A versatile DNA origami based plasmonic nanoantenna for label-free single-molecule SERS

DNA origami technology allows for the precise nanoscale assembly of chemical entities that give rise to new functional materials. We have created a versatile DNA Origami Nanofork Antenna (DONA) by assembling Au or Ag nanoparticle dimers with 1.17 ± 0.67 nm gap size, enabling signal enhancements in surface-enhanced Raman scattering (SERS) of up to 10 11 . This allows for single-molecule SERS measurements, which can even be performed with larger gap sizes to accommodate differently sized molecules, and at various excitation wavelengths. A general scheme is presented to place single analyte molecules into the SERS hot spots using the DNA origami structure exploiting covalent and non-covalent coupling schemes. By using Au and Ag dimers, single-molecule SERS measurements of three dyes and cytochrome c and horseradish peroxidase proteins are demonstrated even under non-resonant excitation conditions, thus providing long photostability during time-series measurement, and enabling unprecedented optical monitoring of single molecules and DNA origami based nanomachines.

The ultimate detection limit in analytical chemistry is the single molecule (SM), ideally without the necessity to label the target molecule. Furthermore, the ability to extract specific information about single molecular events allows for the screening of molecular property distributions, which are otherwise hidden within an ensemble averaged measurement. 1 Up to now, several techniques have been reported that are capable of detecting molecules down to the SM regime , such as fluorescence spectroscopy, 2 scanning probe microscopy, 3 and surface enhanced Raman scattering (SERS). 4 Among them, SERS is a particularly promising technique because in addition to the simple detection of a target molecule, it can also provide detailed chemical and structural information through the Raman vibrational fingerprint of the molecule. However, SERS relies on the strong Raman signal enhancement upon excitation of localised surface plasmon resonances (LSPR), mainly in gold (Au) or silver (Ag) nanostructures. 5,6 The Raman signal enhancement is the strongest in small, nanometer-sized volumes in-between coupled Au or Ag nanostructures, which are referred to as SERS hot spots. Therefore, the detection of single molecules in these hot spots requires a precise control over the arrangement of nanoparticles as well as the placement of target molecules into these hot spots, which imposes severe challenges for nanofabrication. This is why most of the single-molecule SERS measurements reported so far rely on the random adsorption of analyte molecules in the hot spots. 4,7,8 In a complementary approach the analyte molecules are precisely positioned into the hot spots via covalent or noncovalent interactions using DNA. 9 DNA origami is a powerful nanoengineering tool, 10 which is based on the programmed self-assembly into various 2D and 3D shapes. 11,12 DNA origami nanostructures are formed from a long single-stranded scaffold strand and a suitable set of short artificial staple strands. 10 Due to the rich chemical functionalization schemes of DNA, molecules can be positioned along the DNA origami surface with nanometer precision. 13 Furthermore, various kinds of nanoparticles can be arranged on the nanoscale to create sophisticated plasmonic nanostructures and to perform SERS measurements. [14][15][16][17][18] However, so far the DNA origami based architectures made for SERS measurements relied either on the random adsorption of Raman reporter molecules from solution or were limited to dyes or labelled molecules. 15,17,19,20 Here, we introduce a new and versatile threedimensional (3D) plasmonic DNA Origami Nanofork Antenna (DONA) structure representing Au and Ag dimers suitable for SM SERS measurements. The addressable binding sites in the as-designed DONA structures allow for precise placement of basically arbitrary molecules of different dimension in the gap between two Au or Ag nanoparticles, where the electric field enhancement is strong enough to detect single molecules by SERS.

Results and Discussion
To create complex arrangements of Au and Ag nanoparticles on DNA origami nanostructures the particles are coated with thiolated single-stranded DNA that is hybridized to complementary DNA extensions protruding from the DNA origami. 16,21,22 A frequently used nanoparticle configuration for SERS is the nanoparticle dimer. Importantly, even a small offset from the hot spot can lead to a drastic drop in the field enhancement, which calls for precise positioning of molecules in-between the nanoparticles, which however, has been challenging. To overcome this, we have designed a DNA origami nanofork structure forming the basis of the plasmonic DONA structures. It consists of a rectangular base with two arms, as is illustrated in Fig. 1a and Fig. S1 (Supplementary Information, SI).
Two nanoparticles can be bound to the arms such that each particle is connected to both arms. The two arms are connected by a bridge consisting of two DNA double helices. The main purpose of the bridge is to serve as an anchor point for target analyte molecules, which can be precisely placed into the plasmonic gap of the two nanoparticles, at the location where the SERS signal enhancement is the strongest.
The outer dimensions of the DNA origami nanofork are estimated to be 48 nm x 71 nm, based on an inter-helical distance of 2.536 nm for a honeycomb lattice. 23 The distance between the two arms is designed to be about 31 nm. Each arm is equipped with four ssDNA capture strands (capture sequences a = 5'-A24 and b = 3'-(AAC)8 in Fig. 1 and Fig. S1, to bind two differently coated nanoparticles), extending to different sides and designed to bind complementary sequences on Au or Ag nanoparticles. The bridge includes four staple strands, each crossing over once between the two helices to increase the stability and rigidity of the bridge. In addition, the free 3' and 5' ends of these staple strands allow for modifications to be added to the bridge. Further, to increase the precision of the nanoparticle placement, two of the bridge staple strands are equipped with capture strands on each end. Hence, each nanoparticle will be bound by a total of six capture strands. The nanoforks are characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM) as shown in Fig. 1b. The images show clearly the successful formation of the nanoforks including the DNA bridge. In the zoomed-in AFM image the lower part of the base of the DNA origami fork appears elevated, which corresponds to the additional rows of DNA double helices that are also shown in the scheme of the DNA origami structure. The nanoforks are then hybridized with Au or Ag nanoparticles of 60 nm diameter to form the DONAs (Fig. 1c). For optimal SERS measurements a narrow gap between the nanoparticles and precise placement of target molecule therein is required to establish a strong SERS signal enhancement. We have measured the gap distance of the Au DONA structures by TEM, and a typical TEM image is shown in Fig. 1d, along with a histogram of gap sizes. The most probable gap distance is found to be 1.17 nm.
In order to further analyse and optimize the nanofork structure we have performed molecular dynamics simulations using the coarse-grained model oxDNA2. 24,25 The oxDNA model is ideally suited to analyse the effect of bridge length on the distance of the two DNA arms and the general displacement and fluctuation of the DNA bridge, which is important for the precision of the placement of target molecules. We simulated the nanofork with bridge lengths of 120 nucleotides (nt), 90 nt as well as no bridge.  (Fig. 2c). The plot shows that a shorter DNA bridge increases the stiffness of the bridge, and that for the 90 nt bridge a full-width at half maximum (FWHM) of 3.14 nm is found, indicating the precision with which target molecules placed in the bridge could be located at the plasmonic junction. To test the performance of the DONAs for SM SERS we have placed single dye molecules (TAMRA, Cy3.5 and Cy5) at the centre of the DNA bridge using dye-modified staple strands and then assembled Au and Ag DONAs (see Methods and Table S2). In general, to achieve single molecular sensitivity, a system should produce electric (E) field enhancements in the order of 10 9 -10 12 . To investigate this, we calculated the field enhancement (FE) in the gap between the particles with 60 nm diameter as a function of wavelength using the finite difference time domain (FDTD) method (see Methods and SI for details). We have considered different gap sizes ranging from 1.2 nm (corresponding to the mean gap size found in TEM images) to 3.5 nm as shown in Fig. 3a for Au and in Fig. 3b for Ag dimers. The data in Fig. 3a and 3b allow for an analysis of the SERS signal enhancement of nanoparticle dimers with different gap sizes at specific Raman excitation wavelengths and in respect to the most common excitation lines used in Raman spectroscopy (i.e. 785 nm, 633 nm, 532 nm and 488 nm). Although the highest FE is predicted for the narrowest gap size (1.2 nm) around 736 nm for the Au dimer the FDTD simulations indicate that for Au dimers an excitation at 633 nm is advantageous because dimers with different gap sizes give rise to a SERS signal enhancement of 10 9 or higher. For example, the FE at 633 nm excitation wavelength is 255 for a 1.2 nm gap and 312 for a 2.5 nm gap, corresponding to a SERS signal enhancement of around 4.2x10 9 and 9.5x10 9 , respectively, using the E 4 approximation. 26 Besides the plasmonic enhancement, the SERS signal depends also on the optical properties of the analyte molecules, i.e. their optical absorption. The SERS signal can either be further enhanced by the resonance Raman effect by matching the molecule's optical absorption with the LSPR, 27 or nonresonant molecule excitation can be used to avoid photobleaching. spectra and S4 for the absorption spectra). Excitation at 532 nm as used for Ag dimers results in an SM SERS signal with considerably higher signal-to-noise ration than in previous SM SERS studies. 15 Interestingly, when using 633 nm excitation for the corresponding Au DONA, SM SERS of TAMRA can be measured, although TAMRA does not absorb at 633 nm and therefore resonant Raman contributions can be excluded in this case. This indicates that the plasmonic field enhancement at 633 nm is indeed strong enough to detect a single molecule under non-resonant conditions, which is also supported by our FDTD simulations. The same behaviour is observed for Cy3.5, which can be excited by the 532 nm laser, but not sufficiently by the 633 nm laser. Nevertheless, strong and characteristic single-molecule SERS spectra are obtained for both, Au and Ag DONAs. The strongest absorption of Cy5 is observed at around 650 nm (Fig. S4), close to the 633 nm excitation used for the Au dimers.
Consequently, a strong and characteristic single molecule SERS spectrum 28 is obtained for Cy5 using the Au DONAs. In contrast, DONAs containing a pure DNA bridge do not display any fingerprint spectra (see Fig. S9 and S10). Therefore, the success of SM SERS measurements using different dyes, nanoparticle species and excitation wavelengths demonstrate the strength and versatility of DONAs, i.e. the ability to tune the plasmonic properties according to the needs of the molecular systems to be investigated. After demonstrating the potential of DONAs to measure single dye molecules even under nonresonant conditions, we further extend our study to highlight the whole potential of DONAs to directly measure single protein molecules by SERS. This can be achieved with different coupling schemes exploiting either non-covalent or covalent binding of proteins. Here, we have used the heme proteins cytochrome c (cyt c) and horseradish peroxidase (HRP) as model systems to demonstrate their direct, label-free and non-resonant single-molecule SERS detection using DONAs (Fig. 5). Cyt c is found in the inner membranes of mitochondria and is involved in biological electron transfer processes, while HRP is involved in oxidation processes. 34,35 Heme proteins strongly absorb at around 420 nm (Soret or B band) and 550 nm (Q band), 36 however, we use the 633 nm excitation laser for the single molecule measurements, which is non-resonant with the heme proteins. In this way, not only the heme prosthetic group is visible in the spectra, but also the amino acid part of the proteins can be directly detected.

Fig. 5 | Single-molecule SERS measurements of cytochrome c (cyt c) and horseradish peroxidase (HRP). a,
Scheme of the non-covalent and covalent coupling schemes to bind cyt c and HRP to the DNA bridge of the nanofork. Cyt c is coupled non-covalently to pyridine, which is connected to the DNA bridge, while HRP is cross-linked to a DNA staple strand, which is part of the DNA bridge. b, SM SERS spectra of cyt c using Au   Table S2). HRP is covalently coupled to the NHS ester group of a Sulfo-SMCC cross linker via a lysine residue. The maleimide group of the cross linker is then bound to a thiolated staple strand (Table S2), which in turn is incorporated into the DNA bridge of the DNA origami nanofork. Both, cyt c and HRP are bound to the DNA origami nanofork prior to the binding of the 60 nm Au nanoparticles. Fig. 5b and 5c show the single-molecule SERS spectra of cyt c and HRP (blue and red spectra), respectively, recorded at 633 nm excitation along with the corresponding SEM images of Au dimers and a reference spectrum (black spectra). The reference spectra were recorded for cyt c or HRP, respectively, unspecifically bound to clusters of 60 nm Au nanoparticles (see Methods). The SERS spectrum of a single cyt c molecule recorded with a Au DONA matches with the reference spectrum. The characteristic band appearing in the range 1361 -1369 cm -1 is assigned to the stretching vibration of the porphyrin ring (ν4) of the heme group indicating the oxidation state (Fe 2+ ) of the central iron ion. 36,38 The strong signal around 1580 cm -1 is assigned to the ν19 band. 36 Apart from the signals arising from the heme unit, the vibrational ring-breathing mode of the amino acid phenylalanine (Phe) can also be clearly distinguished at 1001 cm -1 . 39 Cyt c possesses four Phe residues, and the possibility to detect these in a single cyt c molecule by SERS using DONAs indicates the high sensitivity of the method. Fig. 5c shows SM SERS spectra of HRP coupled to Au DONAs along with a reference spectrum (black). SERS signals characteristic for HRP can be clearly detected, such as the oxidation state marker at 1633 cm -1 (ν10), indicating the presence of Fe 3+ . 36 To the best of our knowledge, the present data are the first reports of SM SERS spectra of HRP under non-resonant conditions.
In conclusion, the newly designed 3D DNA origami nanofork has successfully been demonstrated for its suitability to assemble both nanoparticles and complex analyte molecules with nanoscale precision for SM SERS measurements. OxDNA simulations were employed to assess the structural dynamics of the nanofork and to choose the most suitable bridge length. The DNA bridge connecting the two arms of the nanofork allows for precise positioning of the target analyte in between the assembled nanoparticles of Au and Ag. This produces SERS hot spots in the scale of few nanometers, creating high enough field enhancement to enable label-free detection of single small molecules and even protein (cyt c and HRP) via SERS, even at larger gap sizes around 2.5 nm. Importantly, the flexible integration of either Au and Ag nanoparticles supports SM SERS measurements both in resonant and non-resonant conditions. Time-dependent SM SERS of TAMRA under non-resonant conditions shows spectral blinking but no significant photobleaching, strikingly reflecting the single-molecular SERS behaviour.
The experimental findings are supported by FDTD simulations that predict field enhancements high enough for single molecule measurements under non-resonant conditions. DNA origami is a remarkable technique that grants control over single molecular entities, thereby enabling novel types of nanomachines. 13,40 Here, we used this principle to build a platform for the direct, chemically-specific and label-free optical detection of single small molecules and single proteins. This opens new potential to study biomolecular reactions at the single molecule level. The DONA structures could be used to monitor enzymatic action in different environments, perhaps even for multi-enzyme reaction cascades. At the same time, the sensing of single small molecules represents one of the greatest challenges in analytical chemistry. Through the study of single reaction intermediates it could prove invaluable for understanding chemical reaction pathways.

DNA origami nanofork folding and purifications
The staple strands used for the assembly of the DNA origami nanofork are shown in Table S1. Depending on the experiment, some strands were substituted with the modified versions of the same strand (see Table S2). The and then cooled down to 20°C over 15 h timespan. The solution was purified from excess oligos using 100 kDa centrifugal filters (Amicon, Ultracel, Centrifugal filter, 100 kDA): the DNA origami solution (100 µl) was added to the filter and 400 µl of Millipore water was mixed in, the filter was spun in 4700 rcf for 5min, the filtrate was discarded and 450 µl of Millipore water was added. The centrifugation was repeated 3 times and the dead volume from the filter was finally recovered. The concentration of TAE and MgCl2 was adjusted to 1x and 5 mM, respectively. A UV-vis spectrometer was used to measure the DNA concentration of the final solution that was used to calculate the DNA origami concentration.
The gold nanoparticles were coated using a modified salt aging method, whereas the coating for silver nanoparticles was done using a pH altering method. 41 For AuNPs, 400 μl of 60 nm AuNP stock solution were centrifuged once (2900 rcf, 5 min), the supernatant was removed and the volume was adjusted to 26.5 μl by MilliQ water. The disulphide bonds in the DPTA-modified coating strands were pre-cleaved using TCEP: 4 µl of the coating strand solution (100 µM) was mixed with 1µl of TCEP (100 mM) and incubated at RT for 10 min. The 5 µl of cleaved DNA strand solution and 3.5 μl of 0.2 % SDS was added to the AuNP solution and incubated 40 min to 1h and at 40 °C. After the incubation, the concentration of NaBr was gradually, in stepwise manner increased to 300 mM using initially 400 mM NaBr buffer and then 1 M buffer, where after each step the solution was incubated for 10 min at 40 °C: initial 4 steps were 1.7 µl (400 mM), the next two 2.1 µl and 2.3 µl (400 mM), followed by 3x 2.3 µl and 1 M and finally 6.5µl and 1 M. After the NaBr addition, 1x TAE and 5 mM MgCl2 was added twice (6 µl and 6.6 µl) in similar stepwise manner. Finally, the excess coating strands were purified by centrifugation: the sample was spun down using 2900 rcf and 5 min, supernatant was removed and buffer added.
During the first two round of centrifugation, 1xTAE, 5 mM MgCl2 and 0.02 % SDS buffer was used and during the final two rounds, the buffer was switched to 1xTAE and 5 mM MgCl2. was deposited on a TEM grid. For DONA deposition, no plasma-treatment was required whereas, for nanoforks, the TEM grids were plasma-treated for 15 s using Diener Electronic Zepto plasma cleaner before deposition of the DNA origami and the staining. In the case of DONAs, 3 µl of DONA solution was pipetted to the grid and incubated for 2-3 min. The excess solution was blotted away and 3 µl of staining solution was added. After 2 min incubation, the excess staining solution was blotted away and the film was washed twice by adding 3 µl of Millipore water to the grid and blotting away the excess liquid. The grid was let to dry in ambient conditions.
In the case of DNA origami nanofork, after the plasma treatment, roughly 3 µl of DNA origami solution was pipetted on a grid and incubated for 5 min. The magnesium and fork concentrations were 20 mM and 0.6 nM, respectively. The excess solution was blotted away and 3 µl of the same staining solution was added. After 2 min incubation, the excess solution was blotted away and the sample was left to dry in ambient condition without any washing. Figure S7 contains extra images of the DNA origami nanoforks.
Sample preparation for SERS, SERS measurements, SEM imaging and SERS-SEM correlation DONAs were deposited on a 7 mm x 7 mm silicon chip labelled with a scratch-marker. A diamond cutter was used to draw a cross-shape on the chip, the surface was scrubbed clean from dust in acetone solution using a cotton stick and finally sonicating the chip in ethanol solution for 30 s. The clean chip was plasma-treated for 10 min.
For the deposition, the magnesium concentration of DONA solution was adjusted to 50 mM, the solution was pipetted to plasma-treated silicon surface and incubated for roughly 1.5 h. The chip was washed with ethanolwater solution (1:1 ratio) and blown dry.
A Witec Alpha 300 Raman microscope was used in the measurements (Witec, Ulm, Germany). For the 488 and 532 nm measurements a grating spectrograph (Blaze 500, grating 600 gr/mm) equipped with Andor DV401-BV CCD-camera was used, and for the 633 nm measurements a spectrograph (Blaze 750, grating 600 gr/mm) equipped with Andor DU401A-BR-DD-352 CCD-camera. The estimated spot sizes were 1.32 µm, 1.44 µm and 1.72 µm for 488 nm, 532 nm and 633 nm, respectively. Raman maps were acquired using varying laser power densities and integration times per point (see SI). After a Raman measurement, the Raman map area was imaged using SEM (Thermo Fisher Phenom ProX Desktop SEM or FEI Quanta 250) where the scratch-markers acted as reference points to identify the area scanned with the Raman microscope. For the dye references, particles coated with dye-polyT oligos were deposited on a silicon surface and point spectra were measured using 532 or 633 nm laser (see SI for more details). For protein samples, the proteins were incubated with the nanoparticles before to adsorb them to the particle surface and cause aggregation. Then the aggregates were deposited on a silicon surface and point measurements were performed using the 633 nm laser and 300 µW (200 µW) laser power and 8 s (12 s) integration time for cyt c (HRP).

FDTD simulations
A detailed overview of the FDTD simulations can be found in the SI. In brief, we used the Lumerical FDTD Solutions software (v8. 19.1584). The model included two gold or silver spheres (refractive index from Johnson and Christy) 42 surrounded by 1.12 nm DNA layer (refractive index 1.7) 43 and on top of a silicon substrate (refractive index from 44 ) with 3 nm silicon dioxide (refractive index 1.44) on top of the Si (see Fig. S21). The DNA layer thickness was measured using AFM (Figs. S19 and S20). Here, we are omitting the bridge and the DNA origami nanofork from the framework. The medium was defined as air. The distance between gold spheres was varied according to the measured distance from Fig. 1 (from 1.2 nm to 3.5 nm), where a water layer was added between DNA layers in the case of 2.5 and 3 nm particle-to-particle distances. The E field distributions were calculated and the field maximum around the gap region was recorded. To solve the E0 case, we removed the DNA layers and the metal spheres. Electric field polarizations along the gap (the gap mode) and perpendicular to gap (the offgap mode) were considered. The results show that the electric field is highly localized within the gap region in the case of the gap mode and the off-gap mode excitation results mainly in plasmonic mode that resembled single particle dipole excitation (Figs. S22-S26).

OxDNA simulations
We performed Langevin molecular dynamics simulations for the three nanofork structures considered (90 nt, 120 nt and no bridge) using the coarse-grained DNA model oxDNA2, 24,25 parametrized to reproduce mechanical and thermodynamic experimental properties of DNA. The initial oxDNA configurations were obtained converting the caDNAno files with the tacoxDNA package. 45 Simulations were carried out with the LAMMPS software implementation. 46 The temperature was set to T = 300 K and the monovalent salt concentration to 1 M. Each system was simulated for about 2 x 10 6 simulation time units, for a total of 2000 configurations sampled.
IB conceived the study, KT and AM prepared and characterized DONAs, KT did the TEM imaging and dye measurements and performed FDTD simulations, AM did sample preparations and measurement involving cyt c, YK did sample preparations and measurement involving HRP, AS performed OxDNA simulations, and all authors analysed the data and contributed to manuscript writing.