Advances in DNA Origami–Templated Nanostructures

The DNA origami mould approach has expanded to palladium nanowires with metallic conductivity (Kemper et al., 2023; Rodriguez-Barea et al., 2026) and gold nanoparticle multimers for ultrasensitive SERS. DNA origami nanopore technology has evolved through three structural phases: hybrid designs with solid-state platforms, vertically inserted nanopores, and horizontally arranged nanopores with functional modifications (aptamers, enzymes) for selective detection. The integration of DNA origami with whispering gallery mode (WGM) sensors has enabled label-free single-molecule DNA hybridization detection (Ghamari et al., 2025).

Sequence-Resolved Single-Molecule Kinetics (ThermoSPARXS)

The development of ThermoSPARXS within this project has enabled the first simultaneous measurement of kon and koff for 79 DNA probe sequences across ten temperatures (26–58°C). The key finding — that koff is strongly temperature- and sequence-dependent while kon is nearly invariant — provides quantitative design rules for engineering thermal switching thresholds. Complementary advances include DNA calorimetric force spectroscopy (Ritort group, 2024–2025) and temperature-jump infrared spectroscopy of dinucleotide hybridisation kinetics (Szostak group, 2023).

Temperature-Dependent Strand Displacement

Within this project, the temperature dependence of toehold-mediated strand displacement has been systematically characterised, revealing a striking non-monotonic behaviour: rates initially increase with temperature but decrease at elevated temperatures due to the interplay between reversible toehold hybridisation and irreversible strand replacement. A Markov chain model successfully describes this behaviour across different toehold lengths (5–10 nt). Invader-target mismatches were shown to enhance temperature sensitivity, providing design handles for thermally responsive DNA devices.

Hybrid Plasmonic DNA Nanopores for SERS

Gold nanoparticles have been synthesised inside DNA origami nanopores through templated seeded growth, creating hybrid plasmonic nanocages. Four cage variants with alkyne reporters at different positions (inner cavity, outer lid, outer mold) were characterised by SERS at 633 nm excitation. The terminal alkyne reporter was detected in all functionalised variants, with spectral response strongly sensitive to reporter position — the inner cage showed intimate metal–DNA coupling while the outer lid cage produced the highest signal intensity. These results establish the DNA origami nanocage as a programmable SERS platform. Systematic studies of molecular orientation effects on SERS using DNA origami antennas (Kanehira et al., 2026) have further advanced the field.

Single-Molecule Thermometry

A ratiometric fluorescence thermometry approach using Alexa 647 / Atto 647N dye pairs has been developed and validated at both bulk and single-molecule levels. A DNA origami-based super-resolution thermometry platform — using DNA-PAINT to resolve individual reporter sites with ~30 nm precision — has been demonstrated, enabling sub-diffraction temperature mapping. These tools provide the thermal characterisation infrastructure needed for all temperature-dependent sensing modalities within the DYNAMO framework.

Convergence and Outlook

The current state of the art is characterised by the convergence of previously separate fields. DNA origami–templated plasmonic nanostructures can now be fabricated with increasing complexity. A comprehensive kinetic framework for DNA hybridization and strand displacement exists to rationally design thermal switching behaviour. Programmable SERS detection within DNA origami nanocages has been demonstrated. And validated thermometry tools enable precise nanoscale temperature characterisation. The remaining challenges include scaling to integrated hybrid devices, translating design rules from surface-tethered to nanopore-confined geometries, and combining optical (SERS/fluorescence) with electrical (ionic current) readout in a single platform.

Selected Output

• N. Kumar, F. Ricci, R. Seidel, P. Irmisch. "Engineering the temperature response of DNA strand displacement." Nature Communications [Under Review]
• N. Kumar, A. Overchenko, A. Sivaraman, C. Bastiaanssen, P. Irmisch, F. Cichos, R. Seidel, C. Joo. "Temperature- and sequence-dependent DNA hybridization kinetics from high-throughput single-molecule measurements." [In Preparation — Nature Chemistry]
• N. Kumar et al. "Probing sequence-specific DNA hybridization dynamics using ThermoSPARXS." Biophysical Journal, 125(4), 287a (2026).
• S. Banerjee, C. Hadlich, M. Scherf, N. Kumar, R. Seidel, J. Kneipp. "SERS detects site-specific labeling of DNA nanocages via hexynyl modifications." [In Preparation]
• N. Kumar, A. Sivaraman, P. Irmisch, D. Renger, C. Joo, R. Seidel. "DNA Origami-Enabled Single-Molecule Ratiometric Thermometry with Nanoscale Spatial Resolution." [In Preparation]

Coupled nanoscale transport and improved EOF resolution

Since the beginning of DYNAMO, the field has progressed toward a deeper understanding of coupled nanoscale transport phenomena[cite: 13]. Recent work has focused on improving the spatial resolution of electroosmotic flow measurements and investigating EOF behavior under different ionic environments and salt concentrations [13,19][cite: 13]. Three-dimensional, optical-tweezer-based flow-field mapping outside glass nanopores has been refined to capture the full out-of-plane structure of voltage-driven flow, revealing polarity-dependent flow fields consistent with competing inner- and outer-wall contributions [13][cite: 13]. Joint experimental and computational studies have further dissected the role of buffer salt concentration and catton identity in setting the magnitude and sign of EOF inside both biological and solid-state pores, showing strongly non-monotonic dependencies that classical Poisson–Nernst–Planck descriptions only partly capture [19,20][cite: 13]. These studies aim to better understand how ion-specific effects and nanoscale confinement influence flow dynamics at solid–liquid interfaces[cite: 13]. At the same time, the use of dark-field optical imaging and high-speed scattering microscopy has enabled more detailed investigation of transport processes with higher spatial and temporal resolution, including single-particle tracking near the pore mouth and inside nanocapillaries [21,22][cite: 13].

Single-molecule DNA hybridization and reconfigurable DNA-origami nanopores

In parallel, recent studies have increasingly focused on the physical mechanisms governing DNA hybridization and melting dynamics rather than only demonstrating structural switching [17,23][cite: 13]. In particular, nucleation and zipping models describing DNA binding and melting are now being investigated experimentally at the single-molecule level through kinetic measurements [23,24][cite: 13]. Hertel et al. (2022) quantified how the stability and number of nucleating interactions determine hybridization rates in the absence of secondary structure [23][cite: 13], while single-molecule fluorescence and DNA-PAINT approaches have provided base-pair-resolved access to association and dissociation kinetics [24][cite: 13]. On the DNA-origami side, fully reversible, mechanically compliant origami nanopores (MechanoPores) that switch between closed, intermediate, and open states by strand displacement have recently been demonstrated and inserted into lipid membranes, achieving size-selective transmembrane transport [25][cite: 13]. These studies provide deeper insight into reversible DNA-origami reconfiguration and may become important for future adaptive nanopore systems with controllable trapping and molecular-transport capabilities[cite: 13].

Outlook and remaining challenges

Current research focuses on understanding the fundamental physics governing nanoscale transport, reversible DNA reconfiguration, and electrohydrodynamic interactions under confined conditions[cite: 13]. Overall, the combination of DNA origami, electroosmotic flow, thermally responsive nanosystems, and single-molecule transport studies represents a promising direction toward adaptive nanopore sequencing technologies[cite: 13]. Coupling EOF and DNA-origami inserts in a single device introduces additional complexity, since the deformability and surface charge of the origami modify the local flow field and ionic environment in ways that are not yet fully predictable [2,25][cite: 13]. Substantial scientific and technological challenges therefore remain — in particular regarding low-noise integration, long-term stability of origami inserts in solid-state membranes, and quantitative modelling of coupled electrohydrodynamic and thermal effects — before fully integrated and scalable sequencing devices can be realized[cite: 13].

Selected Output

• [13] Mc Hugh, J., Thorneywork, A. L., Andresen, K. & Keyser, U. F. 3D flow field measurements outside nanopores. Rev. Sci. Instrum. 93, 053703 (2022)[cite: 13].
• [17] Ouldridge, T. E. et al. DNA hybridization kinetics: zippering, internal displacement and sequence dependence. Nucleic Acids Res. 41, 8886–8895 (2013)[cite: 13].
• [19] Bhattacharya, S. et al. Changes in salt concentration modify the translocation of neutral molecules through a ΔCymA nanopore in a non-monotonic manner. ACS Nano 16, 9355–9366 (2022)[cite: 13].
• [20] Brown, W. et al. Higher ion selectivity with lower energy usage promoted by electro-osmotic flow in the transport through conical nanopores. J. Phys. Chem. C 125, 3269–3276 (2021)[cite: 13].
• [21] Teahan, J. et al. Scanning ion conductance microscopy: surface charge effects on electroosmotic flow delivery from a nanopipette. Anal. Chem. 93, 12281–12288 (2021)[cite: 13].
• [22] Verpillat, F., Joud, F., Desbiolles, P. & Gross, M. Dark-field digital holographic microscopy for 3D-tracking of gold nanoparticles. Opt. Express 19, 26044–26055 (2011)[cite: 13].
• [23] Hertel, S. et al. The stability and number of nucleating interactions determine DNA hybridization rates in the absence of secondary structure. Nucleic Acids Res. 50, 7829–7841 (2022)[cite: 13].
• [24] Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014)[cite: 13].
• [25] Yu, L. et al. Compliant DNA origami nanoactuators as size-selective nanopores. Adv. Mater. 36, 2405104 (2024)[cite: 13].

Plasmon-enhanced spectroscopy at nanopores

Recent years have seen major advances in integrated nanopore sensing electronics, particularly in the development of CMOS ASICs dedicated to low-current biosensing applications[cite: 11]. Modern nanopore readout systems increasingly focus on improving noise performance, scalability, integration density, and compatibility with large sensor arrays[cite: 11].

Within this context, the work developed in the DYNAMO project contributed to the realization of a complete ASIC-based nanopore sensing platform centered around the custom dual-channel ASIC QC01a[cite: 11]. The platform integrates low-noise analog front-end circuitry, DC current handling, data acquisition electronics, PCB implementation, and real-time digital processing[cite: 11].

One of the main innovations of the developed system is the implementation of a PN-junction diode feedback technique in the transimpedance amplifier architecture[cite: 11]. Unlike conventional pseudo-resistors relying on subthreshold channel conduction, the proposed approach uses MOSFET PN-junction conduction to realize ultra-high equivalent resistance with reduced thermal and flicker noise contribution[cite: 11]. The developed system experimentally demonstrated minimum input-referred current noise levels of approximately 1.5 fA/√Hz together with readout bandwidths up to 1 MHz[cite: 11].

The platform was experimentally validated using solid-state nanopore measurements with Lambda DNA translocation experiments performed in 1 M KCl electrolyte solution[cite: 11]. Stable baseline operation and clear transient current blockade events were successfully detected, demonstrating the capability of the system for high-sensitivity nanopore sensing applications[cite: 11].

The developed work also resulted in scientific dissemination and intellectual property generation, including an accepted IEEE Solid-State Circuits Letters (SSC-L) publication and two patent applications related to the developed readout technology[cite: 11]. Additional journal publications are currently under preparation[cite: 11].

Updated field direction

The field has increasingly moved from simple detection of translocation events towards quantitative, interpretable and multi-modal readout. Modern nanopore experiments do not only ask whether a molecule passes through a pore; they ask how the molecule moves, how local features are mapped onto the signal, and how experimental conditions can be adjusted to improve resolution. This is especially important for applications involving molecular barcodes, labelled DNA carriers, short nucleic acids, protein-nucleic-acid complexes and future electro-optical sensing platforms.

Contribution 1: conceptual framework

The first major contribution of the PhD work was a comprehensive review of double-stranded nucleic-acid translocation through solid-state nanopores, now published in Physics of Life Reviews [1]. The review organised the field around the physical processes that determine translocation, including electrophoretic driving, hydrodynamic drag, electro-osmotic effects, polymer conformation, pore geometry, folding and signal formation. This provided a broad framework for interpreting nanopore experiments and for identifying which transport effects matter for future high-resolution sensing.

Contribution 2: molecular labels and readout

A second contribution was an experimental study of how dense molecular labelling affects DNA translocation. Labels are central to many nanopore readout strategies because they can encode sequence position, protein binding or structural information along a carrier molecule. The work showed that dense labelling can leave the global translocation time largely preserved, and the overall velocity profile is preserved across labels. This result is important for designing labelled nanopore assays: labels can be used for obtaining positional and structural information without needing label-specific corrections. [2]

Contribution 3: transport control and electrohydrodynamics

Ongoing work extends this theme by investigating stronger slowdown mechanisms and investigating electro-osmotic flow in nanopore systems with a custom designed electro-optical setup with unprecedented flow resolution, in collaboration with JR(?). Slowing molecules down is directly relevant to the original DYNAMO challenge, because longer residence times increase the time available for electrical or optical interrogation. Electro-osmotic flow measurements are also important because flow can either assist or oppose electrophoretic motion and can vary with salt type, concentration, pH and pore geometry. Understanding these effects helps explain why the same molecule may behave differently under different nanopore conditions. We also demonstrated the passage of multiple DNA&RNA sequences in parallel through two coupled nanopores, one of the key deliverables, demonstrating scalability of the approach.

Relevance to DYNAMO

Taken together, the PhD work contributes to the DYNAMO objective of improved single-molecule nanopore readout by addressing the transport bottleneck that underlies electro-optical detection. The work does not merely optimise one assay; it clarifies the physical conditions required for reliable readout of nucleic acids and molecular features through nanopores. This provides a basis for future implementations in which optical or plasmonic signals are combined with electrical detection to identify short nucleic acids, sequence-dependent features or molecular modifications.

Selected Output

• [1] Brauburger and Keyser, A biophysicist's guide to translocation of double-stranded nucleic acids through solid-state nanopores, Physics of Life Reviews, 2026.
• [2] Brauburger et al., Label type influence on DNA translocation velocity in solid-state nanopores, submitted revisions, ACS Nano, 2026