Strategies for optimizing DNA hybridization on surfaces

Analytical Biochemistry 444 (2014) 41–46 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Review Strategies for optimizing DNA hybridization on surfaces Hadi Ravan a,⇑, Soheila Kashanian b, Nima Sanadgol c, Arastoo Badoei-Dalfard a, Zahra Karami a a Department of Biology, Faculty of Science, Shahid Bahonar University, 76169-14111 Kerman, Iran b Faculty of Chemistry, Sensor and Biosensor Research Center, and Nanoscience and Nanotechnology Research Center, Razi University, Kermanshah, Iran c Department of Biology, Faculty of Science, Zabol University, Zabol, Iran a r t i c l e i n f o a b s t r a c t Article history: Specific and predictable hybridization of the polynucleotide sequences to their complementary counter- Received 16 August 2013 parts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic Received in revised form 27 September 2013 acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA tar- Accepted 30 September 2013 gets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the tar- Available online 9 October 2013 get nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybrid- Keywords: ization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency Nucleic acid hybridization Solid-phase hybridization of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between Solution-phase hybridization DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimiz- ing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorpo- ration of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces. Ó 2013 Elsevier Inc. All rights reserved. The key property of nucleic acids that represents them as a amplification technologies, and a variety of live-cell nucleic acid useful tool in biotechnology and nanotechnology is their ability imaging methodologies [9]. These two types of nucleic acid to hybridize specifically to complementary sequences. This unique hybridizations are distinct from the viewpoint of thermodynamic property of nucleic acids plays important roles in biology, genotyp- and kinetic behaviors [10–12]. Since hybridization in the solid ing diagnostics, and a variety of molecular biology techniques [1– phase occurs at interfacial environments, it is predictable that 3]. DNA hybridization usually occurs between an oligonucleotide the application of an experimental protocol for solution-phase with a known sequence (probe) and an unknown complementary hybridization might not be successful for solid-phase hybridization strand of nucleic acid from solution (target). The hybridization of [4,13]. Therefore, understanding the nature of the differences the probe to the target sequence can be detected using appropriate between the two types of hybridization schemes is crucial for the hybridization indicators. design and development of new nucleic acid hybridization-based Generally, nucleic acid hybridization can be performed under detection systems. two major strategies, that is, hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridiza- tion) and hybridization of the target nucleic acids to randomly Solution-phase versus solid-phase hybridization distributed probes in solution (solution-phase hybridization) [4]. The solid-phase hybridization underpins several modern nucleic Suppose probe strand P, with a hypothetical nucleotide acid analysis techniques, including microarray, micro- and nano- sequence, is hybridized to its target strand T to form duplex PT particle surface-mounted DNA technology, and biosensors [5–7]. under the reaction P + T M PT (Fig. 1). The stability of duplex PT In contrast, the solution-phase strategy is a hybridization scheme can be quantified by defining the change in free energy of the for most common types of nucleic acid detection techniques, hybridization reaction, DGhyb. The DGhyb of the solution-phase including fluorescence in situ hybridization [8], nucleic acid hybridization can be sufficiently deduced from the nucleotide se- quence of the duplex based on the nearest-neighbor model [14]. According to this model, the covalent structure of duplex PT is con- ⇑ Corresponding author. Fax: +98 341 3222032. verted to a collection of unique 4-base quartets, each of which E-mail address: [email protected] (H. Ravan). comprises one base pair and its neighbor base pair (Fig. 1) [15]. 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.09.032 42 Optimizing DNA hybridization on surfaces / H. Ravan et al. / Anal. Biochem. 444 (2014) 41–46 Fig.1. Probe strand P, with a hypothetical nucleotide sequence, is hybridized to its target strand T to form duplex PT. The stability of duplex PT can be quantified based on the nearest-neighbor model. According to this model, the covalent structure of duplex PT is converted to a collection of unique 4-base quartets that each comprise one base pair and its neighbor base pair. The free energy of formation for all possible quartets was experimentally determined by SantaLucia and Hicks [14]. Hydrogen bonds between each pair and base-stacking interactions parameters of these two strategies show that the hybridization at between its adjacent neighbors determine the contribution of an interface is less favorable than hybridization of the same se- energy between each quartet. In addition, one extra assumption, quence in solution [1,4,18]. Several factors have accounted for namely, initiation parameter, which encompasses the thermody- the physicochemical constraints of solid-phase hybridization in namic differences between terminal and internal nearest which interactions between tethered oligonucleotide probes, solid neighbors, is considered for predicting the free energy of entire du- surface, and neighboring DNA molecules play important roles dur- plex [14]. In this model, thermodynamically favorable interactions ing DNA hybridization at interfaces [19]. Based on the nearest- between correctly paired bases in nucleic acids at a certain neighbor model, the interactions between the quartets at a given experimental condition (specific concentration of P and T and experimental condition determine the DGhyb in solution, while in buffer conditions) provide a discriminatory means for separating the solid-phase hybridization, in addition to the aforementioned target from nontarget sequences even with one nucleotide item, penetration of target DNA into a layer of oligonucleotide mismatch. Further study of this model has provided a clear trend strands affects the overall free energy of the hybridization reaction for decreasing the stability of the mismatch base-pair formation, [4]. It has been shown that the surface probe density [20], density for example, DGhyb of mismatches G.G < G.T 6 G.A < T.T 6 A.A of charge on the surface [21,22], confirmation of probe strands < T.C 6 A.C 6 C.C [16]. This means that G.G and C.C hybrids can [23,24], and curvature of the solid surface [25] strongly affect the produce the most stable and the most labile mismatch hybrids in efficiency of nucleic acid hybridization at interfaces. hybridization reactions, respectively. Furthermore, the nearest- neighbor model can predict the effects of mismatch positions with- Factors that affect the efficiency of DNA hybridization on in the probe strand on the overall free energy of the hybridization surfaces reaction [14]. This model sufficiently demonstrates that the inter- nal and penultimate mismatches within the probe strand provide a Surface probe density higher discriminatory power than the terminal mismatches do (Fig. 2) [14,17]. It has been revealed that surface probe density, the number of In contrast, nucleic acid hybridization at an interface is more probe strands per unit area, affects the rate and the efficiency of complex than hybridization in solution. So far, several attempts the target/probe duplex formation. Peterson and co-workers have have been made to formulate the behavior of DNA hybridization shown [20] that at low probe densities (<3  1012 molecules/ on the solid surface; nevertheless, a comprehensive model that cm2) on the planar gold surface, the efficiency of duplex formation demonstrates this behavior has not yet been developed [4]. is 100% and that the DNA hybridization reaction performs under However, investigations on thermodynamic and kinetic fast kinetic, whereas at high probe densities (<5  1012 mole- cules/cm2) the efficiency of duplex formation drops to 10% with a slower hybridization rate. Certainly, the attenuation of DNA hybridization at high probe densities is related to a large electrostatic repulsion between negatively charged probes that suppresses the penetration of the target strands into the probe layer. Density of charge on surface The efficiency of DNA hybridization in the solid-phase format is Fig.2. Three types of mismatch nucleotides in nucleic acid duplexes. affected by changing the charge or electric potential of the surface  Optimizing DNA hybridization on surfaces / H. Ravan et al. / Anal. Biochem. 444 (2014) 41–46 43 [21]. Vainrub and Montgomery Pettitt modeled the dependency of chains extend their structure such that the unfavorable interac- DNA hybridization on surface charge density [26]. They found that tions between neighbor strands are minimized. Since the tethered the melting temperature, Tm—a measure of probe/target affinity oligonucleotide strands at high surface probe density exhibit such representing the temperature at which half of the oligonucleotide properties, paying attention to this behavior is essential. Halperin probes are hybridized to their targets in the equilibrium state— and co-workers have shown [29] that when long target strands increases with increasing surface charge density from 0 to a posi- are hybridized to the tethered oligonucleotide probes, the confor- tive value. Recently, Wong and Melosh [27] in an experimental mation of DNA chains is no longer controlled by the length of the study revealed how an applied voltage on the gold surface controls attached oligonucleotide strands. Indeed, in this case, the length the efficiency of the target/probe duplex formation. They found of the target strands induces the conformation of DNA molecules that, essentially at high probe densities (P1013 molecules/cm2), so that they exhibit a highly stretched brush-like conformation. an increase in the applied voltage from 0 to +300 mV causes a The progress of DNA hybridization in this situation is strongly sup- threefold increase in the efficiency of the duplex formation. Inver- pressed because of surface crowding arising from nonhybridized sely, at a negative voltage (300 mV), the efficiency of DNA duplex tails of the target strands. As a result, the benefits of increasing formation drops to 1% from 10%, which was determined for zero the surface density of the probe decrease when the intended tar- voltage. The change in the efficiency of DNA hybridization can be gets are long [29]. Therefore, it is important to pay attention to this explained in terms of an electrostatic kinetic barrier, which is seen enhanced brush effect in the design of the nucleic acid analyzing at high probe densities [22]. In such cases, negatively charged tools based on the solid-phase hybridization strategy. oligonucleotides on the surface limit the insertion of DNA targets into the probe layer. Application of positive voltages on the surface Curvature of the solid surface reduces this electrostatic barrier, allowing more target strand to penetrate into the probe layer and be hybridized. In contrast, a In general, solid surfaces, according to their geometry, are di- negative voltage enhances this electrostatic barrier, resulting in a vided into planar and curved surfaces. DNA hybridization on planar minimal target/probe duplex formation over experimental time surfaces is seen in a variety of techniques, including microarray scale. [31], biosensor [32], and most of the nucleic acid detection methods based on immunoassay technology [33,34]. In contrast, Conformation of probe strands on surface hybridization on curved surfaces is seen in DNA-mounted micro/ nanostructures, including microspheres, nanoparticles, and Conformation of an array of tethered polyelectrolyte chains on a nanotubes [25,35,36]. Oligonucleotides attached to two different solid surface is determined by the size of the grafted polymer and surface types have manifested distinct behaviors, which can affect the distance between the grafting points [28]. A quantitative the overall efficiency of DNA hybridization [37]. In this study, we parameter for these factors—that reflects the conformation of compare the properties of DNA hybridization on planar and curved polymer chains—is ‘‘reduced tethered density’’ (R), R = rpRg2, (nanoparticle) gold surfaces because the behavior of tethered where Rg is the radius of gyration of tethered polymer at given oligonucleotides on these surfaces has been studied more than that experimental conditions, and r = hdNA/Mn is the grafting density on other surfaces. of the tethered polymer, in which h represents the polymer layer Changes in the size of gold nanoparticles can affect the probe thickness, d is the bulk density of tethered polymer, NA is Avoga- loading capacity and the DNA hybridization property of the dro’s number, and Mn is the polymer’s molecular weight [28]. particles [38]. When gold nanoparticles with different sizes are Generally, tethered polyelectrolyte chains on a surface have compared, the smaller diameter nanoparticles show a higher been recognized to exhibit three conformational regimes, includ- probe-loading capacity than the larger ones, for a given equal ing mushroom-like, crossover, and brush-like states [28,29]. In a surface area. This difference arises because smaller nanoparticles mushroom-like regime, the distance between neighboring poly- have a more curved surface than the larger nanoparticles and the mers is larger than the thickness of the polymer layer (R < 1), oligonucleotide probes attached to these nanoparticles would have allowing the polymer chains to bend their structure and adopt a significantly more distance from one another moving radially away mushroom-like conformation (Fig. 3) [1,28,29]. At low probe from the particle surface (Fig. 4). Since the probe loading on the densities, it is possible for a part of the oligonucleotide probe to gold surface is the result of the interplay between the covalent be adsorbed on the surface and take on a mushroom-like gold/thiol interactions and the electrostatic repulsion between conformation. In this situation, the efficiency of DNA hybridization DNA strands, reducing electrostatic interactions allows more oligo- decreases because of the inaccessibility of probe strands to their nucleotide probes to attach to the highly curved surface [39]. In target [30]. In contrast, in a crossover regime, the distance between addition to this property, the efficiency of DNA hybridization is af- the tethered polymer chains quantitatively approaches the size of fected by the size of the nanoparticles. At a constant surface probe the polymer chains (R  1) (Fig. 3). In this balanced regime, the density, it has been revealed that the binding of target strands to efficiency of DNA hybridization is higher than in the mushroom- the oligonucleotide probes attached to highly curved surfaces like state because the tethered probe strands are in an appropriate (smaller nanoparticles) is more favorable than that for flatter sur- conformation for hybridization. Finally, a polymer brush-like faces. Certainly, relaxation of electrostatic interaction and steric regime occurs when polymer chains attached to the surface are hindrance between neighboring oligonucleotides for highly curved sufficiently near to one another to change the conformation of surfaces allow the target strands to penetrate more easily into the individual polymer chains (R > 1) (Fig. 3). In this regime, polymer probe layer and be hybridized. These size-dependent properties Fig.3. Tethered polyelectrolyte chains on the surface have been recognized to exhibit three conformational regimes, including mushroom-like, crossover, and brush-like states. 44 Optimizing DNA hybridization on surfaces / H. Ravan et al. / Anal. Biochem. 444 (2014) 41–46 enhance the efficiency of the target/probe duplex formation. Schatz and co-workers have shown [41] that DNA hybridization on gold nanoparticles is completely diminished at low concentra- tions of salt (e.g., less than 0.05 M NaCl), while solution-phase hybridization (absence of nanoparticles) of the same sequence is performed under these experimental conditions. The authors have suggested that the reason for the different responses is related to a screening effect of the salt on the surface-immobilized probes [41]. Indeed, the salt ions interact with the negative-charge phosphate groups of the oligonucleotide probes and minimize the electrostatic repulsion between surface-tethered probes. Once the electrostatic repulsion is screened by the electrolyte, the target strands can be easily hybridized with the probe molecules. Fig.4. Effect of solid surface curvature on the probe-loading capacity and DNA- hybridization property of the surface. When gold nanoparticles with different sizes are compared, the smaller diameter nanoparticles show a higher probe loading capacity than the larger ones, for a given equal surface area. This difference arises Strategies for reducing steric hindrance between DNA probes because smaller nanoparticles have more curved surface than the larger nanopar- on the surface ticles and the oligonucleotide probes attached to these nanoparticles would have significantly more distance from one another moving radially away from the Application of a spacer (linker) between solid surface and DNA- particle surface. recognizing sequence Typically, for tethered oligonucleotide probes, a spacer between are well observed for gold nanoparticles below 100 nm diameter. It the solid surface and the recognition sequence is considered. The was also determined that the behavior of the surface of the nano- spacer is important from two aspects: first, it helps the recognition particles above this value is similar to that of planar gold surfaces sequence to be kept away from the solid surface, thus reducing the [39]. steric hindrance of the probe during the hybridization process The efficiency of DNA hybridization on surfaces suffers from [11,42,43]. Second, the spacer helps the oligonucleotide probes to three constraints: (1) the electrostatic repulsion between DNA be maximally loaded on the solid surface during the probe immo- strands on the surface, (2) the steric hindrance between tethered bilization process [44]. The spacer region can be a polynucleotide DNA probes, and (3) the nonspecific adsorption of the attached oli- such as polyadenine or polythymine [45] or an inert polymer such gonucleotides to the solid surface. All three of these parameters as polyethylene glycol [46] or a hexamethylene linker [47]. The in- diminish both the rate and the efficiency of target/probe duplex ert spacers are usually preferred because the charged spacers, like formation. In this regard, several strategies have been developed polynucleotides, exhibit an interstrand electrostatic repulsion due to overcome the problems associated with DNA hybridization on to the negative phosphate backbone, which restricts the maximum surfaces. DNA loading on the surface during the probe immobilization process [44]. Furthermore, the bases of polynucleotide spacers, especially adenine, are nonspecifically adsorbed on the gold sur- face and reduce the hybridization of the target DNA to the probe Strategies for reducing the electrostatic repulsion between DNA molecules [48]. strands on the surface To achieve the highest signal-to-background (S/B) ratio in devices that work based on solid-phase hybridization, the probe Enhancing DNA hybridization on a surface by optimizing the solution density on the surface must be optimized. At low probe densities, pH because of the small number of probe strands attached to the surface, the S/B ratio is low, while at high probe densities, the The efficiency of DNA hybridization on a surface can be opti- electrostatic and steric interactions between the attached probes mized by changing the pH of hybridization solution. Zhang and reduce the efficiency of DNA hybridization. One strategy to co-workers [30] reported that at high probe densities increase the S/B ratio is to work at a surface probe density at which (1.3  1013 molecules/cm2), the efficiency of the DNA hybridiza- the electrostatic and the steric interactions are minimized. tion on the gold surface of the microcantilever biosensor is im- Generally, several strategies have been applied to control the proved by increasing the pH value from 4.5 to 8.5. They have loading of the oligonucleotide probes on surfaces [20,40]. The most demonstrated that a fast and reversible mechanical bending in important strategy includes changing the ionic strength of the the structure of the probe strands occurs in response to pH varia- immobilization solution. Peterson and co-workers have shown tions. At pH 4.5, probe molecules are partially protonated and an [20] that the kinetics of probe immobilization on the gold surface electrostatic attraction between them causes the strands to bend depends strongly on the solution ionic strength. In solutions with toward each other, while at pH 8.5, the probe strands are fully high ionic strength, many more probe molecules adsorbed to the deprotonated and the distance between the probe molecules surface in a short time. Certainly, at a low ionic strength, electro- increases because of electrostatic repulsion. The authors also static repulsions between probes heavily suppress the adsorption observed that at an extreme pH (very low and very high pH), the of DNA strands on the surface, while at a high ionic strength, the efficiency of DNA hybridization is strongly suppressed. They repulsions between the probes are efficiently screened by the salt pointed out that a pH-induced denaturation of the probe/target du- ions, allowing many more probes to adsorb to the surface. plex could be the main reason for decreasing the efficiency of DNA Therefore, adjusting the solution ionic strength can effectively hybridization at an extreme pH value. Therefore, they concluded control the surface probe density. that the efficiency of DNA hybridization can be optimized by The increase in salt concentration, along with an increase in the decreasing the surface steric hindrance as a function of solution oligonucleotide probe loading on the surface, can effectively pH [30].  Optimizing DNA hybridization on surfaces / H. Ravan et al. / Anal. Biochem. 444 (2014) 41–46 45 Strategies for reducing nonspecific adsorption of DNA on the surface It has been shown that the nonspecific adsorption of immobi- lized probes on surfaces, especially for gold nanoparticles, inhibits the formation of probe/target duplex [23]. The most important fac- tors that affected nonspecific adsorption on the surface included the probe’s nucleotide content [48] and the conformation of the oligonucleotide probes on the surface [23]. It has been revealed that the organic bases of the nucleotides in the probe molecules can be preferentially adsorbed to the gold sur- face. The affinity of bases to the gold surface follows the order A > G P C  T, as determined by the competitive adsorption from Fig.5. Incorporation of a polyadenine block into the terminal end of the recognizing solution mixture to the gold surface in homopolymer form sequence to reduce nonspecific DNA adsorption on the surface. The gold surface is [48,49]. Surprisingly enough, the studies have shown that the pref- completely saturated with the polyadenine tail and nonspecific adsorption of the erentially noncovalent adsorption of adenine polynucleotide to the recognizing sequence of the probe is strongly prevented. gold surface is even comparable with thiol/gold covalent interac- tions [50]. Indeed, the nonspecific adsorption of the nucleotides to the gold surface strongly suppressed the base-pairing interac- of the thiol reagent to the gold-labeled oligonucleotide must be tion between oligonucleotide probes and their target [51]. strictly controlled. At very high concentrations or prolonged incu- Furthermore, in addition to the aforementioned property, the bation times it is possible for the immobilized probes on the gold conformation of the oligonucleotide probe influences the surface to be completely replaced by the thiol reagent. Therefore, nonspecific adsorption on the gold surface [23]. The immobilized under optimizing conditions, MCH reacts with gold surfaces and oligonucleotide probes that adopt a compact secondary structure changes the conformation of the immobilized oligonucleotide such as the hairpin structure persist against nonspecific adsorption probes, thereby rendering the hybridization more amenable. on gold nanoparticles. In contrast, the bases of immobilized probes with a flexible random-coil conformation can easily interact with Incorporation of a polyadenine block into the terminal end of the the gold surface; thereby, in this situation the formation of the recognizing sequence to reduce nonspecific DNA adsorption on the target/probe duplex is less favored [23]. The basis for this phenom- surface enon is probably the differences in the structural flexibility of the DNA molecules. When a single-stranded DNA with a random-coil Another strategy for reducing the nonspecific adsorption of the conformation interacts with negative ions, such as citrate ions ad- oligonucleotide probes on the surface is the incorporation of a sorbed on the surface of gold nanoparticles, it can easily uncoil its polyadenine block at the 30 or 50 terminal of the recognizing structure, whereby the negative phosphate groups of the DNA sequence of the oligonucleotide probes. Since the polyadenine strand are sufficiently distant from the ions to minimize the elec- sequence is strongly and preferentially adsorbed on the gold trostatic repulsion. In this situation, the attractive van der Waals surface, it can be used as a binding interface to attach the probe forces between the exposed bases of DNA strands and gold nano- to the gold surface instead of the covalent gold/thiol interaction particles are sufficient to cause the single-stranded DNA to be ad- [40]. In this case, the gold surface is completely saturated with sorbed onto the gold surface [52]. A DNA molecule with a relatively the polyadenine tail, and nonspecific adsorption of the recognizing fixed configuration, such as a hairpin DNA structure, however, has sequence of the probe is strongly prevented (Fig. 5). Certainly, in a stable secondary structure geometry that always presents in a this situation, the recognizing sequence adopts an upright confor- compact configuration with the negative phosphate backbone. mation that is suited to efficient hybridization. In addition to this When the double-stranded part of hairpin DNA is exposed to gold role of the polyadenine, the length of the polyadenine block can nanoparticles, electrostatic repulsion between citrate ions and the spatially control the surface density of the probe molecules. Pei charged phosphate backbone dominates, and the DNA molecule is and co-workers have shown [40] that the probe surface density not adsorbed on the gold nanoparticles [23,52]. decreases when the length of the polyadenine block increases. Indeed, this finding suggests that all adenine bases in the polyade- nine block, independent of the length, are completely adsorbed on Application of thiol reagents to reduce nonspecific DNA adsorption on the gold surface, which allows the distance between recognizing gold surfaces sequences of the oligonucleotides to be controlled by the length of the polyadenine block. At a low surface probe density, the immobilized single-stranded DNA probes can mechanically bend their structure to adsorb on the Summary and conclusion bare patch of the gold surface [53]. In this case, the gold surface undergoes two types of interactions: first, covalent gold/thiol Nucleic acid hybridization at an interfacial environment suffers interactions that occur between the gold surface and thiolated from the interaction between tethered oligonucleotide probes, sur- oligonucleotide probes and second, noncovalent interactions face, and neighboring DNA strands. These interactions diminish between the bare patch of the gold surface and the bases of the both the efficiency and the rate of DNA hybridization on the sur- immobilized oligonucleotide probe [54]. When a thiol reagent such face. In this respect, reducing nonspecific interactions between as mercaptohexanol (MCH–SH) is added to the gold-labeled oligo- the tethered oligonucleotide probes and the surface and decreasing nucleotide suspension, the noncovalent interactions on the surface the electrostatic repulsion between immobilized DNA strands are destabilized and a new covalent gold–S–MCH interaction fills strongly enhance DNA hybridization on surfaces. It is also impor- the bare patch on the surface [55]. Certainly, in this situation, the tant to note that the rational design of new nanodevices based immobilized oligonucleotide probes change their structure and on solid-surface hybridization relies heavily on the development take an upright conformation that is suited to efficient hybridiza- of a comprehensive model that accurately describes the behavior tion. It should be noted that the concentration and exposure time of DNA hybridization on surfaces. 46 Optimizing DNA hybridization on surfaces / H. 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