Analyte detection enhancement by targeted immobilization, surface amplification, and pixelated reading and analysis
This disclosure provides, among other things, a method of sample analysis, that comprises: (a) binding target analytes to capture agents that are attached to a surface of a plate, wherein the plate comprises (i) a sensing amplification layer comprises nanostructures that enhance signals and (ii) the capture agents are attached to said sensing amplification layer; (b) reading the plate with a reading device to produce an image of signals that represent individual binding events; and (c) identifying and counting individual binding events in an area of the image, thereby providing an estimate of the amount of one or more analytes in the sample. A system for performing the method is also provided.
1. A method of sample analysis, comprising:
(a) binding target analytes to capture agents that are attached to a surface of a plate, wherein the plate comprises (i) a sensing amplification layer comprising nanostructures that enhance signals and (ii) the capture agents, wherein the capture agents are attached to said sensing amplification layer;
(b) reading the plate with a reading device to produce an image of signals that represent individual binding events, wherein the reading is done at a resolution sufficient to distinguish between individual binding events and the image shows bright spots that are spatially separated from one another that correspond to individual binding events between a single molecule of target analyte and a single molecule of capture agent;
(c) identifying the bright spots in an area of the image; and
(d) counting the bright spots identified in step (c), thereby providing an estimate of the amount of one or more analytes in the sample.
2. The method of claim 1 , wherein the target analytes comprise proteins, peptides, DNA, RNA, nucleic acid, small molecules, cells, or nanoparticles with different shapes.
3. The method of claim 1 , wherein the capture agent specifically binds to the target analyte.
4. The method of claim 1 , wherein the image shows the position, local intensity, and local spectrum of the signals.
5. The method of claim 1 , wherein the signals are luminescence signals selected from the group consisting of fluorescence, electroluminescence, chemiluminescence, and electrochemiluminescence signals.
6. The method of claim 1 , wherein the signals are Raman scattering signals.
7. The method of claim 1 , wherein the signals are the forces due to local electrical, local mechanical, local biological, or local optical interaction between the plate and the reading device.
8. The method of claim 1 , further comprising, before step (b), labeling the target analytes with a label, either prior to or after they are bound to said capture agent.
9. The method of claim 1 , wherein the reading step (b) is performed by applying a voltage bias between said signal amplification layer and another electrode, thereby providing greater sensitivity.
10. The method of claim 1 , wherein the identifying and counting steps comprise (1) determining the local intensity of background signal, (2) determining local signal intensity for one label, two labels, three labels, and four or more labels; and ( 3 ) determining the total number of labels in the imaged area.
11. The method of claim 1 , wherein the identifying and counting steps comprise (1) determining the local spectrum of background signal, (2) determining local signal spectrum for one label, two labels, three labels, and four or more labels; and ( 3 ) determining the total number of labels in the imaged area.
12. The method of claim 1 , wherein the identifying and counting steps comprise (1) determining the local Raman signature of background signal, (2) determining local signal Raman signature for one label, two labels, three labels, and four or more labels; and (3) determining the total number of labels in the imaged area.
13. The method of claim 1 , wherein the identifying and counting steps comprise determining one or more of the local intensity, spectrum, and Raman signatures.
14. The method of claim 1 , wherein the binding step (a) is accelerated by applying an electric field to the plate, thereby moving the analytes to the sensing amplification layer.
15. The method of claim 1 , wherein the sensing amplification layer has a molecular linking layer that links said capture agents with said sensing amplification layer.
16. The method of claim 1 , wherein the signals are light signals.
17. The method claim 1 , wherein the signals are produced by a fluorescent label, that is associated with the bound analyte, either before or after binding of the analyte to the capture agent.
18. The method of claim 1 , wherein the average distance between the two adjacent signals being read to form the image of signals in reading step is greater than 10 nm.
19. The method of claim 1 , wherein the signals are signals generated by Raman scattering.
20. The method of claim 1 , wherein the capture agent is an antibody.
21. The method of claim 1 , wherein the capture agent is a polynucleotide.
22. A method of sample analysis, comprising;
(a) binding target analytes to capture agents that are attached to a surface of a plate, wherein the surface of the plate comprises (i) a dots-on pillar antenna array (D2PA) comprising nanostructures that enhance signals and (ii) the capture agents, wherein the capture agents are attached to said dots-on pillar antenna array;
(b) reading the plate with a reading device to produce an image of signals that represent individual binding events, wherein the reading is done at a resolution sufficient to distinguish between individual binding events and the image shows bright spots that are spatially separated from one another that correspond to individual binding events between a single molecule of target analyte and a single molecule of capture agent;
(c) identifying the bright spots in an area of the image; and
(d) counting the bright spots identified in step (c), thereby providing an estimate of the amount of one or more analytes in the sample.
23. A method of sample analysis, comprising:
(a) binding target analytes to capture agents are attached to a surface of a plate, wherein the surface of the plate comprises (i) a sensing amplification layer comprising nanostructures that enhance signals and (ii) the capture agents, wherein the capture agents that are attached to said sensing amplification layer and The method of claim 1 , wherein the sensing amplification layer comprises one or a plurality of metallic discs and a flat metallic film, wherein a portion of the metallic disc has a separation from the metallic film and the separation and the dimensions of the disks are less than the wavelength of the light used in sensing;
(b) reading the plate with a reading device to produce an image of signals that represent individual binding events, wherein the reading is done at a resolution sufficient to distinguish between individual binding events and the image shows bright spots that are spatially separated from one another that correspond to individual binding events between a single molecule of target analyte and a single molecule of capture agent;
(c) identifying the bright spots in an area of the image; and
(d) counting the bright spots identified in step (c), thereby providing an estimate of the amount of one or more analytes in the sample.
24. The method of claim 23 , wherein the metallic disk has a shape selected from the group of shapes consisting of round, polygonal, pyramidal, elliptical, elongated bar shaped, and any combination thereof.
25. The method of claim 23 , wherein the separation is 0.5 to 30 nm, and wherein the discs have an average lateral dimension in the range of 20 nm to 250 nm.