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‘‘Smart’’ DNA interfaces Cite this: Chem. Soc. Rev., 2014, 43, 1612

Vinalia Tjong,a Lei Tang,b Stefan Zauscher*bc and Ashutosh Chilkoti*ac This review focuses on surface-grafted DNA, and its use as a molecular building block that exploits its unique properties as a directional (poly)anion that exhibits molecular recognition. The selected examples highlight innovative applications of DNA at surfaces and interfaces ranging from molecular diagnostics

Received 18th September 2013

and sequencing to biosensing.

DOI: 10.1039/c3cs60331h www.rsc.org/csr

1. Introduction Over the past sixty years, insights into the structure and functionality of nucleic acids have expanded their utility beyond that of their canonical role in biology to exciting developments in nucleic acid-based technologies. These applications range from bioimaging, diagnostics and sensing to drug delivery,1–4 and typically involve a solid interface, where they exploit one or more of the unique properties of nucleic acids. Nucleic acids are linear and directional polyanions that exhibit molecular recognition and self-assembly. Single stranded (ss) DNA shows exquisite affinity and specificity for a

Department of Biomedical Engineering, Duke University, Box 90281, Durham, North Carolina 27708, USA. E-mail: [email protected]; Fax: +1 919-660-5409; Tel: +1 919-660-5373 b Department of Mechanical Engineering and Materials Science, Duke University, 144 Hudson Hall Box 90300, Durham, North Carolina 27708, USA. E-mail: [email protected]; Fax: +1 919-660-8963; Tel: +1 919-660-5360 c Center for Biologically Inspired Materials and Materials Systems, Duke University, Durham, North Carolina 27708, USA

Vinalia Tjong

Vinalia Tjong was a graduate student in the Chilkoti Lab. She received her PhD in Biomedical Engineering from Duke University in 2013. Before coming to Duke, she worked as a researcher in the Institute of Materials Research and Engineering, a member of Agency for Science, Technology and Research in Singapore. Her research interests include biointerfaces, biosensors, development of diagnostic assay and devices, and nanomaterial synthesis.

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its complementary strand and double stranded (ds) DNA can be designed to self-assemble into topologically diverse threedimensional structures. Furthermore, in many of these applications, DNA and RNA not only encode information but also serve as polymeric, nanoscale building materials. An attractive feature of nucleic acids as nanoscale building materials is our ability to manipulate their structure and function at the nucleotide level, greatly facilitated by the diversity of unnatural nucleotides and nucleic acid modifying enzymes that are now available. The aim of this review is to highlight important developments in the use of DNA as a ‘‘smart’’ material at interfaces. We do not focus on the use of DNA in the emerging area of structural nanobiotechnology, where, DNA is used to build complex, three-dimensional structures. Several excellent reviews in this area already exist.5 Instead we focus on applications where DNA is immobilized at an interface to impart a useful function. We note that with a few exceptions, we largely focus on DNA in this review, as DNA is generally preferred to RNA in most applications because of its greater stability.

Lei Tang

Lei Tang is a graduate student in the Zauscher Lab. She obtained both BS (2008) and MS (2010) degrees in Materials Science from Tianjin University, China. She then came to United States for her PhD study at Duke University. Her research interests include synthesis and characterization of biologically inspired polymeric materials, particularly functionalized DNA synthesis and its applications in nanobiotechnology.

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We first introduce the unique properties of DNA as a ‘‘smart’’ material, followed by a brief overview of technologies for synthesis of DNA and its modification. We then discuss chemistries by which DNA can be immobilized on a broad range of solid surfaces. Next we showcase recent, innovative applications that harness DNA’s unique properties when integrated with various solid supports. We conclude the review with remarks on the potential for future development and challenges faced by this field of research.

2. DNA as a ‘‘smart’’ polymer In this review, we largely focus on DNA because it is structurally more robust and chemically more stable across a wide range of conditions than RNA. These properties make DNA a useful material for (bio)nanotechnology applications. In addition, DNA can be synthesised by chemical and enzymatic routes, and it exhibits unique physical and chemical properties that can be exploited for diverse applications. 2.1.

DNA structure

Single stranded DNA is a linear polyanion, composed of four major components (Fig. 1): the 5 0 -phosphate terminus, the 3 0 -OH terminus, the phosphate-sugar backbone, and the bases (nucleobase) as side chains (Fig. 1). Compared with other linear macromolecules DNA has several unique attributes, which are briefly discussed next. 2.2.

Molecular recognition capability

Four bases—adenine (A), thymine (T), cytosine (C) and guanine (G)—provide the chemical diversity and information-coding ability of DNA. The A–T and G–C bases between two DNA strands interact through hydrogen bonds and p–p stacking, to form a stable, double stranded DNA helix (Fig. 1). This hybridization process with its exquisite fidelity in molecular recognition makes DNA a versatile, programmable and ‘‘smart’’ polymer. While the specific A–T and G–C interactions are the canonical form of base

Stefan Zauscher

Stefan Zauscher is the Sternberg Family Professor of Mechanical Engineering and Materials Science at Duke University. He received his PhD in Materials Science from the University of Wisconsin-Madison in 2000. Professor Zauscher is an expert in (bio)surface and interface science, where his research is broadly focused on fabrication, manipulation and characterization of surface-confined biomolecular and polymeric micro- and nanostructures.

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Fig. 1 Schematic of a DNA molecule, showing its 5 0 -phosphate terminus, 3 0 -OH terminus, the phosphate-sugar backbone and the four nucleobases (A, T, C and G bases) as side chains. Two complementary strands of DNA hybridize to form a helix that is stabilized by hydrogen bonds and by p–p stacking of the bases.

pairing, also called Watson–Crick base pairing, we note that other non-canonical base pairing can also occur in DNA, such as the formation of G-quadruplexes and i-motif structures that have useful properties, and which are described elsewhere in this review. Although hybridization generates a helical DNA structure that is stable over a wide range of environmental conditions, the helix can be thermally ‘‘melted’’, wherein dsDNA is dissociated by heating above a critical melting temperature (Tm). This is a highly cooperative and reversible process, so that the two complementary single strands re-associate upon cooling below their Tm. DNA is also inherently directional, having a 5 0 -terminus (5th carbon position on the ribose, upstream) and a 3 0 -terminus (3rd carbon position on the ribose, downstream) that chemically encode the orientation of a DNA molecule. In most native DNA, a phosphate group is attached to the 50 -end (hence the 50 -phosphate) and a hydroxyl group is attached to the 3 0 -end (hence the 3 0 -OH). Therefore, in addition to complementarity, the formation of a dsDNA helix is also directional, wherein the 5 0 -end from one DNA strand aligns with the 3 0 -end from another strand.

Ashutosh Chilkoti

Ashutosh Chilkoti is the Theo Pilkington Chair in Biomedical Engineering at Duke University and is currently the Director of the Center for Biologically Inspired Materials and Materials Systems at Duke University. His areas of research include biomolecular engineering with a focus on stimulus responsive biopolymers for applications in protein purification and drug delivery, and biointerface science, with a focus on the development of clinical diagnostics and plasmonic biosensors.

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This directionality is often exploited as a means to control reactions at interfaces. Hybridization also strongly affects the physical properties of DNA. For example, while dsDNA is a rigid rod, characterized by a long persistence length (B50 nm), single stranded DNA (ssDNA) is a much more flexible, worm-like molecule, with a short persistence length (B1 nm).6 2.3.

DNA as a polyanion

The phosphate groups in DNA form the highly negatively charged DNA backbone, which make DNA a polyanion. This negatively charged backbone allows DNA to interact with positively charged surfaces, polymers, dye molecules, surfactants, and nanoparticles, and is a property that has been widely exploited in DNA-based nanotechnology. The use of DNA as a scaffold for the formation of metal nanowires by selective metal deposition, the formation of DNA layers on positively charged surfaces, and signal amplification in DNA sensing by binding of positively charged reporter molecules to DNA are just some examples that take advantage of the anionic properties of DNA. Furthermore, colloids (e.g., gold nanoparticles) can be electrosterically stabilized by adsorption of ssDNA. Finally, as a highly charged molecule, DNA can be manipulated by electric fields. For example, the electrophoretic mobility of DNA in an electric field has been used to improve the immobilization and hybridization efficiency of DNA on surfaces. By applying an electric field, the kinetics of immobilization and hybridization reactions can be enormously accelerated (by a factor of 109), compared to the kinetics in the absence of an electric field.7 This property has been used to control the movement of DNA through nanopores for single molecule DNA sequencing. 2.4.

Nucleic acid modifying enzymes

Another unique characteristic that sets DNA apart from conventional polymers is the ability to manipulate DNA at the molecular level with nucleic acid modifying enzymes.8 These modifying enzymes can digest, cut, ligate, modify and polymerize DNA, and

Table 1

Fig. 2 DNA strands forming secondary structure through intra-chain interaction of the bases. (a), Hoogsteen hydrogen bonds between the four G bases form the G-tetrads that stabilize the G-quadruplex, while in (b) a sequence containing stretches of C bases forms a secondary structure called i-motif when a protonated C forms a noncanonical base pair with an unprotonated C. A molecular beacon comprising a 15 base long loop structure and a 5 base long complementary sequence is shown in (c) and a DNA aptamer that forms a specific secondary structure upon binding to thrombin is shown in (d).

thus serve as a versatile molecular tool kit to manipulate DNA (Table 1). 2.5.

DNA secondary structures

Aside from inter chain base–pair interactions that result in the formation of the double helix, the bases in DNA can also form non-covalent bonds within a chain through p–p stacking, hydrogen bonds, hydrophobic interactions, and van der Waals interactions. These intra-chain interactions can result in the formation of DNA secondary structural motifs, such as the G-quadruplex and the i-motif (C-rich strand) (Fig. 2a and b). Such secondary structures of DNA also have functional utility, e.g., in sensing applications as molecular beacons and aptamers (Fig. 2c and d).

3. DNA synthesis and modification Advances in solid phase chemical synthesis of ssDNA—typically less than 150 bases in length—have enabled the facile synthesis of customized oligodeoxynucleotides (ODNs) and have spurred

Selected nucleic acid modifying enzymes

Function

Enzymes

Activity

Elongating and modifying DNA/RNA:

T4 DNA ligase T4 RNA ligase Taq polymerase Klenow fragment Phi29 DNA polymerase Terminal transferase Poly(A) polymerase

Join DNA, RNA strand Join and label RNA strand Polymerize DNA from template Polymerize DNA from template Polymerize DNA from template Polymerize DNA without template Polymerize ATP without template

Digesting or cutting DNA/RNA

Exonuclease I Exonuclease III DNAse I Restriction endonucleases

Cut ssDNA from 3 0 -end to 5 0 -end Cut dsDNA from 3 0 -end to 5 0 -end Degrades DNA Cut DNA at a specific sequence

Helicase Reverse transcriptase T7 RNA polymerase

Unwind dsDNA to ssDNA Polymerize DNA from RNA template Polymerize RNA from DNA template

Other functions: Unwinding dsDNA Convert RNA to DNA Convert DNA to RNA

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the use of DNA as materials for nanotechnology, therapeutic, and diagnostic applications. For further information on this mature technology, please refer to ref. 9. In this section, we discuss specific technologies and processes that enable applications of DNA on surfaces. 3.1.

Highly parallel, in situ DNA synthesis

For many surface-based applications of DNA, such as microarrays, high ODN multiplexing densities are required. Conventional solid phase chemical synthesis of ssDNA is ill suited for this application, as it is a serial process and requires immobilization of the ODN on a substrate after synthesis. To address these shortcomings, several new in situ approaches have been developed recently. These approaches combine the synthesis and arraying process directly on the glass substrate, and employ photolithography or inkjet printing technologies.10,11 In the photolithographic approach, phosphoramidite monomers with a photolabile protecting group are activated by light, and the coupling reaction only occurs on an area of the chip that is exposed to light. By using photomasks that allow for the selective illumination of different areas on the chip, spots with different ODN sequences can be synthesized in parallel (Fig. 3). An alternative approach uses digital micromirror devices (DMD) to direct light to specific locations on the chip.12 These technologies have been commercialised by Affymetrix (photolithography) and NimbleGen (DMD). Inkjet printing has also been used to deliver the required reagents to specific reaction spots for the parallel in situ synthesis of ODNs on a surface. This technology has been commercialized by Agilent to make microarray chips containing immobilized 60-mer ODN probes. 3.2.

DNA modifications

Rapid progress in the chemical DNA synthesis has enabled the design of DNA molecules with functionalities that differ significantly from those naturally found in DNA. For example, native

Fig. 3 Spatially addressable in situ DNA synthesis using photolithography. Ultraviolet light activates photoprotected hydroxyl groups that are coupled to photolabile nucleotides. By applying different masks in each synthetic cycle, selective areas on the substrate can be activated in parallel. Repeating the cycle of spatially controlled illumination followed by nucleotide coupling generates the desired ODNs with a specific sequence on the chip.

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Fig. 4 Modification of DNA at specific sites, the 50 -terminus, the 30 -terminus, and the 2 0 -position of ribose and an internal modification is highlighted in the scheme.

DNA is susceptible to degradation and hydrolysis. To address this limitation, modifications to the phosphate and sugar moieties of the backbone have been made to improve DNA stability. Furthermore, useful functionalities can be introduced that allow for label attachment, for surface anchoring, for incorporation of metal nucleation sites, and modifications that provide additional active sites on the bases for their chemically orthogonal functionalization with multiple moieties. This flexibility in the chemical synthesis, combined with the availability of nucleic acid modifying enzymes, provides a toolkit that can broadly expand the repertoire of functional nucleic acid-based materials. Site-specific modification. DNA provides multiple sites for modification (Fig. 4). These include (i) the 5 0 - and 3 0 -termini and the 2 0 - and 4 0 -positions of the ribose backbone, (ii) the phosphate groups, (iii) the modification of the four natural bases A, T, C, and G, or (iv) the replacement of the natural bases with unnatural bases that can provide unique reactive handles. In this section, we provide a brief overview of DNA modification strategies. For an extensive review of this topic, the reader is referred to ref. 13. The chain ends at the 5 0 - and 3 0 -termini of DNA are most frequently modified. This modification is important because it can be used: (i) to attach a fluorescent label or quencher, (ii) to add reactive moieties for covalent immobilization of DNA on a surface or for the conjugation of ODN with synthetic polymers, antibodies, proteins and peptides, and lipids, and (iii) to attach hapten molecules, such as biotin. The DNA backbone is another important modification site. For example, the phosphodiester backbone can be replaced by: (i) a phosphorothioate or morpholino backbone to increase in vivo stability (Fig. 5b and c), (ii) a peptide backbone (peptide nucleic acid (PNA)) (Fig. 5d), or (iii) a locked phosphodiester backbone (locked nucleic acid (LNA)) (Fig. 5e), to increase hybridization stability and affinity. Enzymatic modification. In addition to chemical conjugation, DNA can also be modified enzymatically, a feature that distinguishes them from synthetic macromolecules. DNA and RNA modifying enzymes such as ligases, polymerases, and reverse transcriptases can recognize the 5 0 - or 3 0 -termini and

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Fig. 5 Structures of (a) natural and (b–e) modified DNA backbone. Figure is reproduced with permission.13

can add natural or unnatural nucleotides to the chain end. For example, single or multiple fluorescent labels and functional bases such as aminoallyl, biotinylated, azido-derivatized nucleotides, alkynyl nucleotides, or aldehyde nucleotides can be enzymatically added to DNA and RNA.

4. DNA immobilization onto surfaces Immobilizing nucleic acids at interfaces and on surfaces offers several unique opportunities, including access to spatially addressable manipulation and reactions and parallel processing, and enables one to harness nucleic acid–substrate interactions for signal transduction. In microarray technology, for example, ODN or complementary DNA (cDNA) probes are arrayed in spots on a glass slide and used to capture their complementary targets from solution. This enables the highly parallel and quantitative capture and detection of complementary targets from complex mixtures. Grafting DNA onto surfaces also allows one to harness the directionality of DNA chains. For example, DNA polymerases will only recognize either the 5 0 or the 3 0 -terminus, which allows one to selectively modify the desired end of the hybridized target for detection or amplification. Finally, surfaces can also function as signal transduction elements. For example, hybridization of a target by its probe can be transduced by the substrate into a detectable signal, e.g., by fluorescence quenching on gold, or by changes in the conductivity of carbon nanotubes. 4.1.

Surfaces for DNA immobilization

DNA has been grafted onto a diverse range of material surfaces (Table 2), with silicon-based substrate surfaces being the most widely used.14,15 Glass slides, for example, are ubiquitous in microarray technology, where they function as a solid support for the immobilization of DNA with high spatial density. DNA has also been grafted onto optical glass fibers, where the fibers function not only as solid supports but also as signal transduction elements. Furthermore, silicon wafers are used as supports in miniaturized sensor systems, capitalizing on the semiconducting properties of silicon. Gold thin films provide another useful substrate for DNA immobilization.15 First, gold can be easily modified with a range

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of functional groups by the self-assembly of alkanethiols. Second, gold is a useful transducer for DNA hybridization reactions, as it can function as an electrode in electrochemical sensing, as a conducting surface in field effect transistor (FET) devices, or as an optical transducer in surface plasmon resonance (SPR) sensors. Synthetic polymers, including polystyrene (PS), polyacrylamide (PAAM), polymethylmethacrylate (PMMA), polyvinylalcohol (PVA), nylon, polypropylene (PP), and conducting polymers, such as polypyrrole (PPy), can be easily processed as films and readily functionalized, and all have been used as substrates for DNA immobilization. Carbon-based materials, such as carbon nanotubes (CNTs), graphene, and diamond, are important substrates for DNAbased devices, as they have unique opto-electronic properties.16–18 CNTs, for example, provide highly efficient electron transfer, and thus are ideal supports for DNA-based detection systems. Semiconducting single-walled carbon nanotubes (SWNTs) act as optical transducers because they have a nearinfrared band-gap fluorescence that is sensitive to a change in the local dielectric environment. Similarly, graphene, a oneatom thick sheet of ordered sp2-hybridized carbon atoms has been explored as a substrate for DNA immobilization. Compared with CNTs, graphene has some advantages, such as its ease of production and the absence of metallic impurities. Although to date there have been relatively few reports of graphene’s application in biosensors, the electrical conductivity, high surface-to-volume ratio, mechanical strength, and biocompatibility of graphene are all attractive features for biosensing applications. Diamond, an allotrope of carbon, provides a mechanically stable and chemically inert substrate that is particularly attractive for in vivo applications. Because of its semiconducting properties, diamond can function as an electronic or electrochemical transducer in nucleic acid-based biosensors.17,19 Magnetic core–shell particles have also been used as substrates for DNA immobilization in separation and sensing applications. DNA functionalized magnetic core–shell particles allow binding of DNA or other oligonucleotides from a complex mixture by hybridization, followed by magnetic separation and concentration, and release into a new, desired buffer or solvent. Typically, iron oxide in the form of magnetite (Fe2O3) or maghemite (g-Fe2O3), is used to form the magnetic particle core that is embedded in a non-magnetic matrix shell. 4.2.

DNA immobilization strategies

The need to attach DNA on a broad range of material surfaces demands an equally broad range of suitable immobilization strategies (see Table 2). The available methods can be classified broadly into two categories: (i) immobilization to chemically modified surfaces via ‘‘grafting to’’ by physi- or chemisorption and covalent reaction, or by non-covalent molecular recognition (also termed affinity coupling), and (ii) immobilization via ‘‘grafting from’’, as, for example, the in situ synthesis of ODN from solid surfaces (see Section 3.1). Physisorption and chemisorption. Physisorption of DNA can occur through electrostatic interactions, van der Waals

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Substrates and strategies for DNA surface attachment

Substrate

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Glass

Advantages

– Low auto fluorescence – Cost-effective – Resistant to a wide range of conditions – Flexibile for processing and chemical modification

Immobilization Immobilization strategy chemistry

DNA conformation on surfaces

Electrostatic interaction (e.g. via polylysine coating) Non-covalent Hydrophobic interaction Epoxy modified surface Covalent (Organosilane chemistry)

Sulfhydryl modified surface Aminopropyl-modified glass

Gold

– Inert – Generation of surface plasmons – Conducting surface – Fluorescence enhancement/ quenching capability – Availability for chemical modification

Chemisorption

Sulfur–Au interaction (SH–Au)

Entrapment via noncovalent interaction

Polymers

Carbon nanotubes (CNTs), graphene

Diamond

– Cost-effective – Flexibility for processing and chemical modification – Can be used as a coating on other substrates

– – – –

High mechanical strength Excellent chemical and thermal stability Excellent optical and electrical properties Availability for chemical modifications

– High mechanical strength – Excellent chemical and thermal stability – Electronic properties can be altered with dopants

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Non-covalent Adsorption via electrostatic interaction

Covalent

Various chemistries

Non-covalent

p-Stacking effect

Covalent

Covalent linker

Covalent

Covalent linker

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Table 2

Review Article

(continued)

Substrate

Advantages

Magnetic particles

– Capable of being manipulated by magnetic field – Functionalization of the non-magnetic matrix shell for DNA attachment – Stable and specific magnetic signal can be differentiated from other noise in the biological system

Immobilization Immobilization strategy chemistry

Covalent

Covalent linker

Affinity coupling

Avidin (Streptavidin)– biotin interaction

interactions, hydrogen bonds, or hydrophobic interactions. For example, in electrostatic physisorption of DNA, the negatively charged phosphate backbone is adsorbed onto a positively charged surface, such as an aminosilane or poly-L-lysine coating.14,20 Although this immobilization approach is simple and does not require DNA modification, it is limited to charged surfaces and low ionic strength environments, and often requires additional stabilization of the surface-bound DNA, e.g., through crosslinking reactions. Furthermore, physisorption typically provides little control over DNA orientation on the surface. In chemisorption, stronger and directional bonds are formed with the surface, compared to physisorption. For example, the interaction between thiols (–SH) and the Au, leads to the formation of a bond with a bond energy of B1/4 that of a covalent bond, which provides a stronger and more stable surface anchoring than can be achieved by most physisorption processes.15,21 Another example of DNA chemisorption is the p–p stacking interaction of DNA with graphene and carbon nanotubes that occurs between the bases in ssDNA and graphene or the side-walls of CNTs, resulting, for the latter, in a helical wrapping of ssDNA around the nanotube core.18 Covalent attachment. Covalent attachment provides the most stable method for coupling DNA to surfaces. This approach requires modification of DNA—typically at its 5 0 - or 3 0 -end—and the surface, such that each partner presents a reactive pair of functional groups. The coupling between the modified nucleic acid and the activated surface is typically carried out directly or through a linker moiety. The advantage of using a linker is that in addition to presenting appropriate reactive groups, it also provides a physical spacer that can minimize the steric hindrance experienced by the surface-tethered nucleic acid. An increase in the degrees of conformational freedom can promote subsequent reactions, such as hybridization between a surfacegrafted probe and a target molecule. The most common functional groups used for direct coupling of DNA to surfaces include hydroxyl, amine, carboxyl, aldehyde, and epoxy groups, and details of the relevant reaction chemistries have been reviewed elsewhere.22 For indirect coupling,

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DNA conformation on surfaces

bifunctional linkers that can react selectively with the modified DNA and the activated surface are often used (Table 2). More recently, copper-catalyzed azide–alkyne cycloaddition reactions (‘‘click chemistry’’) have been used for the covalent attachment of DNA onto azide- or alkyne-terminated surfaces. The power of click chemistry for DNA attachment (and functionalization) lies in its relative simplicity and high efficiency which enables the immobilization of DNA onto surfaces with high yield.23 Affinity coupling. Affinity coupling—using molecular recognition pairs—is another popular strategy to attach DNA to surfaces. The prototypical approach for affinity coupling is to use the avidin/streptavidin–biotin protein–ligand pair. The (strept)avidin– biotin recognition is one of the strongest non-covalent, biological interactions, with a dissociation constant (Kd) of B1015 M for avidin and B1013 M for streptavidin. In addition to high affinity, the tetrameric biotin binding sites in avidin/streptavidin allow for a ‘‘sandwich-like’’ affinity coupling approach, that links biotin modified DNA and a biotin-modified surface via a (strept)avidin bridge. 4.3. Factors that affect the performance of surfaceimmobilized DNA in hybridization reactions Compared to solution-based reactions, where molecules have many degrees of freedom in terms of their movement and conformation, surface reactions tend to be kinetically slower. Thus, for surface-tethered DNA to reach their full potential in, for example, biosensing applications there are several key factors that need to be considered. DNA conformation on surfaces. The substrate, the immobilization method and the grafting density all affect nucleic acid conformation and flexibility on surfaces (see Table 2). For example, DNA attached to surfaces by electrostatic or hydrophobic interactions tends to adopt a flat conformation in which the phosphate backbone or the hydrophobic bases interact strongly with the substrate surface. On the other hand, covalent attachment by end-grafting, or affinity coupling allow for greater control over the orientation of the nucleic acid chains on the surface, where at sufficiently high grafting densities, the chains can adopt an extended conformation.

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DNA grafting density determines nearest–neighbour interactions, affects nucleic acid–surface interactions, and affects the charge density of the ionizable phosphate groups in the backbone,24 and thus plays an important role in determining the efficiency of subsequent reactions at the substrate–solvent interface. For example, the efficiency by which end-grafted ssDNA probes hybridize with their target strands can be compromised when the grafting density of the probe strands is too high. It was shown that the hybridization efficiency dropped from 95% to 15%, and the hybridization kinetics decreased, when the grafting density of ssDNA probes on a gold surface was increased from 2  1012 to 12  1012 molecules per cm2.25 Spacer molecules for DNA attachment. To reduce steric crowding and its effect on reaction kinetics, spacer molecules or linkers can be introduced between the nucleic acid chains and the solid support. Common spacers include alkyl chains and homo-oligonucleotides.22 Studies of DNA hybridization and aptamer–protein binding show that there is an optimum spacer length that provides the highest hybridization yield or the amount of protein bound. In one example, an increase in the spacer length from zero to eight glycol units led to a 150-fold increase in hybridization efficiency.26 Non-specific interaction on the surface. There are typically two sources of non-specific interactions in nucleic acid hybridization reactions on a surface: (1) cross-hybridization of the probe with non-target DNA that has some sequence complementarity with the immobilized probe and (2) non-specific adsorption of the target directly to the surface. The extent of these side reactions can be minimized by appropriate sequence design for the probe strands, by optimizing the hybridization temperature, salt concentration (e.g. NaCl) and by using additional surface treatments or passivation steps. For example, amine-derivatized surfaces, often used for the adsorption of DNA probe strands, can also attract DNA to the surface through non-specific electrostatic interactions. In this case, surface passivation to deactivate unreacted amines or a blocking step using succinic anhydride or BSA is frequently used to improve hybridization specificity. In other cases, to ensure that the solid support has anti-fouling properties, self-assembled monolayers (SAMs) of thiol-terminated oligoethylene glycol on gold are used to reduce non-specific binding to the surface.27 Surface topography. In addition to surface chemistry, surface topography also affects hybridization efficiency. It was recently shown that DNA hybridization efficiency is greater on nanostructured, rough surfaces compared to a smooth surface.28 This observation can be explained by the increase in effective surface area, which increases the number of target capture sites, and the enhanced accessibility of the DNA probes during hybridization.

5. Exploiting DNA properties at interfaces In the following section we highlight applications that exploit the unique properties of DNA on solid supports. The aim of this section is not to give a comprehensive review, but rather to

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highlight recent developments in the fields of biosensing, sequencing, and biomolecular nanopatterning that share in common the use of surface-grafted DNA as an essential component of their design. We group the examples based on the unique properties of DNA as a ‘‘smart’’ material (see also Section 2): (1) its ability for molecular recognition, (2) its property as a polyanion, (3) its ability to interact with DNA modifying enzymes, and (4) its secondary structure. 5.1.

Molecular recognition

The ability of a DNA strand to hybridize with its complementary strand is the primary mechanism used for DNA analysis (Fig. 6a).29 Here we highlight uses of DNA hybridization that go beyond this canonical configuration of probe and labelled target binding (Fig. 6a). All the selected examples generate signal amplification by eliminating target amplification and labelling steps through creative use of sequence complementarity. A branched DNA (b-DNA) assay (Fig. 6b) was developed to overcome the limitations of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. It has been shown that PCRbased target amplification has variable efficiency depending on the sequence of the target, which potentially introduces errors and systematic bias in the original quantity of the target in the samples. To circumvent the need for PCR, the b-DNA assay uses three innovative design elements (Fig. 6b): (1) the cooperative hybridization of a set of probes (capture probe and capture extender) for a particular target to allow for the efficient and stable capture of messenger RNA (mRNA) target directly from cell lysate and tissue homogenate; (2) a sandwich approach for

Fig. 6 Harnessing DNA hybridization at the interface allows analysis of DNA using creative design strategies. (a) Conventional probe-labelled target hybridization, (b) branched DNA assay utilising cooperative hybridization of a set of probes to one target strand and branched DNA signal amplifiers, (c) supersandwich assay harnessing chain hybridization between the target strand and the signal probe for signal amplification and (d) conformational change upon target binding transduces the binding event into a measurable signal. Fig. 9b–d are reproduced with permission.30,34,35,37

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signal detection via a label extender and a preamplifier to allow direct detection of unlabelled nucleic acid targets; and (3) the binding of multiple b-DNA amplifiers to alkaline-phosphatase labelled ODNs to amplify the hybridization signal.30,31 Although complex ODN sequence design of probes and amplifiers is required for a multiplexed assay, the b-DNA assay has been successfully implemented in the clinic to monitor patients on antiviral therapy for human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV), and to stratify patients for therapy.32 Another, simpler sandwich approach is shown in the inset of Fig. 6c. Here, the target strand is sandwiched between the capture probe and the signal probe, which is labelled with either methylene blue—a redox label for electrochemical biosensors—or with gold nanoparticles for optical detection. To improve the sensitivity of the traditional sandwich configuration, the assay was modified so that a signal probe hybridizes to two regions of a target nucleic acid in such a way that the probe hybridizes more readily with complementary regions on two separate targets rather than on two complementary regions of a single target. This assay format thus triggers a chain hybridization reaction between signal probes and multiple targets on a capture probe, which creates long concatamers (up to 1000 base pairs long as compared to 75 base pairs achieved with the traditional sandwich assay) that contain multiple labels for signal amplification.33 With this ‘‘supersandwich’’ structure, DNA concentrations as low as 100 fM can be detected. Another interesting use of surface-tethered DNA is in reagent-less assays for nucleic acid detection. These approaches harness the conformational change that occurs upon hybridization of the surface-tethered probe strand with its target. Hybridization is detected electrochemically, using the hybridization-triggered change in the proximity of a redox label to the surface electrode. There are several strategies to induce a conformational change by the hybridization of the target molecule to the tethered probe, including target-induced strand displacement (Fig. 6d(i)),34 target-induced loop formation (Fig. 6d(ii)),35 and triplex-forming ODN.36 This assay format is simple, and does not require additional reagents; however, the limit-of-detection (LOD) can be higher (B400 fM to 200 pM) than that of assays that use additional reagents for signal amplification. We note, however, that comparison of LODs across studies is complicated by the definition of LOD used in different studies, and by the unfortunate omission of controls and replicates in some of the studies.

5.2.

DNA as a polyanion

Many applications in nucleic acid-based nanotechnology exploit interactions of the negatively-charged nucleic acid backbone with positively charged moieties. Examples include the use of DNA as a template for nanowire synthesis by selective metal deposition, the electrostatic stabilization of DNA-coated colloidal nanoparticles, the formation of DNA–polymer complexes for gene delivery, and signal amplification in DNA biosensors that

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Fig. 7 Harnessing the polyelectrolyte behaviour of DNA at interfaces allows the analysis of unmodified nucleic acid targets with signal amplification. (a) A multiplexed DNA detection strategy where a DNA capture probe electrostatically binds to a cationic conjugated polythiophene derivative and the formation of dsDNA–polymer triplexes by hybridization generates a fluorescent signal due to a conformational change in polythiophene; (b) an aptamer–polythiophene complex generates a fluorescent signal as the target protein binds to the complex due to conformational change of the aptamer, which allows efficient energy transfer between polythiophene and the conjugated fluorophore; (c) a positively charged cationic conjugated polymer (CCP) binds to a duplex target ssDNA that hybridizes to the immobilized PNA-C* probe, resulting in FRET between CCP and C* reporter; and (d) colorimetric detection of a DNA target that hybridizes to a neutral PNA capture probes, and positively charged gold nanoparticles bind to the duplex. Figures are adapted and reproduced with permission.38–41

arises from the binding or repulsion of positively or negatively charged reporter molecules. In this section, we highlight innovative uses of the polyanion properties of DNA for signal amplification in sensing applications on surfaces. In one example, cationic conjugated polymers (CCPs), such as polythiophene derivatives, have been used as fluorescent reporter molecules for rapid and sensitive DNA detection (Fig. 7a). The cationic reporter molecules bind to anionic ssDNA probe strands, and upon hybridization with their complementary target, the DNA, and thus the conjugated polythiophene reporter molecules undergo a large conformational change, which enhances their fluorescence. This approach has exquisite sensitivity and is capable of detecting even a single base–pair mismatch.38 An analogous approach, in which polythiophene forms a complex with DNA aptamers, has been used to detect protein targets (Fig. 7b).39 Other applications employ peptide nucleic acid (PNA) capture probes and cationic reporters which include CCPs,41 enzymes,42 and gold nanoparticles.40 PNA is used as the capture probe because of its neutral peptide backbone. Unlike ODNs, hybridization of a ssPNA probe with a DNA target generates localized negative charges that correspond to the amount of DNA target bound (Fig. 7c and d). This net negative charge can be detected using CCPs (Fig. 7c), or positively charged gold nanoparticles (Fig. 7d), to produce a fluorescence or colorimetric signal, respectively. In colorimetric detection, positively charged gold nanoparticles selectively attach to the hybridized, negatively charged DNA, and allow visualization of the captured target DNA, and after treatment with a metal enhancement solution, even with the naked eye.40

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These examples demonstrate that the creative combination of the molecular recognition capability and the negatively charged backbone of DNA can be exploited to produce a range of surface-tethered, biomolecular sensors, whose signals can be amplified by various schemes. 5.3.

DNA synthesis and modification with enzymes

Various nucleic acid-modifying enzymes (Table 1) can be used to elongate, cut, digest, or modify nucleic acids with molecular precision that cannot be achieved by chemical methods. Although these enzymes have been widely used in molecular biology, they have only recently been adopted as tools to synthesize and modify nucleic acids on surfaces. Spatially localized, enzyme catalysed nucleic acid reactions have been used for nanopatterning DNA on surfaces,21,43 for signal amplification,44–46 for on-chip protein expression,47–49 and for single molecule DNA sequencing.50–52 In a demonstration of top–down, subtractive nanolithography of DNA, an ODN SAM was self-assembled on gold, and the spatially addressable deposition of DNAse I by an AFM tip, locally confined the enzymatic digestion of the DNA, leading to subtractive lithography of the surface-immobilized ODN with nanometer lateral resolution (Fig. 8).21 In a bottom–up demonstration of additive nanolithography, a thiol-terminated ODN was chemisorbed to gold islands—patterned on a silicon substrate—and a template independent polymerase, terminal deoxynucleotidyl transferase (TdT), catalysed the in situ polymerization of the ODN primer to yield ssDNA polymer brushes that led to amplification of the initial nanopatterns in the Z-direction.43 Nucleic acid modifying enzymes can also be used for signal amplification in the detection of nucleic acids or protein targets on surfaces. A range of enzymes, including TdT,44,45 Klenow fragment,46 Taq polymerase,53 Phi29 DNA polymerase,54–58 Poly(A) polymerase (PaP), and T4 RNA ligase are used to modify DNA through ligation or polymerization of nucleotides that contain reporter molecules, such as fluorophores, reactive linkers or haptens. For example, in an immuno-PCR assay53

Fig. 8 Nanopatterning of DNA by enzymatic lithography. (a) Top–down subtractive DNA lithography created by delivering DNAse I locally, using an AFM tip. Upon activation by addition of co-factor Mg2+, DNAse I selectively digests DNA. (b) Bottom–up, additive pattern lithography by selective immobilization of ODN initiators on gold substrates, and subsequent DNA polymerization by TdT. Figures are adapted and reproduced with permission.21,43

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(Fig. 9a), detection of a protein analyte is accomplished through the use of a detection antibody–DNA conjugate. The protein analyte interacts specifically with the antibody, and the DNA conjugated to the antibody acts as a signal amplifier of protein– antibody interaction by PCR amplification of the DNA tag. A similar strategy is used in the immuno-RCA (rolling circle amplification) assay (Fig. 9b),54 where binding of the detection antibody–DNA conjugate to the protein analyte is amplified by RCA. The RCA reaction grows a concatenated DNA strand from the detection antibody, which is detected by hybridization of multiple fluorescently labelled ODNs to the concatemer. Signal amplification by reporting the binding of a nucleic acid target to a tethered probe on the surface can also be achieved by a template-dependent polymerase reaction. For example, in the RNA-primed, array-based Klenow enzyme assay, binding of microRNA (miRNA) to a tethered DNA probe is followed by digestion of unhybridized probes, and Klenow polymerase catalysed extension of the miRNA target with a biotin-labelled nucleotide. To make the detection independent of the target sequence, which is necessary for this method to be broadly useful, all probes are designed to begin with an oligo A sequence at their 5 0 -end that is tethered to the surface, thus allowing labelling of all miRNA targets—irrespective of their sequence—at their 3 0 -terminus with a complementary biotin-labelled dATP (Fig. 9c).46 Recently, a simpler templateindependent approach—surface-initiated, enzymatic polymerization (SIEP) of DNA—was reported that can detect DNA, mRNA and microRNA from complex mixtures (Fig. 9d). This template independent reaction extends the hybridized DNA and RNA target strand from its 3 0 -OH terminus, while incorporating multiple fluorescent nucleotides, and amplifies the hybridization

Fig. 9 Signal amplification methods harnessing the surface reaction and specificity of nucleic acid modifying enzymes for sensitive protein and nucleic acids detection. Protein target signal amplification using (a) immuno-PCR and (b) immuno-RCA with the antibody–DNA conjugate as a signal amplifier, while hybridization of the nucleic acid target is amplified through DNA extension from the 3 0 -terminus of the bound target strands by a template-dependent Klenow fragment catalysed labelling of the hybridized target (c), or a template-independent TdT catalysed polymerization of DNA from the 3 0 -end of the hybridized target (d). Figures are adapted and reproduced with permission.44,46,53,54

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Fig. 10 Generation of a protein microarray through immobilization of encoding cDNA sequences can be achieved by immobilizing (a) plasmid DNA or (b) PCR-amplified DNA fragments, followed by exposure to a cellfree expression reagent. The protein of interest contains an immobilization tag which allows for on-chip synthesis and direct capture on the solid support, thus enabling the assembly of high density protein arrays. Figures are adapted and reproduced with permission.47,48

signal directly on-chip.44,45 Both of these strategies allow for labelling of the unmodified nucleic acid target directly on-chip, without the need for sample pre-processing. Furthermore, the labels are incorporated only upon hybridization of the target, thereby minimizing a non-specific hybridization signal. An innovative use of the DNA polymerases in conjunction with surface-tethered DNA molecules is in the on-chip expression of proteins for protein array applications (Fig. 10). In this strategy, DNA molecules (in the form of DNA fragments47 or plasmids48) that encode proteins of interest, are tethered to a surface. The spatial control over DNA deposition on the surface results in local mRNA production through the action of the T7 RNA polymerase. In the presence of a cell-free translation mixture, the proteins, encoded by the mRNA, are then locally expressed on the surface. Perhaps the most important commercial application of surface-specific DNA reactions is in the field of massively parallel DNA sequencing technology. These technologies rely on immobilized DNA molecules or DNA polymerases on a surface and use template-dependent DNA extension to generate a spatially specific signal to read out the DNA sequence. This format allows for millions, or even billions, of sequencing reactions to occur simultaneously and be recorded independently. Here, we highlight two of the latest, third generation, single molecule sequencing technologies, one developed by Helicos BioSciences and the other by Pacific Biosciences (Fig. 11). In the Helicos Biosciences platform, the DNA or RNA to be sequenced is first fragmented. The fragments (B200–250 bp) are then modified with a common polyA adaptor, which allows for hybridization with spatially distributed and covalently immobilized polyT primers on a solid support. Next, the surface bound DNA or RNA template is reacted with Taq polymerase or E. coli DNA polymerase I to incorporate fluorescent nucleotides (Virtual Terminator (VT) nucleotides), that contain

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Fig. 11 Single molecule sequencing by technologies developed by (a) Helicos Biosciences and (b, c) Pacific Biosciences. In (a), each cycle of nucleotide addition produces an on or off signal from a specific spot that is recorded with respect to the input nucleotide, and in (b), each well produces a fluorescent signal when the corresponding nucleotide is incorporated by the enzyme, as shown in (c). Figures are adapted and reproduced with permission.59

a fluorescent dye and chemically cleavable inhibiting groups. The fluorescent dye produces the signal once the nucleotide is being polymerised while the inhibiting groups can be cleaved so that the addition of the next nucleotide according to the template sequence can be activated. The use of the proprietary VT nucleotides allows for step-wise synthesis and hence sequencing (Fig. 11a).52,59 In the Pacific Biosciences platform, spatially distributed single molecules of Phi29 DNA polymerase are immobilized on a surface of a zero-mode waveguide (ZMW) detector (Fig. 11b). The DNA polymerase can then capture the primed DNA or RNA to be sequenced.50 Using the DNA or RNA sequence as a template, the enzyme synthesizes a new strand and incorporates fluorescently labelled, phosphor-linked nucleotides. The proximity of these nucleotides to the waveguide detector as they are incorporated generates fluorescent pulses that can be detected (Fig. 11c). The serial generation of these fluorescent pulses corresponds to the sequence of the bases that have been incorporated, and allows for real-time sequencing of the DNA or RNA template. 5.4. DNA secondary structure formation: a switch and a catalyst The intramolecular interactions of bases within ssDNA can lead to the formation of secondary structures, such as hairpin loops, G-quadruplexes and i-motifs (Fig. 2). This section highlights the applications of four major types of surface-tethered DNA molecules with specific secondary structures, as molecular beacons, aptamers, catalytic nucleic acids (DNAzyme), and biomolecular switches.

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A molecular beacon (MB) is a ssDNA molecule that contains a stem-and-loop structure (Fig. 2c).60 The loop region is designed to contain a probe sequence that is complementary to the target DNA sequence, while the bases flanking the probe sequence on either side, are designed to form the stem-duplex through hybridization. The ends of a MB are typically labelled to enable detection of the binding of a target sequence to the ¨rster resonance energy transfer probe. For example, for Fo (FRET)-based detection, one end of the stem strand contains a fluorophore, while the other end carries a quencher moiety. When the hairpin loop structure is formed, the two ends are in close proximity, leading to quenching of fluorescence. Upon binding of the target, the stem dissociates, and the loop opens up to permit a more stable association with the target. This moves the quencher sufficiently far away from the fluorophore, which leads to a fluorescence signal (Fig. 12a). This approach of immobilization of the MB on a surface is attractive as it allows reagentless target detection with a subnanomolar limit of detection (LOD).61 Similar approaches, utilising immobilized hairpin-loop structures on surfaces, are illustrated in Fig. 12b and c. In these examples, the surface not only functions as a support, but also plays an important role in signal transduction. In one implementation of this approach, analogous to the MB approach, a gold surface is used as a quencher (Fig. 12b). In another approach (Fig. 12c), a hairpin loop is end-labelled with ferrocene and tethered to a gold electrode, and the change in electron transfer efficiency upon target hybridization is detected by cyclic voltammetry. A LOD of 10 pM has been reported for this approach. An aptamer is a short single stranded DNA or RNA ODN that binds to a target molecule with high affinity and specificity. Aptamers can be generated to bind a diverse range of small organic or inorganic molecules and macromolecules such as proteins by an in vitro selection and enrichment process called systematic evolution of ligands by exponential enrichment (SELEX).70 A powerful feature of an aptamer is that it changes its 3-D conformation upon binding with its target molecule which allows an aptamer to function as a receptor and a transducer of molecular binding. Surface-tethered aptamers have been used as receptors in various label-free sensors, including quartz crystal microbalance (Fig. 12d), surface plasmon resonance, ellipsometry, fibre optic based sensors, and field effect transistor based sensors. Interesting strategies that exploit the 3-D conformational change of aptamers upon target binding are illustrated in Fig. 12e and f. For example, for the electrochemical detection of thrombin,64 an electrode-bound thrombin aptamer (capture probe) is hybridized with a probe DNA strand, that is tagged with methylene blue. This labelled probe hybridizes to the thrombin-binding portion and the linker sequence of the immobilized capture probe (Fig. 12e), forming a rigid duplex that prevents the redox tag from approaching the electrode surface. In the presence of thrombin, the thrombin-binding portion of the capture aptamer probe forms a stable G-quadruplex conformation, releasing the end of the signal probe that is tagged with methylene blue. This consequently allows the redox tag to

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Fig. 12 Harnessing DNA secondary structures (molecular beacons, aptamers, and DNAzymes) at an interface allows for the analysis of nucleic acid targets, and the detection of small molecules (e.g., cocaine), ions (Pb2+) and proteins (e.g., thrombin, IgE), as illustrated in (a) to (h). In (i) and (j), the sensitivity of the i-motif to changes in pH is exploited. A pH change translates into a conformational change that controls (i) the wettability of a surface, or (ii) the in-plane surface forces which cause a bending response of a microcantilever. Figures are adapted and reproduced with permission.61–69

come into close proximity of the electrode surface, producing a readily detectable, Faradaic current. Another example is the detection of cocaine (Fig. 12f), where DNA aptamers, again tagged at one end with methylene blue, are tethered to gold electrode surfaces via thiol linkers. In the absence of cocaine, the immobilized aptamer is partially unfolded, but once cocaine is presented to the surface, the aptamer folds into a three-way junction that binds cocaine, altering electron transfer of the redox label to the electrode, which can be readily detected electrochemically.65 Some DNA molecules can fold into complex secondary structures that involve formation of a G-quadruplex, which

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imparts catalytic properties to them. The interactions of these nucleic acid enzymes, called DNAzymes and ribozymes (Fig. 2a), with surface tethered DNA can be exploited for different applications. As shown in Fig. 12g, a sandwich assay for the detection of a DNA target employs a G-rich DNAzyme that functions as the detection probe and binds to hemin. This yields a biocatalytic complex with peroxidase activity. In the presence of peroxide (H2O2) and luminol, the biocatalytic properties of the DNAzyme–hemin complex produce chemiluminescence, which can be used to quantify the amount of DNA bound to the immobilized capture probe. A similar strategy has also been used for the analysis of telomerase activity in cancer cells.66 An important mechanism that can be used to regulate the catalytic activity of DNAzymes is the use of cofactors. Through in vitro selection, various cofactor-dependent DNAzymes have been generated. One example of such a cofactor is Pb2+. As Pb2+ is an important, toxic pollutant, its property as a cofactor can be harnessed for lead detection, using Pb2+-induced DNAzyme activity (Fig. 12h).67 An innovative, single step electrochemical approach for detecting Pb2+ is shown in Fig. 12h. In this approach, the DNAzyme is turned on in the presence of Pb2+, which then cleaves a base–pair of a rigid, surface-tethered DNA complex. Strand dissociation is electrochemically detected through the relaxation of surface-tethered methylene blue, resulting in nanomolar LOD and excellent selectivity for Pb2+. Another useful DNA secondary structure is the C-rich i-motif, which is sensitive to a change in pH. Protonation of C-bases and their noncanonical base pairing with unprotonated C-bases, leads to the formation of an interdigitated, quadruple helix that is stable at pH 4.5–5.0 (Fig. 2b). This pH sensitivity has been harnessed to tune surface wettability. In this application, a C-rich ODN, with a hydrophobic Bodipy fluorophore attached to one end of the ODN strand, was tethered to a gold surface through thiol–Au bonds (Fig. 12i).68 At low pH (state I), the tethered ODN forms the i-motif, which exposes the hydrophilic phosphate backbones on the surface, making the surface hydrophilic. When the pH is raised (state II), the i-motif is destabilized and the tethered chains adopt an extended conformation, which exposes the hydrophobic Bodipy moieties on the surface, switching the surface from hydrophilic to hydrophobic. As the chain conformation in state II is dynamic, addition of DNA strands that are complementary to the tethered strands leads to a rigid duplex conformation (state III) that stabilizes the hydrophobicity of the surface. Tethering ssDNA to a surface that contains the i-motif sequence, has also been used to actuate micro cantilevers.69 In this application, the C-rich strands (X) are tethered to a gold-coated microcantilever (Fig. 12j). Under acidic conditions, the steric interactions between the strands, which now carry the self-folded i-motif, lead to cantilever bending. When a complementary, duplex-forming strand (Y) is added under basic conditions, the duplex (X–Y) forms, and its more ordered, extended conformation reduces cantilever bending. This conformational change, and hence the bending of the cantilever, is reversible, and the extent of cantilever bending can be tuned by control of pH and ionic strength.

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6. Conclusions and future outlook This review summarizes the unique properties of DNA as a molecular building block and its innovative applications at surfaces and interfaces. We highlight DNA’s capacity for molecular recognition, its properties as a polyanion, the ability to manipulate DNA with molecular precision using the tools of molecular biology, and the ability of DNA to fold into useful secondary structural motifs, all properties that are ultimately useful for a broad range of interfacial applications. Current developments in the chemical synthesis of oligo- and polynucleotides and various enzymatic methods of DNA modification are presented to give readers a broad perspective on the progress in the field of nucleic acid chemistry. Because this review is focused on the application of DNA at surfaces, we introduce important specific reaction chemistries by which DNA molecules have been tethered to a broad range of surfaces and substrates where they can form ‘‘smart’’, functional layers. We then highlight interesting and recent examples that harness the ‘‘smart’’ properties of DNA for surface-specific applications. We believe that the recent rapid development in the chemical synthesis and modification of DNA will further enable the rational design of DNA as molecular building blocks that transcend their purely biological function. The ability to attach and manipulate engineered DNA on surfaces will continue to evolve and yield new breakthrough applications ranging from diagnostics to bioelectronics.

Acknowledgements This research was supported by NSF (grant CBET-1033621) to A.C. and S.Z. and by the NSF’s Research Triangle MRSEC (DMR-1121107).

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