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
Büber, Ece; Schröder, Tim; Scheckenbach, Michael; Dass, Mihir; Franquelim, Henri G; Tinnefeld, Philip
@article{pmid36735241,
title = {DNA Origami Curvature Sensors for Nanoparticle and Vesicle Size Determination with Single-Molecule FRET Readout},
author = {Ece Büber and Tim Schröder and Michael Scheckenbach and Mihir Dass and Henri G Franquelim and Philip Tinnefeld},
doi = {10.1021/acsnano.2c11981},
issn = {1936-086X},
year = {2023},
date = {2023-02-01},
urldate = {2023-02-01},
journal = {ACS Nano},
abstract = {Particle size is an important characteristic of materials with a direct effect on their physicochemical features. Besides nanoparticles, particle size and surface curvature are particularly important in the world of lipids and cellular membranes as the cell membrane undergoes conformational changes in many biological processes which leads to diverging local curvature values. On account of that, it is important to develop cost-effective, rapid and sufficiently precise systems that can measure the surface curvature on the nanoscale that can be translated to size for spherical particles. As an alternative approach for particle characterization, we present flexible DNA nanodevices that can adapt to the curvature of the structure they are bound to. The curvature sensors use Fluorescence Resonance Energy Transfer (FRET) as the transduction mechanism on the single-molecule level. The curvature sensors consist of segmented DNA origami structures connected via flexible DNA linkers incorporating a FRET pair. The activity of the sensors was first demonstrated with defined binding to different DNA origami geometries used as templates. Then the DNA origami curvature sensors were applied to measure spherical silica beads having different size, and subsequently on lipid vesicles. With the designed sensors, we could reliably distinguish different sized nanoparticles within a size range of 50-300 nm as well as the bending angle range of 50-180°. This study helps with the development of more advanced modular-curvature sensing devices that are capable of determining the sizes of nanoparticles and biological complexes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Particle size is an important characteristic of materials with a direct effect on their physicochemical features. Besides nanoparticles, particle size and surface curvature are particularly important in the world of lipids and cellular membranes as the cell membrane undergoes conformational changes in many biological processes which leads to diverging local curvature values. On account of that, it is important to develop cost-effective, rapid and sufficiently precise systems that can measure the surface curvature on the nanoscale that can be translated to size for spherical particles. As an alternative approach for particle characterization, we present flexible DNA nanodevices that can adapt to the curvature of the structure they are bound to. The curvature sensors use Fluorescence Resonance Energy Transfer (FRET) as the transduction mechanism on the single-molecule level. The curvature sensors consist of segmented DNA origami structures connected via flexible DNA linkers incorporating a FRET pair. The activity of the sensors was first demonstrated with defined binding to different DNA origami geometries used as templates. Then the DNA origami curvature sensors were applied to measure spherical silica beads having different size, and subsequently on lipid vesicles. With the designed sensors, we could reliably distinguish different sized nanoparticles within a size range of 50-300 nm as well as the bending angle range of 50-180°. This study helps with the development of more advanced modular-curvature sensing devices that are capable of determining the sizes of nanoparticles and biological complexes.
@article{pmid36594816,
title = {Gold Nanorod DNA Origami Antennas for 3 Orders of Magnitude Fluorescence Enhancement in NIR},
author = {Kateryna Trofymchuk and Karol Kołątaj and Viktorija Glembockyte and Fangjia Zhu and Guillermo P Acuna and Tim Liedl and Philip Tinnefeld},
doi = {10.1021/acsnano.2c09577},
issn = {1936-086X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {ACS Nano},
abstract = {DNA origami has taken a leading position in organizing materials at the nanoscale for various applications such as manipulation of light by exploiting plasmonic nanoparticles. We here present the arrangement of gold nanorods in a plasmonic nanoantenna dimer enabling up to 1600-fold fluorescence enhancement of a conventional near-infrared (NIR) dye positioned at the plasmonic hotspot between the nanorods. Transmission electron microscopy, dark-field spectroscopy, and fluorescence analysis together with numerical simulations give us insights on the heterogeneity of the observed enhancement values. The size of our hotspot region is ∼12 nm, granted by using the recently introduced design of NAnoantenna with Cleared HotSpot (NACHOS), which provides enough space for placing of tailored bioassays. Additionally, the possibility to synthesize nanoantennas in solution might allow for production upscaling.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
DNA origami has taken a leading position in organizing materials at the nanoscale for various applications such as manipulation of light by exploiting plasmonic nanoparticles. We here present the arrangement of gold nanorods in a plasmonic nanoantenna dimer enabling up to 1600-fold fluorescence enhancement of a conventional near-infrared (NIR) dye positioned at the plasmonic hotspot between the nanorods. Transmission electron microscopy, dark-field spectroscopy, and fluorescence analysis together with numerical simulations give us insights on the heterogeneity of the observed enhancement values. The size of our hotspot region is ∼12 nm, granted by using the recently introduced design of NAnoantenna with Cleared HotSpot (NACHOS), which provides enough space for placing of tailored bioassays. Additionally, the possibility to synthesize nanoantennas in solution might allow for production upscaling.
@article{pmid35089694,
title = {Quantitative Single-Molecule Measurements of Membrane Charges with DNA Origami Sensors},
author = {Sarah E Ochmann and Tim Schröder and Clara M Schulz and Philip Tinnefeld},
doi = {10.1021/acs.analchem.1c05092},
issn = {1520-6882},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Anal Chem},
abstract = {Charges in lipid head groups generate electrical surface potentials at cell membranes, and changes in their composition are involved in various signaling pathways, such as T-cell activation or apoptosis. Here, we present a DNA origami-based sensor for membrane surface charges with a quantitative fluorescence read-out of single molecules. A DNA origami plate is equipped with modifications for specific membrane targeting, surface immobilization, and an anionic sensing unit consisting of single-stranded DNA and the dye ATTO542. This unit is anchored to a lipid membrane by the dye ATTO647N, and conformational changes of the sensing unit in response to surface charges are read out by fluorescence resonance energy transfer between the two dyes. We test the performance of our sensor with single-molecule fluorescence microscopy by exposing it to differently charged large unilamellar vesicles. We achieve a change in energy transfer of ∼10% points between uncharged and highly charged membranes and demonstrate a quantitative relation between the surface charge and the energy transfer. Further, with autocorrelation analyses of confocal data, we unravel the working principle of our sensor that is switching dynamically between a membrane-bound state and an unbound state on the timescale of 1-10 ms. Our study introduces a complementary sensing system for membrane surface charges to previously published genetically encoded sensors. Additionally, the single-molecule read-out enables investigations of lipid membranes on the nanoscale with a high spatial resolution circumventing ensemble averaging.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Charges in lipid head groups generate electrical surface potentials at cell membranes, and changes in their composition are involved in various signaling pathways, such as T-cell activation or apoptosis. Here, we present a DNA origami-based sensor for membrane surface charges with a quantitative fluorescence read-out of single molecules. A DNA origami plate is equipped with modifications for specific membrane targeting, surface immobilization, and an anionic sensing unit consisting of single-stranded DNA and the dye ATTO542. This unit is anchored to a lipid membrane by the dye ATTO647N, and conformational changes of the sensing unit in response to surface charges are read out by fluorescence resonance energy transfer between the two dyes. We test the performance of our sensor with single-molecule fluorescence microscopy by exposing it to differently charged large unilamellar vesicles. We achieve a change in energy transfer of ∼10% points between uncharged and highly charged membranes and demonstrate a quantitative relation between the surface charge and the energy transfer. Further, with autocorrelation analyses of confocal data, we unravel the working principle of our sensor that is switching dynamically between a membrane-bound state and an unbound state on the timescale of 1-10 ms. Our study introduces a complementary sensing system for membrane surface charges to previously published genetically encoded sensors. Additionally, the single-molecule read-out enables investigations of lipid membranes on the nanoscale with a high spatial resolution circumventing ensemble averaging.
@article{Ochmann2021,
title = {DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity},
author = {Sarah E. Ochmann and Himanshu Joshi and Ece Büber and Henri G. Franquelim and Pierre Stegemann and Barbara Saccà and Ulrich F. Keyser and Aleksei Aksimentiev and Philip Tinnefeld},
doi = {10.1021/acs.nanolett.1c02584},
year = {2021},
date = {2021-10-01},
urldate = {2021-10-01},
journal = {Nano Letters},
volume = {21},
number = {20},
pages = {8634--8641},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Pfeiffer2021,
title = {Single antibody detection in a DNA origami nanoantenna},
author = {Martina Pfeiffer and Kateryna Trofymchuk and Simona Ranallo and Francesco Ricci and Florian Steiner and Fiona Cole and Viktorija Glembockyte and Philip Tinnefeld},
doi = {10.1016/j.isci.2021.103072},
year = {2021},
date = {2021-09-01},
urldate = {2021-09-01},
journal = {iScience},
volume = {24},
number = {9},
pages = {103072},
publisher = {Elsevier BV},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Glembockyte2021,
title = {DNA Origami Nanoantennas for Fluorescence Enhancement},
author = {Viktorija Glembockyte and Lennart Grabenhorst and Kateryna Trofymchuk and Philip Tinnefeld},
doi = {10.1021/acs.accounts.1c00307},
year = {2021},
date = {2021-08-01},
urldate = {2021-08-01},
journal = {Acc Chem Res},
volume = {54},
number = {17},
pages = {3338--3348},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Trofymchuk2021,
title = {Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope},
author = {Kateryna Trofymchuk and Viktorija Glembockyte and Lennart Grabenhorst and Florian Steiner and Carolin Vietz and Cindy Close and Martina Pfeiffer and Lars Richter and Max L. Schütte and Florian Selbach and Renukka Yaadav and Jonas Zähringer and Qingshan Wei and Aydogan Ozcan and Birka Lalkens and Guillermo P. Acuna and Philip Tinnefeld},
doi = {10.1038/s41467-021-21238-9},
year = {2021},
date = {2021-02-01},
urldate = {2021-02-01},
journal = {Nature Communications},
volume = {12},
number = {1},
publisher = {Springer Science and Business Media LLC},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Hemmig2018,
title = {Optical Voltage Sensing Using DNA Origami},
author = {Elisa A. Hemmig and Clare Fitzgerald and Christopher Maffeo and Lisa Hecker and Sarah E. Ochmann and Aleksei Aksimentiev and Philip Tinnefeld and Ulrich F. Keyser},
doi = {10.1021/acs.nanolett.7b05354},
year = {2018},
date = {2018-02-01},
journal = {Nano Letters},
volume = {18},
number = {3},
pages = {1962--1971},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Kaminska2018a,
title = {Strong plasmonic enhancement of single molecule photostability in silver dimer optical antennas},
author = {Izabela Kaminska and Carolin Vietz and Álvaro Cuartero-González and Philip Tinnefeld and Antonio I. Fernández-Domínguez and Guillermo P. Acuna},
doi = {10.1515/nanoph-2017-0081},
year = {2018},
date = {2018-02-01},
journal = {Nanophotonics},
volume = {7},
number = {3},
pages = {643--649},
publisher = {Walter de Gruyter GmbH},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Kaminska2018,
title = {Strong Plasmonic Enhancement of a Single Peridinin–Chlorophyll a–Protein Complex on DNA Origami-Based Optical Antennas},
author = {Izabela Kaminska and Johann Bohlen and Sebastian Mackowski and Philip Tinnefeld and Guillermo P. Acuna},
doi = {10.1021/acsnano.7b08233},
year = {2018},
date = {2018-01-01},
journal = {ACS Nano},
volume = {12},
number = {2},
pages = {1650--1655},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Ochmann2017,
title = {Optical Nanoantenna for Single Molecule-Based Detection of Zika Virus Nucleic Acids without Molecular Multiplication},
author = {Sarah E. Ochmann and Carolin Vietz and Kateryna Trofymchuk and Guillermo P. Acuna and Birka Lalkens and Philip Tinnefeld},
doi = {10.1021/acs.analchem.7b04082},
year = {2017},
date = {2017-11-01},
journal = {Analytical Chemistry},
volume = {89},
number = {23},
pages = {13000--13007},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Wei2017,
title = {Plasmonics Enhanced Smartphone Fluorescence Microscopy},
author = {Qingshan Wei and Guillermo Acuna and Seungkyeum Kim and Carolin Vietz and Derek Tseng and Jongjae Chae and Daniel Shir and Wei Luo and Philip Tinnefeld and Aydogan Ozcan},
doi = {10.1038/s41598-017-02395-8},
year = {2017},
date = {2017-05-01},
journal = {Scientific Reports},
volume = {7},
number = {1},
publisher = {Springer Science and Business Media LLC},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Vietz2017a,
title = {Broadband Fluorescence Enhancement with Self-Assembled Silver Nanoparticle Optical Antennas},
author = {Carolin Vietz and Izabela Kaminska and Maria Sanz Paz and Philip Tinnefeld and Guillermo P. Acuna},
doi = {10.1021/acsnano.7b01621},
year = {2017},
date = {2017-04-01},
journal = {ACS Nano},
volume = {11},
number = {5},
pages = {4969--4975},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
