Paolo. The authors thank Emanuele Enrico and Luca Boarino for their support.
- Vieu C, Carcenac F, Pepin A, Chen Y, Mejias M, Lebib A, Manin-Ferlazzo L, Couraud L, Launois H: Electron beam lithography: resolution limits and applications. Appl Surf Sci 2000, 164: 111–117. 10.1016/S0169-4332(00)00352-4View ArticleGoogle Scholar
- Tseng AA, Chen K, Chen CD, Ma KJ: Electron beam lithography in nanoscale fabrication: recent development. IEEE Trans Electron Packag Manuf 2003, 26: 141–149. 10.1109/TEPM.2003.817714View ArticleGoogle Scholar
- Rocchia M, Borini S, Rossi AM, Boarino L, Amato G: Submicrometer functionalization of porous silicon by electron beam lithography. Adv Mater 2003, 15: 1465–1469. 10.1002/adma.200304919View ArticleGoogle Scholar
- Borini S, D'Auria S, Rossi M, Rossi AM: Writing 3D protein nanopatterns onto a silicon nanosponge. Lab Chip 2005, 5: 1048–1052. 10.1039/b505089hView ArticleGoogle Scholar
- Cullis AG, Canham LT, Calcott PDJ: The structural and luminescence properties of porous silicon. J Appl Phys 1997, 82: 909–965. 10.1063/1.366536View ArticleGoogle Scholar
- Rao AV, Ozanam F, Chazalviel JN: In situ Fourier-transform electromodulated infrared study of porous silicon formation - evidence for solvent effects on the vibrational linewidths. J Electrochem Soc 1991, 138: 153–159. 10.1149/1.2085526View ArticleGoogle Scholar
- Stievenard D, Deresmes D: Are electrical properties of an aluminum-porous silicon junction governed by dangling bonds. Appl Phys Lett 1995, 67: 1570–1572. 10.1063/1.114942View ArticleGoogle Scholar
- Stewart MP, Buriak JM: Chemical and biological applications of porous silicon technology. Adv Mater 2000, 12: 859–869. 10.1002/1521-4095(200006)12:12<859::AID-ADMA859>3.0.CO;2-0View ArticleGoogle Scholar
- Dancil KPS, Greiner DP, Sailor MJ: A porous silicon optical biosensor: detection of reversible binding of IgG to a protein A-modified surface. J Am Chem Soc 1999, 121: 7925–7930. 10.1021/ja991421nView ArticleGoogle Scholar
- Letant SE, Hart BR, Kane SR, Hadi MZ, Shields SJ, Reynolds JG: Enzyme immobilization on porous silicon surfaces. Adv Mater 2004, 16: 689- + .View ArticleGoogle Scholar
- Rossi AM, Wang L, Reipa V, Murphy TE: Porous silicon biosensor for detection of viruses. Biosens Bioelectron 2007, 23: 741–745. 10.1016/j.bios.2007.06.004View ArticleGoogle Scholar
- Borini S, Amato G, Rocchia M, Boarino L, Rossi AM: Electron-beam irradiation of porous silicon: application to micromachining. J Appl Phys 2003, 93: 4439–4441. 10.1063/1.1560853View ArticleGoogle Scholar
- Borini S, Rocchia M, Rossi AM, Boarino L, Amato G: Electron beam irradiation of porous silicon for application in micromachining and sensing. Physica Status Solidi a-Applications and Materials Science 2005, 202: 1648–1652. 10.1002/pssa.200461210View ArticleGoogle Scholar
- Petrova EA, Bogoslovskaya KN, Balagurov LA, Kochoradze GI: Room temperature oxidation of porous silicon in air. Materials Science and Engineering B-Solid State Materials for Advanced Technology 2000, 69: 152–156.View ArticleGoogle Scholar
- Rossi AM, Amato G, Camarchia V, Boarino L, Borini S: High-quality porous-silicon buried waveguides. Appl Phys Lett 2001, 78: 3003–3005. 10.1063/1.1370536View ArticleGoogle Scholar
- De Stefano L, Rea I, Nigro MA, Della Corte FG, Rendina I: A parametric study of laser induced ablation-oxidation on porous silicon surfaces. J Phys Condens Matter 2008, 20: 265009. 10.1088/0953-8984/20/26/265009View ArticleGoogle Scholar
- Wouters D, Schubert US: Nanolithography and nanochemistry: probe-related patterning techniques and chemical modification for nanometer-sized devices. Angew Chem Int Ed 2004, 43: 2480–2495. 10.1002/anie.200300609View ArticleGoogle Scholar
- Boarino L, Enrico E, De Leo N, Celegato F, Tiberto P, Sparnacci K, Laus M: Macro and quasi-mesoporous silicon by self-assembling and metal assisted etching. Physica Status Solidi a-Applications and Materials Science 2011, 208: 1403–1406. 10.1002/pssa.201000316View ArticleGoogle Scholar
- Chen MY, Sailor MJ: Charge-gated transport of proteins in nanostructured optical films of mesoporous silica. Anal Chem 2011, 83: 7186–7193. 10.1021/ac201636nView ArticleGoogle Scholar
- Noinville S, Revault M, Baron MH, Tiss A, Yapoudjian S, Ivanova M, Verger R: Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study. Biophys J 2002, 82: 2709–2719. 10.1016/S0006-3495(02)75612-9View ArticleGoogle Scholar
- Su TJ, Lu JR, Thomas RK, Cui ZF: Effect of pH on the adsorption of bovine serum albumin at the silica water interface studied by neutron reflection. J Phys Chem B 1999, 103: 3727–3736. 10.1021/jp983580jView ArticleGoogle Scholar
- De Stefano L, Rea I, De Tommasi E, Rendina I, Rotiroti L, Giocondo M, Longobardi S, Armenante A, Giardina P: Bioactive modification of silicon surface using self-assembled hydrophobins from Pleurotus ostreatus. European Physical Journal E 2009, 30: 181–185. 10.1140/epje/i2009-10481-yView ArticleGoogle Scholar
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