Single-molecule sensing with DNA origami

Biosensing with plasmonic signal amplification

Signal amplification strategies are essential for improving sensitivity, speed, and robustness of sensing assays for point-of-care diagnostic applications. One strategy to achieve this relies on physical fluorescence signal amplification by plasmonic nanostructures that act as optical nanoantennas concentrating the incident excitation light into zeptoliter volumes and enhancing the radiative decay rate of fluorescent molecules. In our lab, we exploit the unique positioning precision of DNA origami to directly place the diagnostic elements in the plasmonic hotspots of silver and gold dimer nanoantennas (Figure 1). Using fluorescence-based diagnostic assays in the hotspots of such nanoantennas we can achieve the signal amplification reaching several hundred-fold.

Figure 1. Illustration of DNA origami nanoantenna. DNA origami platform is utilized to position plasmonic nanoparticles and create plasmonic hotspots. Placing diagnostic elements in the hotspot region of the nanoantenna allows to enhance the fluorescence signal up to few hundred fold.

Our recent progress in the development of DNA nanoantenanas for diagnostics includes the development of addressable NAnoantennas with Cleared plasmonic HOtspotS (NACHOS). NACHOS enabled us to incorporate the diagnostic assay specific to DNA of antibiotic resistant bacteria directly into the hotspot of dimer DNA nanoantenna as well as demonstrate the detection of single molecules on a cheap and low-tech diagnostic platform such as a portable smartphone microscope (Figure 2).

Figure 2. a) DNA origami structure used to scaffold addressable NanoAntennas with Cleared HOtSpots (NACHOS); b) Structure of a full DNA origami nanoantenna containing two 60-nm gold nanoparticles; c) Fluorescence enhancement values obtained for NACHOS containing two 100-nm silver nanoparticles; d) sketch of the portable smartphone-based microscope used to detect single fluorescent molecules with the help of NACHOS; e) picture of the portable microscope (left) and images as well as single-molecule trajectories acquired on the smartphone camera (right).

Molecular voltage sensors

The electrical potential of the cell membrane is important for many signaling pathways, e.g. neuronal activity or apoptosis. Using DNA nanotechnology, we are developing a novel family of voltage sensitive probes with fluorescent read-out where we can design the different sensor elements individually and modularly to combine them into a nanometer-sized device. The sensor is based on FRET and employs a new principle using a hydrophobic ATTO647N dye that anchors the sensor unit in the membrane (Figure 3a). A donor dye is placed on negatively charged DNA linkers that are attached to the DNA origami. Depending on the potential, the flexible element with the donor changes its position relative to the FRET acceptor in the membrane yielding a FRET change that is read out on single FRET pairs. Besides transmembrane potentials, a similar principle can also be employed to study surface potentials induced by the composition of lipids with different charges in large unilamellar vesicles (Figure 3b).

Figure 3. Working principles of DNA origami FRET-based sensor for a) transmembrane potential b) surface charge.

In addition to the single-molecule sensing approaches described above we are also exploring the possibility of using DNA origami method to build a general platform for the development of tunable fluorescence sensors. We aim at tuning and controlling the dynamic range of the single-molecule sensors, introducing signal amplification mechanisms and expanding the utility of the biosensors beyond the limits dictated by the bio-recognition interaction itself.

Publications

Quantitative Single-Molecule Measurements of Membrane Charges with DNA Origami Sensors

Ochmann, Sarah E; Schröder, Tim; Schulz, Clara M; Tinnefeld, Philip

Quantitative Single-Molecule Measurements of Membrane Charges with DNA Origami Sensors Journal Article

In: Anal Chem, 2022, ISSN: 1520-6882.

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DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity

Ochmann, Sarah E.; Joshi, Himanshu; Büber, Ece; Franquelim, Henri G.; Stegemann, Pierre; Saccà, Barbara; Keyser, Ulrich F.; Aksimentiev, Aleksei; Tinnefeld, Philip

DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity Journal Article

In: Nano Letters, vol. 21, no. 20, pp. 8634–8641, 2021.

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Single antibody detection in a DNA origami nanoantenna

Pfeiffer, Martina; Trofymchuk, Kateryna; Ranallo, Simona; Ricci, Francesco; Steiner, Florian; Cole, Fiona; Glembockyte, Viktorija; Tinnefeld, Philip

Single antibody detection in a DNA origami nanoantenna Journal Article

In: iScience, vol. 24, no. 9, pp. 103072, 2021.

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DNA Origami Nanoantennas for Fluorescence Enhancement

Glembockyte, Viktorija; Grabenhorst, Lennart; Trofymchuk, Kateryna; Tinnefeld, Philip

DNA Origami Nanoantennas for Fluorescence Enhancement Journal Article

In: Acc Chem Res, vol. 54, no. 17, pp. 3338–3348, 2021.

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Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope

Trofymchuk, Kateryna; Glembockyte, Viktorija; Grabenhorst, Lennart; Steiner, Florian; Vietz, Carolin; Close, Cindy; Pfeiffer, Martina; Richter, Lars; Schütte, Max L.; Selbach, Florian; Yaadav, Renukka; Zähringer, Jonas; Wei, Qingshan; Ozcan, Aydogan; Lalkens, Birka; Acuna, Guillermo P.; Tinnefeld, Philip

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope Journal Article

In: Nature Communications, vol. 12, no. 1, 2021.

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Hemmig, Elisa A.; Fitzgerald, Clare; Maffeo, Christopher; Hecker, Lisa; Ochmann, Sarah E.; Aksimentiev, Aleksei; Tinnefeld, Philip; Keyser, Ulrich F.

Optical Voltage Sensing Using DNA Origami Journal Article

In: Nano Letters, vol. 18, no. 3, pp. 1962–1971, 2018.

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Kaminska, Izabela; Vietz, Carolin; Cuartero-González, Álvaro; Tinnefeld, Philip; Fernández-Domínguez, Antonio I.; Acuna, Guillermo P.

Strong plasmonic enhancement of single molecule photostability in silver dimer optical antennas Journal Article

In: Nanophotonics, vol. 7, no. 3, pp. 643–649, 2018.

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Kaminska, Izabela; Bohlen, Johann; Mackowski, Sebastian; Tinnefeld, Philip; Acuna, Guillermo P.

Strong Plasmonic Enhancement of a Single Peridinin–Chlorophyll a–Protein Complex on DNA Origami-Based Optical Antennas Journal Article

In: ACS Nano, vol. 12, no. 2, pp. 1650–1655, 2018.

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Ochmann, Sarah E.; Vietz, Carolin; Trofymchuk, Kateryna; Acuna, Guillermo P.; Lalkens, Birka; Tinnefeld, Philip

Optical Nanoantenna for Single Molecule-Based Detection of Zika Virus Nucleic Acids without Molecular Multiplication Journal Article

In: Analytical Chemistry, vol. 89, no. 23, pp. 13000–13007, 2017.

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Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

Plasmonics Enhanced Smartphone Fluorescence Microscopy Journal Article

In: Scientific Reports, vol. 7, no. 1, 2017.

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Vietz, Carolin; Kaminska, Izabela; Paz, Maria Sanz; Tinnefeld, Philip; Acuna, Guillermo P.

Broadband Fluorescence Enhancement with Self-Assembled Silver Nanoparticle Optical Antennas Journal Article

In: ACS Nano, vol. 11, no. 5, pp. 4969–4975, 2017.

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