DNA origami adsorption at single-crystalline TiO₂ surfaces

Abstract

The immobilization of DNA origami nanostructures on solid surfaces is an important prerequisite for their application in many biosensors. So far, DNA origami immobilization has been investigated in detail only on a few surfaces such as mica, SiO₂, and graphite. TiO₂ is a conductive oxide with extensive applications in photocatalysis, energy conversion, and (bio)sensing. Despite its great importance, however, TiO₂ has not been investigated as a substrate for DNA origami immobilization yet. Here, we systematically investigate the adsorption of 2D DNA origami triangles on single-crystalline TiO₂ surfaces under various experimental conditions. Interestingly, the effect of the Mg²⁺ concentration on DNA origami surface coverage is found to depend on the orientation of the TiO₂ surface. On TiO₂(110) and TiO₂(111), 10 mM Mg²⁺ yields a higher surface coverage than 5 mM. However, the inverse is observed for the TiO₂(001) surface, where the lower Mg²⁺ concentration leads to an increase in surface coverage by up to 75%. This is explained by the interplay between Mg²⁺ binding to the DNA phosphates and Mg²⁺ adsorption at the TiO₂ surfaces, which in the case of TiO₂(001) results in a maximum density of Mg²⁺ salt bridges already at a low Mg²⁺ concentration. At higher concentrations, both the surface and the DNA phosphates are getting saturated with Mg²⁺ ions, which introduces electrostatic repulsion at the TiO₂–DNA interface and thus lowers the surface coverage. Our results demonstrate that DNA origami surface coverage at different TiO₂ surfaces can be controlled by the Mg²⁺ concentration. However, the same mechanism may also play a role in DNA origami adsorption at other single-crystalline oxide surfaces.

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Introduction

DNA origami nanostructures¹ have become a widely employed molecular tool in biosensing.^2–5 These nanostructures are fabricated by folding a single-stranded DNA scaffold into an arbitrary, user-defined shape via hybridization with a set of short synthetic oligonucleotides called staple strands.^6,7 It allows for the precise fabrication of two- and three-dimensional nanostructures, which can be modified with sub-nanometer precision to display controlled arrangements of DNA motifs,⁸ small molecule ligands,⁹ antibodies,¹⁰ enzymes,¹¹ fluorescent dyes,¹² and various inorganic nanoparticles.^13–17 This versatility is exploited in various biosensor concepts, in which the DNA origami nanostructures may serve as both recognition elements and transducers. DNA origami biosensors can therefore implement diverse detection strategies based on fluorescence,¹⁷ surface-enhanced Raman scattering,¹⁸ circular dichroism,¹⁴ surface plasmon resonance,¹⁹ and electrochemistry,²⁰ among others. Since many sensor concepts require the immobilization of the DNA origami nanostructures on solid surfaces, controlling DNA origami adsorption at relevant materials interfaces has become an important technological issue.^21–31 Most of the previous works focused on SiO₂ surfaces,^{21,23–26,28,30,31} while a few investigated also carbon-based materials.^22,27,29

TiO₂ has unique photocatalytic and electronic properties, making it a key candidate for diverse applications such as environmental remediation, photovoltaics, and sensing. TiO₂ is used as a photocatalyst in water splitting,^32–34 in CO₂ reduction,^35–37 and in solar hydrogen³⁸ and energy harvesting³⁹ strategies, thus playing a crucial role in sustainable energy solutions. Furthermore, its high surface reactivity, stability, and tunable electronic properties under ultraviolet (UV) light irradiation render TiO₂ a highly interesting material for the development of sensors for the detection of gases, chemicals, and biological molecules.^40–42 For such applications, understanding and ultimately controlling the interaction of TiO₂ surfaces with relevant molecules is an important factor. Biomolecules have received particular attention in this regard because of the important applications of TiO₂-based materials in biosensing as well as regenerative medicine. Most studies in this area have focused on proteins because of their ubiquitous presence in physiological media and their great relevance as diagnostic biomarkers.^43–50 However, there are also DNA-based sensing concepts utilizing TiO₂ surfaces, nanoparticles, and nanowires.^51–60

Despite its great importance in biosensing and biomedicine, TiO₂ so far has not been investigated as a substrate for DNA origami adsorption. This study aims to close this gap and investigates the adsorption of two-dimensional DNA origami triangles at single-crystalline TiO₂ surfaces. Atomic force microscopy (AFM) is used to quantify DNA origami surface coverage in dependence of the DNA origami and Mg²⁺ concentrations, as well as the incubation time. We observe that DNA origami adsorption is influenced by the orientation of the TiO₂ surface, with the TiO₂(001) surface exhibiting a higher DNA origami surface coverage at 5 mM than at 10 mM Mg²⁺. For TiO₂(110) and TiO₂(111), the situation is reversed. These observations are attributed to the interplay between Mg²⁺ binding to the DNA and Mg²⁺ adsorption at the TiO₂ surfaces, which is stronger on the TiO₂(001) surface due to its larger content of surface oxygens resulting in increased basicity.

Materials and methods

DNA origami assembly and purification

DNA origami triangles¹ were assembled using the single-stranded M13mp18 scaffold DNA (BAYOU BIOLABS) and 208 staple strands (Eurofins). The staples and scaffold were mixed at a 10 : 1 molar ratio in 1× TAE buffer (pH 8.5, Carl Roth) supplemented with 10 mM MgCl₂ (Carl Roth) in a 100 μL reaction volume. The mixture was placed in a thermocycler (Primus 25 Advanced, PEQLAB), heated to 80 °C, gradually cooled to room temperature, and stored at 4 °C. To remove the unbound staples, the samples were purified using 100 kDa molecular weight cutoff filters (Amicon Ultra, Millipore) with 1× TAE supplemented with 10 mM MgCl₂ as the solvent. For Mg²⁺-free experiments, the assembly buffer was exchanged during spin filtering against Mg²⁺-free Tris buffer (40 mM, pH 8.5) as described previously,⁶¹ resulting in a residual Mg²⁺ concentration around 10 μM. A UV-vis spectrophotometer (Implen Nanophotometer P330) was used to measure the concentration of assembled DNA origami nanostructures based on their absorption at 260 nm.⁶²

Substrate preparation

TiO₂(001), TiO₂(110), and TiO₂(111) wafers were purchased from Crystal GmbH. The substrates were soaked in Hellmanex III solution (Hellma GmbH) for two hours, rinsed thoroughly with HPLC-grade water (Carl Roth) and subsequently dried under a stream of argon. The cleaned substrates were treated with an O₂ plasma (Diener Zepto, Diener Electronic) for 1 minute to create a hydrophilic, hydroxyl-rich surface.⁶³ This was verified by contact angle measurements (see Fig. S1†). Afterwards, the substrates were examined using AFM. If contaminants were detected in the AFM images, the cleaning process was repeated until a clean surface was achieved.

Si(100) wafers (Siegert Wafer) with native surface oxide were immersed in preheated RCA-1 solution (1 : 1 : 5 25% NH₃, 35% H₂O₂, H₂O) at 75 °C for 15 min to remove organic residues and create a hydrophilic hydroxyl-rich surface.⁶⁴ Then, they were rinsed thoroughly with HPLC-grade water and subsequently dried with stream of argon.

Contact angle measurements

The contact angle measurements were performed using an OCA 15 plus contact angle system (Dataphysics Instruments) with the sessile drop method, applying 5 μl of HPLC-grade water (Carl Roth) on the surface.

DNA origami adsorption

The assembled DNA origami triangles were diluted to concentrations of 0.5 nM, 1 nM, 2.5 nM, and 5 nM in 1× TAE buffer supplemented with 5 mM and 10 mM MgCl₂, respectively. For Mg²⁺-free experiments, the samples were diluted in pure Tris buffer. 50 μL samples were pipetted onto the substrates and incubated for 1, 5, 10, and 30 minutes, respectively. After incubation, the substrates were gently rinsed with HPLC-grade water and dried under a stream of argon.

AFM imaging

AFM imaging was performed in air using a Bruker Dimension Icon operated in ScanAsyst mode with SCANASYST-AIR cantilevers (Bruker) with a nominal tip radius of 2 nm and a JPK Nanowizard 3 operated in intermittent contact mode with HQ:NSC18/Al BS cantilevers (MikroMasch) with a tip radius <8 nm. Images were acquired with a scan size of 2 × 2 μm² at a resolution of 1024 × 1024 pixels.

Image processing

The images were processed using the open-source software Gwyddion.⁶⁵ To calculate the DNA origami surface coverage, a suitable height threshold was applied to each image using the Mark by Threshold tool. The value of the threshold was adjusted individually for each image to mask only the DNA origami nanostructures but not the surface. The surface coverage was subsequently calculated using the Grain Summary tool.

X-ray photoelectron spectroscopy (XPS)

XPS measurements were conducted in ultra-high vacuum (base pressure better than 10⁻¹⁰ mbar) using an Omicron ESCA+ system (Omicron NanoTechnology) with a monochromatic Al Kα (1486.7 eV) X-ray source and a hemispherical energy analyzer. The source-analyzer angle was 102°, while the take-off angle of the detected photoelectrons was set to 30° with respect to the surface plane. A pass energy of 100 eV, a step size of 0.5 eV, and a dwell time of 0.1 s were used for survey spectra. A pass energy of 20 eV, a step size of 0.1 eV, and a dwell time of 0.5 s were used for high-resolution core-level spectra. Neutralization was done using simultaneous irradiation with a low energy electron beam (2 eV). The O 1s peaks were fitted using the UNIFIT 2019 program, using a convolution of Gaussian and Lorentzian line shapes and Shirley-type backgrounds. In the fit, the Lorentzian component was fixed to 0.1 eV, while the Gaussian width was left free. The stoichiometry was determined by the normalization to 100% of the background-subtracted areas divided by the appropriate normalization factors.

Results and discussion

DNA adsorption at TiO₂ surfaces has mostly been studied at acidic pH close to or below the isoelectric point (IEP) of the TiO₂ surface.^59,60,66 Under such conditions, the TiO₂ surface is electrically neutral or positively charged, so that there is no electrostatic repulsion between the surface and the negatively charged DNA. DNA origami nanostructures, however, are usually synthesized in TAE buffer with a pH of 8.5, at which the TiO₂ surface is negatively charged.⁶⁷ Therefore, adsorption of the negatively charged DNA origami nanostructures at this pH requires the presence of a sizeable amount of divalent cations in solution to act as salt bridges at the TiO₂–DNA interface. Mg²⁺ ions are the most obvious candidates for this as they are usually added to the TAE buffer during DNA origami assembly to screen electrostatic interhelix repulsion.⁶⁸ To verify the role of salt bridges in DNA origami adsorption at TiO₂ surfaces, assembled DNA origami triangles (2.5 nM) were transferred into pure Tris buffer (pH 8.5, residual Mg²⁺ concentration about 10 μM)⁶¹ and incubated for 10 min on the TiO₂(001) surface (IEP 5.5–5.8).⁶⁷ As can be seen in the AFM image shown in Fig. S3,† no DNA origami triangles are detected on the surface after incubation. In contrast, incubation under equivalent conditions but in the presence of 5 mM Mg²⁺ leads to a large number of adsorbed DNA origami triangles (see Fig. 1), which supports the assumption that DNA origami adsorption at the TiO₂(001) at basic pH requires salt bridges. Therefore, we investigated the adsorption of DNA origami triangles at the TiO₂(001) surface for different Mg²⁺ concentrations of 5 mM and 10 mM, different DNA origami concentrations between 0.5 and 5 nM, and different incubation times between 1 min and 30 min, respectively.

Fig. 1 AFM images (2 × 2 μm²) of DNA origami triangles adsorbed on TiO₂(001) surfaces in the presence of 5 mM Mg²⁺. The DNA origami concentrations and incubation times are indicated.

Fig. 1 shows AFM images of DNA origami nanostructures adsorbed at TiO₂(001) in the presence of 5 mM Mg²⁺. At constant DNA origami concentration, longer incubation times in general lead to a larger DNA origami surface coverage. The same is observed at a fixed incubation time upon increasing the DNA origami concentration. For DNA origami concentrations of 2.5 nM and higher, this leads to the formation of multilayers at incubation times exceeding 5 min.

Interestingly, increasing the Mg²⁺ concentration to 10 mM does not result in any strong variations in the overall trends (see Fig. 2). However, upon close inspection of the corresponding AFM images, it appears that for long incubation times and high DNA origami concentrations (such as 30 min at 5 nM), the obtained surface coverage is slightly decreased at 10 mM Mg²⁺. This behavior is rather surprising because for SiO₂ surfaces, which also are negatively charged in 1× TAE buffer, higher Mg²⁺ concentrations usually result in larger surface coverage.^26,30

Fig. 2 AFM images (2 × 2 μm²) of DNA origami triangles adsorbed on TiO₂(001) surfaces in the presence of 10 mM Mg²⁺. The DNA origami concentrations and incubation times are indicated.

For better comparison, the surface coverage after DNA origami adsorption was calculated for each condition and is presented in Fig. 3. As can be seen, the overall trends visually observed in the AFM images are well reproduced in the quantitative data. Increasing both incubation time and DNA origami concentration leads to higher surface coverage. More importantly, however, the data in Fig. 3 also reveals that the differences in the surface coverage obtained for the different Mg²⁺ concentrations depend on incubation time and DNA origami concentration. At short incubation times below 10 min and low DNA origami concentrations below 2.5 nM, the Mg²⁺ concentration does not have a pronounced effect on surface coverage. At high DNA origami concentrations and long incubation times, however, the surface coverage obtained in the presence of 5 mM Mg²⁺ is notably higher than that obtained at 10 mM Mg²⁺. Under these conditions, the increase in surface coverage due to the reduction in Mg²⁺ concentration ranges from 22 to 75% with no notable trend.

Fig. 3 DNA origami surface coverage on the TiO₂(001) surface at different conditions. Values represent averages over 3 to 12 AFM images recorded at different positions on the surfaces. Error bars represent the standard deviations. See Fig. S4–S35† for the thresholded images.

Next, we sought to investigate whether this peculiar effect of the Mg²⁺ concentration is universal for TiO₂. Therefore, we performed additional experiments with alternative substrates. DNA origami triangles were incubated in both Mg²⁺ concentrations also on TiO₂(110) and TiO₂(111) surfaces under conditions that on TiO₂(001) resulted in higher adsorption at 5 mM Mg²⁺, i.e., 2.5 nM DNA origami triangles incubated for 10 min. For comparison, a silicon wafer with native surface oxide was used as a substrate for adsorption as well. The corresponding AFM images in Fig. 4 reveal a different trend for TiO₂(110) and TiO₂(111). Here, more DNA origami triangles are adsorbed at 10 mM Mg²⁺ than at 5 mM. In contrast, no apparent differences between the two Mg²⁺ concentrations are observed for the SiO₂ surface.

Fig. 4 AFM images (2 × 2 μm²) of DNA origami triangles (2.5 nM) adsorbed in the presence of 5 mM and 10 mM Mg²⁺, respectively, at TiO₂(001), TiO₂(110), TiO₂(111), and SiO₂ for 10 min.

These qualitative observations are substantiated by the calculated surface coverage (Fig. 5). For both the TiO₂(110) and the TiO₂(111) surface, the obtained surface coverage increases by about 34% upon increasing the Mg²⁺ concentrations from 5 mM to 10 mM. For the SiO₂ surface, the two Mg²⁺ concentrations result only in negligible differences. This demonstrates that this peculiar behavior of a lower Mg²⁺ concentration resulting in stronger DNA origami adsorption is specific for the TiO₂(001) surface.

Fig. 5 DNA origami surface coverage on the different TiO₂ and the SiO₂ surfaces after 10 min adsorption of 2.5 nM DNA origami triangles in the presence of 5 mM and 10 mM Mg²⁺, respectively. Values represent averages over 3 to 12 AFM images recorded at different positions on the surfaces. Error bars represent the standard deviations. See Fig. S22, S26, and S36–S41† for examples of the thresholded images.

As mentioned above, the IEP of the TiO₂(001) surface lies between 5.5 and 5.8.⁶⁷ In contrast, the SiO₂ surface has an IEP around 3.9.⁶⁹ Interestingly, the IEP of the TiO₂(110) surface lies in-between those values, i.e., between 4.8 and 5.5.⁶⁷ In addition, the TiO₂(001) surface has a lower density of cationic sites than TiO₂(110), i.e., 4.8 vs. 6.0 nm⁻².⁶⁷ For the TiO₂(111) surface, the situation is more complex as this surface is composed of two different domains with cation densities of 3.5 and 5.3 nm⁻², respectively.⁷⁰ Because of these differences, it has been observed before that the adsorption rates of various ions on single-crystalline TiO₂ surfaces depend on the crystal orientation.^71–73 Therefore, we assume that the observed differences in the influence of Mg²⁺ concentration on DNA origami adsorption are rooted in an orientation-dependence of Mg²⁺ adsorption.

To verify this hypothesis, we have analyzed the chemical composition of the three TiO₂ surfaces after the cleaning procedure by XPS (see Fig. S42 and S43†). The results reveal that the TiO₂(001) surface indeed exhibits a higher fraction of surface oxygens than the other two surfaces, as observed in both the O 1s : Ti 2p ratio and the O 1s surface : bulk ratio (see Table 1). This is in agreement with previous observations and can be attributed to the fact that the TiO₂(001) surface has a low stability and undergoes extensive reconstruction.⁶⁷

Table 1 O 1s : Ti 2p ratios and O 1s surface : bulk (S : B) ratios of the three TiO₂ surfaces as determined by XPS

	TiO2(001)	TiO2(110)	TiO2(111)
O 1s : Ti 2p	3.4	2.3	2.5
O 1s S : B	1.1	0.6	0.7

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Adsorption of negatively charged DNA origami nanostructures at the negatively charged TiO₂ surfaces is controlled by the interplay of Mg²⁺ adsorption at the surface and Mg²⁺ binding to the DNA backbone phosphates. Efficient adsorption requires Mg²⁺ ions to bind simultaneously to a phosphate group and a negative surface charge. Maximum DNA origami adsorption is observed when this condition is met for all phosphate groups in contact with the surface. For the TiO₂(001) surface, the higher fraction of surface oxygens results in an increased basicity, which in turn leads to stronger Mg²⁺ adsorption. Therefore, surface saturation with Mg²⁺ is observed already at a concentration of 5 mM. While a higher Mg²⁺ concentration of 10 mM will not reduce the Mg²⁺ surface coverage, it will lead to more Mg²⁺ ions binding to the DNA phosphate groups already in bulk solution. Upon DNA origami adsorption, this leads to electrostatic repulsion between phosphate-bound and surface-bound Mg²⁺ ions and thus to a lower surface coverage. In this picture, the TiO₂(110) and TiO₂(111) surfaces adsorb fewer Mg²⁺ ions than the TiO₂(001) surface at the same Mg²⁺ concentration. This was verified by XPS, which revealed that after 10 min exposure to 5 mM MgCl₂ solution, the TiO₂(110) surface exhibits an about three times lower concentration of adsorbed Mg²⁺ than the TiO₂(001) surface (see Fig. S44†). This reduced Mg²⁺ adsorption results in an insufficient density of salt bridges, so that electrostatic repulsion between the negatively charged phosphate groups and the negative surface charges weakens DNA origami adsorption. At 10 mM, however, a higher number of phosphate groups already carry Mg²⁺ ions that upon adsorption can form salt bridges with the still unoccupied negative surface charges. At still higher Mg²⁺ concentrations, also those TiO₂ surfaces will get saturated with adsorbed Mg²⁺ ions, which then again hinders adsorption. However, at such high Mg²⁺ concentrations, complete charge neutralization and partial charge inversion of the DNA origami nanostructures may occur and lead to DNA origami aggregation.^74,75

Conclusions

In summary, we have investigated the adsorption of DNA origami triangles at single-crystalline TiO₂ surfaces at different Mg²⁺ concentrations in dependence of incubation time and DNA origami concentration. The surface coverage of the adsorbed DNA origami triangles was quantified by AFM. While our results show that the DNA origami surface coverage on the TiO₂(001) surface increases with incubation time and DNA origami concentration, they also reveal that maximum surface coverage at high DNA origami concentrations and long incubation times is achieved at a rather low Mg²⁺ concentration of 5 mM. At a higher Mg²⁺ concentration of 10 mM, surface coverage is reduced considerably. Intriguingly, this behavior is observed only for the TiO₂(001) surface, whereas for the TiO₂(110) and TiO₂(111) surfaces, maximum surface coverage is observed at 10 mM Mg²⁺. We attribute this peculiar behavior of the TiO₂(001) surface to the interplay between Mg²⁺ adsorption at the surface and Mg²⁺ binding to the DNA phosphates. Efficient DNA origami adsorption requires a large number of salt bridges at the DNA–TiO₂ interface in the form of Mg²⁺ ions that are bound to both a DNA phosphate and a negatively charged surface site. If the Mg²⁺ concentration is too high, phosphates and surface sites will be both occupied by Mg²⁺ ions, introducing electrostatic repulsion. At the TiO₂(001) surface with its higher basicity, this situation occurs at lower Mg²⁺ concentrations than at the TiO₂(110) and TiO₂(111) surfaces. Our results thus demonstrate that DNA origami surface coverage at different TiO₂ surfaces can be controlled precisely by careful adjustments of the Mg²⁺ concentration, which has important implications for various applications as the electrochemical and photochemical reactivity of TiO₂ is known to depend on its surface orientation.^76,77 However, the same general mechanism may also play a role in the adsorption of DNA origami nanostructures at other single-crystalline oxide surfaces.

Data availability

Data for this article, i.e., raw AFM images and XPS spectra, are available at Zenodo at https://doi.org/10.5281/zenodo.15369685.

Author contributions

Xiaodan Xu: methodology, validation, formal analysis, investigation, visualization, writing – original draft, writing – review and editing; Sandra Gołębiowska: formal analysis, investigation, visualization, writing – review and editing; Teresa de los Arcos: formal analysis, writing – review and editing, supervision; Guido Grundmeier: conceptualization, resources, writing – review and editing, supervision; Adrian Keller: conceptualization, methodology, writing – review and editing, supervision.

Conflicts of interest

There are no conflicts to declare.

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Footnote

† Electronic supplementary information (ESI) available: Contact angle measurements; additional AFM images with and without applied thresholds; XPS results. See DOI: https://doi.org/10.1039/d5lf00109a

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