Method for modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte and a probe for remote detection of the analyte using SERS

Abstract

A method for modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte is provided. The method of the present invention comprises the steps of: providing a liquid sample to be analyzed using SERS; and adding an oxygen scavenger to the liquid sample to remove dissolved oxygen from the liquid sample. A probe for remotely sensing an analyte in a liquid sample using SERS is also provided. The probe of the present invention comprises a detection chamber having a window transparent to SERS excitation light and Raman scattering signals, and a pipe having first and second ends, wherein the first end of the pipe is movably connected to the detection chamber and the second end of the pipe is configured to be positioned in contact with the liquid sample.

Description

Technology Field

[0001] related Cross-application for applications

[0002] This application claims the benefit of U.S. Provisional Application No. US 62 / 890,216, filed August 22, 2019, under 35 U.S.C. § 119(e). All of the above documents are incorporated herein in their entirety by reference.

[0003] Field of invention

[0004] The present invention relates to a method for modifying a liquid sample to be analyzed using surface-enhanced Raman spectroscopy (SERS). More specifically, the present invention relates to a method for modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte. The present invention also relates to a probe for remote sensing of an analyte using SERS. Background Technology

[0005] Raman spectroscopy involves the attenuation or enhancement of light energy inelastically scattered by molecules through changes in the molecules' vibrational energy states. Since the energy change of scattered light relative to incident light corresponds to the energy difference between the molecules' vibrational states, molecules can be identified using the spectrum of inelastically scattered light—namely, the Raman spectrum—and the intensity of the inelastically scattered light can be used to quantify the number of said molecules. Because inelastic Raman light scattering is weak, a significant concentration of the effective analyte molecules is required, and thus this technique has low sensitivity. However, there are methods that can be used to significantly enhance the intensity of the inelastically scattered light.

[0006] Surface-enhanced Raman spectroscopy (SERS) increases the probability that light is scattered inelastically rather than elastically, thereby increasing the intensity of the inelastically scattered light. SERS achieves this by adsorbing molecules of the analyte onto a plasmonic material and then irradiating them with incident light. Although the precise mechanism of enhancement is currently debated in the academic community, the plasmonic material must be composed of rough or nanostructured nanoscales, and the enhancement of signal intensity is 10 10 to 10 11 It is known that it can be as high. In particular, vibrational modes detectable by surface-enhanced Raman spectroscopy can differ from the vibrational modes of free molecules, and therefore, Raman spectra and surface-enhanced Raman spectra can differ for the same molecule.

[0007] The sensitivity and reproducibility of SERS can be enhanced by engineering an optical hotspot (e.g., a nanoprism tip or within a nanogap between adjacent nanoparticles), which is the location of the nanostructure exhibiting the strongest electromagnetic field. While the hotspot generates the highest signal amplification for SERS, sensitivity is limited because the analyte molecule must simultaneously be located within the hotspot, the sampling area of ​​the Raman laser beam, and the detector's field of view. This sampling area or volume is typically a negligible portion of the total sample volume. Therefore, in practice, the detection limit of SM-SERS is at the nM or pM level (10 -9 -10 -12 M). Conventional surface-enhanced Raman techniques can dramatically reduce the detection limit of analyte molecules, but they generally fail to achieve the "single molecule" detection limit that is optimistically advertised. Recently, this limit has been broken by combining pre-enrichment of the analyte with SERS detection using superhydrophobic or nonhydrophobic surfaces, which has increased detection sensitivity to the fM level.

[0008] Substrate surface oxidation can affect the SERS detection limit. When silver nanoparticles (AgNPs) or silver films are oxidized by ambient air to form a sub-monolayer of Ag2O, the SERS enhancement factor (EF) is up to 10 5 It has been reported to decrease to [amount]. This is analyte-dependent and quantitatively correlated with the thickness of the Ag2O layer. The Ag2O layer effect is attributed to the reduction in analyte adsorption and metal-to-analyte electron transfer, which impairs both the chemical and electromagnetic enhancement of SERS. However, while the substrate oxidation effect on SERS EF was later observed to be less significant, noise levels increase along with a corresponding decrease in signal intensity when combined with atmospheric carbon pollution. SERS detection can also be achieved using shell-separated nanoparticle-enhanced Raman spectroscopy (SHINERS), specifically AuNPs coated with thin silica or alumina layers. The success of SHINERS indicates that thin dielectric oxide layers may not result in a drastic reduction in SERS EF.

[0009] According to the present invention, the following method and probe are provided:

[0010] 1. A method for modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte, comprising the steps of: providing the liquid sample to be analyzed using SERS; and adding a scavenger to the liquid sample to remove dissolved oxygen from the liquid sample.

[0011] 2. The method of item 1, wherein the analyte is a thiolated compound, amine, pesticide, persistent organic pollutant, transition metal complex, peptide, protein, nucleic acid, polysaccharide, or hormone.

[0012] 3. A method according to Item 1 or Item 2, wherein the analyte is a transition metal complex, peptide, protein, nucleic acid, persistent organic contaminant, pesticide, or hormone.

[0013] 4. A method in which, in any one of items 1 to 3, the analyte is p-aminothiophenol (pATP) or p-nitrothiophenol (pNTP).

[0014] 5. A method according to any one of items 1 to 4, wherein the liquid in the sample is water or an organic solvent, e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, a hydrocarbon, or a mixture thereof.

[0015] 6. A method in which, in any one of items 1 through 5, the liquid is water.

[0016] 7. A method comprising, if necessary, a step of modifying the analyte to enable it to interact with the surface of the plasmonic material used for SERS detection in any one of items 1 to 6.

[0017] 8. A method according to item 7, wherein modification of the analyte is performed by attaching a thiol group to the analyte, so that the analyte is chemically adsorbed on an Ag or Au plasmon surface through the thiol.

[0018] 9. A method according to item 7, wherein the modification of the analyte is performed by physisorption through hydrophobic / hydrophilic interactions or van der Waals forces; chemiosorption through other bonding atoms; indirect detection through chemical reaction with a pre-adsorbed reporter molecule; chelation of metal ions by a pre-adsorbed chelating agent; indirect detection through affinity such as the binding of biomolecules to antigens; and indirect detection by the possession of a plasmonic nanoparticle possessing a surrogate SERS-active reporter molecule through a specific interaction with the analyte such as an antigen-antigen interaction.

[0019] 10. A method in which, in any one of items 1 to 9, the oxygen remover is sodium sulfite, hydrazine, ascorbic acid, or ascorbyl palmitate.

[0020] 11. A method in which an excess amount of oxygen remover is added to any one of items 1 through 10.

[0021] 12. A method in which, in any one of items 1 to 11, a sufficient oxygen scavenger is added to remove at least 98%, 99%, 99.5%, or 99.9% of DO from a liquid sample, preferably at least 99.5% or 99.9%.

[0022] 13. A method in which, in any one of items 1 to 12, a sufficient oxygen remover is added such that the concentration of residual DO is at most about 0.020 mM, about 0.010 mM, about 0.005 mM, or about 0.002 mM, preferably at most about 0.005 mM or about 0.002 mM.

[0023] 14. In any one of items 1 through 13, after the liquid sample is modified, the LOD for a given analyte is at least about 10 8 Pear, preferably about 10 9 Ship, or even up to about 10 10 A method that reduces by a factor of two.

[0024] 15. In any one of items 1 through 14, after the liquid sample is modified, the LOD for the analyte using SERS is at least about 10 9 or about 10 10 A method that improves by the amount of a pear.

[0025] 16. A method in which, in any one of items 1 through 15, the LOD for the analyte using SERS is at most about 1 pM, about 100 fM, about 10 fM, about 1 fM, about 100 aM, about 10 aM, about 1 aM, about 100 zM, or about 10 zM.

[0026] 17. A method in which, in any one of items 1 through 16, the LOD for the analyte is at most about 10 zM.

[0027] 18. A method comprising, in any one of items 1 through 17, further a step of contacting the liquid sample with a plasmonic material.

[0028] 19. A method according to item 18, wherein at least a portion of the analyte is adsorbed or adsorbed on the surface of a plasmonic material.

[0029] 20. The method according to item 18 or 19, wherein the plasmonic material is a material composed of a conductive, semiconducting and / or dielectric material, a combination thereof, or a composite thereof.

[0030] 21. A method according to any one of items 18 through 20, wherein the plasmonic material is gold, silver, copper, or a mixture thereof.

[0031] 22. A method according to any one of items 18 to 21, wherein the plasmonic material is gold or silver, preferably silver.

[0032] 23. A method according to any one of items 18 to 22, wherein the plasmonic material has a rough plasmonic surface, is composed of plasmonic nanoparticles, or has a plasmonic surface and is composed of plasmonic nanoparticles.

[0033] 24. The method of item 23, wherein the roughness of the rough plasmon surface is about 5 nm to about 50 nm, preferably about 10 nm to about 20 nm.

[0034] 25. The method of item 23 or item 24, wherein the roughness of the rough plasmonic surface is at least about 5 nm; at least about 10 nm; or at least about 20 nm; and / or up to about 200 nm; up to about 100 nm; or up to about 50 nm.

[0035] 26. A method according to any one of items 23 to 25, wherein the plasmonic material is composed of plasmonic nanoparticles.

[0036] 27. A method according to any one of items 23 to 26, wherein the plasmonic nanoparticle comprises gold, silver, or copper nanoparticles (including silica-coated gold, silver, or copper nanoparticles).

[0037] 28. A method according to any one of items 23 to 27, wherein the plasmon nanoparticles are silver or gold nanoparticles (coating member), more preferably silver nanoparticles (coating member).

[0038] 29. A method according to any one of items 23 through 28, wherein the plasmon nanoparticles are prismatic, ellipsoidal (including spherical), plate-like, or rod-like.

[0039] 30. A method according to any one of items 23 to 29, wherein the diameter D50 of the plasmon nanoparticle is about 2 nm to about 500 nm.

[0040] 31. A method according to any one of items 23 to 30, wherein the diameter D50 of the plasmonic nanoparticle is at least about 2 nm, at least about 10 nm, at least about 25 nm, or at least about 50 nm, and / or up to about 500 nm, up to about 250 nm, up to about 200 nm, up to about 100 nm, up to about 75 nm, or up to about 50 nm.

[0041] 32. A method according to any one of items 23 through 31, wherein the plasmon nanoparticles aggregate and come into very close proximity (and / or contact) with each other.

[0042] 33. A method in which the plasmonic nanoparticles are aggregated according to any one of items 23 through 32 by adding an aggregating agent, e.g., an aggregating molecule (i.e., a molecule capable of linking nanoparticles and inducing aggregation) or an electrolyte (e.g., a salt), adjusting the pH, adding an organic solvent such as an alcohol, or a combination of these approaches.

[0043] 34. A method according to any one of items 23 to 33, wherein the plasmon nanoparticles are aggregated through the addition of an aggregating agent such as a sulfate or a sulfite.

[0044] 35. A method according to item 33 or 34, wherein the coagulant and the oxygen remover are different substances.

[0045] 36. The method according to item 33 or 34, wherein the coagulant is an oxygen remover, preferably a sulfite, preferably sodium sulfite.

[0046] 37. A method in which, in any one of items 23 to 36, the nanoparticles are aggregated through the addition of a dissolved salt and / or pH adjustment.

[0047] 38. A method in which, in any one of items 32 through 37, the pH of the liquid sample is adjusted to about 4 before coagulation.

[0048] 39. A method according to any one of items 32 through 38, wherein the plasmonic nanoparticles are added to a liquid sample prior to the aggregation of the nanoparticles and prior to the removal of dissolved oxygen using an oxygen remover.

[0049] 40. A method according to any one of items 23 to 39, wherein the plasmonic nanoparticles are stabilized by loosely bound small capping molecules, preferably citrate, ethanol, ethylene glycol, and / or polyethylene glycol, more preferably citrate and / or ethanol.

[0050] 41. A method according to any one of items 23 through 40, wherein a chelating agent for a molecule, e.g., a metal ion, which interacts with a specific analyte to generate a distinct signal, is pre-adsorbed onto a plasmonic nanoparticle or a rough plasmonic surface before coming into contact with the analyte.

[0051] 42. A method comprising the step of measuring the SERS spectrum of the liquid sample in any one of items 1 to 41.

[0052] 43. The method of item 42, wherein the measurement step comprises the step of irradiating a plasmonic material with light, generally a laser, and the step of detecting a Raman signal scattered by said plasmonic material.

[0053] 44. A probe for remote sensing of an analyte in a liquid sample using SERS, comprising: a detection chamber having a window transparent to SERS excitation light and Raman scattering signals; and a pipe having first and second ends, wherein the first end of the pipe is fluidly connected to the detection chamber and the second end of the pipe is configured to be in contact with the liquid sample, wherein the pipe between the first end and the second end and the detection chamber comprise plasmonic nanoparticles immersed in a deoxygenated solvent, wherein the plasmonic nanoparticles are in very close proximity to each other to allow uninterrupted propagation of a plasmonic field from the detection chamber to the second end of the pipe and from the second end of the pipe to the detection chamber, and / or the inner wall of the detection chamber and the inner wall of the pipe between the first end and the second end are coated with a plasmonic layer, wherein the plasmonic layer is continuous from the detection chamber to the second end of the pipe, thereby allowing for uninterrupted propagation of a plasmonic field from the detection chamber to the second end of the pipe and to the rear A probe that allows for the uninterrupted propagation of a plasmon field, wherein the piping and detection chamber are filled with a deoxygenating solvent, and the deoxygenating solvent comprises an oxygen remover that removes dissolved oxygen from the deoxygenating solvent.

[0054] 45. A probe according to item 44, wherein the plasmon nanoparticles are as defined in any one of items 23 to 41 and / or the plasmon layer is a layer of plasmon material comprising a rough plasmon surface as defined in any one of items 23 to 25.

[0055] 46. ​​In item 44 or 45, the piping between the detection chamber and the first end and the second end comprises a probe containing plasmon nanoparticles immersed in a deoxygenating solvent.

[0056] 47. A probe in which, in any one of items 44 through 46, the plasmon nanoparticles aggregate along the length of the piping as defined in any one of items 32 through 39 using any technique defined in any one of items 33 through 39.

[0057] 48. A probe according to any one of items 44 to 47, wherein the solvent is water or an organic solvent, such as methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, a hydrocarbon, or a mixture thereof.

[0058] 49. A probe in which the solvent is water, in any one of items 44 through 48.

[0059] 50. In any one of items 44 through 49, the oxygen remover is a probe as defined in any one of items 1 through 43.

[0060] 51. In any one of items 44 to 50, the concentration of the added oxygen remover as well as the concentration of the residual DO are as defined in any one of items 1 to 43.

[0061] 52. A probe in which, in any one of items 44 through 51, the second end of the piping is open or closed (preferably open).

[0062] 53. A probe according to any one of items 44 through 52, wherein the inner wall of the pipe is coated with a plasmon layer and the pipe is filled with a deoxygenating solvent.

[0063] 54. A probe in any one of items 44 through 53, wherein the second end of the piping is opened and the detection chamber and piping are refilled with a deoxygenating solvent (and plasmon nanoparticles if plasmon nanoparticles are used) from, for example, a pump or storage unit, and the piping and detection chamber are maintained in a state filled with a deoxygenating solvent (and plasma nanoparticles so that the plasmon nanoparticles are kept close to each other if plasmon nanoparticles are used).

[0064] 55. In item 54, the pump or the storage unit is directly connected to the first end of the piping, or the probe is connected to the detection chamber in a manner that replaces any deoxygenated solvent / plasmon nanoparticle flowing out from the second end of the piping.

[0065] 56. In any one of items 44 through 55, the capillary force retains the deoxygenating solvent (and, if plasmonic nanoparticles are used) inside the tubing even though the second end of the tubing is open, the probe.

[0066] 57. A probe according to any one of items 44 through 56, wherein the second end of the pipe is open and the second end of the pipe is at least partially coated with a plasmon layer.

[0067] 58. A probe according to any one of items 44 through 57, wherein the second end of the pipe comprises plasmon nanoparticles and the end wall, the inner wall facing the second end of the pipe, and the outer wall facing the second end are coated with a plasmon layer.

[0068] 59. In any one of items 44 through 58, the inner wall of the pipe is coated with a plasmon layer, and the plasmon layer also covers the end wall and the outer wall toward the second end of the pipe, a probe.

[0069] 60. A probe in any one of items 44 through 59, wherein the second end of the piping is also capped with a plasmon layer.

[0070] 61. A probe in any one of items 44 through 60, wherein the piping is made of metal, quartz, glass, or a polymer plastic such as polyethylene, polypropylene, polystyrene, polyetheretherketone, polyvinyl chloride, polytetrafluoroethylene, or polydimethylsiloxane.

[0071] 62. A probe in any one of items 44 through 61, wherein the piping is a polyetheretherketone (PEEK) polymer piping or a glass capillary.

[0072] 63. A probe according to any one of items 44 to 62, wherein the inner diameter of the piping is about 0.08 mm to about 1 cm, preferably about 0.3 mm to about 0.5 mm.

[0073] 64. A probe according to any one of items 44 through 63, wherein the inner diameter of the piping is at least about 0.08 mm; at least about 0.1 mm; at least about 0.2 mm; or at least about 0.3 mm; and / or up to about 1 cm; up to about 5 mm; up to about 2 mm; up to about 1 mm; up to about 0.75 mm; or up to about 0.5 mm.

[0074] 65. A probe in any one of items 44 to 64, wherein the inner diameter of the pipe is about 0.3 mm or about 0.5 mm.

[0075] 66. A probe, wherein in any one of items 44 through 65, the length of the piping is at least about 5 cm; at least about 10 cm; at least about 20 cm; at least about 50 cm; or at least about 75 cm; and / or up to about 50 m; up to about 20 m; up to about 10 m; up to about 5 m; up to about 2 m; or up to about 1 m.

[0076] 67. A probe in any one of items 44 through 66, wherein the length of the piping is about 1 m or about 2 m.

[0077] 68. A probe in any one of items 44 through 67, wherein the cross-section of the piping is circular, elliptical, or rectangular.

[0078] 69. A probe, wherein “proximity” in any one of items 44 through 68 means a distance of up to about 15 nm, preferably up to about 10 nm, most preferably up to about 5 nm (between plasmonic nanoparticles, between plasmonic nanoparticles and a plasmonic layer, or between one of these and an analyte).

[0079] 70. A probe in any one of items 44 to 69, wherein the density of the plasmon nanoparticles varies along the length of the pipe.

[0080] 71. In any one of items 44 through 70, the probe wherein the detection chamber and the first end of the piping are movably connected to each other through a hole defined by the detection chamber.

[0081] 72. In any one of items 44 to 71, the detection chamber is a probe made of glass, plastic, or quartz, more preferably glass or quartz.

[0082] 73. A probe wherein, in any one of items 44 to 72, the analyte contained in the liquid is disposed in contact with a plasmonic material defined in any one of items 18 to 41 contained in the probe (e.g., in the case of a closed second end, a plasmonic layer completely covering the second end; or in the case of an open second end, a plasmonic layer coating the second end or plasmonic nanoparticles contained in a deoxygenated solvent).

[0083] 74. A probe wherein, in any one of items 44 to 73, the liquid sample is disposed in contact with a plasmonic material as defined in any one of items 18 to 41, and preferably adsorbed onto the plasmonic material.

[0084] 75. In any one of items 44 to 74, the probe wherein the liquid sample is preferably deoxygenated using an oxygen remover as defined in any one of items 1 to 43.

[0085] 76. In any one of items 44 to 75, the amount of the oxygen remover, as well as the residual DO concentration in the liquid sample, is as defined in any one of items 1 to 43, a probe.

[0086] 77. A probe in any one of items 44 through 76, wherein the liquid sample is the liquid sample defined in any one of items 1 through 43.

[0087] 78. A probe in which, in any one of items 44 through 77, the liquid sample is modified using a method as defined in any one of items 1 through 43.

[0088] 79. A probe in any one of items 44 through 78, wherein the analyte is adsorbed to a surface of a plasmonic material which may be a surface of a nanoparticle within the pipe, a plasmonic layer coated on a second end of the pipe, and / or a plasmonic layer capping the second end of the pipe.

[0089] 80. In any one of items 44 through 79, the analyte is generated according to the method defined in any one of items 1 through 43 and is in a deoxygenated liquid sample comprising aggregated plasmonic nanoparticles or a rough plasmonic surface as defined in any one of items 23 through 41; and a probe in contact with the nanoparticles in the tubing; a plasmonic layer coated on a second end of the tubing; and / or a plasmonic layer capping the second end of the tubing.

[0090] 81. In any one of items 44 through 80, a molecule that interacts with a specific analyte to generate a distinct signal, e.g., a chelating agent for a metal ion or an item that interacts with a biomolecule, a peptide, or an aptamer is a probe that is pre-adsorbed to a plasmonic nanoparticle of a tubular structure, a plasmonic nanoparticle of a liquid sample, a plasmonic layer coated on a second end of the tubular structure, and / or a plasmonic layer capping the second end of the tubular structure.

[0091] 82. In any one of items 44 through 81, the probe is configured to work with a surface-enhanced Raman spectrometer for remote sensing, which can be used to detect an analyte by applying incident laser irradiation to a sample holder or detection chamber and measuring scattered light using a detector.

[0092] 83. In item 82, a probe in which the backscattered light, i.e., light scattered 180° with respect to the incident light, is detected.

[0093] 84. A method in which, in any one of items 1 through 43, the measurement step is performed using a probe defined in any one of items 44 through 83.

[0094] 85. A method in which, in any one of items 1 through 43, the SERS system is as defined in any one of items 44 through 83. Brief explanation of the drawing

[0095] In the attached drawing: FIG. 1a is a longitudinal cross-sectional view of the second end of a pipe (g) according to an embodiment of the probe of the present invention. FIG. 1b is a longitudinal cross-sectional view of the second end of a pipe (g) containing plasmon nanoparticles according to an embodiment of the probe of the present invention. FIG. 1c is a longitudinal cross-sectional view of a second end of a pipe (g) in which the inner wall of the pipe (g) is coated with a plasmon layer according to another embodiment of the probe of the present invention. FIG. 1d is a longitudinal cross-sectional view of the second end of a pipe (g) comprising plasmon nanoparticles coated with a plasmon layer, the end wall, the inner wall facing the second end of the pipe (g), and the outer wall facing the second end. FIG. 1e is also a cross-sectional view of the second end of a tube (g) in which the inner wall is coated with a plasmon layer covering the end wall and the outer wall toward the second end of the tube (g). FIG. 1f is a cross-sectional view of the second end of a tube (g) containing plasmon nanoparticles covered with a plasmon layer. FIG. 1g is a longitudinal cross-sectional view of the second end of a pipe (g) in which the inner wall is coated with a plasmon layer covering the second end of the pipe (g). Figure 2 shows a schematic diagram of a conventional surface-enhanced Raman spectrometer. FIG. 3 illustrates a schematic diagram of a surface-enhanced Raman spectrometer for remote sensing including the probe of the present invention. Figure 4 shows transmission electron microscope images of silver nanoparticles and aggregates used in the experiment, specifically, Figure 4 shows a) individual, b) and c) aggregates, and d) nano-gaps between nanoparticles. Figure 5 illustrates the generation of photocurrent through an integrated plasmon field. Specifically, Figure 5 a) shows an experimental schematic diagram illustrating the relative positions of the incident radiation and the honeycomb electrode surface. Figure 5 b) shows the photocurrent generated by NIR (λ=785 nm) irradiation at a distance of 1 cm from the electrode surface at a constant ion intensity, with or without dissolved oxygen. Figure 6 illustrates surface plasmon coupling within and between silver nanoparticle assemblies. Specifically, in Figure 6, the charge distribution (top) and electric field (bottom) are shown for silver nanoparticle assemblies investigated under the following conditions: a) uncoated nanoparticles, b) uncoated nanoparticles with two oxygen molecules in the nanogap, c) uncoated nanoparticles with oxygen molecules bound to the surface of the left nanoparticle, d) uncoated nanoparticles with oxygen molecules bound to the surfaces of the left and right nanoparticles, respectively, e) Ag2O-coated nanoparticles (2 nm thick), f) Ag2O-coated nanoparticles with two oxygen molecules in the nanoparticles, g) Ag2O-coated nanoparticles with oxygen molecules bound to the right nanoparticles, and h) Ag2O-coated nanoparticles with oxygen molecules bound to the left and right nanoparticles, respectively. Figure 7 illustrates surface plasmon coupling within and between silver nanoparticle assemblies. The charge distribution (top) and electric field (bottom) for the investigated ac) silver nanoparticles and df) silver nanoparticle assemblies coated with silver oxide (thickness 2 nm) are plotted as a function of distance between the far right nanoprism and the rest of the assembly under anaerobic (deoxygenated) conditions. The distances in question are a) 10, b) 13, c) 17, d) 8, e) 9.8, and f) 17 nm. Figure 7 also illustrates electromagnetic field transfer through silver nanorods in g) the absence and h) the presence of dissolved oxygen, with silver nanorods having diameters and lengths of 4 and 50 nm, respectively. Figure 7 also shows electromagnetic field transfer through a dimer composed of two kl) nanoprisms in the absence of nanospheres and nanospheres i) and k) and the presence of j) and l). In addition, Fig. 7 shows the electromagnetic field transfer between m) a nanoparticle tetrad composed of three nanoprisms and a nanosphere, and n) a nanoparticle tetrad composed of two nanoprisms and two nanospheres in the absence of dissolved oxygen. FIG. 8 shows surface-enhanced Raman spectra in the presence and absence of dissolved oxygen, and specifically, FIG. 8 shows surface-enhanced Raman spectra of a) p-aminothiophenol and b) p-nitrothiophenol at 1 fM with dissolved oxygen removed and without dissolved oxygen removed (blanks contain Na2SO3). Figure 9 shows the oxygen removal effect on the detection limit of surface-enhanced Raman spectroscopy; specifically, Figures 9a) and b) show the surface-enhanced Raman spectra of p-aminothiophenol at various concentrations without dissolved oxygen removal, and c) shows the intensity of the CS stretching mode as a function of the logarithm of concentration. Figures 9d) and e) show the surface-enhanced Raman spectra of p-aminothiophenol at various concentrations with dissolved oxygen removal. Additionally, Figure 9 shows the intensities of f) CH and g) CS stretching modes as a function of the logarithm of concentration. Figure 10 shows the characteristics of silver nanoparticles and citrate <1000 cm⁻¹ -1 It shows surface-enhanced Raman interference with the analyte. Specifically, Fig. 10a) shows the size distribution of silver nanoparticles measured via dynamic light scattering. Also, in Fig. 10, the Raman spectra of b) silver nanoparticles and the analytes c) p-aminothiophenol and d) p-nitrothiophenol are shown. FIG. 11 shows the range of sensitivity enhancement by removing dissolved oxygen, and specifically, FIG. 11 shows the surface-enhanced Raman spectra of a thiolated oligonucleotide sequence in the presence (10 pM) and b) absence (8 zM) of dissolved oxygen. Figure 12 shows a) a transmission electron microscope image of spherical silver nanoparticles and b) oxygen removal-dependent signal enhancement of adsorbed p-aminothiophenol (for spherical nanoparticles). Figure 13 shows the surface-enhanced Raman spectra of p-nitrothiophenol at various concentrations with oxygen removal by a) ascorbic acid and b) hydrazine. Figure 14 shows the correlation between dissolved oxygen concentration and surface-enhanced Raman scattering intensity. Specifically, Figure 14 shows the relative surface-enhanced Raman spectra and signal intensity as a function of dissolved oxygen concentration when oxygen is removed by a) and b) the addition of Na2SO3 and c) and d) argon spraying. Figure 15 shows the effect of dielectric oxide surfaces on the enhancement of oxygen removal in surface-enhanced Raman spectroscopy, and specifically, Figure 15 shows the cyclic voltammetry of a) Ag2NO3 and silver nanoparticles with adsorbed p-aminothiophenol in b) the presence and c) absence of dissolved oxygen. Figure 16 a) shows the absorbance spectrum of gold nanorods and b) shows the surface-enhanced Raman spectra in the presence and absence of dissolved oxygen (the Raman spectrum of cetyltrimethylammonium bromide (CTAB) capping agent is provided for reference). Figure 17 a) shows a transmission electron microscope image of silica-coated silver nanoparticles and, for reference, b) surface-enhanced Raman spectra obtained in the presence of p-aminothiophenol and c) oxygen removal with 50 aM p-nitrothiophenol. Figure 18 shows the remote detection of an analyte adsorbed on a silver mirror at the bottom of a quartz NMR tube; specifically, Figure 18 shows the surface-enhanced Raman spectra of a) p-aminothiophenol and b) p-nitrothiophenol transmitted from the silver mirror through silver nanoparticles and measured at a distance of 10 cm from the incident laser and detector field of view. Figure 18 also shows the surface-enhanced Raman spectra of c) and d) CS stretching band intensity of p-aminothiophenol adsorbed on the silver mirror and transmitted through silica-coated silver nanoparticles as a function of distance. Figure 19 shows the remote detection of an analyte adsorbed on deoxygenated and aggregated silver nanoparticles in a 96-well microtiter plate, specifically, Figure 19 shows a surface-enhanced Raman spectrum measured using a glass capillary pre-filled with deoxygenated and aggregated pristine silver nanoparticles (incident laser 6 cm away from the inserted end of the capillary). Figure 19 shows the spectra of a) p-aminothiophenol and b) p-nitrothiophenol as a function of integration time. FIG. 20 illustrates remote detection of an analyte through integrated plasmonic coupling over 1 m; specifically, FIG. 20 shows a) a schematic diagram of remote detection of an analyte at the distal end of a 1 m long PEEK capillary using the probe of the present invention. Additionally, FIG. 20 illustrates the detection of b) p-aminothiophenol and c) p-nitrothiophenol from the configuration given in a). Specific details for implementing the invention

[0096] A method for increasing the SERS signal intensity of an analyte in a sample by modifying a liquid sample containing an analyte, and a probe for remote detection of an analyte in a liquid sample using SERS are provided.

[0097] The inventors have discovered that dissolved oxygen (DO) in a liquid sample is a major factor limiting the propagation of the plasmon field throughout the liquid sample, thereby limiting SERS sensitivity. Specifically, the inventors have found that by removing DO using a chemical deoxygenator, increased SERS sensitivity can be achieved, reaching the detection of analytes at the zM level, for example, in water. Importantly, these favorable results cannot be obtained by other methods of oxygen removal, such as spraying with an oxygen-free gas, which do not achieve sufficient DO removal. Furthermore, when using a deoxygenator, the inventors detected analytes outside the detector's field of view, which exhibits completely unexpected long-range signal propagation along the plasmonic material, allowing for remote detection of the analyte, for example, using the probe described herein.

[0098] How to modify liquid samples

[0099] In a first aspect of the present invention, a method is provided for modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte. The method of the present invention comprises the steps of: (a) providing a liquid sample to be analyzed using SERS; and (b) adding an oxygen scavenger to the liquid sample to remove dissolved oxygen from the liquid sample.

[0100] Liquid sample

[0101] The analyte used in the sample may generally be any analyte measured using SERS, i.e., molecules and ions containing two or more atoms of organic or inorganic matter. It is also known that single-atom ions can be detected indirectly using SERS, for example, through the formation of a vibrationally active complex. In such cases, the substance under study is technically a single atom, but the analyte detected by SERS (i.e., the vibrationally active complex) is polyatomic. In the examples, the analyte is a thiol compound, amine, pesticide, persistent organic pollutant, transition metal complex, peptide, protein, nucleic acid, polysaccharide, or hormone. In a preferred embodiment, the analyte is a transition metal complex, peptide, protein, nucleic acid, persistent organic pollutant, pesticide, or hormone. As an example, the analyte may be p-aminothiophenol (pATP) or p-nitrothiophenol (pNTP).

[0102] The liquid of the sample is preferably water, or an organic solvent such as methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, hydrocarbons, or mixtures thereof. In a preferred embodiment, the liquid is water.

[0103] In the examples, the method of the present invention further includes the step of modifying the analyte as needed so that it can interact with the surface of the plasmonic material used for SERS detection. Such modifications are well known to those skilled in the field of SERS spectroscopy. A common technique is to attach thiol groups to the analyte so that the analyte is chemisorbed on an Ag or Au plasmonic surface via these thiols. Other techniques include physisorption via hydrophobic / hydrophilic interactions or van der Waals forces; chemisorption via other bonding atoms; indirect detection via chemical reactions with pre-adsorbed reporter molecules; chelation of metal ions by pre-adsorbed chelating agents; indirect detection via affinity such as antibody-antigen binding of biomolecules; and indirect detection by holding plasmonic nanoparticles that possess surrogate SERS active reporter molecules via specific interactions with the analyte, such as antibody-antigen interactions.

[0104] The liquid sample to be modified using the method of the present invention must be analyzed using SERS. Since the method of the present invention is intended to increase the SERS signal intensity of the analyte in the liquid sample, the liquid sample contains the analyte to be analyzed. It should be understood that if the liquid sample does not contain the analyte (e.g., a blank), the desired increase in SERS signal intensity naturally does not occur.

[0105] Oxygen scavenger

[0106] As previously mentioned, the inventors have discovered that SERS sensitivity can be increased by removing DO using a chemical oxygen scavenger. Therefore, the oxygen scavenger may be any oxygen scavenger known in the art for reducing DO in a liquid. In the examples, the oxygen scavenger is sodium sulfite, hydrazine, ascorbic acid, or ascorbyl palmitate.

[0107] It is desirable to remove as much dissolved oxygen as possible from a liquid sample. Therefore, it is desirable to add an excess amount of oxygen scavenger to ensure that as much DO as possible is removed. In the examples, sufficient oxygen scavenger is added to remove at least 98%, 99%, 99.5%, or 99.9% of the DO from the liquid sample, preferably at least 99.5% or 99.9%. In the examples, sufficient oxygen scavenger is added so that the concentration of residual DO is at most about 0.020 mM, about 0.010 mM, about 0.005 mM, or about 0.002 mM, preferably at most about 0.005 mM or about 0.002 mM.

[0108] Additional selection steps

[0109] As mentioned above, liquid samples must be analyzed using SERS. This spectroscopic technique relies on localized surface plasmon resonance (LSPR). LSPR occurs when the vibrations of free-conducting electrons in a plasmonic material resonate with incident light (typically a laser). Both the position and intensity of LSPR bands depend on the properties of the plasmonic material and are highly sensitive to the dielectric properties of the surrounding medium. In particular, the position and intensity of LSPR bands change when various moiety is attached to (or in proximity to) the plasmonic material. In fact, when light is incident on a plasmonic material, the electromagnetic field is locally amplified (due to the excitation of localized surface plasmons), creating what is known as a "hot spot" where this field enhancement is higher. When molecules of the analyte come into contact with (or are in proximity to) the plasmonic material (e.g., adsorbed to the surface), the molecules interact with these hot spots, altering the resonance conditions of the surface plasmon waves. Due to these changes in resonance conditions, a SERS signal is generated that can be measured by a SERS spectrometer detector, allowing for the detection and quantification of the analyte.

[0110] Liquid samples and Plasmon Contact of substances

[0111] When a liquid sample is analyzed using SERS, in the examples, the method further includes the step of bringing the above-modified (or to be modified above) liquid sample into contact with a plasmonic material (also known as a SERS substrate). This is done to ensure that the analyte is in very close proximity to the surface of the plasmonic material (thus, it can interact with the amplified electric field generated by the plasmonic material upon SERS laser irradiation, thereby obtaining the benefits of the SERS enhancement factor unique to SERS technology). Various methods for boosting this SERS enhancement factor (and thus the measured signal) are very well known to those skilled in the art.

[0112] In the examples, at least a portion of the analyte is adsorbed (or becomes adsorbed) on the surface of the plasmonic material. As mentioned above, a general technique to achieve this is to allow the chemisorption of the analyte onto the plasmonic material, for example, to allow the chemisorption of an analyte containing thiol groups onto the surface of an Ag or Au plasmonic material.

[0113] By modifying a liquid sample by the method of the present invention, the limit of detection (LOD) for the analyte is unexpectedly and significantly reduced compared to the unmodified liquid sample. In the examples, the LOD for a given analyte / plasmonic material is typically at least about 10 8 , preferably about 10 9 , or even up to about 10 10 It is reduced by a multiple of . In a preferred embodiment, by the method of the present invention, the LOD is at least about 10 for an analyte using SERS 9 or about 10 10It is improved by a multiple of . In the examples, the LOD of the analyte using SERS is therefore up to about 1 pM, about 100 fM, about 10 fM, about 1 fM, about 100 aM, about 10 aM, about 1 aM, about 100 zM, or about 10 zM. In a preferred example, the LOD of the analyte is up to about 10 zM. Of course, the exact LOD of the modified liquid sample depends on the plasmonic material used and the analyte, as mentioned above.

[0114] Based on the experimental results described below, oxygen removal generates effective coupling of SERS fields, and this plasmonic coupling enables the propagation of the electromagnetic (EM) field throughout the sample volume without significant signal energy loss (i.e., with limited quenching). The plasmonic field generated by the photoexcitation of the plasmonic material travels throughout the sample, reaches the plasmonic material in contact or near the analyte, and then travels in the reverse direction to return the Raman scattering signal of the analyte to the detector. Without DO removal, it was found that the high electron affinity of oxygen traps electrons and prevents the effective generation, coupling, propagation, and integration of SERS fields along the plasmonic material, which was observed experimentally and through computational modeling as described below. Conversely, DO removal enables plasmonic coupling and SERS field transmission. This is the cause of the increase in the SERS signal intensity of the analyte.

[0115] Typically, molecular oxygen suspended in nanogaps or adsorbed on the surface of plasmonic materials causes charge redistribution and extinguishes plasmonic fields and coupling. However, as shown in the experiments below, this is avoided by using the method of the present invention. Even in the case of Ag2O-coated nanoparticles, where the plasmonic field is somewhat reduced compared to uncoated silver nanoparticles, the introduction of DO significantly quenched the EM field and prevented coupling. In the experiments below using plasmonic nanoparticles, the DO quenching effect is independent of representative parameters characteristic of the nanogaps, including the nanogap distance, the number of nanoparticles within the nanoassembly (2, 3, and 4 nanoparticles), and the characteristics of the plasmonic nanoparticles (size, shape, position, orientation, and surface chemistry). This modeling demonstrated that while DO extinguishes the SERS EM field, in the absence of an EM field, the EM field can propagate throughout the sample via adjacent plasmonic nanostructures and their aggregates through plasmonic coupling.

[0116] As explained above, the conventional understanding of SERS is based on signal amplification using individual nanostructures or plasmonic field coupling of adjacent nanoparticles (e.g., nanogaps). This is the "hot spot" theory. However, in this experiment (see below), it was demonstrated that large-scale plasmonic field coupling (or integrated surface-plasmon resonance) is possible during DO removal with an oxygen scavenger. This integrated field can effectively transmit SERS signals from single molecules that are in close proximity to or in contact (preferably adsorbed) with individual plasmonic materials (e.g., plasmonic nanoparticles) throughout the entire liquid sample; for instance, all nanoparticles can activate SERS and act as "hot spots" for the adsorbed analyte or transmit SERS signals to perform actual single-molecule detection.

[0117] In the method of the present invention, signal enhancement due to oxygen removal has been observed for various plasmonic materials, including silica-coated plasmonic nanoparticles such as silver nanoparticles and AgNPs, i.e., SHINERS technology (see examples below), where LOD is 500 nM to 50 aM (10 10 It was observed that the overall sensitivity of silica-coated plasmon nanoparticles is generally lower than that of exposed (uncoated) plasmon nanoparticles (when comparing silica-coated AgNPs and uncoated AgNPs, 10 4 (times lower), it appears that DO, rather than the surface oxide layer, is the primary factor in extinguishing SERS signals; that is, although absolute sensitivity varies by particle, the relative signal enhancement upon DO removal is always 10 9 to 10 10 It is a double. As an example, the effect of DO from the surface oxidation effect was decoupled using AuNRs (gold nanorods) and silica-coated AgNPs.

[0118] Accordingly, in the method of the present invention, the plasmonic material may be any plasmonic material used in SERS. Such materials are known to those skilled in the art and are extensively described in the scientific literature. As is known to those skilled in the art, plasmonic materials for SERS consist of materials capable of exhibiting localized surface plasmon resonance when irradiated with a SERS laser. Such materials may be conductive, semiconductive, and / or dielectric materials, combinations thereof, or composites thereof. Non-limiting examples of preferred materials capable of exhibiting localized surface plasmon resonance when irradiated with a SERS laser include gold, silver, and copper, as well as such materials having surfaces covered by various thin dielectric oxide layers, including thin layers of silica or alumina as reported in the prior art, for example. Preferred plasmonic materials are gold, silver, and copper, preferably gold and silver, more preferably silver.

[0119] In addition, it is well known that plasmonic materials may have a rough surface (referred to herein as a "rough plasmonic surface"), be composed of nanoparticles (referred to herein as "plasmonic nanoparticles"), or be composed of a combination thereof. In a preferred embodiment, the plasmonic material is a plasmonic nanoparticle.

[0120] For clarity, when referring to a "rough plasmonic surface," the degree of roughness must be sufficient to generate the desired local surface plasmon resonance. Those skilled in the art will understand the meaning of the expression "rough plasmonic surface" in the context of SERS. For clarity, in the examples, the roughness (Ra) of the rough plasmonic surface is about 5 nm to about 50 nm, preferably about 10 nm to about 20 nm. In the examples, the roughness of the plasmonic surface is at least about 5 nm; at least about 10 nm; or at least about 20 nm; and / or up to about 200 nm; up to about 100 nm; or up to about 50 nm. For clarity, the rough plasmonic surface does not necessarily need to undergo a separate "roughening" step; the fabricated surface may already have the roughness defined above.

[0121] Those skilled in the art will understand that while the above roughness parameters relate to rough plasmonic surfaces, other plasmonic materials, such as plasmonic nanoparticles, may include regions of high curvature, such as edges and vertices, where the electromagnetic field is generally highest.

[0122] Common examples of plasmonic nanoparticles include gold, silver, or copper nanoparticles (including silica-coated gold, silver, or copper nanoparticles). In a preferred embodiment, the plasmonic nanoparticles are silver or gold nanoparticles (without coating), more preferably silver nanoparticles (without coating).

[0123] As is known to those skilled in the art, plasmonic nanoparticles may have various (nano)-shapes, sizes, forms, and size distributions. In particular, the size and shape of the nanoparticles may be customized to increase the SERS enhancement factor for a given analyte. In preferred embodiments, the plasmonic nanoparticles are prismatic, ellipsoidal (including spherical), plate-shaped, or rod-shaped. In the embodiments, the D50 of the diameter of the plasmonic nanoparticles is a size of about 2 nm to about 500 nm. In the embodiments, the D50 of the diameter of the plasmonic nanoparticles is at least about 2 nm, at least about 10 nm, at least about 25 nm, or at least about 50 nm, and / or up to about 500 nm, up to about 250 nm, up to about 200 nm, up to about 100 nm, up to about 75 nm, or up to about 50 nm.

[0124] In a preferred embodiment of the method of the present invention, plasmonic nanoparticles are aggregated and brought very close (and / or in contact) with one another. Indeed, as shown in the example below, the use of an oxygen scavenger allows for long-distance signal propagation along the plasmonic material, which in turn allows for remote detection of the analyte. Plasmononic nanoparticles can be aggregated using techniques known in the art. It is known to those skilled in the art that there are numerous approaches to aggregate nanoparticles, including adding an aggregating molecule (i.e., a molecule capable of linking nanoparticles and inducing an aggregating agent) or an aggregating agent such as an electrolyte (e.g., a salt), adjusting the pH, adding an organic solvent such as an alcohol, or combining these approaches. In a preferred embodiment of the present invention, nanoparticles are aggregated through the addition of a dissolved salt and / or pH adjustment.

[0125] In the examples, plasmonic nanoparticles are aggregated through the addition of a coagulant such as a sulfate or a sulfite. In the examples, the coagulant and the oxygen scavenger are different substances. In the examples, the coagulant is an oxygen scavenger, which is preferably a sulfite, preferably sodium sulfite. In some examples, the pH of the liquid sample is adjusted to about 4 before aggregation. For example, SiO2-coated AgNPs can have their pH adjusted to about 4 before aggregation by adding a sulfite or a sulfate.

[0126] Additionally, in a preferred embodiment, the plasmonic nanoparticles may be stabilized by loosely bound small capping molecules, preferably citrate, ethanol, ethylene glycol and / or polyethylene glycol, more preferably citrate and / or ethanol. These capping molecules can act as reducing agents and structural indicators, which means they can determine the particle shape during synthesis and stabilize the nanoparticles formed later. These capping molecules (capping ligands) can also be easily replaced by thiolated analytes while simultaneously enhancing the optical properties of the plasmonic material (functioning as a SERS substrate).

[0127] In a preferred embodiment, plasmonic nanoparticles are added to a liquid sample before nanoparticle aggregation and before the removal of dissolved oxygen using an oxygen scavenger.

[0128] In the examples, molecules that interact with a specific analyte to generate a distinct signal, such as chelating agents for metal ions, can be pre-adsorbed onto plasmonic nanoparticles or a rough plasmonic surface before contacting the analyte.

[0129] SERS Spectrum measurement

[0130] When a liquid sample is modified according to the method defined above and then comes into contact with a plasmonic material, the method of the present invention may further include the step of measuring the SERS spectrum of the liquid sample.

[0131] This step may be performed using any SERS technology known in the art using any SERS system known in the art. Generally, this step includes irradiating the plasmonic material with light, typically a laser, and detecting the Raman signal scattered by the plasmonic material.

[0132] In a preferred embodiment, the measurement step is performed using the probe of the present invention defined in a subsequent section.

[0133] In a preferred embodiment of the present invention, the SERS system is as defined in the following section.

[0134] SERS For remote sensing used probe

[0135] In a second aspect of the present invention, a probe is provided for remote detection of an analyte in a liquid sample using SERS. The probe

[0136] A detection chamber having a window transparent to SERS excitation light and Raman scattering signals, and

[0137] A pipe having first and second ends, wherein the first end of the pipe is movably connected to the detection chamber and the second end of the pipe is configured to come into contact with a liquid sample, and

[0138] The piping and detection chamber between the first and second ends above contain plasmon nanoparticles contained in a deoxygenation solvent, and

[0139] The plasmon nanoparticles are in very close proximity to each other to allow the uninterrupted propagation of a plasmon field from the detection chamber to the second end of the piping and from the second end of the piping to the detection chamber, and / or

[0140] The inner wall of the detection chamber and the inner wall of the piping between the first end and the second end are coated with a plasmon layer, and

[0141] The plasmon layer is continuous from the detection chamber to the second end of the piping, thereby allowing uninterrupted propagation of the plasmon field from the detection chamber to the second end of the piping and backward, and

[0142] The above piping and detection chamber are filled with a deoxygenation solvent, and

[0143] The above deoxygenating solvent includes an oxygen remover that removes dissolved oxygen from the above deoxygenating solvent.

[0144] A plasmonic layer is a layer of plasmonic material containing a rough plasmonic surface. In this section, the plasmonic material (including plasmonic nanoparticles and rough plasmonic surfaces) is the same as that described in the previous section (e.g., identical in material, roughness, shape, etc.).

[0145] While the probe is in use, SERS excitation light will enter the detection chamber through a transparent window. This SERS excitation light shines on the nanoparticles behind the window of the detection chamber, generating a plasmon field. Because the plasmon nanoparticles are very close to each other (or the plasmon layer is continuous) throughout the probe (tubation and detection chamber) and because the plasmon nanoparticles are in a deoxygenated solvent containing an oxygen scavenger, this plasmon field will propagate uninterruptedly from the detection chamber to the second end of the tubing. There, the plasmon field will interact with the analyte to generate a signal plasmon field, which will propagate back along the tubing toward the detection chamber. Finally, the signal plasmon field exits the detection chamber through the transparent window and is eventually detected by a photodetector.

[0146] As mentioned, the probe of the present invention is intended for remote sensing using SERS. In particular, by bringing the second end of the tube into contact with a liquid sample, the SERS spectrum of the sample can be obtained even though the sample is at a specific distance (i.e., the length of the tube) from the spectrometer. This allows the spectrum of the sample to be measured without bringing the sample close to the spectrometer, and the sample can be measured even in a space too small for the spectrometer (since it is only necessary for the piping to enter the space where the liquid sample is held).

[0147] The size and length of the probe depend on the dimensions of the piping and detection chamber, details of which are defined below. For example, using longer and narrower piping naturally results in a longer and narrower probe. However, those skilled in the art will understand that the dimensions of the probe can be selected and adjusted according to its intended application. For example, if the probe is used to measure liquid samples contained in a nuclear reactor, the dimensions of the piping (and thus the probe) are specified so that the liquid samples within the reactor can be safely reached and measured.

[0148] In a preferred embodiment, the detection chamber and the piping between the first end and the second end comprise plasmonic nanoparticles immersed in a deoxygenated solvent. In the embodiment, the plasmonic nanoparticles aggregate along the length of the piping as defined in the previous section using any of the techniques discussed in the previous section.

[0149] The deoxygenation solvent used may be any solvent known in the art. In the examples, the solvent is water or an organic solvent, such as methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, hydrocarbons, or a mixture thereof. In a preferred example, the solvent is water. As previously stated, the solvent is deoxygenated, and the solvent comprises an oxygen scavenger that removes dissolved oxygen from it, meaning that its dissolved oxygen has been removed using the oxygen scavenger. The oxygen scavenger is as defined in the previous section. Additionally, the concentration of the added oxygen scavenger and the concentration of residual DO are as defined in the previous section.

[0150] As mentioned in the previous section, the removal of dissolved oxygen creates effective coupling of the SERS field, and this plasmonic coupling enables the propagation of the electromagnetic field without significant signal energy loss. Therefore, using a deoxygenated solvent, the electromagnetic field propagates along the length of the piping.

[0151] pipe

[0152] The piping of the probe of the present invention enables the propagation of a plasmon field from the detection chamber to a second end of the piping and from the second end of the piping to the detection chamber. A signal plasmon field can be generated at the second end in various ways. One example is when the analyte contained in the liquid sample comes into contact (preferably adheres) with a plasmonic material, such as nanoparticles contained in a deoxygenating solvent, at the second end of the piping. Another example is when the liquid sample generates a plasmon field on its own (e.g., when the liquid sample contains plasmonic nanoparticles modified and aggregated using the method defined above). The latter example implies that both the liquid sample and the deoxygenating solvent contained in the tube have been deoxygenated using an oxygen remover.

[0153] FIG. 1a is a longitudinal cross-sectional view of the second end of a pipe (g) according to an embodiment of the probe of the present invention. In this drawing, the inner wall (12) and the outer wall (16) of the pipe (g) are identified together with the end wall (14) of the end of the pipe (g).

[0154] The second end of the pipe may be open or closed (it is preferable to be open). FIGS. 1B and FIGS. 1C illustrate an embodiment in which the second end of the pipe (g) is open. FIGS. 1B is a cross-sectional view of the second end of the pipe (g) containing plasmon nanoparticles (18) contained in a deoxygenating solvent according to an embodiment of the probe of the present invention. FIGS. 1C is a cross-sectional view of the second end of the pipe (g) in which the inner wall (12) of the pipe (g) is coated with a plasmon layer (20) and the pipe (g) is filled with a deoxygenating solvent (23) according to another embodiment of the probe of the present invention.

[0155] When the piping is opened, the deoxygenating solvent (and plasmon nanoparticles, if used) may flow out from the second end of the piping in some cases. Accordingly, in some embodiments where the second end of the piping is open, the detection chamber and the piping are refilled with the deoxygenating solvent (and plasmon nanoparticles, if used) from, for example, a pump or reservoir, so that the piping and the detection chamber remain filled with the deoxygenating solvent (and plasmon nanoparticles, if used, so that the plasmon nanoparticles are kept close to each other). The pump or reservoir may be connected directly to the first end of the piping or connected to the detection chamber in such a way that the deoxygenating solvent / plasmon nanoparticles flowing out from the second end of the piping are replaced. In other cases, particularly when the diameter of the piping is small, capillary forces may retain the deoxygenating solvent (and plasmon nanoparticles, if used, even though the second end of the piping is open). In another case, it may be sufficient to simply ensure that there is no air leakage in the detection chamber and piping so that the deoxygenating solvent (and plasmonic nanoparticles, if plasmonic nanoparticles are used) remains inside the piping even though the second end of the piping is open (this is similar to removing a straw from a cup of water with the top of the cup blocked, and the water remains in the straw while the top of the straw is blocked).

[0156] In a preferred embodiment in which the second end of the piping is open, the second end of the piping is at least partially coated with a plasmon layer. An example showing such a coating is illustrated in FIGS. 1d and 1e. FIG. 1d is a longitudinal cross-sectional view of the second end of the piping (g) containing plasmon nanoparticles (18), wherein the end wall (14), the inner wall facing the second end of the piping (g), and the outer wall facing the second end (24) are coated with a plasmon layer (26). This plasmon layer (26) is in very close proximity (preferably in contact) with the plasmon nanoparticles and the deoxygenated solvent, thereby ensuring the propagation of a plasmon field between the nanoparticles and the layer. FIG. 1e is a longitudinal section of the second end of a pipe (g) in which the inner wall (12) is coated with a plasmonic layer (28) (similar to that shown in FIG. 1c), but the plasmonic layer (28) also covers the outer wall and the end wall (14) toward the second end (24) of the pipe (g). These plasmonic layers (26, 28) also come into contact with a liquid sample that allows for a desired interaction between the analyte and the plasmonic field.

[0157] In an alternative embodiment, the second end of the tubing may also be covered with a plasmonic layer. An embodiment illustrating such capping is shown in FIG. 1f and FIG. 1g. FIG. 1f is a cross-sectional view of the second end of the tubing (g) containing plasmonic nanoparticles (18) covered with a plasmonic layer (30). The inner side of the plasmonic layer (30) is in very close proximity (preferably in contact) with the plasmonic nanoparticles and the deoxygenated solvent, thus ensuring the propagation of a plasmonic field between the nanoparticles and the layer. FIG. 1g is a cross-sectional view of the second end of the tubing (g) in which the inner wall (12) is coated with a plasmonic layer (32) (similar to that shown in FIG. 1c), but the plasmonic layer (32) also covers the second end of the tubing (g). When in use, the outer surface of these plasmonic layers (30, 32) comes into contact with the liquid sample, allowing for the desired interaction between the analyte and the plasmonic field. In fact, any analyte in contact with or near the plasmonic layer will result in the generation of a signal plasmonic field that is transmitted to a detection chamber where the SERS signal can be measured by a spectrometer. With this configuration, there is no need to continuously replenish the plasmonic nanoparticles contained in the deoxygenated solvent because nothing will escape from the second end (unlike a configuration where the second end is open).

[0158] The tubing may be manufactured from any tubing material known in the art that does not have an adverse effect on the generation and transmission of the plasmon field. Generally, the choice of tubing material may be influenced by the intended use of the probe. For example, if the probe is used in a situation where the tubing must be bent, a more flexible material may be more suitable. In the embodiments, the tubing is manufactured from metal, quartz, glass, or polymeric plastics such as polyethylene, polypropylene, polystyrene, polyetheretherketone, polyvinyl chloride, polytetrafluoroethylene, or polydimethylsiloxane. In a preferred embodiment, the tubing is a polyetheretherketone (PEEK) polymer tubing or a glass capillary.

[0159] Generally, a narrower tubing is advantageous. By using tubing with a smaller inner diameter, fewer plasmon nanoparticles will fill the tubing while submerged in the deoxygenating solvent; capillary action within the tubing will increase, and the transmission of the plasmon field along the length of the tube will be improved because it will be easier to pack the plasmon nanoparticles together in close proximity. Additionally, tubing with a smaller outer diameter results in a smaller size (meaning the tubing can fit into a smaller space). Furthermore, the inner diameter of the tubing must be suitable for scattering detection. In the examples, the inner diameter of the tubing is between about 0.08 mm and about 1 cm, preferably between about 0.3 mm and about 0.5 mm. In the examples, the inner diameter of the tubing is at least about 0.08 mm; at least about 0.1 mm; at least about 0.2 mm; or at least about 0.3 mm; and / or up to about 1 cm; up to about 5 mm; up to about 2 mm; up to about 1 mm; up to about 0.75 mm; Or up to about 0.5 mm. In a preferred embodiment, the inner diameter of the pipe is about 0.3 mm or about 0.5 mm.

[0160] As previously mentioned, extending the length of the tubing increases the length of the probe, allowing the SERS spectrum of the sample to be measured at a greater distance from the SERS spectrometer. In the embodiments, the length of the tubing is at least about 5 cm; at least about 10 cm; at least about 20 cm; at least about 50 cm; or at least about 75 cm; and / or up to about 50 m; up to about 20 m; up to about 10 m; up to about 5 m; up to about 2 m; or up to about 1 m. In a preferred embodiment, the length of the tubing is about 1 m or about 2 m.

[0161] Those skilled in the art will understand that piping does not necessarily have to have a circular cross-section. In fact, the cross-section can be of any shape (elliptical, rectangular, etc.).

[0162] Close proximity

[0163] Those skilled in the art are well aware that the intensity of a plasmon field generated on the surface of a plasmonic material decreases (somewhat exponentially) as the distance from the surface increases.

[0164] Here, in the context that the analyte is in very close proximity to the plasmonic nanoparticle or plasmonic layer, "proximity" similarly refers to the analyte being close enough to the layer / nanoparticle to interact with the plasmonic field of the nanoparticle / layer to generate a signal plasmonic field.

[0165] Here, in the context of plasmonic nanoparticles, "proximity" means that the nanoparticles are close enough for the plasmonic field of the nanoparticle to excite the next nanoparticle and thus generate a plasmonic field on the next nanoparticle, and this propagation of the plasmonic field is repeated uninterrupted from the detection chamber to the second end of the tubing and from the second end of the tubing to the detection chamber. Of course, "proximity" includes cases where the particles touch each other. In the context of a plasmonic layer being close to a plasmonic nanoparticle, "proximity" similarly means that the nanoparticle and the layer are close enough for the plasmonic field of the nanoparticle to excite the plasmonic field of the layer to generate a plasmonic field on the layer (and vice versa).

[0166] As explained above, the exact distance over which the plasmon field is transmitted depends on the plasmon material used. For example, as further discussed below, computational modeling demonstrated that for clean (uncoated) silver nanoparticles and silver nanoparticles coated with silver oxide, the plasmon field transmission terminates at distances of 17 nm and 9.8 nm, respectively. Closer distances will transmit the plasmon field further because they have stronger resonances. Therefore, in the examples, "proximity" refers to a maximum distance of about 15 nm, preferably a maximum distance of about 10 nm, and most preferably a maximum distance of about 5 nm (between plasmon nanoparticles, between plasmon nanoparticles and a plasmon layer, or between one of these and the analyte).

[0167] It must be understood that plasmon nanoparticles can come into very close proximity to each other in a manner where the density of plasmon nanoparticles varies along the length of the tubing, and importantly, that SERS signals can be transmitted along the length of the tube.

[0168] Because the plasmonic nanoparticles are in very close proximity to each other (or because the plasmonic layer is continuous), the signal plasmonic field generated at the second end of the pipe can be transmitted along the pipe to the first end, where the SERS signal can be measured by a spectrometer. It is important to reiterate here that the above propagation (between plasmonic nanoparticles or along the plasmonic layer) does not occur if dissolved oxygen is not removed using an oxygen scavenger. In such cases, the plasmonic field dissipates over a very short distance, even when using oxygen reduction techniques such as gas dispersal.

[0169] Detection chamber (Detection chamber)

[0170] The detection chamber is an element of the probe that is flexibly connected to the first end of the pipe. The probe's detection chamber is configured to be used with a surface-enhanced Raman spectrometer, so that incident light from the surface-enhanced Raman spectrometer can be applied to the detection chamber. The detection chamber is also where the incoming SERS signal from the pipe is measured by the surface-enhanced Raman spectrometer within the field of view of the spectrometer detector. This is why the detection chamber is transparent to SERS laser light and Raman scattered light; by doing so, the SERS laser can be applied to the detection chamber, and the incoming SERS signal can be measured within the field of view of the detector.

[0171] The detection chamber and the first end of the piping are preferably fluidly connected to each other through an opening defined by the detection chamber so that a deoxygenating solvent (and plasmonic nanoparticles, if plasmonic nanoparticles are used) can flow between the detection chamber and the piping; or the plasmonic layer is continuous (i.e., uninterrupted) from the detection chamber to the piping. The detection chamber also contains plasmonic nanoparticles immersed in the deoxygenating solvent or has an inner wall coated with a plasmonic layer, so that the SERS signal transmitted to the first open end of the piping can propagate throughout the detection chamber, where it can be measured within the field of view of the detector.

[0172] Those skilled in the art will understand that the detection chamber may have any shape and structure suitable for use in SERS and may be manufactured of any material suitable for SERS. In a preferred embodiment, the detection chamber is made of glass, plastic, or quartz, more preferably glass or quartz.

[0173] Liquid sample

[0174] The liquid sample may be any liquid sample to be measured by the probe. In the embodiments, the analyte contained in the liquid may be placed in contact with a plasmonic material contained in the probe (defined in the previous section) (e.g., a plasmonic layer completely covering the second end in the case of a closed second end, or a plasmonic layer coating the second end in the case of an open second end, or plasmonic nanoparticles contained in a deoxygenated solvent). In the embodiments, the liquid sample may be placed in contact with the plasmonic material as defined in the previous section. In these embodiments, the plasmonic material (and the liquid sample accordingly) may be located on a surface and / or a container configured to be in contact with the second end of the tubing. In a preferred embodiment, the analyte is adsorbed onto the plasmonic material.

[0175] In a preferred embodiment, the liquid sample was preferably deoxygenated using an oxygen scavenger as defined in the previous section. The amount of oxygen scavenger and the DO concentration remaining in the liquid sample may be as defined in the previous section.

[0176] In the examples, the liquid sample is the liquid sample defined in the previous section. In a preferred embodiment, the liquid sample was modified using the method of the present invention.

[0177] Those skilled in the art will understand that since the probe is intended to be used for SERS measurements, the liquid sample may be any liquid sample on which SERS measurements can be performed.

[0178] As described above, the probe of the present invention is configured to come into contact with a liquid sample. For example, the second end of the probe may be immersed in the liquid sample or the second end may be applied to the surface of the liquid sample. For clarity, this is the tip of the second end of the tubing (whether closed or not) positioned to come into contact with the liquid sample.

[0179] SERS Used with a spectrometer probe

[0180] This section will describe how to configure a probe to operate with a SERS spectrometer. Figure 2 illustrates a schematic diagram of a conventional surface-enhanced Raman spectrometer. The system in Figure 2 consists of a transparent sample holder, a laser light source, a detector, a fiber optic cable connecting the laser light source and the detector to the sample holder, and a computer that interprets and displays data obtained from the detector. However, Figure 3 illustrates a diagram of a surface-enhanced Raman spectrometer for remote sensing that includes most of the same components as the surface-enhanced Raman spectrometer given in Figure 2. However, instead of a sample holder, the probe has a detection chamber where incident light is applied and scattered light is detected, which is connected to a tip (a second end of the tubing) that comes into contact with a liquid sample through the tubing (e.g., immersed in the liquid sample or applied to the surface of the liquid sample). The detection chamber shown in Figure 3 is connected to a pump and a reservoir to fill the tubing with fluid.

[0181] In addition, it should be noted that the probe of the present invention can be used in SERS spectrometer systems, such as those used for remote testing in the experimental section below.

[0182] When in use, the probe of the present invention is positioned to come into contact with the analyte. That is, the second end of the tubing comes into contact with the analyte. In a preferred embodiment, the analyte is adsorbed onto the surface of a plasmonic material, which may preferably be the surface of nanoparticles within the tubing, a plasmonic layer coated on the second end of the tubing, and / or a plasmonic layer covering the second end of the tubing. In an alternative embodiment, the analyte is in a deoxygenated liquid sample comprising plasmonic nanoparticles generated and aggregated according to the method of the present invention or a rough plasmonic surface, either of which comes into contact with nanoparticles within the tubing; a plasmonic layer coated on the second end of the tubing; and / or a plasmonic layer covering the second end of the tubing.

[0183] In the examples, molecules that interact with a specific analyte to generate a distinct signal, such as chelating agents for metal ions or antibodies, peptides, or aptamers that interact with biomolecules, may be pre-adsorbed on plasmonic nanoparticles of the tube, plasmonic nanoparticles of the liquid sample, a plasmonic layer coated on the second end of the tubing, and / or a plasmonic layer covering the second end of the tubing.

[0184] Both the surface-enhanced Raman spectrometer of FIG. 2 (when using a liquid sample modified according to the method of the present invention) and the surface-enhanced Raman spectrometer for remote sensing shown in FIG. 3 (including the probe of the present invention) can be used to detect an analyte by applying incident laser irradiation to a sample holder or detector and measuring scattered light using a detector. In a preferred embodiment, backscattered light, i.e., light scattered 180° relative to the incident light, is detected, but light scattered at any angle relative to the incident light may be detected.

[0185] Dramatic signal amplification is observed in the surface-enhanced Raman spectrometer of Fig. 2 (when using a liquid sample modified according to the method of the present invention) and the surface-enhanced Raman spectrometer for remote sensing shown in Fig. 3 (including the probe of the present invention) when oxygen is removed using a chemical oxygen remover; oxygen removal also enables the remote sensing capability of the surface-enhanced Raman spectrometer using the probe of the present invention.

[0186] Advantages of the present invention

[0187] In developing the method of the present invention, the inventors discovered a surprising result that the SERS spectrum of the analyte increases dramatically after DO removal using an oxygen scavenger.

[0188] In the examples, in addition to the previously discussed advantages, the probe of the present invention as well as the method of the present invention may exhibit one or more of the following advantages:

[0189] The limit of detection (LOD) for the analyte can reach 10 zM (sensitivity is 10 compared to 10 pM for aerobic (non-deoxygenated) samples). 9 increase).

[0190] Signal enhancement is LOD from 500nM to 50aM(10 10 AgNPs reduced by a factor of 1 / 2 can also be observed when using silica-coated nanoparticles such as SHINERS technology.

[0191] Absolute sensitivity varies depending on the particle, but the relative signal enhancement upon DO removal (compared to unmodified liquid samples) is consistently 10 9 -10 10 It could be between the boats.

[0192] There is no longer a need to irradiate the sample under analysis (i.e., it no longer needs to be within the beam diameter of the incident light source) nor within the field of view of the detector, which allows for a wide variety of potential applications of the method and probe of the present invention, such as environmental monitoring, for example.

[0193] The detection time appears to be determined by the movement of the plasmon field, rather than by the diffusion of the analyte into the detector's field of view. The SERS signal can be obtained immediately upon irradiation at the other end of the tubing. This rapid response provides high temporal resolution along with high sensitivity, enabling the monitoring of rapid chemical reactions (e.g., chemical reaction studies or detection of short-lived species at the single-molecule level).

[0194] The method of the present invention is cost-effective and requires only a standard Raman spectrometer without an expensive SERS detector. This will significantly reduce the capital cost of conducting SERS experiments and promote this research globally, particularly in developing countries and resource-constrained regions. It is believed that the unprecedented detection sensitivity, fast response time, and remote sensing capabilities can generate many applications in medical diagnostics, environmental monitoring, and national security.

[0195] definition

[0196] In the context of describing the invention (particularly in the context of the following claims), the terms "a," "an," "the," and similar designations shall be interpreted as including both singular and plural forms, unless otherwise indicated in this specification or clearly contradicted by the context.

[0197] The terms "comprising," "having," "including," and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited thereto") unless otherwise noted.

[0198] References to ranges of values ​​in this specification are intended merely to serve as a shortened method of individually referring to each individual value within the range, unless otherwise indicated in this specification, and each individual value is incorporated into the specification as if individually cited in this specification. All subsets of values ​​within the range are also incorporated into the specification as if individually cited in this specification.

[0199] All methods described in this specification may be performed in any appropriate order unless otherwise indicated in this specification or clearly contradictory in the context.

[0200] All examples or use of exemplary language provided herein (e.g., "such as") are merely for better illustrating the invention and do not limit the scope of the invention unless otherwise claimed.

[0201] No language in the specification shall be interpreted as representing an element not claimed to be essential to the practice of the invention.

[0202] The term "about" used herein has a conventional meaning. In the embodiments, this may mean plus or minus 10% or plus or minus 5% of the qualified numerical value.

[0203] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains.

[0204] Other objects, advantages, and features of the present invention will become more apparent from reading the following non-limiting description of specific embodiments of the invention given merely as examples, with reference to the accompanying drawings.

[0205] Exemplary Examples Explanation of

[0206] The present invention is further illustrated in detail by the following non-limiting examples.

[0207] Experimental Procedure

[0208] Surface-enhanced Raman spectrum is 785 nm solid-state Nd-YAG laser (linewidth <3.2 cm -1 The backscattered light (180°) was recorded using a standard Sunshine TG-Raman fiber spectrometer (Changchun New Industries Optoelectronics Tech. Co., Ltd., Changchun, China). The following is a detailed experimental procedure including sample preparation, analysis, and computational modeling.

[0209] Materials and Reagents

[0210] All reagents were commercially available and used without further purification. Silver nitrate (AgNO3, 99.9%), sodium borohydride (NaBH4, 99%), trisodium citrate (99%), 4-aminothiophenol (pATP, 97%), 4-nitrothiophenol (pNTP, 80%), tetraethyl orthosilicate (TEOS, 98%), sodium hydroxide (99%), sodium sulfate (99%), sodium sulfite (99%), and ammonium hydroxide (30%) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Ascorbic acid (>99%) was purchased from Alfa Aesar (Ward Hill, MA, USA). Hydrazine hydrate was purchased from EMD Millipore (Burlington, MA, USA). Anhydrous ethanol (EtOH) and hydrogen peroxide (H2O2, 30 wt%) were purchased from VWR (Mississauga, ON, Canada). Single-stranded thiolized DNA 5-mers, namely 5a, 5c, 5g, and 5t, were purchased from Eurofins MWG Operon (Huntsville, AL, USA). Nanopure water (18 MΩ cm) was obtained using a Barnstead Nanopure system (Thermo Fisher Scientific, Waltham, MA, USA).

[0211] Silver nanoparticle synthesis (Silver Nanoparticle Synthesis)

[0212] Silver nanoparticles (AgNPs) stabilized by small, loosely bound capping molecules, namely citrate and ethanol, were used as SERS substrates due to their excellent optical properties and the easy displacement of capping ligands by thiolized analytes. They were prepared using the method reported in *A Systematic Study of the Synthesis of Silver Nanoplates: Is Citrate a "Magic" Reagent?* by Q. Zhang, N. Li, J. Goebl, Z. Lu, Y. Yin, J. Am. Chem. Soc. 133, 18931-18939 (2011), the entirety of which is incorporated herein by reference. The generated AgNPs exhibited a maximum absorbance at 590 nm, and TEM imaging revealed triangular nanoparticles with an average diameter of 17 ± 2 nm (see Fig. 4 for transmission electron micrographs of the silver nanoparticles and aggregates used in the experiment below). As prepared, AgNPs had a concentration of 0.3 nM, which was concentrated to 7 nM by centrifugation at 12,000 rpm for 20 minutes. Unless otherwise specified, all references to AgNPs in the experiments below refer to the AgNPs prepared above.

[0213] Preparation of Silica-coated Silver Nanoparticles

[0214] Equal volumes of AgNP (7 nM) and TEOS (1 μM) were sonicated in 1.5 mL of EtOH in a polypropylene microcentrifuge tube for 10 minutes. After centrifugation at 12,500 rpm for 20 minutes, no aggregation was observed. The pH was adjusted to 4 before adding the aggregator for SERS measurement. Unless otherwise specified, all references to silica-coated AgNP in the experiments below refer to the silica-coated AgNP prepared above.

[0215] Electrochemical Measurements

[0216] Current measurement experiments were performed using a bipotentiostat (CS2350, Wuhan Corrtest Instruments Corp., Ltd., Wuhan, China). Screen-printed gold honeycomb working and counter electrodes were used in conjunction with an external Ag / AgCl reference electrode (Pine Research Instrumentation, Durham, NC, USA). The honeycomb electrode had 19 holes 0.50 mm in diameter and 1.5 mm deep separated by 0.75 mm, allowing AgNPs to pass through the working electrode. The solution was stirred at 1200 rpm. For all potential range measurements, the initial potential was 0 V, and the low and high potentials were -0.9, -1, and -1.7 V, and +0.9, +1, and +1.7 V, respectively. Current enhancement was independent of the applied voltage, and no contribution due to electrode instability was observed. The pulse width was 10 seconds at a frequency of 10 Hz. A quartz cuvette was used as a sample holder (no background interference was observed). The sample was irradiated with a 785 nm laser outside the electrode region. Refer to Fig. 5a for an experimental schematic showing the system described above used for electrochemical measurements. For clarity, these electrochemical experiments were designed to demonstrate enhanced plasmon field transfer through the excitation of AgNPs with a laser. The analyte was absent or no spectrum was generated.

[0217] Surface Enhanced Raman Spectroscopy

[0218] SERS signals were measured in 5 x 100 mm quartz NMR tubes (0.5 mm quartz thickness) to evaluate the detection sensitivity for various analytes adsorbed on AgNPs. Samples were aggregated by adding sulfites or sulfates and other additives as indicated in the experiment. In particular, SiO2-coated AgNPs required pH adjustment to ~4 prior to aggregation with the addition of sulfites or sulfates. Samples were analyzed using a solid-state Nd-YAG laser (785 nm, linewidth <3.2 cm). -1It was excited using (500 mW). Laser stability is 5-8 cm. -1 The resolution was ±5%. A 785 nm fiber laser probe was connected to a laser source with an output power of 450 mW and a beam diameter of 2.5 μm. The sample power (~5 mW) was recorded using a Thorlabs PM100 optical wattmeter. Backscattered (180°) light was collected using a standard Sunshine TG-Raman fiber spectrometer (Changchun New Industries Optoelectronics Tech. Co., Ltd., Changchun, China). The SERS spectrum was at 1000 cm⁻¹ due to strong spectral interference from capping molecules, namely citrate and ethanol. -1 It was collected from the excess. However, to support the conclusions summarized below, 200-1650cm -1 Sample spectra between them are provided. All spectra are displayed as the average of three measurements, each consisting of 1505 data points.

[0219] Sample Preparation and Single-Molecule Detection Probability Analysis

[0220] Careful sample preparation through serial dilution was used to obtain accurate and precise measurements of samples containing extremely low concentrations of the analyte (pNTP). All pre-washed glass vessels (e.g., 100 mL volumetric flasks and quartz cuvettes) were immersed in freshly prepared aqua regia for 2 hours, rinsed with nanopure water until neutral, and then rinsed with anhydrous ethanol. The analyst preparing the samples tested the accuracy and precision of the volume delivered by the 20 μL micropipette by weighing the volume of water delivered using an analytical balance. The obtained value was 20.00 ± 0.09 μL (n=10). The stock solution was carefully prepared by a series of 5000-fold dilutions (20 μL to 100 mL) from an initial stock prepared with >100.0 mg of pNTP to obtain four significant figures in the measurement, namely the initial stock solution (13.22 mM), which was prepared by dissolving 102.7 mg of pNTP in 50 mL of anhydrous ethanol with vortex mixing and sonication to ensure complete dissolution of the particles. The number of dilution steps was minimized to mitigate error propagation in the final concentration obtained. The first serial dilution (8 μM) was prepared by diluting 60.5 μL of the initial stock into a 100 mL Class A volumetric flask (100.00 ± 0.08 mL at 25°C). This was inverted several times to mix. 20 μL of the previous solution was diluted in a new 100 mL volumetric flask, and this procedure was repeated three times to produce solutions with nominal concentrations of 1.6 nM, 320 fM, and 64 aM. 160 and 32 zM stock solutions for sample preparation were prepared by diluting 37.5 or 7.5 μL of the 64 aM solution in a new 15 mL centrifuge tube immediately before use.

[0221] Two batches of samples (100 samples each) were prepared at two concentrations such that the respective estimated probability of containing a single analyte molecule was 60 or 12%. The sample concentrations required to contain statistical means of 0.60 and 0.12 molecules in 25 μL were 40 and 8 zM, respectively. Samples were prepared by adding 10 μL of pNTP stock solution (160 or 32 zM), 10 μL of 7 nM AgNP solution, and 20 μL of nanopure water to a 600 μL microcentrifuge tube. For rapid screening, 40 μL of each sample was aggregated and deoxygenated with Na2SO3 powder, and then 25 μL was recovered and completely siphoned by capillary force into a standard melting point capillary (1.5-1.8 x 100 mm Corning 9530-1 Pyrex capillary) for SERS measurement.

[0222] Similar sample preparation procedures were applied to other analytes containing four homopolymer oligoDNA sequences with different concentrations of pATP and pNTPs. However, while the pNTP samples above were prepared at a 5000-fold dilution, the samples defined in this paragraph were prepared at a 100-fold serial dilution. Then, the analyte such as pATP was mixed with an AgNP solution to obtain target analyte concentrations (e.g., 75 μL each of pATP and AgNP solutions and 150 μL of nanopure water). Each concentration was prepared in three independent replicates to obtain the standard deviation. 10 mg of Na2SO3 or Na2SO4 powder was added to each solution (e.g., pATP+AgNP solution) (300 μL) to produce solutions with the same ionic strength but with or without dissolved oxygen, respectively. Unless otherwise specified, the solutions containing the analytes used in the experiments discussed below were prepared according to the above method with the necessary adjustments to achieve the target concentrations.

[0223] Error Propagation in Sample Preparation by Serial Dilution

[0224] Assuming an error of ±0.1 mg in the analytical balance and an error of ±0.1 mL in the 50 mL tube volume, there is a potential maximum error of 0.22% in the concentration of the initial pNTP stock solution. For each dilution, the relative standard deviation of the concentration is determined by the square root of the relative standard deviations of pipetting (0.47%) and volumetric flasks (0.08%), which is approximately 0.48%. This error is compounded across the initial and three consecutive dilution steps, which are treated as independent events. In other words, the deviations are random and unsystematic, generating a concentration error of approximately 1.9%. Subsequently, there are additional dilution steps to form the 160 and 32 zM stock solutions for analysis. Assuming an error of ±0.1 mL in the 15 mL tube and the same relative deviation in pipetting, an additional 0.81% error occurs. The nanoparticle adsorption step involves three pipetting events in which errors occur in the final concentration of the analytical solution. Assuming similar pipetting errors for the addition of 10 and 20 μL, an additional ~0.43% error in concentration occurs. Since each dilution step is an independent event, the total relative error in concentration is expected to be ~2.1%. Therefore, the concentration of the solution to be analyzed should be 40 ± 0.85 or 8 ± 0.17 zM, which corresponds to a detection probability of 58.7–61.3% or 11.7–12.3% in a 25 μL sample, respectively.

[0225] Results and Significance of the Probability Analysis

[0226] Samples (25 μL, 100 each) were analyzed at 40 and 8 zM, respectively, with nominal probabilities of 60% and 12% containing a single pNTP molecule after nanoparticle aggregation and deoxygenation by adding Na2SO3 to standard melting point capillaries. pNTPs were detected in 55 and 8 out of 100 samples at concentration levels of 40 and 8 zM, respectively. The measured frequencies were compared to the predicted frequencies using Fisher's exact test to determine if there was a non-random association between the predicted and measured frequencies. There were no statistically significant differences for the 60% and 12% probabilities, with p=0.57 and 0.48, respectively. Therefore, the experimentally measured detection frequencies at both concentrations were within the expected values ​​for single molecule detection. For clarity, spectra were not generated for this probability analysis.

[0227] Remote Sensing

[0228] To test the efficacy of the probe of the present invention and to determine the degree of plasmonic coupling within and between plasmonic nanoparticle assemblies, two experiments were devised. These experiments aimed to separate the analyte molecules from the incident light source and field of view of the detector by adsorbing the analyte onto a silver mirror surface and completely removing any unadsorbed or loosely bound analyte through extensive rinsing (these experiments are presented in FIGS. 18 to 20, and the results are discussed below). This was achieved using a 10 cm long quartz NMR tube or a 1 m long PEEK capillary coupled to a short section of a quartz NMR tube.

[0229] First, to fabricate a silver mirror surface on the end of the tubing, a Tollen reagent solution was prepared using 0.1 M AgNO3. This solution was precipitated by first adding dilute NaOH to form Ag2O. The resulting Ag2O was redissolved by adding concentrated aqueous NH3 dropwise to form an Ag(NH3)2 complex. The Tollen reagent solution was added to the surface of the PEEK tubing on a quartz NMR tube or a polypropylene microcentrifuge tube to cover half of the desired surface area (~5 mm) for silver coating. Then, an equal volume of 0.1 M glucose was added, and the mirror was developed. After coating the surface with silver, it was rinsed approximately 30 times each with nano-pure water and EtOH to remove excess reagent or other adsorbed substances before drying at ambient temperature. Care must be taken during handling, as the adhesion of silver to the polymer surface is weak and it can be easily rubbed off.

[0230] Subsequently, 100 nM of the analyte, such as pATP, was added to cover the silver mirror surface. Then, the NMR tube and PEEK tubing were rinsed 30 times each with EtOH and nanopure water, respectively, to remove unbound analytes from the silver mirror surface (in the case of PEEK tubing, this was performed at the end opposite the silver mirror to prevent the analyte from migrating into the tubing). To detect the analyte adsorbed on the distal end (second end) of the glass NMR tube or PEEK tube, the thoroughly rinsed apparatus was filled with an aggregated and deoxygenated AgNP solution immediately before the addition of an excess amount of Na2SO3. An optical fiber bundle connected to the incident laser source and detector was aligned with the end of the cuvette or tubing opposite the adsorbed analyte, and Raman spectra were recorded. As discussed in more detail below, the analyte was detected when the AgNPs were aggregated with Na2SO3 and deoxygenated, but not when aggregated only with Na2SO4.

[0231] Blind Remote Sensing from Nanoparticle Aggregates

[0232] Samples for remote sensing using the probe of the present invention were prepared for a blind test by creating stock solutions using 0 or 2 μM of pNTPs and 3.5 nM of AgNPs in 50 vol% ethanol (400 μL). The solution was sonicated for 30 minutes to confirm that the pNTPs and AgNPs were well mixed and that the pNTPs were adsorbed onto the NP surfaces. To remove residual unadsorbed pNTPs that could diffuse through the solution, the sample was centrifuged at 12,700 rpm for 30 minutes to precipitate the NPs, and all solution except 20 μL was removed (to avoid disturbing the precipitated NPs). The sample was diluted with 1500 μL of ethanol and sonicated for an additional 30 minutes. This process was repeated for a total of two washes (reducing the maximum possible free pNTP to ~356 pM) prior to final centrifugation, solvent removal (to 20 μL), and resuspension in 50 wt% ethanol with a final maximum possible free pNTP concentration of ~18 pM. 20 μL of the sample was randomly assigned to 20 microcentrifuge tubes and diluted with 20 μL of water (for a final maximum free pNTP concentration of ~9 pM in the case where pNTPs were not adsorbed, and an estimated positive control AgNP adsorption concentration of 1 μM). The number and identity of the positive and negative controls were unknown to the analyst, and the analyst used Na2SO3 prior to measurement. The samples were simultaneously aggregated and deoxygenated by adding powder. Glass capillaries pre-filled with the aggregated and deoxygenated AgNP solution were aligned with a Raman laser and immersed in the aggregated sample solution (6 cm from the incident light). Raman spectra were recorded by a blind analyst and assigned to positive and negative controls. The sample assignments were compared with their identities, and all samples (positive controls n=9, negative controls n=11) were correctly identified. For clarity, no spectra were generated in this blind test.

[0233] calculate modeling Theoretical Interpretation for Computation Modeling

[0234] To understand how Ag2O and O2 affect the SERS signal response, the electromagnetic field (E-Field) enhancement of surface plasmon nanocavities was modeled using a boundary element method (BEM) approach. The surface integral depends on the scalar and vector potentials of the interfacial charge and current associated with the frequency-dependent local dielectric function. The surface charge is assumed to be located at the center of individual infinitesimal triangles (including index j).

[0235] The E-field is a scalar and a vector potential, respectively. It is expressed as (electric scalar potential), and A (magnetic vector potential).

[0236]

[0237] After applying boundary conditions, the electrical displacement (D) at the interface surface(s) of the two media 1 and 2 is proposed as follows. (n s Normal vector and ε a Genetic function of medium(a):

[0238]

[0239] The introduction of compact matrix notation was adopted for spatial convolutions. The electric displacement of a system without an oxygen or oxide layer yields the following as the sum of the electric displacements for each interface of each AgNP:

[0240]

[0241] In the equation, Ag represents AgNPs and AgxO represents the oxide layer. In a system with an oxide layer, the electrical displacement of each AgNP is as follows:

[0242]

[0243] When molecular oxygen (O2) is present in the medium, each AgNP has the following electrical displacement.

[0244]

[0245] Those skilled in the art will understand that detailed descriptions of the BEM method and its application to NP surface plasmons can be found in the literature. AgNPs, Ag x The dielectric constants for O, oxygen, and water can also be found in the literature. The modeling results are as shown in Figures 6 and 7, which are described above and below.

[0246] In relation to Figure 7, it should be noted that the electromagnetic field transmittance is lost at 17 and 9.8 nm for each of the clean silver oxide-coated silver nanoparticles.

[0247] Results and Discussion

[0248] Results and Discussion on Surface-Enhanced Raman Spectroscopy and Single-Molecular Detection Probability Analysis of Prepared Samples

[0249] For the first series of measurements, p-aminothiophenol and p-nitrothiophenol solutions of various concentrations with and without dissolved oxygen were prepared using the method defined above. AgNPs were used as plasmonic materials (SERS substrates) and sodium sulfite was used as an oxygen scavenger.

[0250] In Fig. 8, the spectrum labeled "blank" is for a sample containing 7 nM of AgNPs aggregated with Na2SO3 (angular, average size 17 nm as mentioned above). The liquid sample for the spectrum labeled "Na2SO3" (deoxygenated using 10 mg of sodium sulfite) and Na2SO4 (containing 10 mg of sodium sulfate, and thus containing dissolved oxygen) each contain 1 fM of the analyte, with Na2SO3 or Na2SO4 acting as the aggregator. As mentioned above, the spectra were obtained using a solid-state Nd-YAG laser (785 nm, line width <3.2 cm). -1It was recorded on a 5 x 100 mm quartz NMR tube irradiated with 500 mW (beam diameter 2.5 μm). Backscattered (180°) light was collected with a standard Sunshine TG-Raman fiber spectrometer. All spectra were expressed as the average of three measurements, each consisting of 1505 points (integration time = 100 ms). The analytes were p-aminothiophenol (Fig. 8a) and p-nitrothiophenol (Fig. 8b).

[0251] In Fig. 9, the same measurement parameters as in Fig. 8 were used, but various concentrations of p-aminothiophenol analytes were used. The measurement parameters taken for Fig. 9 were otherwise identical to those in Fig. 8. The spectra in Figs. 9a through 9c are for samples containing dissolved oxygen (10 mg sodium sulfate), whereas the spectra in Figs. 9d through 9f are for samples deoxygenated using 10 mg sodium sulfite. Figs. 9b and 9e show the spectra separated from Figs. 9a and 9d, respectively.

[0252] By using thiophenols, namely p-aminothiophenol (pATP) and p-nitrothiophenol (pNTP), as the major analytes and prismatic AgNPs as plasmonic materials (SERS substrates) for proof-of-principle studies, it was observed that the SERS spectra of aqueous pATP and pNTP increased sharply after DO removal using sodium sulfite (Na2SO3; see Figures 8 and 9 described in more detail above). The peak in Figure 8 is associated with in-plane deformation of the aromatic ring, and at higher concentrations, this peak should overlap with the N=N band of the azo dimer.

[0253] As mentioned, for all experiments, triangular prism AgNPs were used unless otherwise specified. In all experiments performed, unless otherwise noted, sodium sulfite acted as a DO scavenger, which simultaneously aggregated AgNPs by screening surface charges from the citrate capping agent (sodium sulfate, Na2SO4, was used as a control for sodium sulfite to aggregate AgNPs in aerobic, i.e., non-deoxygenated experiments); the spectral window was ≥1000 cm⁻¹ due to the overwhelming signal of the citrate. -1 It was limited to (see Figs. 4 and 10, described in more detail above).

[0254] As mentioned, Figure 4 shows transmission electron microscope images of AgNPs deposited on a copper grid, Figures 4 a) and b) before aggregation and c) and d) after aggregation with 10 mg of sodium sulfite and extensive rinsing to remove salt crystals.

[0255] Figure 10a shows the AgNP size distribution via dynamic light scattering, while Figures 10b–d show the Raman spectra for b) AgNP, c) p-aminothiophenol, and d) p-nitrothiophenol. Specifically, Figure 10b is the blank spectrum of the prepared AgNP, where the large peak is associated with the citrate capping agent. Figures 10c and d are the Raman spectra of pATP and pNTP at high concentrations (100 mM) to show the peaks available for detection (1000 cm⁻¹). -1 It should be noted that peaks below this level are obscured by the strong signal of surface-bound citrate.)

[0256] The limit of detection (LOD) for pATP is CH bending (1135-1142 cm -1 ) and CS expansion (1070-1080 cm) -1 Based on the ) band, 10 zM (sensitivity of 10 compared to 10 pM of aerobic samples) 9Reached an increase (although not all vibration modes are equally sensitive and some become undetectable as the analyte concentration drops).

[0257] A similar increase in SERS enhancement factor (EF) was observed for other analytes, namely four homopolymer oligoDNA sequences prepared using the method described above (see Figures 11a and b, described in more detail above). Specifically, Figures 11a and b show the SERS spectra of oligonucleotides using the same measurement parameters as used in Figure 8 for oxygenated (analyte at 10 pM) and deoxygenated (analyte at 8 zM) samples, respectively.

[0258] Similar results were observed when spherical AgNPs were used instead of prismatic AgNPs. DO concentration (measured electrochemically) is inversely proportional to SERS signal intensity regardless of whether physical removal is used, such as chemical oxygen removal, e.g., inert gas, e.g., N2 or Ar, or spraying (see Figures 12, 13, and 14 described in more detail above). In Figure 12b (Figure 12a showing the TEM of spherical AgNPs), the SERS spectrum was obtained using the same measurement parameters as in Figure 8, except that spherical AgNPs were used instead of prismatic AgNPs.

[0259] In Figures 13a and 13b, various SERS spectra were obtained using the same measurement parameters as in Figure 8, but with different analyte concentrations of p-nitrothiophenol and different oxygen scavengers. Specifically, Figure 13a shows the SERS spectrum for pNTPs deoxygenated with 10 mg of ascorbic acid, while Figure 13b shows the SERS spectrum for pNTPs deoxygenated with hydrazine (50 mM).

[0260] In Figures 14a and b, SERS spectra were obtained using the same measurement parameters as used in Figure 8 (analyte pATP concentration 100 fM), except that measurements were performed to monitor oxygen concentration in the electrochemical system, and different amounts of oxygen scavenger (Na2SO3) were used to test various DO concentrations.

[0261] Similarly, in Figures 14c and 14d, SERS spectra were obtained using the same measurement parameters used in Figure 8, except that measurements were performed in an electrochemical system to monitor oxygen concentration (analyte pATP concentration 100 fM), and argon gas was used instead of an oxygen scavenger. To test various DO concentrations, the spraying time was gradually increased.

[0262] In Fig. 13, it can be seen that hydrazine and ascorbate enabled the same zM LOD for pNTPs as Na2SO3. As can be seen in Fig. 14, argon spraying removed DO and increased SERS signal intensity, but the level of DO removal was still much lower than the maximum achieved using an oxygen scavenger. It should be noted that the spectrum obtained in Fig. 14c using the lowest oxygen concentration (0.013 mM, nearly 10 times higher than the 0.0019 mM DO concentration obtained using Na2SO3) required 20 minutes of argon spraying along with simultaneous sonication. As mentioned, in Fig. 14, dissolved oxygen concentration was measured electrochemically. Additionally, Fig. 14 presents the average of the three spectra, and the data standard deviations of b) and d) are smaller than the data markers.

[0263] The effect of AgNP surface oxidation on SERS enhancement was investigated (Fig. 15, described in more detail above), as DO removal also reduced Ag2O formation on the AgNP surface compared to aerobic (i.e., un-deoxygenated) samples. Fig. 15a shows the voltammetry of Ag+ (obtained through the addition of Ag2NO3), while Figs. 15b and c show the voltammetry of AgNP in a sample containing DO and a sample deoxygenated using 10 mg of sodium sulfite, respectively.

[0264] Similarly, Fig. 16a shows the UV-Vis spectrum of gold nanorods (AuNR), whereas Fig. 16b shows the SERS spectrum of cetyltrimethylammonium bromide (CTAB) with and without oxygen removal from AuNR, and the Raman spectrum of concentrated CTAB (100 mM). For clarity, the spectrum labeled "Na2SO3" is for a sample deoxygenated using 10 mg of sodium sulfite, and the spectrum labeled "Na2SO4" contains 10 mg of sodium sulfite and thus contains dissolved oxygen. Also, the amount of CTAB in the samples with the spectra labeled "Na2SO3" and "Na2SO4" is the same as the residual surface (CTAB) of commercially supplied AuNR.

[0265] As mentioned, Fig. 17a shows the TEM of silica-coated AgNPs, while Fig. 17b shows the SERS spectrum of pATP for deoxygenated aggregated silica-coated AgNPs (at pH 4) at various concentrations, and Fig. 17c shows the SERS spectrum of pNTP (50 aM) for pATP (50 aM) and deoxygenated aggregated silica-coated AgNPs (at pH 4).

[0266] Based on these results, the formation of a dielectric oxide layer (in this case, Ag2O formation) appears to be a minor factor compared to the presence of DO. At LODs of 500 nM to 50 aM (10 10Signal enhancement was also observed when using the SHINERS technique, i.e., silica-coated AgNPs reduced by a factor of 10. The overall sensitivity of silica-coated AgNPs was 10 times that of exposed AgNPs. 4 Although the DO is lower than the surface oxide layer, the SERS signal (10 10 10 4 It appears to be a major factor in eliminating the (b) element; that is, while absolute sensitivity varies depending on the particle, the relative signal enhancement upon DO removal is always 10 9 -10 10 It is a pear. Using AuNRs and silica-coated AgNPs, the effect of DO was separated from the surface oxidation effect.

[0267] This finding challenges the existing understanding of SERS, where the analyte must be simultaneously irradiated within the detector's field of view. Because the laser beam is too narrow (2.5 μm when collimated), the analyte is 5.4 x 10⁻⁶ based on the volume ratio of the optical path to the sample. -8 It is assumed that the value is 1 and the analyte is uniformly distributed within the liquid sample. Since this is nearly zero, the analyte should not be detectable based on conventional SERS theory. However, numerous independent analyses were performed at the zM level (n > 300 by different analysts on different days using AgNPs manufactured in different batches, including the blind experiments discussed above), and consistently reproducible results were obtained. As previously mentioned, to verify whether single molecule detection is real, statistical analysis was performed on two sets of 100 samples at concentrations where the probability of a single molecule in the total sample volume was <1. The predicted detection frequency and the actual detection frequency were compared using Fisher's exact test, and it was confirmed that there was no statistically significant difference between the measured and predicted detection frequencies (p = 0.57 and 0.48 for 60% and 12% detection probabilities, respectively).

[0268] Based on these observations, oxygen removal appears to generate effective coupling of SERS fields, and this plasmonic coupling enables the propagation of electromagnetic (EM) fields across the entire volume of the liquid sample without significant signal energy loss. The plasmonic field generated by the photoexcitation of AgNPs travels throughout the sample, reaches the AgNPs along with the adsorbed analyte, and returns the Raman scattering signal of the analyte to the detector. Without DO removal, the high electron affinity of oxygen traps electrons and prevents the effective generation, coupling, propagation, and integration of SERS fields between AgNPs and their aggregates, as observed experimentally and through computational modeling. However, DO removal enables plasmonic coupling of SERS field transmission with plasmonic materials (in this case, AgNP aggregates).

[0269] The plasmon field was calculated around model AgNP aggregates, demonstrating the expected plasmon coupling between nanoparticles enhanced within the nanogap (see Figures 6 and 7 described in more detail above). Molecular oxygen floating in the nanogap or adsorbed on the AgNP surfaces resulted in charge redistribution and annihilation of the plasmon field and coupling. When modeling Ag2O-coated nanoparticles, the plasmon field was somewhat reduced, but the introduction of DO significantly annihilated the EM field and prevented coupling. The DO quenching effect was independent of representative parameter characteristics for the nanogap, including the nanogap distance, the number of nanoparticles in the nanoassembly (2, 3, and 4 AgNPs), and nanoparticle properties (size, shape, position, orientation, and surface chemistry). This modeling demonstrates that DO annihilates the SERS EM field, whereas in the absence of the EM field, it can propagate throughout the sample via adjacent AgNPs and their aggregates through plasmon coupling.

[0270] SERS signal propagation through liquid samples, such as aqueous solutions, was also studied electrochemically. DO removal significantly enhanced the photocurrent induced by laser irradiation of suspended AgNPs not physically attached to the gold electrode (see Fig. 5, described in more detail above). As mentioned in the methodology section, Fig. 5a shows the experimental schematic used, where the photocurrent was measured on a honeycomb gold electrode of AgNP solutions aggregated by Na2SO3 (dissolved oxygen removal) and Na2SO4 (oxygen retention) through light irradiation of the AgNP solution (not the electrode). Meanwhile, Fig. 5b shows photocurrent generation under the listed conditions via NIR (λ = 785 nm) irradiation at a distance of 1 cm from the electrode surface in the presence or absence of dissolved oxygen at a constant ionic intensity. As can be seen in Fig. 5b, the current generated by irradiation was increased by both the addition of salts (sodium sulfate or sulfite) and the increase in conductivity. However, sodium sulfite also removed dissolved oxygen and significantly enhanced photocurrent generation.

[0271] Remote Sensing and Blind Remote Sensing Results and Discussion

[0272] Further evidence supporting long-range SERS field transmission in liquid samples such as aqueous solutions comes from the remote detection of analytes by anaerobic SERS using the probe of the present invention.

[0273] First, the analyte adsorbed on the silver mirror coating the distal end of an NMR tube filled with a deoxygenated, clean AgNP solution shows that other vibrational bands pointing further away and in different directions decrease rapidly (see Fig. 18, described in more detail above), but when measured at a distance of 10 cm from the silver mirror, the CS stretching band (1070–1080 cm⁻¹) near and perpendicular to the Ag surface... -1 It maintained ~70% of the signal strength of ).

[0274] For clarity, in Fig. 18, the spectrum was recorded using the same measurement parameters as in Fig. 8; however, the analyte was adsorbed onto the silver mirror at the base of the NMR tube without scattering through the liquid sample. AgNPs were added and aggregated with Na2SO3 (dissolved oxygen removed) as described in Fig. 8, but were added and aggregated in the detection chamber along the tube.

[0275] As previously mentioned, to fabricate a silver mirror surface on the end of the piping, a Tollen reagent solution was prepared with 0.1 M AgNO3, which was precipitated by first adding dilute NaOH to form Ag2O. The obtained Ag2O was redissolved by adding concentrated aqueous NH3 dropwise to form an Ag(NH3)2 complex. The Tollen reagent solution was added to the bottom of a quartz NMR tube to cover half of the desired surface (~5 mm) to be coated with silver. Then, an equal volume of 0.1 M glucose was added, and the mirror was developed. After coating the surface with silver, it was rinsed approximately 30 times each with nano-pure water and EtOH to remove excess reagent or other adsorbed substances before drying at ambient temperature.

[0276] Using the above settings and parameters, the following SERS spectrum was obtained (see Fig. 18).

[0277] Fig. 18a: Adsorbed p-aminothiophenol measured at positions 0 and 10 cm away from the mirror.

[0278] Fig. 18b: Adsorbed p-nitrothiophenol and blank measured at a distance of 10 cm from the silver mirror.

[0279] Fig. 18c: Spectrum of adsorbed p-aminothiophenol as a function of measurement distance from the mirror.

[0280] Similarly, FIG. 18d shows the CS stretching strength of p-aminothiophenol as a function of distance from the mirror.

[0281] As can be seen in Fig. 18, propagation through silica-coated AgNPs was less efficient. It should be noted that in Fig. 18, the blanks of all experiments do not show significant signals beyond the noise level. The average of the three spectra is presented, and the standard deviation of d) is smaller than the size of the data marker.

[0282] Second, remote SERS was applied for high-throughput detection in a 96-well microtiter plate aggregated with DO removal and sodium sulfite after the analyte was pre-adsorbed onto AgNPs in a 96-well plate (see Fig. 19, described in more detail above). The measurement parameters used in Fig. 19 were similar to those in Fig. 18, but the analyte was pre-adsorbed onto deoxygenated aggregated AgNPs within the wells of the microtiter plate. Capillary melting point tubes pre-filled with deoxygenated aggregated AgNPs were immersed in the wells. Spectra were measured through a capillary tube located 6 cm away from the wells. These spectra are provided as a function of integration time for p-aminothiophenol (Fig. 19a) and p-nitrothiophenol (Fig. 19b).

[0283] For clarity, a 6 cm glass capillary filled with a deoxygenated, clean aqueous solution of AgNP aggregates was placed perpendicular to each well to transmit the SERS signal upward where the incident light irradiation was perpendicular to the capillary (and parallel to the microtiter plate). This was verified by the blind test defined above, in which 20 wells were randomly filled with either analytes or blanks and detected with 100% accuracy. In Figure 19, it should be noted that the blanks in all experiments did not show a significant signal beyond the noise level.

[0284] Finally, remote SERS were verified using a 1 m long PEEK capillary with a silver mirror at the distal end (see Fig. 20, described in detail above). The measurement parameters used in Fig. 20 were similar to those in Fig. 18, but the silver mirror was grown at one end of the 1 m long PEEK capillary. The other end was connected to the length of the NMR tube (Fig. 20a), and the system was flushed with a solvent. Blank measurements were performed by passing deoxygenated aggregated AgNPs through the tube, after which the analyte was adsorbed onto the silver mirror, the apparatus was rinsed, and the experiment was repeated. Fig. 20a shows a schematic diagram of the setup defined above, while Fig. 20b shows the SERS spectrum using p-aminothiophenol as the analyte, including oxygen removal and oxygen retention, and Fig. 20c shows the SERS spectrum using p-nitrothiophenol as the analyte, where the sample was deoxygenated using 10 mg of sodium sulfite. For clarity, in FIG. 20a, the spectrum labeled "Na2SO3" was deoxygenated using 10 mg of sodium sulfite, and the spectrum labeled "Na2SO4" contains dissolved oxygen because it contains 10 mg of sodium sulfate.

[0285] It should be noted that in Figures 20b and c, no analyte was detected for the blank or without oxygen removal.

[0286] The above experiment established long-range transmission of surface plasmon fields in liquid samples such as aqueous solutions, but the length of signal transmission varies depending on the particles; specifically, clean AgNPs can transmit over at least 1 m, whereas less effective silica-coated AgNPs are reduced over ~6 cm. This is consistent with the results of computational modeling (Fig. 6), where impaired SERS field intensity and propagation using silica-coated AgNPs are 10 times that of clean AgNPs (10 zM). 4 Generates a much higher LOD (50aM).

[0287] The conventional understanding of SERS is based on signal amplification using individual nanostructures or, for example, plasmon field coupling between adjacent plasmon nanoparticles—that is, nanogaps. "Hotspot" theory is sufficient to interpret all experimental observations where SERS signal amplification is based on local surface plasmon resonance, which is, for example, individual EM fields around individual or small clusters of plasmon nanoparticles. However, current experiments have demonstrated that large-scale plasmon field coupling (or integrated surface-plasmon resonance) is possible upon DO removal. This integrated field enables, for example, the effective transmission of SERS signals from single molecules adsorbed on individual plasmon nanoparticles across the entire sample; for instance, all plasmon nanoparticles become SERS active, acting as "hotspots" for the adsorbed analyte or transmitting SERS signals to enable actual single-molecule detection. Here, it was shown that such plasmon field transport occurs over long distances even in oxygen-free water. AgNP aggregation is still preferred in anaerobic SERS, which is advantageous for plasmon field transfer (see Figures 7a-f described in more detail above, where it can be observed that electromagnetic field transfer is lost at 17 and 9.8 nm for novel and silver oxide-coated silver nanoparticles, respectively). In SHINERS experiments, excellent SERS sensitivity (50 aM) is achieved only when silica-coated AgNPs aggregate by adding sodium sulfite and lowering the pH of the solution, where sodium sulfite alone is sufficient for DO removal but cannot aggregate silica-coated AgNPs.

[0288] One notable advantage of current anaerobic (i.e., deoxygenated) SERS measurements is that the detection time is determined not by the diffusion of the analyte into the detector's field of view, but rather by the transport of the plasmon field (particularly because these are surface-bound to a fixed silver mirror or are slowly diffusing nanoparticle aggregates). In 1m remote SERS detection using the probe of the present invention, when the analyte is fixed to a silver mirror at one end of a PEEK tube and immobile, a SERS signal is obtained immediately upon irradiation at the other end of the tube. This ultrafast response provides high temporal resolution along with high sensitivity, allowing for the monitoring of rapid chemical reactions, such as studying chemical reactions at the single-molecule level or detecting short-lived species. This technique is also cost-effective and requires only a standard Raman spectrometer without the need for expensive SERS detectors. This will significantly reduce the capital cost of conducting SERS experiments and facilitate this research globally, particularly in developing countries and resource-constrained regions. It is believed that unprecedented detection sensitivity, fast response time, and remote sensing capabilities can generate many applications in medical diagnosis, environmental monitoring, and national security.

[0289] The scope of the claims should not be limited by the preferred embodiments described in the examples, and the broadest possible interpretation consistent with the description in its entirety should be given.

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Claims

Claim 1 A method for modifying a liquid sample containing an analyte to increase the surface-enhanced Raman spectroscopy (SERS) signal intensity of the analyte, comprising the steps of: providing said liquid sample to be analyzed using SERS; and adding a scavenger to said liquid sample to remove dissolved oxygen from said liquid sample, wherein a sufficient amount of scavenger is added such that the concentration of residual dissolved oxygen during SERS measurement is at most 0.020 mM. Claim 2 A method according to claim 1, wherein a sufficient oxygen remover is added so that the concentration of residual dissolved oxygen becomes a maximum of 0.010 mM. Claim 3 In claim 1, after the liquid sample is modified, the detection limit for a given analyte is at least 10 8 A method that reduces by a factor of two. Claim 4 A method according to claim 1, wherein the detection limit for the analyte using SERS is a maximum of 1 pM. Claim 5 A method according to claim 1, further comprising the step of contacting the liquid sample with a plasmonic material for SERS. Claim 6 In claim 5, the method wherein the plasmonic material is composed of plasmonic nanoparticles. Claim 7 A method according to claim 6, wherein the plasmon nanoparticles are aggregated by adding an aggregator, adjusting the pH, adding an organic solvent, or a combination of these approaches. Claim 8 In claim 7, the method wherein the coagulant is an oxygen remover. Claim 9 A method according to claim 6, wherein the plasmonic nanoparticles are added to the liquid sample prior to the aggregation of the nanoparticles and prior to the removal of dissolved oxygen using an oxygen remover. Claim 10 A method according to claim 1, further comprising the step of measuring the SERS spectrum of the liquid sample. Claim 11 As a probe for remote sensing of an analyte in a liquid sample using Surface Enhanced Raman Spectroscopy (SERS), the probe comprises a detection chamber having a window transparent to SERS excitation light and Raman scattering signals, and a pipe having first and second ends, wherein the first end of the pipe is movably connected to the detection chamber and the second end of the pipe is configured to be disposed in contact with the liquid sample, and the pipe between the first and second ends and the detection chamber comprise plasmon nanoparticles immersed in a deoxygenated solvent, wherein the plasmon nanoparticles are in close proximity to each other, thereby allowing uninterrupted propagation of a plasmon field from the detection chamber to the second end of the pipe and / or, the inner wall of the detection chamber and the inner wall of the pipe between the first and second ends are coated with a plasmon layer, wherein the plasmon layer is continuous from the detection chamber to the second end of the pipe, thereby allowing uninterrupted propagation of a plasmon field from the detection chamber to the pipe A probe that allows uninterrupted propagation of a plasmon field to the second end and inversely, wherein the piping and detection chamber are filled with a deoxygenating solvent, and the deoxygenating solvent includes an oxygen scavenger that removes dissolved oxygen therefrom, and there is sufficient oxygen scavenger present so that the concentration of residual dissolved oxygen during SERS measurement is at most 0.020 mM. Claim 12 In claim 11, the probe comprises a detection chamber and a piping between the first and second ends containing plasmon nanoparticles contained in a deoxygenating solvent. Claim 13 In claim 11, the probe in which the plasmon nanoparticles aggregate along the length of the piping. Claim 14 A probe according to claim 11, wherein the second end of the piping is open, and when used, the piping and the detection chamber are kept filled with plasmon nanoparticles and a deoxygenating solvent, and when used, the detection chamber and the piping are refilled with plasmon nanoparticles and a deoxygenating solvent so that the plasmon nanoparticles are kept close to each other. Claim 15 In claim 11, a probe wherein the second end of the pipe is open and the second end of the pipe is at least partially coated with a plasmon layer. Claim 16 In claim 11, the probe, wherein the second end of the piping is also capped with a plasmon layer. Claim 17 In claim 11, the probe, wherein the liquid sample is deoxygenated using an oxygen remover. Claim 18 In claim 11, the probe, wherein the liquid sample is modified using the method of claim 1. Claim 19 In claim 11, a probe in which the analyte is adsorbed onto the surface of a plasmonic material which is the surface of a nanoparticle in the pipe, a plasmonic layer coated on a second end of the pipe, and / or a plasmonic layer capping the second end of the pipe. Claim 20 In claim 11, the probe is configured to work with a surface-enhanced Raman spectrometer for remote sensing, which can be used to detect an analyte by applying incident laser irradiation to a sample holder or detection chamber and measuring scattered light using a detector. 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