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Chapter 2
Aptamer Arrays
Eva Baldrich
Abstract
In less than 40 years, aptamers have consolidated their role in biosensor development. Chemically related to nucleic acid probes, production of aptamers against targets of various sizes and compositions places them as ideal capture elements, alternative to more consolidated molecules such as antibodies. Thanks to their chemical simplicity and production, as well as their unique characteristics, aptamers have been successfully integrated in several innovative approaches. The incorporation of aptamers into the existing microarray technologies has lead to the reporting of various detection strategies, including direct fluores-cence detection of fluorescent reporters, fluorescence anisotropy, FRET, SPR imaging, and electrochemical detection.
Key words: Aptamer, Aptamer array, Aptasensor, Reagentless detection
Aptamers are artificial nucleic acid ligands, selected in vitro from DNA/RNA random pools against specific nonnucleic acid tar-gets. The reported aptamers have shown equal or higher affinity and specificity for their targets than their equivalent antibodies. Furthermore, aptamers have been selected against a vast variety of targets, including small molecules, drugs, peptides, and hor-mones, and also complex objectives such as proteins, spores, and whole cells, showing surprising versatility compared to other biorecognition components (1–6). The fact that aptamers are selected and produced in vitro eludes the use of animals and related ethical concerns, ensures no batch-to-batch variation, and allows selection under nonphysiological conditions and toward small molecules and toxins. The whole procedure is potentially automated and easier, quicker, and cheaper than antibody
p roduction. From an integration point of view, aptamers are 1. Introduction
1.1. Aptamers:
Description,
Advantages,
and Drawbacks Ali Khademhosseini et al. (eds.), Biological Microarrays: Methods and Protocols , Methods in Molecular Biology, vol. 671,DOI 10.1007/978-1-59745-551-0_2, © Springer Science+Business Media, LLC 2011
36Baldrich
smaller and less complex than antibodies (5–25 kDa vs. 150 kDa),
and are easier to modify during or after synthesis, favoring immo-
bilization and labeling. In addition, the unique chemical and
structural characteristics of nucleic acids permit aptamer revers-
ible denaturation and thus the design of truly reusable devices.
The main concerns regarding the real applicability of aptamers,
related to inherent nucleic acids properties such as sensitivity to
nuclease attack and chemical simplicity, are being circumvented
in the shape of spiegelmers and chemically modified aptamers.
The fact that each single DNA/RNA sequence can adopt multi-
ple conformation, reducing assay efficiency and increasing cost,
can be also minimized by careful folding characterization and
assay optimization.
The first reports on aptamer arrays exploited either optical fiber
arrays (7, 8), or agarose beads deposited in the wells of microma-
chined flow chips (9). Later works, however, used glass slides to
produce aptamer arrays in a variety of assay formats that, taking
advantage of detection strategies already proved for DNA, per-
mitted detection of an assortment of targets (Fig. 1). Gold and
1.2. Aptamer Arrays中医
推拿按摩床
a
d
Fig. 1. Scheme of different fluorescence aptameric array assay formats. (a) Target capture, followed by labeling with a reactive fluorophore. (b) Sandwich assay using aptamer and labeled antibody as capture and detector biocomponents.
(c) Competition between native and labeled target variants. (d) Molecular beacon format exploiting the use of a labeled aptamer immobilized on surface; target binding induces changes in fluorophore emission.
37
Aptamer Arrays coworkers introduced the use of photoaptamers that, remaining covalently bound to their targets after photoactivated cross-linking, allowed of highly stringent washes and efficient removal of non-specific binders (10–12). In view of the enhanced signal-to-noise ratios and low detection limits registered, the SomaLogic team developed a 17-plex photoaptamer array on activated glass slides (10). Detection was based in a sandwich format, using either NH-reactive fluorophores or fluorophore-labeled antibodies, with detection limits below 10 fM for several analytes measured in 10% serum.Ellington et al. reported on a number of arrays manufactured using the lysozyme, ricin, IgE, and thrombin RNA/DNA aptam-ers on streptavidin slides (13–15). As a novelty, they defined a “universal buffer” in which the four aptamers retained acceptable affinity for the fluorophore-labeled analytes at concentrations over seven orders of magnitude (10–107 pg/mL).In a different approach, the team at the company Archemix immobilized fluorescein-labeled RNA/DNA aptamers on strepta-vidin slides (16). In this format, target binding is directly mea-sured as changes in fluorescence polarization anisotropy in a completely reagentless format. The system detected and quanti-fied four different proteins in the presence of serum and bacterial cell lysates. Alternatively, Lin self-assembled the aptamer into high-density nanoarrays, following modification with a fluores-ce
nt nucleotide analog near the target-binding site (17). Target binding generated increase in fluorescence, measurable by confo-cal fluorescence microscope imaging down to the low nanomolar range. Finally, Bera Aberem spotted onto silanized slides an aptamer, Cy3-labeled and hybridized with a chromic, cationic, water-soluble polythiophene (18). In the absence of target, the polymer quenched Cy3 emission; target binding induced poly-mer displacement and fluorescence increase.Alternative approaches include the development of surface plasmon resonance imaging (SPRi) and electrochemical aptamer arrays (19–21).
1. Microscope slides, modified so as to incorporate on surface reactive groups (amino, aldehyde, maleimide, thiol, and epoxy) or components (streptavidin and biotin) are provided, among others, by Xenopore Corp. (Hawthorne, NJ), Pierce–Thermo Fisher Scientific Inc. (Rockford, IL), Corning (Corning, NY), Nanocs Inc. (New York, NY), Nalge Nunc International (Rochester, NY), Genetix (New Milton, UK),
2. Materials
2.1. Physical Support
38Baldrich
and Erie Scientific (Portsmouth, NH). In some cases, customized coated glass slides can be produced. 2. Streptavidin-coated agarose and silica beads can be obtained from Sigma-Aldrich and Pierce-Thermo Fisher Scientific Inc. (Rockford, IL). 3. Gold-coated slides and substrates can be purchased from a number or companies, including Aldrich, Platypus Technologies (Madison, WI), Phasis (Geneva; Switzerland), Nanocs Inc. (New York, NY), and Asylum Research (Santa Barbara, CA); and Gentel Biosciences, Inc. (Madison, WI) produces gold-coated substrates especially optimized for SPRi. 4. Electrodes of different geometries, composition, and com-plexity can be obtained from providers such as BASi (West Lafayette, IN), DropSens (Oviedo, Spain), BVT Technologies, a.s. (Brno, Czech Republic), Palm Instruments BV (Houten; The Netherlands), Applied BioPhysics (Troy, NY), and Princeton Applied Research (Oak Ridge, TN). 1. Once its sequence is known, an aptamer is produced by clas-sical DNA/RNA synthesis. In the lack for synthesis facilities, the aptamer can be ordered to any company commercially
providing oligonucleotides. In this case, double check the sequence ordered and ensure that appropriate spacers/linkers are being added to the extreme chosen for aptamer immobi-lization/modification (see Note 1).
2. For novel aptamers, at least three companies produce cus-tomized aptamers: RNA-tec (Leuven,
Belgium), AptaRes
(Luckenwalde, Germany), and Nascacell (Munchen, Germany). 3. Reconstitute lyophilized aptamers to 100–500 m M using ster-
毛皮加工ile water or binding buffer. Store frozen in small aliquots in order to avoid repeated thaw-freeze cycles (see Note 2). 4. The most widely used binding buffers, which often corre-测试
网页游戏 spond to the solutions employed in aptamer SELEX, are the following ones:
– PBS (10 mM phosphate buffer, 138 mM NaCl, 2.7 mM KCl, pH 7.4).
– PBS, 1–5 mM MgCl 2.
– 5 mM NaH 2PO 4, 5 mM KH 2PO 4, 2 mM MgCl 2.
– 10–100 mM Tris–HCl, pH 7.5, 50–150 mM NaCl,
0–5 mM MgCl 2.
– 20 mM Tris–acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2, 1 mM MgCl 2.
– 10–50 mM Hepes, pH 7.4, 0–150 mM NaCl, 0–5 mM KCl, 0–5 mM MgCl 2.
2.2. Aptamer
Production
and Manipulation
39Aptamer Arrays 5. When possible, sterile plasticware, pipette tips with filter, and separate pipettes (specific for work with DNA/RNA) should be used. If working with RNA aptamers, additional measures should be considered such as using RNAse-free material, treating all solutions and surfaces with an RNAse inhibitor (such as DEPC), and working in a space physically separated and labeled for work with RNA. 1. Use an aptamer produced or modified with an amino/thiol/biotin group at one of the extremes, incorporating the appro-priate spacer/linker (see Note 1).
2. Unless otherwise stated, use the following solutions. Binding buffer: sterile binding buffer of choice (see Subheading 2.2).
Blocking buffer: binding buffer supplemented with 0.1% Tween (see Note 3). Washing buffer: binding buffer contain-ing 0.05–0.1% Tween.
3. Amine-silane-based aptamer immobilization. Activation
solution (1): 0.05 M dioxane solution of carbonyldiimida-zole. Activation solution (2): 5% (v/v) glutaraldehyde in PBS, pH 7. Washing solution (1): dioxane and diethyl ether. Washing solution (2): PBS, pH 7. Chemical block-ing: 1 M ethanolamine, pH 8.5, prepared in sterile water or binding buffer.
气囊止血带
4. Thiol-silane-based aptamer immobilization. Chemical block-ing: 0.1 M mercaptoethanol prepared in sterile water or bind-ing buffer. dna探针
5. Self-assembly of thiolated aptamer on gold surfaces. Use
0.1 M in KH 2PO 4, pH 3.8.
ddtsf6. Aptamer labeling with amine-reactive reagents. Reaction buffer: freshly prepared 0.1 M tetraborate buffer, pH 8.5
(see Note 4). Alternatively, 0.1 M sodium bicarbonate buf-fer pH 8–9 can be used (see Note 5). For labels/reagents insoluble in water, dissolve in dimethyl sulfoxide (DMSO) or in dimethyl formamide (DMF). Caution : DMF is a pos-sible carcinogen and should be manipulated wearing
protections. 7. Aptamer conjugation to COOH-bearing electroactive labels. Reaction buffer: 10 mM PBS or HEPES (4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid), pH 7.4. For labels/reagents insoluble in water, dissolve in DMSO or in DMF. Caution : DMSO
and DMF are possible carcinogens and should be manipulated wearing protections. Cross-linkers: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)
and N -hydroxy succinimide (NHS). 8. Aptamer precipitation. 3 M NaCl, cold ethanol, and cold 70% (v/v) ethanol.2.3. Aptamer
Immobilization
and Labeling
