Sol–Gel Derived Nanoporous Compositions for Entrapping Small

Article pubs.acs.org/ac

Sol−Gel Derived Nanoporous Compositions for Entrapping Small Molecules and Their Outlook toward Aptamer Screening Ji-Young Ahn,† SangWook Lee,‡ Minjoung Jo,§ Jeehye Kang,† Eunkyung Kim,† Ok Chan Jeong,⊥ Thomas Laurell,*,†,‡ and Soyoun Kim*,† †

Department of Biomedical Engineering, Dongguk University, Seoul, Korea Department of Measurement Tech. & Ind. Electrical Engineering, Lund University, Sweden § PCL Inc., Korea ⊥ Department of Biomedical Engineering, Inje University, Korea ‡

S Supporting Information *

ABSTRACT: This paper reports for the first time the application of sol− gel microarrays for immobilizing nonsoluble small chemicals (Bisphenol-A; BPA). Also, known problems of sol−gel adhesion to conventional microtiter well plate substrates are circumvented by anchoring the sol− gel microspots to a porous silion surface so-called, PS-SG chips. We confirmed low molecular weight chemical immobilization inside a sol−gel network using fluorescein. BPA and the BPA specific aptamer were utilized as a model pair to verify the affinity specific interaction in the PS-SG selection system. The aptamer interacted specifically with BPA in the sol− gel spots, as shown in microarrays forming the letters “L”, “U”, “N”, and “D”. Moreover, the bound aptamer was released by heat, recovered, and verified by gel electrophoresis. The developed PS-SG chip platform will be used for screening aptamers against numerous small molecules such as toxins, metabolites, or pesticide residues.

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strong anchoring of the sol−gel matrix to the substrate while a broad range of sol−gel solvent compositions can be employed. Porous silicon (PS) can be produced by anodic etching of monocrystalline silicon and offers a wide range of porous compositions based on the electrochemical process conditions. Previous work on PS applied for protein immobilization has targeted either covalent immobilization of enzymes6−9 or surface adsorption of antibodies.10,11 These PS compositions however are not optimally suited for sol−gel based anchoring and aptamer selection. The work in this paper describes modified porous silicon morphology as a sol−gel anchoring substrate, which allows an arbitrary formulation of sol−gel compositions suitable for the entrapment of low molecular species. The present work also builds on optimized sol−gels for small molecule encapsulation with the goal to conveniently isolate high affinity aptamers against low molecular chemicals. Aptamers are molecular recognition probes with a high affinity for considerably different molecules, ranging from large targets such as proteins, peptides, and complex molecules of drugs to organic small molecules or even metal ions.12−16 It has generally been recognized that aptamer affinity is comparable to or even higher than that of antibodies.17,18 Typically,

ol−gel entrapment of biomolecules, such as proteins and enzymes, has been of considerable interest over the past decade.1−3 The nanostructural composite made by the sol−gel process provides an aqueous environment that preserves biological activity and may enhance the biomolecule stability.4 Upon entrapment, biomolecules are encased by hydrated silica in pores tailored to the size of the embedded molecules. The sol−gel matrix constrains the motion of the encapsulated biomolecules and may prevent irreversible structural deformation. That is, the entrapment may be able to yield weak intermolecular interaction between the host sol−gel matrix and certain exposed residues of molecules. Kim et al.5 reported on a screening strategy for a sol−gel formula to immobilize various targets, depending on their molecular weight or/and diameters. They examined many aspects of sol−gels including adhesiveness, droplet morphology, transparency, breakage, and entrapment phenomenon and successfully immobilized their targets within sol−gel networks, including aptamers. Although sol−gels have been extensively used to entrap macromolecules, successful entrapments of low molecular weight chemicals with full access for affinity ligands have not been reported. Sol−gel formulations may be designed to entrap small molecules, but commonly, this compromises the adhesiveness of the sol−gel matrix to conventional substrates such as microtiter well plate polymers. In order to address the problem of substrate adhesion, we have developed a new porous silicon substrate that offers a © 2012 American Chemical Society

Received: September 27, 2011 Accepted: January 27, 2012 Published: January 27, 2012 2647

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Scheme 1. Concept of the PS-SG Chip Platforma

a

Schematic of the PS-SG in vitro selection aptamer chip concept, where small target molecules are entrapped in a spotted sol−gel microarray (a) and (b) subsequently incubated with binders (aptamers). In this step, aptamers can enter into the sol−gel network and diffuse to find their targets. After washing and heating, selectively bound aptamers can be recovered.

aptamers have been engineered through repeated rounds of in vitro selection, or equivalently, SELEX (systematic evolution of ligand exponential enrichment).15,19−22 In order to adapt SELEX into an appropriate tool for selecting high affinity aptamers targeting small molecules, current methods such as nitrocellulose membrane extraction or affinity chromatography cannot be utilized. Specifically, we demonstrate for the first time the formulation of a sol−gel composition that entraps a small molecule, Bisphenol A (BPA), which was microarrayed and anchored to a PS substrate. The combined porous silicon sol−gel (PS-SG) system was used as a platform for specific aptamer binding (Scheme 1). We report successful binding and retreival of a BPA specific aptamer to its target inside the sol−gel matrix. This paper demonstrates the first combination of PS processing and sol−gel microchip arrays (PS-SG chips), that may open a generic route to the development of affinity ligands, i.e., aptamers, against small molecules.

(hydrofluoric acid), depending on a multitude of process parameters (current density, etching time, crystal orientation, silicon dopant type, doping level, illumination, electrolyte composition, temperature, and surface roughness).6,24 The detailed outline of porous silicon fabrication has been described elsewhere.11,25 Briefly, a two-compartment electrochemical cell with sapphire glass (Melles Griot BV) on one side was used to allow for illumination during anodizing. The wafers were etched at a constant current of 2 mA/cm2 for 10 min. The backside of the silicon was illuminated during the anodization period using a 100 W halogen lamp (Osram, Germany) at a distance of 10 cm from the window of the back of the electrochemical cell. The porous silicon surfaces were analyzed by scanning electron microscope (SEM) imaging, using a JEOL JSM-6700F field emission scanning electron microscope (JEOL, Japan). Before SEM analysis, a 10 nm thick layer of platinum was deposited on the samples using a PT (platinum) sputtering coater adjacent to the SEM. The deposition rate is about 0.04 nm/s under 10 mA electric powers. The sample was deposited 250 s to fabricate 10 nm thickness layers on the surface. Sol−Gel Manufacturing for Immobilization of Chemicals. Sol−gel droplets were spotted onto the surfaces of the porous silicon chips. The sol−gel materials for immobilizing chemicals were utilized according to the the manufacturer’s recommendation (SolB complete kit, PCL Inc., Korea, www. pclchip.com). The fluorescent indicator was prepared with two different sol−gel formulations to investigate the immobilization performance. In detail, using two sol−gel formulations (Formula-A: SolB I 27.5%; SolB II 10%; SolB H 12.5%; SolB S 12.5%; DW 25%, and Formula-B: SolB I 25%; SolB II 7.5%; SolB III 5%; SolB H 12.5%; SolB S 12.5%; DW 25%), the fluorescent indicator was mixed with visually homogeneous SolB reagents to prepare the sol−gel microarrays. Formula A was designed to entrap smaller molecules (metabolites) whereas Formula B was designed to entrap larger molecules (proteins).5,26 Microarraying. The sol−gel mixture containing chemical (target analyte, BPA) was arrayed with negative (without analyte) and/or positive control (Cy3-labeled antibody) in a 96-well plate using a sciFlexarrayer S11 (Scienion AG, Germany) non-contact dispensing machine, and a single spot volume was calculated automatically using autodrop volume

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EXPERIMENTAL SECTION Materials. The following materials were used in these experiments: BPA (4,4′-dihydroxy-2,2-diphenylpropane, SigmaAldrich, USA) was dissolved in 50% dimethylformamide (DMF) at a final concentration of 100 mM. Fluorescein sodium salt (Sigma, F6377) was purchased as a fluorescent indicator and was dissolved in 1× PBS and sequentially diluted. All reagents used were protected from light and stored at 4 °C. Anti-Bisphenol A (BPA) single strand DNA (ssDNA) aptamer23 was synthesized by IDT Inc. (USA)[5′-GGG CCG TTC GAA CAC GAG CAT GCC GGT GGG TGG TCA GGT GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC AGG ACA GTA CTC AGG TCA TCC TAG-3′, underline indicates PCR amplified products, 86 bp]. Cy3 labeled secondary antibody (anti-Rabbit IgG) was purchased from Jackson ImmunoResearch Laboratories, Inc (USA) for positive controls in Figure 4. Fabrication of Porous Silicon Surface. Porous silicon (PS) is an anisotropic, nanocrystalline silicon architecture of high surface area. It can be fabricated directly from monocrystalline silicon through a galvanostatic electrochemical or photochemical etching procedure in the presence of HF 2648

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Figure 1. Entrapment of chemicals in sol−gels with different formulations. The fluorescent indicator [Fluorescein, 376.27 Da] was spotted with two different SolB formulas (Formula-A and Formula-B) in the wells of a microplate. Each sample, diluted with 1× PBS buffer, was mixed with FormulaA and B sol−gel solutions and, after spotting, was maintained in the dark at room temperature and over 60% humidity. The sol−gel arrays with Fluorescein at levels of 1525, 781, 391, and 195 nM were printed. The fluorescein embedded sol−gel arrays were soaked in PBS solution for different time intervals (0, 1, 3, and 5 h). After brief drying, resultant wells were scanned and analyzed.

application that became possible upon sol−gel entrapment of small molecules. A fluorescent indicator [Fluorescein sodium salt, 376.27 Da] was spotted with two different sol−gel formulas (Formula-A and Formula-B). We hypothesized that if the sol−gel entrapped fluorescein indicator retained its intensity in the spots after PBS buffer washing, a successful entrapment of small molecules had been accomplished within the given sol−gel formulation. Diluted fluorescein indicators of different concentrations were mixed with sol−gels and arrayed on a 96-well type microplate (1525, 781, 391, and 195 nM per single spot). After gelation for 16 h, 100 μL of PBS solution was added to the microwells for 1, 3, and 5 h. Figure 1 summarizes the fluorescence intensity of the fluorescein indicator. The sol− gel composition with Formula-A maintained its fluorescence intensity over the entire wash interval, while the fluorescence using Formula-B decreased significantly over the initial 1 h wash interval. This may have been caused by poor entrapment and hence diffusion of the fluorophore out of the sol−gels. The experiment demonstrated that the nature of the sol−gel based on Formula-A improved the entrapment of small molecules inside the pores and/or cages and was thus used for further studies on aptamer binding to small molecules in the sol−gel network. Tailored Porous Silicon Surfaces Improve the Anchoring of Sol−Gels. Our initial motivation to utilize porous silicon substrates for sol−gel applications was driven by protein microarray studies, showing that it is possible to tailor properties for effective protein physisorption, as well as the fact that porous silicon surfaces were known to display low immunoassay background signals.25,28,29 It was also noted that small chemical-containing sol−gel droplets spotted onto plastic microwell surfaces displayed poor adhesion to the microwell surfaces and all of the spots were washed out with buffer solution at the first equilibrium/wetting step, frequently encountered when generating the data for the sol−gel formulation optimization in Figure 1. This might have been an effect of the solvents used, such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO), for solvating the smallmolecule chemicals. A porous silicon surface was therefore developed as a new substrate for physical anchoring of the sol−gel droplets. Porous silicon (Si) surfaces were fabricated by anodic dissolution of monocrystalline silicon in hydrofluoric acid (Figure S-1,

detection software. BPA (100 nM) embedded sol−gel material was spotted onto the porous silicon surfaces, delivering approximately 4 nL per single droplet in each array. The spot to spot distances were set to 400 μm, and 8 × 8 arrays were spotted on each chip. Aptamer Assay. For the binding assay, BPA aptamers were labeled with Cy3 using terminal deoxynucleotidyl transferase (Fermentas, Canada) and Cy3-dUTP (GeneChem Inc., Korea). The following reaction mixture was prepared: 4 μL of 5× reaction buffer, 0.5 nmol of ssDNA aptamer, 1 nmole of Cy3-dUTP, and 30 U of TdT enzymes in a 20 μL reaction volume. After a 4 h incubation at 37 °C, labeled DNA aptamers were precipitated by ethanol at −70 °C. For the binding assay, 100 pmole BPA aptamers dissolved in 50 μL of 1× PBS were boiled at 95 °C for 10 min and allowed to cool to room temperature over 3 h. The BPA embedded PS-SG chips were treated with blocking buffer (20 μg/mL tRNA in 1× PBS) for 2 h and incubated with 2 μM of Cy3-labeled BPA aptamer for 1 h. After three times washing for 15 min and brief drying for 10 min, the resultant sol−gel arrays were scanned and analyzed with a fluorescence scanner. Fluorescence Measurements. For the chemical entrapment assay, after curing the fluorescein embedded sol−gel arrays for 13 h at room temperature with 60% humidity, sol− gel droplets were exposed to 1× PBS for wetting and washed for three different time periods (1, 3, 5 h) with wash solution (0.2% Tween-20 (Sigma Aldrich) in 1× PBS). After brief drying for 10 min, resultant wells were scanned with a fluorescence scanner (Typhoon FLA9000, GE Healthcare) and analyzed. Aptamer Retrieval by Heat Treatment. After heat treatment, released aptamers were recovered by ethanol precipitation and 15 cycles of amplification. The PCR cycling conditions were as follows: an initial heating to 94 °C for 5 min; 15 cycles of 94, 55, and 72 °C each for 30 s; and final elongation for 7 min. The PCR products (86 bp) were confirmed by electrophoresis on a 2% agarose gel.

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RESULTS AND DISCUSSION

Immobilization of Small Molecules in a Sol−Gel Matrix. The confinement of proteins and antibodies in unique sol−gel formulations were established in recent years by studying a protein chip.4,5,27 In this paper, we investigate a new 2649

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Figure 2. Preparation of sol−gel droplet-integrated porous silicon chip, PS-SG chip. PS-SG microarray (upper left, sol−gel array chip); scanning electron micrograph (SEM) of a single sol−gel spot (upper center); close-up SEM of the sol−gel spot surface displaying the porous sol−gel network (upper right); sol−gel spot boundary showing the anchoring of the sol−gel to the porous silicon surface (bottom left); and close-up view (SEM) of the micro/nanoporous silicon surface network (bottom right).

Figure 3. Schematic of the protocol for demonstrating the specific interaction between BPA and aptamers. BPA and its specific aptamers were used as a model system for proving the aptamer interaction with a small molecule entrapped within a sol−gel. The sequential procedure for the assay is shown in (a). In (b), BPA entrapped sol−gels were printed to reveal the characters of L, U, N, D, and blank sol−gels were spotted to complete the 8 × 8 array. After 1 h of incubation and washing, all assayed chips were scanned.

Supporting Information). For preparing a sol−gel microarray chip, superporousified (highly porous) silicon wafers were

diced and a small molecule mixed with a sol−gel (Formula-A) was spotted onto the chip surface. However, while a 2650

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Figure 4. Retrieval of bound aptamers from the porous silicon chips. (a) The BPA entrapped sol was spotted as an “H” surrounded by controls with a Cy-3 labeled antibody (control spots). After assaying, two chips were soaked with 300 μL of PBS solution. One chip was heated for 1 min, and the other was heated for 5 min; the solutions were transferred for PCR amplification. It should be noted that bound aptamers were fully released from the target embedded sol−gels during 5 min of heating whereas the chip undergoing 1 min heating still displayed a remaining aptamer signal. (b) Retrieved aptamers were confirmed by PCR amplification.

2). While the adhesiveness of the BPA-containing sol−gels was strongly enhanced, the nonspecific aptamer trapping voids were alleviated. Specific Aptamer Interaction to Chemicals. We previously reported a specific aptamer against BPA.23,30 Using this model pair, we investigated the specific interaction between the aptamer and BPA on the PS-SG chip assembly as described above. Sol−gel droplets were spotted in 8 × 8 arrays on porous silicon chips, encoding a pattern of BPA containing sol−gel droplets spelling “L,” “U”, “N”, and “D” surrounded by negative control sol−gel droplets (without BPA). Cy3-labeled anti-BPA aptamers (100 pmole) were incubated in each assay well for 1 h, and the chips were scanned with a fluorescence scanner. As shown in Figure 3, anti-BPA aptamers specifically bound to BPA inside the sol−gel spots, also all of the 64 sol−gel droplets, were strongly attached to the modified porous surface during

superporous chip surface with a combined micro- and nanomorphology has good properties for increasing probe density in protein microarray applications,28,29 this was not an optimal morphology for the sol−gel arrays. That is, voids between the condensed sol−gel matrix and the surface in the micropores of the porous silicon were observed homogeneously in the superporous surface (Figure S-2, Supporting Information). In these voids, aptamers may be nonspecifically trapped during the assay and thus generate a highly fluorescent background. In Figure 2, the condition for porosification of the silicon substrate was thus modified, generating a thinner porous layer with smaller pores that provided improved sol−gel assay conditions. Compared to the original macro/nanoporous surface structure (Figure S-2, Table S-1, Supporting Information), the new sol−gel array adapted chip surface provided a shallow porosity as demonstrated by the SEM images (Figure 2651

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SG platform to encapsulate small molecules with retained access for aptamers can open the route to develop target specific aptamers for a wide range of small molecules. The described approach for small molecule aptamer in vitro selection allows for the development of aptamer based affinity probes against, e.g., pesticides, toxins, and metabolites where a subsequent step is the development of sol−gel based sensing systems33−35 where the target specific aptamers are encapsulated in sol−gel microarrays and serve as the target molecule capture probe, Scheme 1. The conventional way would be to implement a sandwich assay system; however, sandwich assays are extremely difficult to realize for small molecule targets.30 We therefore anticipate that a reverse assay strategy in a competitive binding format will be most feasible.

the overall assay time period (for blocking, 2 h; for incubating, 1 h; for washing, 30 min). The results show that small target chemicals can indeed be incorporated within a sol−gel matrix, yet allowing aptamers to access the immobilized chemical and bind specifically. This suggests that sol−gels hold a complex porous structure with nanopores for entrapping target chemicals and larger diffusion routes to enable binding probes like aptamers to enter and diffuse through the sol−gel matrix. Aptamer Retrieval by Heat Treatment. In in vitro selection of aptamers, several methods to extract aptamers from aptamer−target complexes have been reported.20,21,31 The complete retrieval of bound aptamers can reduce the number of cycles needed in the SELEX process and thus save resources. The means of releasing bound aptamers might be selected, depending on the characteristics of the capturing probe molecules. In this paper, we demonstrate “heat elution” of aptamers from their targets. The structure of aptamers is generally destructed by heat, and hence, they loose binding affinity to their targets. We monitored the fluorescent signals of bound aptamers on sol−gel spots after heat treatment. As shown in Figure 4, aptamer signals disappeared completely after heating for 5 min at 95 °C, demonstrating that bound aptamers can be released by heat, collected, and subsequently amplified. Figure 4b shows an aptamer (86 bp) recovered from a PS-SG chip. This indicates that the proposed PS-SG chip manipulation has the possibility to be applied in chemical SELEX experiments without any harsh denaturing release for recovery of aptamers.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82-2-2260-3840. Fax: +82-2-2260-3840. E-mail: [email protected] (S.K.); [email protected] (T.L.).

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ACKNOWLEDGMENTS J.-Y.A. and S.W.L. contributed equally to this work. This work was supported by KME/KEITI as "Eco-Innovation Project (E211-41003-0007-0)". The Swedish Research Council (Grant no. 621-2009-5361) and STINT Institutional Grant IG2010 2068 are greatly acknowledged for their financial support. KME (Conversing technology project), MKE/KEIT (10035501) and KEIT (S1072576) supported this work. This work was also supported by Seoul R&BD program (SS100003). M.J. was supported by grant from the International Collaborative Research and Development program (GT-2009-ME-DI0076). J.-Y.A. would like to acknowledge the support of NRF (KRF-2009-353-D00004).

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CONCLUSIONS AND PERSPECTIVES A most important benefit of the reported small molecule sol− gel microarray anchoring and aptamer binder extraction is the simple encapsulation of small molecules without the development of a chemical coupling method for each species where the small molecule still is accessible for affinity specific binding. The introduction of porous silicon as a substrate to anchor sol− gel microarrays is essential for the sol−gel encapsulated small molecule aptamer selection since sol−gels for small molecules do not adhere to conventional microtiter well plate substrates nor can these withstand the organic solvents commonly used in the sol−gel immobilization step. Furthermore, the PS-SG chip platform is compatible with the industrial standard for microtiter plate formats, i.e., the 96 or 384 ANSI/SBS format, and is hence amenable to robotic fluid handling. Our further development of the PS-SG platform is directed toward in vitro selection of aptamers against small molecules, where the SELEX (systematic evolution of ligand exponential enrichment) protocol will be followed. SELEX is a screening technique that involves the progressive selection of highly specific ligands, or aptamers, by repeated rounds of partitioning and amplification from a large combinatorial nucleic acid library.16 In conventional chemical SELEX processes, a small molecule target should be physically attached to a stationary support by a linker molecule or large carrier molecule in a primary step. In this case, the physical and chemical properties of the supports should also be considered in terms of their suitability against the target molecules, 32 and coupling efficiency must additionally be checked for every selection round to maintain persistent conditions. Although several aptamers have been successfully isolated by conventional methods, many compounds cannot be successfully immobilized for SELEX processing.14−16,20 The generic properties of the PS-

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