Methods and systems of enhancing electromagnetic radiation signals from extracellular vesicles
Systems, methods, and devices are described herein for detecting and/or monitoring target extracellular vesicles (“EVs”), e.g., to detect and/or monitor cancer treatment, such as breast cancer, in a subject. The methods can include obtaining a nano-plasmonic array including nanostructures configured to amplify one or more specific wavelengths of electromagnetic radiation, flowing a liquid sample over the nano-plasmonic array, optionally labeling target EVs captured on the nano-plasmonic array with one or more reporter groups, projecting electromagnetic radiation onto the labeled target EVs captured on the nano-plasmonic array, and capturing an image of the target EVs by receiving electromagnetic radiation emitted, scattered, or reflected by the labeled target EVs or by reporter groups on the labeled target EVs.
1 . A method of fluorescence imaging of individual target extracellular vesicles (EVs) on a substrate, the method comprising:
obtaining a nano-plasmonic array comprising,
a substrate,
a plurality of nanostructures arranged in an array on a surface of the substrate, wherein the nanostructures comprise nanorods, nanodisks, nanopillars, nanogrooves, or any combination thereof, and
one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands specifically bind to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures;
flowing a liquid sample over the nano-plasmonic array at a flow rate that enables EVs in the liquid sample, if any, to bind to the affinity ligands thus capturing the EVs on the nano-plasmonic array;
labeling target EVs among EVs captured on the nano-plasmonic array with one or more different fluorescent reporter groups;
exposing the labeled target EVs captured on the nano-plasmonic array to a first electromagnetic radiation thereby causing the target EVs, or fluorescent reporter groups on the target EVs, or both target EVs and fluorescent reporter groups on the target EVs, to emit, scatter, or reflect one or more fluorescent signals;
receiving all or a portion of the one or more fluorescent signals, wherein the nanostructures in the nano-plasmonic array are arranged and dimensioned to amplify the fluorescent signals, thereby amplifying the fluorescent signals from individual target EVs on the substrate; and
obtaining an image of one or more individual target EVs by receiving the amplified fluorescent signal.
2 . The method of claim 1 ,
(i) wherein the one or more affinity ligands bind non-specifically to at least one surface marker on the EVs, or to at least one intravesicular marker inside the EVs, or to at least one surface marker on the EVs and to at least one intravesicular marker inside the EVs; and the fluorescent reporter groups are bound to capture agents that specifically bind to at least one surface marker on the target EVs, or to at least one intravesicular marker inside the target EVs, or to at least one surface marker on the target EVs and to at least one intravesicular marker inside the target EVs, or
(ii) wherein the one or more affinity ligands specifically bind to at least one surface marker on the target EVs, or to at least one intravesicular marker inside the target EVs, or to at least one surface marker on the target EVs and to at least one intravesicular marker inside the target EVs, and the fluorescent reporter groups are bound to capture agents that bind to at least one surface marker on the target EVs, or to at least one intravesicular marker inside the target EVs, or to at least one surface marker on the target EVs and to at least one intravesicular marker inside the target EVs, either specifically or non-specifically.
3 . The method of claim 1 , wherein the plurality of nanostructures are arranged to form a periodic array of nanostructures on the substrate, wherein the periodic array of nanostructures is arranged and dimensioned to amplify the fluorescent signals emitted, scattered, or reflected by EVs bound to the nanostructures, or EVs bound to the substrate near the nanostructures, or EVs bound to the nanostructures and EVs bound to the substrate near the nanostructures, or to amplify the fluorescent signals emitted, scattered, or reflected by the fluorescent reporter groups attached to the EVs.
4 . The method of claim 1 , wherein the liquid sample is from a subject, wherein the fluorescent reporter groups are bound to capture agents that specifically bind to tumor-derived target EVs, and wherein the method further comprises analyzing the obtained image to detect whether the liquid sample comprises tumor-derived target EVs, thereby detecting or monitoring cancer in the subject.
5 . The method of claim 4 , further comprising
identifying EVs by size and discarding any EVs or other components larger than one micron;
selecting target EVs from the identified EVs based on positivity for target EV markers to generate selected target EVs;
specifying selected target EVs as originating from specific organs or tissues by positivity for organ- or tissue-specific markers to generate specific, selected target EVs; and
analyzing individual specific, selected target EVs based on tetraspanin biomarkers on the surface of the specific target EVs, based on intravesicular biomarkers within the specific target EVs, or based on both tetraspanin and intravesicular biomarkers.
6 . The method of claim 4 , wherein the fluorescent reporter groups comprise a first fluorescent label.
7 . The method of claim 4 , wherein the reporter groups comprise antibodies that specifically bind to a biomarker on the surface of the target EVs.
8 . The method of claim 7 , wherein the antibodies comprise at least two different types of antibodies, wherein antibodies of a first type bind to EpCAM and antibodies of a second type bind to HER2.
9 . The method of claim 7 , wherein the antibodies comprise at least four different types of antibodies, wherein antibodies of a first type bind to MUC1, antibodies of a second type bind to EGFR, antibodies of a third type bind to EpCAM, and antibodies of a fourth type bind to HER2.
10 . The method of claim 6 , further comprising labeling target EVs that include the fluorescent reporter groups comprising the first fluorescent label with a second fluorescent label that is different from the first fluorescent label.
11 . The method of claim 4 , wherein the cancer is breast cancer.
12 . The method of claim 1 , wherein the nanostructures comprise metal nanoparticles bound to metal nanopillars.
13 . The method of claim 12 , wherein the metal nanoparticles comprise gold, silver, aluminum, or platinum and the metal nanopillars comprise gold, silver, aluminum, or platinum.
14 . The method of claim 13 , wherein the metal nanoparticles are gold and the metal nanopillars are gold.
15 . The method of claim 1 ,
wherein the fluorescent reporter groups comprise multiple different fluorescent reporter groups; and
wherein obtaining an image of one or more individual target EV comprises conducting multichannel fluorescence imaging to form the image.
16 . The method of claim 1 , wherein the method enables the discrimination of individual EVs based on their cellular origins.
17 . The method of claim 16 , wherein the method enables the discrimination of individual target EVs originating from tumors in breast, brain, or immune system tissues.