Micro/Nanoscale Parallel Patterning of Functional Biomolecules, Organic Fluorophores and Colloidal Nanocrystals
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We describe the design and optimization of a reliable strategy that combines self-assembly and lithographic techniques, leading to very precise micro-/nanopositioning of biomolecules for the realization of micro- and nanoarrays of functional DNA and antibodies. Moreover, based on the covalent immobilization of stable and versatile SAMs of programmable chemical reactivity, this approach constitutes a general platform for the parallel site-specific deposition of a wide range of molecules such as organic fluorophores and water-soluble colloidal nanocrystals.
KeywordsMolecular self-assembly Lithographic techniques DNA Green fluorescent proteins Colloidal nanocrystals
Green fluorescent protein
Considerable efforts have recently been devoted by the nanoscience community to develop reliable patterning methodologies for the spatially controlled deposition of a wide range of molecules, exploiting their spontaneous organization in the form of 2D or 3D matrices onto substrates of different materials (semiconductors, metals, plastics) . Notably, in the case of biological species (such as DNA, proteins, antibodies, cells), the creation of patterned active substrates, enabling precise positioning of biomolecules with nanoscale resolution over large areas, may provide new attractive diagnostic tools to perform more efficient analyses in high throughput . The peculiar self-assembling capabilities of biomolecules may lead to the development of novel bio/inorganic or organic/inorganic active interfaces , likely preserving biological functionality upon immobilization, and favoring the possibility to characterize biomolecular interaction events at single molecule level . Interestingly, such hybrid active interfaces may conjugate the specificity and reactivity of the biological “soft machines” to particular electronic or optical characteristics of the “hard substrates” (such as smart plastic films enabling recognition of target biomolecules by optical excitation ) and can potentially be applied to a variety of research fields, ranging from biosensors and diagnostics [6, 7] to optoelectronics and microfluidics [8, 9].
To date, several nanofabrication techniques, including plasma deposition and electron beam lithography (EBL) [10–13], micro-contact printing [14, 15], dip-pen nanolithography [16–18], and screen printing , have been exploited for the production of micro- and nanostructured biomolecular substrates. In particular, interesting examples of selective surface patterning, based on the use of optical lithography  or PECVD  coupled to the formation of different silane-based self-assembled monolayers (SAMs), have recently been reported, demonstrating the selective nanopositioning of proteins and colloidal nanocrystals (NCs) or metallic nanoparticles and NCs. However, while each of the above-mentioned techniques has some remarkable advantages, it seems they are rather complementary and often present significant drawbacks usually associated to possible losses of biomolecular functionality, as well as to the precise spatial control and/or uniformity of the nanostructured active substrate to immobilize probe molecules. In this frame, we show here the design and optimization of a reliable strategy to obtain patterned bioactive surfaces by combining EBL technique to molecular self-assembling. We demonstrate the possibility to obtain precise micro- and nanopositioning of functional biomolecules (such as DNA and proteins), as well as the simultaneous patterning of organic fluorophores and water-soluble colloidal nanocrystals.
In this work, we have demonstrated a very versatile and simple method based on the spatially controlled self-assembling of silane molecules onto EBL-nanopatterned substrates for the site-selective nanopositioning of different molecular species. The strategy has a wide applicability as SH− and NH2− silanes reactivity is well established and routinely used to link, through specific activating spacers (e.g., GTA, EDC), the most frequent reactive chemical groups of biomolecules and fluorophores (i.e., COOH−, NH2−, SH−) [23–26]. The patterned substrates may enable very precise and efficient interactions with a wealth of molecules, including biomolecules, organic fluorophores and water-soluble nanocrystals, leading to the development of hybrid scaffolds of smart bio/inorganic or organic/inorganic active interfaces. Importantly, such hybrid self-assembled structures can be applied in several fields, ranging from diagnostics to optoelectronics. In the case of biomolecules, for instance, such bioactive substrates may open up interesting possibilities to implement novel purification methods onto a solid substrate (e.g., for antibodies or antigens), but also for cell patterning/sorting applications and for single cells studies.
Micro- and Nanopatterned SAMs: Fabrication and Characterization
Silicon dioxide substrates were treated with a NH4OH/H2O2/H2O (1:1:5) solution at 70 °C for 10 min, rinsed with distilled water for 5 min, immersed in HCl/H2O2/H2O (1:1:5) solution at 70 °C for 10 min, rinsed again with distilled water for 5 min, and finally dried with a nitrogen stream. PMMA was then deposited onto the SiO2substrates by spin coating, and subsequently baked at 180 °C for 2 min onto an hotplate. EBL was performed by a Leica Lion LV1 system. The exposed PMMA was then developed in a methyl isobutylketone/isopropyl alcohol solution at room temperature. The samples were subsequently dried under a nitrogen stream and treated with oxygen plasma (5 s, 70 mTorr, 25 Watt) in order to remove the PMMA debris and to promote the activation of the SiO2-exposed areas, thus improving the surface reactivity for the following SAM deposition. PMMA nanostructures were analyzed by Holographic Microscopy (Lyncèe Tec DHM 1000, transmission mode). Afterward, samples were treated for 5 min with a freshly prepared aqueous solution of APTES (0.5% v/v) (Sigma–Aldrich) in order to deposit a cationic layer yielding surface-exposed primary amino groups. Samples were then washed with deionized water for 10 min, dried with a nitrogen stream and stored in a vacuum desiccator overnight, to evaporate physisorbed APTES molecules. The surface density of active chemical groups on the substrate was assessed and quantified by fluorescence measurements, exploiting fluorescein-5-isothiocyanate (FITC, BioChemika, 10 mg/mL stock solution in DMSO) covalent binding to primary amino groups, and comparison with FITC calibration curve (data not shown). Standard solutions of APTES, ranging from 0.005 to 10% (v/v), were spotted onto the silicon substrates and then incubated with FITC (0.1 mg/mL, over night incubation at 4 °C in the dark). We found that the highest fluorescence signal was obtained with the 0.5% (v/v) value, approximately corresponding to a surface density of exposed amino groups of 22 pmol/cm2. The remaining PMMA onto the silanized samples were then removed by soaking samples in hot acetone (10 min), isopropylic alcohol (10 min) and dried with nitrogen flow.
The aminosilanized substrates were activated with a homo-bifunctional linker (glutaraldehyde), suitable to react with aminated probes, such as 5′-aminated ssDNA, antibodies, etc. We used solutions of 2.5% (v/v) glutaraldehyde in 100 mM phosphate buffer, pH 7. The reactions were carried out at 4 °C in the dark for 2 h. Afterward, substrates were abundantly washed with MilliQ water (2 washing cycles for 10 min at ambient temperature), dried with N2flow and desiccated in an oven for 3 min at 60 °C.
Hybridization onto Micro- and Nanopatterned DNA Arrays
Solutions of 0.03 μM ssDNA probes (5′-NH2-CGC AGG ATG GCA TGG GGG AG-3′) in 1× TE buffer, pH 8.0 were spotted on the micro- and nanopatterned surfaces. After incubating the sample for 2 h in humidified wells at 37 °C, DNA-modified substrates were washed for 3 min with 1× TE solutions and left to dry in air. Also in this case, the surface density of ssDNA was optimized and quantified by fluorescence measurements, using Cy3-labeled ssDNA (5′-NH2—CGC AGG ATG GCA TGG GGG AG—Cy3-3′) and relative calibration curve (data not shown). We investigated the concentration range of probe DNA from 10 to 0.001 μM, finding an optimal value of 0.03 μM, approximately corresponding to ~0.9 picomol/cm2of probe DNA immobilized onto the silicon substrate. Prior to hybridization experiments, activated samples were blocked with NaBH4. Hybridization experiments were carried out by investigating the hybridization efficiency of target DNA complementary to the probe (5′-CTC CCC CAT GCC ATC CTG CG-3′), as compared to single-mismatch sequences (5′-CTC CCC CATA CC ATC CTG CG-3′). In both cases, we used a 10 μM concentration of target DNA in 1× PCR buffer. Finally, in order to detect the fluorescence signal after the hybridization events, we used a solution of 0.5× SYBR Green I in 1× PCR buffer, a typical intercalating dye highly specific for double strand DNA (1 h incubation at room temperature). After the reaction, samples were washed twice for 10 min with water and gently dried with a stream of nitrogen. High resolution fluorescence imaging was carried out by confocal microscopy (Leica TCS-SP5 AOBS). AFM imaging was performed in air at ambient conditions (20–25 °C, atmospheric pressure, ∼50% of humidity) by using a CP-II scanning probe microscope or a Nanoscope IV MultiMode SPM (Veeco, Santa-Barbara, CA), equipped with 5 μm scanners.
Protein Micro- and Nanoarrays
Functional micro- and nanoarrays of antibodies and proteins were produced by a similar procedure, spotting aqueous solutions of 1 μg/mL of antibodies (AntiTurboGFP, Evrogen) in 100 mM phosphate buffer onto the glutaraldehyde-activated micro- and nanopatterns (prepared as described above). Reactions were carried out for 4 h in the dark. After antibodies’ incubation, the generated Schiff bases were stabilized, and secondary amines linkage with terminal formyl groups were produced by soaking the samples in NaBH4for 1 h at room temperature. Afterward, samples were washed in the same buffer with the addition of 0.02% of Tween20 (or 2 mg/mL BSA) in order to prevent the formation of aspecific binding sites. The surface density of antibody molecules was optimized by investigating a wide concentration range of antibodies solutions (from 30 to 0.1 μg/mL), finding an optimal value of 1 μg/mL. Such experiments were performed by exploiting the corresponding fluorescent antigen, i.e., GFP protein. The patterned substrates were always washed by means of a gentle agitation in phosphate buffer for 10 min, and then left to interact with solutions containing GFP proteins (10 μg/mL, rTurboGFP, Evrogen, 1 h in the dark). After gently washing with buffer (10 min, 2 cycles), samples were immediately characterized by confocal microscopy and AFM.
Parallel Patterning of Organic Fluorophores and Water-Soluble NCs
For these experiments, PMMA micro- and nanostructured substrates were subjected to two sequential different silanes deposition. Initially, samples were incubated with solutions of 0.5% MPTS (v/v) in isopropyl alcohol solution for 1 h at room temperature and then washed three times for 5 min with absolute EtOH, contemporaneously sonicating in order to remove silane aggregates. PMMA was then removed by liftoff and samples were dried under nitrogen stream. Samples were subsequently subjected to the multistep covalent immobilization of 0.5% APTES and 2.5% GTA, following the procedures described above, producing alternating regions with different exposed chemical reactive groups. An aqueous mixture containing the two different fluorophores (0.01% of fluorescein-5-maleimide, λex/em492/530 nm, 0.5% stock solution in DMSO, and 0.06 μM of amino-terminated water-soluble nanocrystals, Evident Technologies, λem610 nm) was spotted onto the active substrates for 4 h in the dark at room temperature. These samples were then washed thoroughly with water, dried with a nitrogen stream and immediately analyzed by confocal microscopy for imaging and spectral analysis.
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The authors gratefully acknowledge Barbara Sorce and Loretta L. del Mercato for their help during experiments, and E. D’Amone and V. Fiorelli for the expert technical assistance. This work was supported by the Italian Ministry of Research through MIUR “FIRB” project (RBLA03ER38_001).