Analysis of DNA hybridization regarding the conformation of molecular layer with piezoelectric microcantilevers

Abstract

Lead Zirconate Titanate (PZT)-embedded microcantilevers were fabricated with dimensions of 30 × 90 × 3 μm³ (width × length × thickness). A thicker PZT layer improved the actuation and enabled long-term data acquisition in common aqueous buffers with a frequency resolution of 20 Hz. A quantitative assay was conducted in the range of 1–20 μM and the resonant frequency was found to increase with the concentration of target DNAs and the probe DNAs were almost saturated at 20 μM. Back-filling with ethyleneglycol-modified alkanethiol was shown to facilitate the hybridization efficiency and stabilize the surface reaction, resulting in a signal enhancement of 40%. We report for the first time how secondary structures in oligonucleotide monolayer change the surface property of a dynamic mode microcantilever and subsequently affect its oscillating behavior. Using fabricated microcantilevers, the real time changes in resonant frequency upon hybridization were measured by utilizing different probe and target sets. The results revealed that the microcantilevers experienced a resonant frequency upshift during the hybridization with complementary DNAs if a dimer structure was present between DNA probes. A resonant frequency downshift was observed for DNA probes that did not contain any complex secondary structures. In addition, the results demonstrate the potential of using these microcantilevers to extract structural information of oligonucleotides.

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Introduction

Microcantilevers, which are devices that can transduce force acting on the probe tip or surface property change into a signal of deflection or resonant frequency, have wide applications such as imaging instrument and sensor arrays. Intensive studies have been conducted on not only the development of the microcantilever itself but also their applications.^1–6

The microcantilever acts in two modes: static mode or deflection mode, and dynamic mode or resonant mode. In the static mode, the entire beam structure bends up and down upon an applied external force or surface stress change. In the dynamic mode, a signal is generated upon a change in the resonant frequency of an oscillating microcantilever. For quantitative analysis, microcantilever biosensors have been successfully used for the detection of a number of protein biomarkers. A typical example was the detection of prostate specific antigen (PSA). Majumdar et al. quantitatively detected PSA over a range of 0.2 ng mL⁻¹ to 60 μg mL⁻¹ using a static mode microcantilever, and Kim et al. used dynamic mode microcantilever to detect the same protein at a concentration as low as 10 pg mL⁻¹ in air and 1 ng mL⁻¹ in liquid.^7–9 For the detection of DNA hybridization using microcantilevers, most efforts have been focused on lowering the limit of detection. Mutharasan et al. fabricated piezoelectric-excited, millimetre-sized cantilevers and detected hybridization of 10-mer DNA at femtomolar concentrations.¹⁰ Others have attempted to use mass enhancing strategies. For example, Kim et al. used silica nanoparticle labeled HBV (Hepatitis B Virus) target DNA and the detection limit was improved up to the femtomolar level compared with the picomolar level without labels for signal enhancement.¹¹ In addition, Dravid et al. used gold nanoparticle labeled oligonucleotides followed by a silver enhancement step and successfully detected 50 pM of DNA targets.¹²

Despite the studies mentioned above, only a relatively small number of studies have attempted investigating the biophysical behavior of oligonucleotides with microcantilevers. The most recent report on the analysis of DNA with resonating cantilevers highlighted the effect of hydrodynamic loading on single-stranded and double-stranded DNA.¹³ Manipulative techniques have recently advanced to the point where studies are under way to rationally and elaborately designing these programmable DNAs to behave as so-called DNA nanomachines.^14–19 Among these recent studies, there have been several reports on the development of nanodevices that can be driven by certain DNA sequences that induce a structural switch.^19–24 An example of this were studies on the i-motif.^21–24 The environmental pH can cause the oligonucleotide structure to reversibly switch between a compact four-stranded structure to a loose single-stranded structure. This structural change in turn changed the surface property of the microcantilever causing it to bend. Thundat et al. investigated DNA hybridization reactions using full match target and partial mismatch targets. When the mismatch locations were changed, the static mode microcantilevers showed interesting deflection reversals.²⁵ The authors of the study attributed this to a change in conformational entropy. Another case of signal reversal was observed in DNA layers localized at the fixed end of the microcantilevers.²⁶

In the present work, DNA probe and target sets were carefully designed to have distinct structures and their impacts on the behavior of Lead Zirconate Titanate (PZT)-embedded microcantilever were compared. Interestingly, the resonant frequency of the microcantilever was altered in opposite directions when different DNA target-probe sets were used. Based on this finding, we believe this approach is a much simpler way to investigate oligonucleotide structures and holds promise in the exploration of new applications of the PZT-embedded microcantilever.

Materials and methods

Chemicals

Modified oligonucleotides were synthesized from Bioneer (Daejeon, South Korea) and TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) was from the same company. The sequences used were listed in Table 1. For simplicity, each sequence was labeled using a distinct combination of several letters. Triethylene glycol-modified alkanethiol, HS-(CH₂)₁₁-(OCH₂CH₂)₃–OH, was kindly donated by Doctor Yeo's laboratory (Konkuk University, Korea). All other reagents, if not indicated otherwise, were purchased from Sigma (MO 63103, USA) and used as supplied.

Table 1 Reagent information

Nomination	Molecular information
HP0 (T0 HBV Probe)	5′-HS-(CH2)6-CTTTCCTTCTATTCGAGATCTCCTCGA-3′
HP10 (T10 HBV Probe)	5′-HS-(CH2)6-(CH2)18-T10CTTTCCTTCTATTCGAGATCTCCTCGA-3′
HT (HBV Target)	5′-Cy3-TCGAGGAGATCTCGAATAGAAGGAAAG-3′
RP10 (Reference Probe)	5′-HS-(CH2)6-T10TCAGGCTGCTGTCCGATCCATTCACTA-3′
RT (Reference Target)	5′-TAGTGAATGGATCGGACAGCAGCCTGA-3′
NC (non-complementary target)	5′-Cy3-AATGAAGGGTGGGTCCACCGGTCTA-3′
Back-filler	HS-(CH2)11-(OCH2CH2)3-OH

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PZT-embedded microcantilever fabrication

The PZT-embedded microcantilevers consisting of Ta/Pt/PZT/Pt/SiO₂ on a SiNx supporting layer were fabricated using a micromachining process as depicted in previous papers published by our lab.^8,9,11,27,28 To prevent possible leakage in aqueous solution during the experimental process, the entire surface of the microcantilever was conformal-coated with 500 μm of parylene (PACO Inc., Incheon, Republic of Korea) using a parylene coating system NPCR-400 (Nuricell Inc., Seoul, Republic of Korea). For the in situ experiment in aqueous buffer, microcantilevers were embedded with 1.7 μm of the PZT layer to ensure that they were able to safely oscillate in an aqueous environment where the damping effect could impose severe negative effects. The size of the microcantilever was 30 × 90 × 3 μm³ (width × length × thickness). Signal acquisition was conducted with Laser Doppler Vibrometer MLD-221D (LDV) (NEO ARK Co., Japan)

Functionalization of PZT-embedded microcantilever and bioassay

Two types of microcantilever surfaces, the DNA probe with and without the back-filling process, were prepared for target binding. For surface functionalization, gold deposited microcantilevers were incubated with 10 μM of HP10 for 3 h at room temperature. Excess reagents were then washed away with DI water. During the back-filling process, probe modified microcantilevers were incubated in 5 mM of back-filler HS-(CH₂)₁₁-(OCH₂CH₂)₃–OH for 1.5 h at room temperature, and washing was performed as described above.

Hybridization of HT to the microcantilever functionalized with HBV probe was monitored with LDV (ESI Fig. 1†). Microcantilevers were fixed in a liquid cell with a total volume of 40 μL to monitor their resonant frequency transition. First, the resonant frequency in stabilizing buffer was monitored for about 30 min. Subsequently, the stabilizing buffer was replaced by analyte solution and measurements were carried out for about 30 min. Finally, the analyte solution was pulled out and washing buffer was injected in. Again, the signal was processed as described above until the signal tended to stabilize.

Using microcantilevers that had been optimized as above, quantitative assays were performed as follows. In detail, functionalized microcantilevers were incubated in 1 μM, 5 μM, 10 μM and 20 μM of the target DNA solution, and shifts in the resonance frequency were monitored as a function of time in each solution.

To investigate the origin of “unique” signals from target binding, two sets of control experiments were carried out. The experimental conditions and procedures were all the same as described above but the reagents were replaced: one set with HP0 and HT, the other with RP10 and its corresponding target RT. In the latter one, the DNA sequence was carefully designed so as to avoid complex DNA secondary structures from forming such as hairpins or dimers.

Results and discussion

Surface functionalization with or without back-filling process

PZT-embedded microcantilevers were customized for in situ measurements in liquid. The thickness of the PZT layer was doubled compared with the previous thickness, which improved the quality factor of the frequency spectrum.⁸ The microcantilevers displayed stable responses in most of the aqueous buffers. A frequency resolution of 20–70 Hz was achieved and this value depended on the types of buffer and flow conditions. All DNA experiments were carried out in TE buffer to help promote hybridization at room temperature. In these experiments, the frequency resolution was 30 Hz. (ESI Fig. 2†)

The alignment of DNA probes on the surface of the microcantilever was optimized to investigate subtle variations in the molecular layer. DNA probes were immobilized directly on the microcantilevers using Au-thiol bonding, which promoted efficient signal transduction from the molecular layer to the microcantilever. To screen the non-specific interaction between oligonucleotides and Au surface, HS-(CH₂)₁₁-(OCH₂CH₂)₃-OH was inserted into the inter-DNA space. HS-(CH₂)₁₁-(OCH₂CH₂)₃-OH was revealed to quickly form self assembled monolayer on gold surface exposing the ethylene glycol moiety against gold surface, which was found to be hydrophilic and efficient in preventing nonspecific binding of “contaminating” proteins and cells.^29–31 We have evaluated the effect of back-filling by comparing two microcantilevers with and without Triethylene glycol-modified alkanethiol respectively (Fig. 1). When the two molecular layers were exposed to target DNAs, the molecules became reorganized due to specific binding to DNAs. This process induced temporal transition in the resonant behavior of microcantilevers as shown in Fig. 2. The frequency shift in the case with back-filling was 40% more than the other case after hybridization was completed.

Fig. 1 Schematic diagram of the cantilever surface for hybridization with target DNA. (a) Cantilever surface functionalized with HP10 only. (b) Cantilever surface functionalized with HP10 and back-filler.

Fig. 2 LDV monitoring and fluorescent imaging (inset) of the response of different cantilever surfaces to 5 μM HT hybridization. (a) Cantilever surface functionalized HP10 only. (b) Cantilever surface functionalized with HP10 and back-filler.

A higher signal shift from the back-filled surface could be explained based on the findings reported in previous studies.^32–38 It was well known that, besides sulfur–gold linkage, the nitrogen atom that is present in the purine and pirimidine bases of nucleotides can interact mildly with gold and lead to nonspecific adsorption of oligonucleotides onto the surface.^32,33 Subsequent exposure of this functionalized surface to thiolated molecules, HS-(CH₂)₁₁-(OCH₂CH₂)₃–OH, helps to improve the sensing capability of the microcantilever in two ways. First, it improved the hybridization efficiency by anchoring the thiolated end of the probe DNAs to the gold surface and preventing other interactions. In the case of the nitrogen–gold interaction, DNA probes tend to lie down on the surface, which is not preferable for further formation of a helix with DNA targets. However, during exposure to the back-fillers, the DNA probes were erected by intercalation of the smaller thiolated molecules between them, which allowed them to become more accessible to DNA targets. The advantage of back-filling was confirmed in the hybridization of fluorescent-labeled target DNA. Fluorescent images of the microcantilevers in Fig. 2 showed that the florescence was stronger on the back-filled surface. Based on these findings, it was inferred that the back-filling step either enhanced hybridization or increased the distance between the DNAs and the Au surface. Secondly, back-fillers can cover up bare regions on the gold surface and reduce nonspecific interactions of target DNAs. The ethylene glycol moiety of the back-filler is a well-known inert chemical that prevents adsorption of biomolecules. Because of the results described above, the back-filling process was used in all of the following experiments.

Quantitative assay

One of the more interesting results of this study was that the resonance frequency shifted upward upon binding of complimentary DNAs (Fig. 2). This result is in contrast with most previous experiments on DNA hybridization with mechanical resonating sensors. Therefore, we attempted to determine the reason for this result through quantitative and biophysical analysis.

Quantitative assays were conducted under the optimal conditions described above. Functionalized microcantilevers were exposed to a series of HT concentrations and the resonant frequency shift was observed in real-time. The frequency shift before and after the hybridization step was plotted as a function of the concentration of target DNA (Fig. 3). The upshift in the resonant frequency gradually increased with HT concentration up to a plateau. As shown in Fig. 3, when the HT concentration was increased from 1 ∼ 20 μM, the corresponding resonant frequency upshift increased from 120 Hz to 270 Hz. Negligible resonant frequency shifts were observed in the control experiment, where noncomplementary DNAs were injected instead of the targets, indicating that the signal originated from the specific hybridization of HP10 and HT. In addition, these findings demonstrated that the more the specific binding to HT was, the larger the change in surface property, which in turn led to observable signals as shifts in resonant frequency. As the curve approached a plateau around a HT concentration of 20 μM, the probe DNAs HP10 were saturated with the targets.

Fig. 3 LDV monitoring of hybridization of various concentrations of HT and NC with HP10. (a) Quantitative detection of HT hybridization with HP10. (b) Control experiment: LDV measurement of NC hybridization. (c–e) Fluorescent images of the cantilever surface after incubation with 1 μM HT, 5 μM HT and 5 μM NC. HT and NC were fluorescent-labelled.

The effect of DNA structures

This consistent reversal in frequency shift inspired us to go a step further and look more closely at the DNA sequences used in this study. A recent study showed that, under certain circumstances, metastable states in which non-complementary DNAs hybridize through partial base-pairing or single-stranded DNAs form special secondary structures via self base-pairing. Chaikin et al. dexterously utilized the difference in thermodynamic stability of those structures and successfully used them to guide the self-assembly of microparticles.³⁹

A simulation of the HP10 structures was performed with the help of DINAMelt Server (http://www.bioinfo.rpi.edu/applications/hybrid/quikfold.php) (Fig. 4). According to the results of this simulation, the HP10 probes showed a level of intermolecular interaction as high as 8 base pairing with a ΔG value of −5.4 kcal mol⁻¹ (25 °C, [NaCl] = 10 mM, [DNA] = 10 μM). This thermodynamic stability of partial hybridization implied the existence of dimer structures that may have formed between the HP10 strands on the microcantilever surface. Presumably, the amount of dimers on the surface is much higher than that obtained from the above calculation, where the reaction was assumed to occur in homogenous liquid. Since the oligonucleotides were proximately confined on the surface of microcantilevers, their collision rate was much higher than the molecules free in solution. Thus, a majority of the DNA probes existed in the form of dimers rather than free single strands at room temperature. For comparison with the HP10 probes, a set of DNA probe and target sequences, named as RP10 and RT, was designed to prevent the formation of complex structures (simulation data were shown in Fig. 4). The RP10 and RT contained the same number of nucleotides and linkers with the longest possible consecutive base pairs of 4. Typically, complex structures cannot be formed through only 4 base pair matches at room temperature.

Fig. 4 Simulation result and thermodynamic parameters of HP10 and prediction of RP10 secondary structure.

The microcantilever surface was functionalized with reference probe RP10, using the same protocol outlined above. As in Fig. 5, gradual downshift in resonant frequency was observed upon hybridization with its complementary DNA strand, RT. Thus, the exceptional phenomenon observed for the HP10 and HT system was most likely due to the structural property of the HP10 probes, which conceivably formed dimer structures through the 8 base pairs. The linkage between DNAs affected the mechanical property of the molecular layer and possibly absorbed the oscillating motion of the microcantilevers leading to a lower resonant frequency. Then, via hybridization of these probes with HT, the dimer structures were gradually disassembled and inter-DNA networks became rare. A less dissipating layer of independent helix structures was formed on the microcantilever surface.

Fig. 5 Control experiment set #1: sequence effect. (a) Schematic diagram of hybridization of HT with HP10, and RT with RP10. (b) Black circle: LDV monitoring of hybridization between HT (5 μM) with HP10; White circle: LDV monitoring of hybridization of RT (5 μM) with RP10.

Mass loading vs. structural change

According to our previous work, the mass sensitivity of the cantilever in this experiment is less than 0.2 pg/Hz.⁴⁰ The maximum mass added to the cantilever during hybridization is ∼0.6 pg assuming that the RP10 probes are saturated with RT. (Molecular weight of is RT 9.1 kD and the density of RP10 is 2.5 × 10¹² molecules cm⁻².) 250 Hz decrease cannot be explained by added mass of RPs. Thundat et al. recently reported that the anomalously large decrease of resonance frequency in deionized water can be attributed to the hydrodynamic loading to a DNA layer.¹³ The calculated response to the added mass and measurements showed difference in magnitude up to 2 orders of magnitude and it agreed qualitatively with the decrease of resonance frequency during hybridization to RP10.

However, the apparent increase in resonant frequency during hybridization indicated that the effect of the structural transition in HP10 was more dominant than the mass addition of HT or the hydrodynamic loading of liquid. Sader et al. proposed that the change in surface elasticity, instead of surface stress, could explain the cantilever's frequency shift upon chemical binding in liquid.⁴¹ Since the mechanical property of the molecular layer is related not only to the individual single molecules but also to the interaction between the molecules, the information on the elasticity of molecular monolayers is very difficult to analyze.²⁶ However, the case of HP10 and HT is the good example demonstrating the strong effect of interconnection in molecular layers on resonating cantilevers.

The effect of DNA length

To gain further insight into the DNA conformation and microcantilever resonance, the organization of the molecular layer was controlled by reducing the length of the DNA linkers. The confined distance between short probes was likely to limit the possibility of partial base-pairs forming between neighboring oligonucleotides. HP0, in which the 10 thymine bases were removed, was immobilized on the microcantilever surface. After back-filling, the same concentration of HT, 5 μM, was injected and the resonant frequency shift was observed as shown in Fig. 6. In contrast to the case of longer HP10, a downshift in the resonant frequency of 200 Hz was observed upon hybridization with HT.

Fig. 6 Control experiment set #2: spacer effect. (a) Schematic diagram of hybridization of HT with HP10 and HP0. (b) Black circle: LDV monitoring of hybridization of HT (5 μM) with HP10; White circle: LDV monitoring of the hybridization of HT (5 μM) with HP0.

We measured the average density of DNA on the microcantilever surface as described in other studies. Briefly, fluorescent dye labeled DNAs were immobilized on the gold surface viathiol groups and then the dyes were released from the gold surface by treatment with deoxyribonuclease I. Thus, the number of DNAs on gold surface could be directly measured through the amount of released fluorescent dye.⁴² The surface density of the DNA probe in this report was fixed at 2.52 × 10¹² molecules cm⁻². The average distance between DNA probes was 7.24 ± 0.12 nm. HP0 were shorter than HP10 in length (about 5.5 nm shorter assuming one base-pair of 0.55 nm), thus, formation of the dimer structures was much more difficult. (ESI Fig. 3†) The inter-probe distance was hardly encompassed with the shorter linkers in HP0.

As a result, most of the HP0 was isolated as a single strand on the microcantilever surface before the targets arrived. The typical transition from free single strands to double helix resulted in a gradual decrease in the resonance frequency as has been reported in many other studies. Consequently, DNAs with the same active sequence, HP10 and HP0, could form qualitatively different molecular layers and completely different reaction mechanisms were responsible for the formation of the molecular layer. Thus, the in situ measurement of microcantilever resonance could be used to determine the presence of secondary structures in an oligonucleotide layer.

Conclusion

Various microcantilevers were used to quantitatively analyze oligonucleotides and were shown to be highly sensitive. However, most of the microcantilevers are also very sensitive to the structure and organization of molecular layers. Since the signal reversal was observed on microcantilevers, reasonable explanation has been attempted but their quantitative analysis is still elusive in the most recent research. Through comparative experiments with designed DNA probes in this study, signal reversal was demonstrated between distinct sequences and within probe identical sequences. A dissipative molecular layer composed of oligonuleotides with internal base-pairing became more rigid with decreasing number of linkage between DNA probes. The upshift of resonant frequency upon hybridization of target DNA was ascribed to the changes in surface elasticity. These results also cautioned that the complex secondary structures may alter the quantitative information from the microcantilever sensors, which could be problematic if ignored. Thus, special care is needed to design oligonucleotide probes and organize the recognition layer on the surface.

In contrast, microcantilevers are sensitive tools to study complex and temporal transitions in the DNA/RNA structure on the surface. In this work, oligonucleotide molecular layers were constructed with various characteristics. PZT-embedded microcantilevers were used to monitor structural transitions in real time and were able to recognize the difference in molecular layers depending on the secondary structures of the oligonucleotides.

The sensitive response of PZT-embedded microcantilever to biomolecular (DNA/RNA) structure implies its wide potential applications. For example, basic scientific research will benefit from this development such as the study of biophysics of DNA or RNA molecules. In addition, it also shows the feasibility of detecting small molecules by use of DNA or RNA aptamers. Our previous electrical detection technique is currently being improved for liquid phase operation.⁴³ In the near future, this will allow for the complete integration of a portable microcantilever sensing system for more cost effective and productive analysis.

Acknowledgements

The authors are very grateful for the financial support from the KIST institutional program, the Intelligent Microsystem Center sponsored by the Ministry of Knowledge Economy, as a component of the 21st century's Frontier R&D Projects (Grant MS-01-133-01) and the National Core Research Center for Nanomedical Technology sponsored by Korea Science and Engineering Foundation (Grant R15-2004-024-00000-0).

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Footnotes

† Electronic supplementary information (ESI) available: Fig. S1 to S3. See DOI: 10.1039/c0lc00122h

‡ Published as part of a LOC themed issue dedicated to Korean Research: Guest Editors: Professor Je-Kyun Park and Kahp-Yang Suh.

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