Modular, multifunctional nanoparticle-based bioconjugate for realtime visualization of cellular membrane potential
Inventors
Delehanty, James B. • Stewart, Michael H. • Nag, Okhil • Deschamps, Jeffrey R. • Susumu, Kimihiro • Oh, Eunkeu • Field, Lauren D. • Efros, Alexander L. • Huston, Alan L. • Medintz, Igor L. • Dawson, Philip E.
Assignees
Publication Number
US-10705092-B2
Publication Date
2020-07-07
Expiration Date
2038-01-29
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Abstract
A construct for detecting cellular membrane potential includes a nanoparticle operable as an electron donor; a modular peptide attached to the nanoparticle, the peptide comprising a nanoparticle association domain, a motif configured to mediate peptide insertion into the plasma membrane, and at least one attachment point for an electron acceptor positioned at a controlled distance from the nanoparticle; and an electron acceptor. The nanoparticle can be a quantum dot and the electron acceptor can be C60 fullerene. Emission correlates with cellular membrane potential.
Core Innovation
The invention provides a modular, multifunctional nanoparticle-based electron donor-acceptor bioconjugate designed for realtime visualization of changes in cellular membrane potential. The construct includes a photoluminescent nanoparticle electron donor, a modular peptide attached to the nanoparticle comprising a nanoparticle association domain, amino acid motifs to mediate peptide insertion into the plasma membrane, and at least one attachment point for an electron acceptor positioned at a controlled distance from the nanoparticle, and the electron acceptor itself. The rate of electron transfer between the donor and acceptor is modulated by changes in membrane potential, which is reported as a measurable change in donor photoluminescence.
The described nanoparticle can be a quantum dot, and the electron acceptor can be C60 fullerene, covalently attached via the peptide. The modular peptide enables control over the distance between the electron donor and acceptor, affecting electron transfer efficiency. Experimental results show that the photoluminescence quenching of the nanoparticle donor correlates with changes in membrane potential induced by potassium chloride and depends on the donor-acceptor separation distance designed into the peptide.
The problem addressed by the invention is the lack of effective techniques for ascertaining cellular membrane potential. Existing opto-electrical sensors such as voltage-sensitive dyes suffer from poor solubility, nonspecific labeling, poor photostability, and cytotoxicity. Molecular wire approaches require complex molecular synthesis, incorporate poorly photostable fluorophores, and involve intricate electron transfer pathways. This invention aims to overcome these limitations by providing a modular, peptide-assembled nanoparticle bioconjugate that enables efficient, tunable, and photostable optical sensing of membrane potential changes.
Claims Coverage
The patent contains one independent method claim describing the use of a modular nanoparticle-peptide-electron acceptor construct for detecting cellular membrane potential, focusing on specific peptide sequences and nanoparticle/acceptor combinations.
Method of detecting cellular membrane potential using a modular nanoparticle-peptide-acceptor construct
The method involves providing a construct comprising a nanoparticle operable as an electron donor, a modular peptide attached to the nanoparticle which includes a nanoparticle association domain, a motif configured to mediate peptide insertion into the cellular plasma membrane, and at least one attachment point for an electron acceptor positioned at a controlled distance from the nanoparticle; contacting a cell with the construct; and detecting visual emission from the construct wherein the emission correlates with cellular membrane potential, with the modular peptide selected from SEQ ID No: 1, 2, or 3.
Use of quantum dot as the nanoparticle electron donor
The nanoparticle operable as the electron donor in the construct is a quantum dot.
Use of C60 fullerene as the electron acceptor
The electron acceptor attached to the peptide at a controlled distance from the nanoparticle is C60 fullerene.
The independent claim describes a method employing a modular nanoparticle-peptide-electron acceptor system with defined peptide sequences, specifying the nanoparticle as a quantum dot and the electron acceptor as C60 fullerene, enabling optical detection of cellular membrane potential through emission changes correlated to membrane potential modulations.
Stated Advantages
Amenability to both covalent and noncovalent attachment strategies enabling flexible modular design and iterative peptide development.
Rapid self-assembly of peptide to nanoparticle surface with high affinity, avoiding complex covalent chemistries requiring purification.
Fast and efficient cellular membrane labeling within 20 minutes.
Electron transfer is entirely distance-dependent, allowing tunable control of quenching efficiency by controlling donor-acceptor separation distance.
Exceptional photostability of quantum dot-based constructs permits much longer imaging times compared to voltage-sensitive dyes.
Large two-photon action cross section of quantum dots makes them suitable for deep tissue imaging.
The ratio or valence of peptide binding can be controlled to tune electron transfer rate and donor quenching efficiency.
Documented Applications
Stable, long-term imaging of changes in membrane potential in cultured cells, tissue slices, whole tissues, or animals.
Imaging and optical recording of electrical activity in electrically active cells such as neurons and muscle cells.
Use in quantum dot-based LED cells for tuning quantum dot luminescence in the presence of an electric field.
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