Multifunctional nanoparticle bioconjugates for photoacoustic-based recording 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. • Rasheed, Nashaat • Chitnis, Parag V. • Cressman, John R.
Assignees
Publication Number
US-10780185-B2
Publication Date
2020-09-22
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. Photoacoustic emission from the construct correlates with cellular membrane potential.
Core Innovation
The invention describes a modular, multifunctional nanoparticle-based electron donor-acceptor bioconjugate constructed to enable real-time detection of changes in cellular membrane potential. The construct includes a photoluminescent nanoparticle electron donor, a modular peptide that features domains for nanoparticle association, membrane insertion, and specific attachment points for an electron acceptor at controlled distances from the nanoparticle, and an electron acceptor such as C60 fullerene. The rate of electron transfer between the donor and acceptor, modulated by membrane potential changes, alters donor photoluminescence and produces a photoacoustic emission correlatable with the cellular membrane potential.
The problem addressed is the need for improved techniques to ascertain cellular membrane potential. Existing methods like electrochromic voltage-sensitive dyes suffer from poor aqueous solubility, nonspecific labeling, poor photostability, and cytotoxicity, while molecular wires require complex synthesis and exhibit limited photostability. These limitations restrict the utility of current opto-electrical sensors in imaging cellular electrical activity, especially for neurons and electrically active cell types.
The invention solves these problems by employing a nanoparticle-peptide-acceptor assembly that self-assembles rapidly with high affinity without requiring complex covalent chemistry, enables precise control over donor-acceptor distance and electron transfer rates, and supports both photoluminescence and photoacoustic sensing modalities. The photoacoustic sensing improves signal detection in tissue by converting optical signals into acoustics, which traverse tissue with less scattering than luminescence. This multifunctional bioconjugate thus overcomes limitations of prior voltage sensors by providing sensitive, tunable, and stable detection of membrane potential changes.
Claims Coverage
The patent includes one independent claim which covers a method of detecting cellular membrane potential using a specifically constructed quantum dot-based bioconjugate assembly and the detection of photoacoustic emission correlated with membrane potential.
Construct comprising a quantum dot donor linked to a modular peptide and electron acceptor
The method employs a construct that includes a quantum dot operable as an electron donor, a modular peptide attached to the quantum dot with a nanoparticle association domain, a motif mediating peptide insertion into a cellular plasma membrane, and at least one attachment point for an electron acceptor positioned at a controlled distance from the quantum dot, and an electron acceptor comprising C60.
Detection of photoacoustic emission correlated with cellular membrane potential
The method detects photoacoustic emission from the construct upon contacting a cell, where the photoacoustic emission correlates with the cell's membrane potential, allowing membrane potential measurement through photoacoustic sensing.
Selection of modular peptide sequences
The modular peptide in the construct is specifically selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, which comprise defined sequences enabling controlled attachment and membrane insertion.
Photoacoustic emission in absence of aggregation
The photoacoustic emission detected from the construct occurs without aggregation of the bioconjugate to ensure accurate correlation with membrane potential.
Application to cells in tissue or living animals
The method can be applied to cells within tissues, including tissues in living animals, extending the utility beyond isolated cells.
The independent claim covers a method for detecting cellular membrane potential using a quantum dot-based electron donor coupled with a modular peptide and C60 electron acceptor, detecting correlated photoacoustic emission without construct aggregation, applicable to cells in tissues and living animals, with defined peptide sequences enabling precise assembly and membrane insertion.
Stated Advantages
Amenable to both covalent and noncovalent attachment strategies allowing flexible assembly.
Modular peptide design permits iterative development and controlled tuning of donor-acceptor distance.
Noncovalent assembly of peptide to quantum dot is rapid (10 min) with high nanomolar affinity.
Construct enables rapid labeling of cell membranes (within 20 minutes total) with high specificity.
Electron transfer is completely distance-dependent, allowing precise control unlike existing molecular wires.
Valence or ratio of peptides on nanoparticle surface can be controlled to tune electron transfer efficiency.
Quantum dot constructs exhibit exceptional photostability, supporting imaging over 100 times longer than voltage-sensitive dyes.
Large two-photon action cross section of quantum dots enables improved deep tissue imaging.
Photoacoustic sensing allows improved imaging in tissue due to better ultrasound tissue penetration compared to luminescence.
Documented Applications
Imaging and optical recording of electrical activity in one or more cultured cells.
Imaging and recording membrane potential changes in tissue slices, whole tissues, and living animals.
Applications to electrically active cells such as neurons and muscle cells.
Potential use in quantum dot-based LED cells where tuning of luminescence by electric fields is desired or required.
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