Methods of modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte, and probes for detecting a remote analyte using SERS

Abstract

Methods are provided for modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte. The methods of the invention include the steps of providing a liquid sample to be analyzed using SERS, adding an oxygen scavenger to the liquid sample to remove dissolved oxygen from the liquid sample. Also provided is a probe for remotely detecting an analyte in a liquid sample using SERS. The probe of the invention includes a detection chamber having a window that is transparent to SERS excitation light and Raman scattered signals, and a conduit having a first end and a second end, the first end of the conduit being flowably connected to the detection chamber, the second end of the conduit being in contact with the liquid sample.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit under 35 U.S.C. § 119(e)(1) of U.S. Provisional Application Serial No. 62 / 890,216, filed August 22, 2019. All of the above-identified documents are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present invention relates to methods of modifying a liquid sample to be analyzed using surface-enhanced Raman spectroscopy (SERS). More specifically, the present invention relates to methods of modifying a liquid sample containing an analyte to be analyzed to increase the SERS signal intensity of the analyte. The present invention also relates to probes for detecting a remote analyte using SERS. BACKGROUND

[0004] Raman spectroscopy involves the attenuation or enhancement of the energy of photons that are inelastically scattered by a molecule through changes in the vibrational energy states of the molecule. Since the change in the energy of the scattered light relative to the incident light corresponds to the energy difference between vibrational states of the molecule, the spectrum of the inelastically scattered light, i.e., the Raman spectrum, can be used to identify the molecule, and the intensity of the inelastically scattered light can be used to quantify the amount of the molecule. Inelastic Raman light scattering is very weak, so that analyte molecules must reach a significant concentration to be effectively detected, and thus this technique is low in sensitivity. However, there are several technical approaches that can be used to greatly increase the intensity of the inelastically scattered light.

[0005] Surface-enhanced Raman spectroscopy (SERS) increases the intensity of inelastically scattered light by increasing the probability of inelastic scattering of photons rather than elastic scattering. It does this by first adsorbing analyte molecules onto a plasmonic material, and then illuminating by an incident light beam. The exact mechanism of the signal enhancement is currently a matter of debate in the scientific community, but it is known that the plasmonic material must be rough or composed of nanostructures on the nanometer scale, and the enhancement of the signal intensity can be as high as 10 10 -10 11 times. Notably, the vibrational modes of molecules that can be detected by SERS can be different from the vibrational modes of free molecules. Thus, the Raman spectrum and the surface-enhanced Raman spectrum of the same molecule can be different.

[0006] The sensitivity and reproducibility of SERS can be improved by designing optical hotspots, which are located at positions where the nanostructures show the strongest electromagnetic field, such as inside the nanoparticle tips or the nanogaps between adjacent nanoparticles. The highest SERS signal amplification occurs at the hotspots. However, its sensitivity is limited because the analyte molecules to be analyzed must be located at the hotspots, within the sampling area of the Raman laser beam, and in the field of view of the detector at the same time. This sampling volume usually occupies a negligible fraction of the entire sample volume. Therefore, in practice, the detection limit of SM-SERS is at the nM or pM level (10 -9 --10 -12 Molar / Liter). Although conventional surface-enhanced Raman techniques can significantly reduce the detection limit of analyte molecules, they usually cannot achieve the optimistic single-molecule detection limit. Recently, this limitation has been broken by combining SERS detection with pre-concentration of the analyte using superhydrophobic or omniphobic surfaces, and the detection sensitivity has been improved to the fM level.

[0007] Surface oxidation of the substrate material can affect the SERS detection limit. It has been reported that when silver nanoparticles (AgNPs) or silver films are oxidized by ambient air to form a sub-monolayer of Ag2O, the SERS enhancement factor (EF) decreases by 10 5 times. This decrease is often analyte-dependent and is quantitatively related to the thickness of the Ag2O layer. The Ag2O layer effect is due to the reduction of analyte adsorption and the reduction of electron transfer between the metal and the analyte, which affects the chemical and electromagnetic enhancement of SERS. However, it was later observed that the effect of substrate material oxidation on SERS EFs was less significant, but when combined with atmospheric carbon pollution, the noise level increased with a corresponding decrease in signal strength. SERS detection can also be achieved with AuNPs coated with a thin layer of silica or alumina, i.e., shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS). The success of SHINERS suggests that a thin dielectric oxide layer may not cause a sharp drop in SERS EFs. SUMMARY

[0008] According to the current invention, there is provided:

[0009] 1. A method of modifying a liquid sample containing an analyte to increase the SERS signal intensity of said analyte, said method comprising the steps of:

[0010] providing said liquid sample to be analyzed using SERS; and

[0011] adding an oxygen scavenger to said liquid sample to remove dissolved oxygen from said liquid sample.

[0012] 2. The method according to item 1, wherein the analyte is a thiolated compound, an amine, a pesticide, a persistent organic pollutant, a transition metal complex, a peptide, a protein, a nucleic acid, a polysaccharide or a hormone.

[0013] 3. The method according to item 1 or 2, wherein the analyte is a transition metal complex, a peptide, a protein, a nucleic acid, a persistent organic pollutant, a pesticide or a hormone.

[0014] 4. The method according to any one of items 1 to 3, wherein the analyte is p-aminothiophenol (pATP) or p-nitrothiophenol (pNTP).

[0015] 5. The method according to any one of items 1 to 4, wherein the liquid in the sample 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.

[0016] 6. The method according to any one of items 1 to 5, wherein the liquid is water.

[0017] 7. The method according to any one of items 1 to 6, wherein the method further comprises a step of modifying the analyte as needed to enable its interaction with the surface of the plasmonic material used for the SERS detection.

[0018] 8. The method according to item 7, wherein the modification of the analyte is done by binding a thiol group to the analyte, whereby the analyte will be chemisorbed on the Ag or Au plasmonic surface through this thiol group.

[0019] 9. The method according to item 7, wherein the modification of the analyte is done by physical adsorption through hydrophobic / hydrophilic interactions or van der Waals forces, chemical adsorption through other binding atoms, indirect detection by chemical reaction with a pre-adsorbed reporter molecule, chelation of metal ions by a pre-adsorbed chelating agent, indirect detection by affinity, such as antibody-antigen binding of biomolecules, indirect detection by retaining plasmonic nanoparticles with SERS-active reporter molecules through specific interactions of the analyte, such as antigen-antibody reactions.

[0020] 10. The method according to any one of items 1 to 9, wherein the oxygen scavenger is sodium sulfite, hydrazine, ascorbic acid or ascorbyl palmitate.

[0021] 11. The method according to any one of items 1 to 10, wherein an excess of oxygen scavenger is added.

[0022] 12. The method of any one of claims 1-11, wherein sufficient oxygen scavenger is added to remove at least 98%, 99%, 99.5%, or 99.9%, preferably at least 99.5% or 99.9%, of the DO in the liquid sample.

[0023] 13. The method of any one of claims 1-12, wherein sufficient oxygen scavenger is added such that the concentration of the remaining 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.

[0024] 14. The method of any one of claims 1-13, wherein the LOD of a given analyte is reduced by at least about 10 8 fold, preferably about 10 9 fold, or and even as high as about 10 10 fold after modifying the liquid sample.

[0025] 15. The method of any one of claims 1-14, wherein the LOD of the analyte using SERS is reduced by at least about 10 9 fold or about 10 10 fold after modifying the liquid sample.

[0026] 16. The method of any one of claims 1-15, wherein the LOD of 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.

[0027] 17. The method of any one of claims 1-16, wherein the LOD of the analyte is at most about 10 zM.

[0028] 18. The method of any one of claims 1-17, wherein the method further comprises the step of contacting the liquid sample with a plasmonic material.

[0029] 19. The method of claim 18, wherein at least a portion of the analyte is adsorbed on or becomes adsorbed on the surface of the plasmonic material.

[0030] 20. The method of claim 18 or 19, wherein the plasmonic material is a substance made of an electrically conductive, semiconductive, and / or dielectric material, combinations thereof, or composites thereof.

[0031] 21. The method of any one of claims 18-20, wherein the plasmonic material is gold, silver, copper, or mixtures thereof.

[0032] 22. The method of any one of clauses 18 to 21, wherein the plasmonic material is gold or silver, preferably silver.

[0033] 23. The method of any one of clauses 18 to 22, wherein the plasmonic material has a rough plasmonic surface, is comprised of plasmonic nanoparticles, or both.

[0034] 24. The method of clause 23, wherein the roughness of the rough plasmonic surface is between about 5 nm and about 50 nm, preferably between about 10 nm and about 20 nm.

[0035] 25. The method of clause 23 or 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 at most about 200 nm, at most about 100 nm, or at most about 50 nm.

[0036] 26. The method of any one of clauses 23 to 25, wherein the plasmonic material is comprised of plasmonic nanoparticles.

[0037] 27. The method of any one of clauses 23 to 26, wherein the plasmonic nanoparticles comprise gold, silver, or copper nanoparticles (including silica-coated gold, silver, or copper nanoparticles).

[0038] 28. The method of any one of clauses 23 to 27, wherein the plasmonic nanoparticles are silver or gold nanoparticles (uncoated), preferably silver nanoparticles (uncoated).

[0039] 29. The method of any one of clauses 23 to 28, wherein the plasmonic nanoparticles are pyramidal, spheroid (including spherical), platelet, or rod-shaped.

[0040] 30. The method of any one of clauses 23 to 29, wherein the D50 size of the diameter of the plasmonic nanoparticles is between about 2 nm and about 500 nm.

[0041] 31. The method of any one of clauses 23 to 30, wherein the D50 size 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 at most about 500 nm, at most about 250 nm, at most about 200 nm, at most about 100 nm, at most about 75 nm, or at most about 50 nm.

[0042] 32. The method according to any one of items 23 to 31, wherein the plasmonic nanoparticles are condensed such that they are in close proximity (and / or contact) with each other.

[0043] 33. The method according to any one of items 23 to 32, wherein the plasmonic nanoparticles are condensed by adding a condensing agent, such as a condensing molecule (i.e. a molecule capable of bridging nanoparticles and inducing condensation) or an electrolyte (e.g. a salt), adjusting the pH, adding an organic solvent, such as an alcohol, or a combination of these methods.

[0044] 34. The method according to any one of items 23 to 33, wherein the plasmonic nanoparticles are condensed by adding a condensing agent such as a sulphate or a sulphite.

[0045] 35. The method according to item 33 or 34, wherein the condensing agent and the oxygen scavenger are different materials.

[0046] 36. The method according to item 33 or 34, wherein the condensing agent is the oxygen scavenger, which is preferably a sulphite, preferably sodium sulphite.

[0047] 37. The method according to any one of items 23 to 36, wherein the nanoparticles are condensed by adding a dissolved salt and / or adjusting the pH.

[0048] 38. The method according to any one of items 32 to 37, wherein the pH of the liquid sample is adjusted to about 4 prior to condensation.

[0049] 39. The method according to any one of items 32 to 38, wherein the plasmonic nanoparticles are added to the liquid sample prior to condensation of the nanoparticles and prior to removal of dissolved oxygen using an oxygen scavenger.

[0050] 40. The method according to any one of items 23 to 39, wherein the plasmonic nanoparticles are stabilised by small, loosely bound capping molecules, which are preferably citrate, ethanol, ethylene glycol and / or polyethylene glycol, more preferably citrate and / or ethanol.

[0051] 41. The method according to any one of items 23 to 40, wherein molecules that interact with a specific analyte to produce a unique signal are pre-adsorbed to the plasmonic nanoparticles or the rough plasmonic surface prior to contact with the analyte, which molecules are for example chelators of metal ions.

[0052] 42. The method of any one of items 1 to 41, wherein the method further comprises the step of measuring a SERS spectrum of the liquid sample.

[0053] 43. The method of item 42, wherein the step of measuring comprises irradiating the plasmonic material with light, typically from a laser, and detecting Raman signals scattered by the plasmonic material.

[0054] 44. A probe for remote detection of an analyte in a liquid sample using SERS, the probe comprising:

[0055] a detection chamber having a window transparent to SERS excitation light and Raman scattered signals; and

[0056] a conduit having a first end and a second end, the first end of the conduit being flowably connected to the detection chamber, and the second end of the conduit being configured to be in contact with a liquid sample,

[0057] wherein:

[0058] the detection chamber and the conduit between the first and second ends contain plasmonic nanoparticles immersed in an oxygen scavenging solvent, wherein the plasmonic nanoparticles are in close proximity to each other, thereby allowing unhindered propagation and return of plasmonic fields from the detection chamber to the second end of the conduit, and / or

[0059] the inner walls of the detection chamber and the conduit between the first and second ends are coated with a plasmonic nanolayer, wherein the plasmonic nanolayer is continuous from the detection chamber to the second end of the conduit, thereby allowing uninterrupted propagation and return of plasmonic fields from the detection chamber to the second end of the conduit, and wherein the conduit and the detection chamber are filled with the oxygen scavenging solvent; and

[0060] wherein the oxygen scavenging solvent contains an oxygen scavenger that removes dissolved oxygen from the oxygen scavenging solvent.

[0061] 45. The probe of item 44, wherein the plasmonic nanoparticles are as defined in any one of items 23 to 41, and / or the plasmonic layer is a plasmonic material layer comprising a rough plasmonic surface as defined in any one of items 23 to 25.

[0062] 46. The probe of item 44 or 45, wherein the detection chamber and the conduit between the first and second ends contain plasmonic nanoparticles immersed in an oxygen scavenging solvent.

[0063] 47. The probe according to any one of items 44 to 46, wherein the plasmonic nanoparticles are condensed along the longitudinal direction of the conduit using a technique as defined in any one of items 33 to 39, such as defined in any one of items 32 to 39.

[0064] 48. The 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.

[0065] 49. The probe according to any one of items 44 to 48, wherein the solvent is water.

[0066] 50. The probe according to any one of items 44 to 49, wherein the oxygen scavenger is as defined in any one of items 1 to 43.

[0067] 51. The probe according to any one of items 44 to 50, wherein the concentration of added oxygen scavenger and the concentration of residual DO are as defined in any one of items 1 to 43.

[0068] 52. The probe according to any one of items 44 to 51, wherein the second end of the conduit is open or closed (preferably open).

[0069] 53. The probe according to any one of items 44 to 52, wherein the inner wall of the conduit is coated with a plasmonic layer and the conduit is filled with oxygen- depleted solvent.

[0070] 54. The probe according to any one of items 44 to 53, wherein the second end of the conduit is open and the detection chamber and the conduit are refilled with oxygen- depleted solvent (and plasmonic nanoparticles, if used) from, e.g., a pump or a reservoir device, such that the conduit and the detection chamber remain filled with oxygen- depleted solvent (and plasmonic nanoparticles, if used, keeping the plasmonic nanoparticles in close proximity to each other).

[0071] 55. The probe according to item 54, wherein the pump or the reservoir device is directly connected to the first end of the conduit or to the detection chamber in such a way that any oxygen- depleted solvent / plasmonic nanoparticles flowing out of the second end of the conduit are replaced.

[0072] 56. The probe according to any one of items 44 to 55, wherein capillary forces keep the oxygen- depleted solvent (and plasmonic nanoparticles, if used) within the conduit, despite the second end of the conduit being open.

[0073] 57. The probe of any one of clauses 44-56, wherein the second end of the conduit is open and the second end of the conduit is at least partially coated with a plasma layer.

[0074] 58. The probe of any one of clauses 44-57, wherein the second end of the conduit comprises a plasma nanoparticle and an end wall, the inner wall toward the second end of the conduit, and the outer wall toward the second end is coated with a plasma layer.

[0075] 59. The probe of any one of clauses 44-58, wherein the inner wall of the conduit is coated with a plasma layer and the plasma layer also covers the end wall and outer wall toward the second end of the conduit.

[0076] 60. The probe of any one of clauses 44-59, wherein the second end of the conduit is also sealed with a plasma layer.

[0077] 61. The probe of any one of clauses 44-60, wherein the conduit is made of metal, quartz, glass, or a polymeric plastic, such as polyethylene, polypropylene, polystyrene, polyether ether ketone, polyvinyl chloride, polytetrafluoroethylene, or polydimethylsiloxane.

[0078] 62. The probe of any one of clauses 44-61, wherein the conduit is a polyether ether ketone (PEEK) polymer tube or a glass capillary tube.

[0079] 63. The probe of any one of clauses 44-62, wherein the inner diameter of the conduit is between about 0.08 mm and about 1 cm, preferably between about 0.3 mm and about 0.5 mm.

[0080] 64. The probe of any one of clauses 44-63, wherein the inner diameter of the conduit 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 at most about 1 cm; at most about 5 mm; at most about 2 mm; at most about 1 mm; at most about 0.75 mm; or at most about 0.5 mm.

[0081] 65. The probe of any one of clauses 44-64, wherein the inner diameter of the conduit is about 0.3 mm or about 0.5 mm.

[0082] 66. The probe according to any one of items 44 to 65, wherein the length of the conduit 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 at most about 50 m; at most about 20 m; at most about 10 m; at most about 5 m; at most about 2 m; or at most about 1 m.

[0083] 67. The probe according to any one of items 44 to 66, wherein the length of the conduit is about 1 m or about 2 m.

[0084] 68. The probe according to any one of items 44 to 67, wherein the cross-section of the conduit is circular, elliptical or rectangular.

[0085] 69. The probe according to any one of items 44 to 68, wherein "in close proximity" means a distance of at most about 15 nm, preferably at most about 10 nm, more preferably at most about 5 nm (between plasmonic nanoparticles, between plasmonic nanoparticles and a plasmonic layer, or between one of these and an analyte).

[0086] 70. The probe according to any one of items 44 to 69, wherein the density of the plasmonic nanoparticles varies along the longitudinal direction of the conduit.

[0087] 71. The probe according to any one of items 44 to 70, wherein the detection chamber and the first end of the conduit are fluidically connectable to each other through a hole defined by the detection chamber.

[0088] 72. The probe according to any one of items 44 to 71, wherein the detection chamber is made of glass, plastic or quartz, more preferably of glass or quartz.

[0089] 73. The probe according to any one of items 44 to 72, wherein the analyte comprised in the liquid is contacted with a plasmonic material as defined in any one of items 18 to 41 comprised in the probe (e.g. a plasmonic layer completely coating the second end in case of a closed second end; or a plasmonic layer coating the second end or plasmonic nanoparticles immersed in the oxygen scavenging solvent in case of an open second end).

[0090] 74. The probe according to any one of items 44 to 73, wherein the liquid sample is contacted with, preferably adsorbed on, a plasmonic material as defined in any one of items 18 to 41.

[0091] 75. The probe according to any one of items 44 to 74, wherein the liquid sample has been deoxygenated, preferably using an oxygen scavenger as defined in any one of items 1 to 43.

[0092] 76. The probe according to any one of items 44 to 75, wherein the amount of oxygen scavenger in the liquid sample and the residual DO concentration is as defined in any one of items 1 to 43.

[0093] 77. The probe according to any one of items 44 to 76, wherein the liquid sample is a liquid sample as defined in any one of items 1 to 43.

[0094] 78. The probe according to any one of items 44 to 77, wherein the liquid sample has been modified using a method as defined in any one of items 1 to 43.

[0095] 79. The probe according to any one of items 44 to 78, wherein the analyte becomes adsorbed to the surface of the plasmonic material, which can be the surface of the nanoparticle in the conduit, a plasmonic layer coated on the second end of the conduit and / or a plasmonic layer capping the second end of the conduit.

[0096] 80. The probe according to any one of items 44 to 79, wherein the analyte is in a deoxygenated liquid sample produced according to a method as defined in any one of items 1 to 43, and comprises condensed plasmonic nanoparticles or rough plasmonic surfaces as defined in any one of items 23 to 41, any of which are in contact with the nanoparticle in the conduit, a plasmonic layer coated on the second end of the conduit and / or a plasmonic layer capping the second end of the conduit.

[0097] 81. The probe according to any one of items 44 to 80, wherein molecules that produce a unique signal upon interaction with a specific analyte are pre-adsorbed on the plasmonic nanoparticles within the conduit, on the plasmonic nanoparticles in the liquid sample, on a plasmonic layer coated on the second end of the conduit and / or on a plasmonic layer capping the second end of the conduit, the molecules being for example chelators for metal ions or antibodies, peptides or aptamers that interact with biological molecules.

[0098] 82. The probe according to any one of items 44 to 81, wherein the probe is configured to work with a surface enhanced Raman spectrometer for remote detection, the surface enhanced Raman spectrometer being operable to detect an analyte by shining an incident laser light to a sample holding chamber or detection chamber and measuring scattered light using a measurer.

[0099] 83. The probe according to item 82, wherein backscattered light, i.e. light scattered 180° with respect to the incident light, is detected.

[0100] 84. The method according to any one of items 1 to 43, wherein the measuring step is performed using the probe as defined in any one of items 44 to 83.

[0101] 85. The method according to any one of items 1 to 43, wherein the SERS system is as defined in any one of items 44 to 83. BRIEF DESCRIPTION OF DRAWINGS

[0102] In the drawings:

[0103] Figure 1A Longitudinal cross-sectional view of the second end of a conduit g for an embodiment of a probe according to the present application.

[0104] Figure 1B Longitudinal cross-sectional view of the second end of a conduit g containing plasmonic nanoparticles for an embodiment of a probe according to the present application.

[0105] Figure 1C Longitudinal cross-sectional view of the second end of a conduit g for another embodiment of a probe according to the present application, wherein the inner wall of the conduit g is coated with a plasmonic layer.

[0106] Figure 1D Longitudinal cross-sectional view of the second end of a conduit g containing plasmonic nanoparticles, wherein the end wall, the inner wall facing the second end of the conduit g and the outer wall facing the second end are all coated with a plasmonic layer.

[0107] Figure 1E Longitudinal cross-sectional view of the second end of a conduit g, wherein the inner wall is coated with a plasmonic layer that also covers the end wall and the outer wall facing the second end of the conduit g.

[0108] Figure 1F Longitudinal cross-sectional view of the second end of a conduit g containing plasmonic nanoparticles and being capped with a plasmonic layer.

[0109] Figure 1G Longitudinal cross-sectional view of the second end of a conduit g, wherein the inner wall is coated with a plasmonic layer that also caps the second end of the conduit g.

[0110] Figure 2 Schematic representation of a conventional surface-enhanced Raman spectrometer.

[0111] Figure 3 Schematic representation of a surface-enhanced Raman spectrometer for remote detection comprising a probe of the present application.

[0112] Figure 4 is a transmission electron micrograph of silver nanoparticles and aggregates used in the present experiments; in particular, Figure 4 shows A) individual; B) and C) aggregates of silver nanoparticles; D) micrograph of nanogap between silver nanoparticles.

[0113] Figure 5 shows the integrated plasmonic electromagnetic field generated photocurrent; in particular, Figure 5A ) shows an experimental schematic showing the relative position of the incident radiation and the honeycomb electrode; Figure 5B shows the photocurrent generated by NIR (λ = 785 nm) irradiation at a distance of 1 cm from the electrode surface with and without dissolved oxygen at constant ionic strength.

[0114] Figure 6 shows the surface plasmon coupling within and between silver nanoparticle aggregates. In particular, Figure 6 shows the charge distribution (top) and electric field (bottom) of irradiated silver nanoparticle aggregates under the following conditions: A) uncoated nanoparticles; B) uncoated nanoparticles with two oxygen molecules in the nanogap; C) uncoated nanoparticles with an oxygen molecule bound to the surface of the left nanoparticle; D) uncoated nanoparticles with an oxygen molecule bound to the surface of each nanoparticle; E) Ag2O-coated (2 nm thickness) nanoparticles; F) Ag2O-coated nanoparticles with two oxygen molecules in the nanogap; G) Ag2O-coated nanoparticles with an oxygen molecule bound to the surface of the right nanoparticle; and H) Ag2O-coated nanoparticles with an oxygen molecule bound to the surface of each nanoparticle.

[0115] Figure 7 shows the surface plasmon coupling within and between silver nanoparticle aggregates. It shows the charge distribution (top) and electric field distribution (bottom) of irradiated A-C) silver nanoparticle aggregates and D-F) silver nanoparticles coated with silver oxide (2 nm thickness) as a function of the distance between the rightmost nanoprism and the rest of the aggregate under oxygen-free (de-aerated) conditions. The distances are A) 10, B) 13, C) 17, D) 8, E) 9.8, and F) 17 nm. Figure 7 also shows the transmission of the electromagnetic field through silver nanorods with diameters of 4 nm and 50 nm in the absence of G) dissolved oxygen and H) in the presence of dissolved oxygen. Figure 7 also shows the transmission of the electromagnetic field through dimers consisting of I-J) a nanoprism and a nanosphere and K-L) two nanoprism in the absence of I and J) and in the presence of K and L) dissolved oxygen. In addition, Figure 7 shows the transmission of the electromagnetic field between a tetramer of nanoparticles in the absence of dissolved oxygen. The tetramer consists of M) three nanoprisms plus one nanosphere and N) two nanoprisms plus two nanospheres.

[0116] Figure 8 shows surface enhanced Raman spectra in the presence or absence of dissolved oxygen. Specifically, Figure 8 shows surface enhanced Raman spectra of A) p-aminothiophenol and B) p-nitrothiophenol at 1 fM in the absence and presence of dissolved oxygen (blank contains Na2SO3).

[0117] Figure 9 shows the effect of oxygen scavenging on the detection limit of surface enhanced Raman spectroscopy. Specifically, Figure 9A A) and B) show surface enhanced Raman spectra of p-aminothiophenol at different concentrations in the presence of dissolved oxygen; C) shows the function between the intensity of the C-S stretching mode and the logarithm of the concentration. Figure 9D A) and E) show surface enhanced Raman spectra of p-aminothiophenol at different concentrations in the absence of dissolved oxygen. In addition, Figure 9 shows F) the C-H stretching mode and G) the C-S stretching mode intensity as a function of the concentration.

[0118] Figure 10 shows silver nanoparticle characteristics and the interference of citrate salts on the surface enhanced Raman of the analyte at less than 1000 cm -1 Specifically, Figure 10A A) shows the silver nanoparticle size distribution measured by dynamic light scattering. In addition, shown in Figure 10 are B) the Raman spectra of the silver nanoparticles and the analytes C) p-aminothiophenol and D) p-nitrothiophenol.

[0119] Figure 11 shows the range of enhanced sensitivity by removing dissolved oxygen. Specifically, Figure 11 shows surface enhanced Raman spectra of the mercaptated oligonucleotide sequence in the presence of A) dissolved oxygen (10 pM) and B) absence (8 zM).

[0120] Figure 12 shows A) a transmission electron microscope image of spherical silver nanoparticles and B) oxygen removal dependent signal enhancement of adsorbed (on the spherical silver nanoparticles) p-aminothiophenol.

[0121] Figure 13 shows surface enhanced Raman spectra of p-nitrothiophenol at different concentrations in the absence of oxygen using A) ascorbic acid and B) hydrazine.

[0122] Figure 14 shows the correlation between dissolved oxygen concentration and surface enhanced Raman scattering intensity. Specifically, Figure 14 shows the function of the relative surface enhanced Raman spectra and its signal intensity with the dissolved oxygen concentration in the presence of A) and B) Na2SO3 addition and C) and D) argon gas displacement oxygen removal, respectively.

[0123] Figure 15 shows the effect of oxygen removal enhancement of dielectric oxide surfaces on surface enhanced Raman spectroscopy. Specifically, Figure 15 shows the cyclic voltammograms of silver nanoparticles adsorbed with p-aminothiophenol in A) Ag2NO3 and in the presence of B) dissolved oxygen and C) absence of dissolved oxygen.

[0124] Figure 16A B) shows their surface enhanced Raman spectra in the presence and absence of dissolved oxygen (the Raman spectrum of cetyltrimethylammonium bromide (CTAB) coating is provided as a reference).

[0125] Figure 17A B) shows their surface enhanced Raman spectra after oxygen removal in the presence of p-aminophenylthiol, and C) their surface enhanced Raman spectra with 50 aM p-nitrothiophenol as a reference.

[0126] Figure 18 shows remote detection of analytes adsorbed on silver mirrors at the bottom of a quartz NMR tube. Specifically, Figure 18 shows surface enhanced Raman spectra of A) p-aminothiophenol and B) p-nitrothiophenol transmitted from the silver mirror through silver nanoparticles, measured at a distance of 10 cm between the incident laser and the field of view of the detector. Figure 18 also shows surface enhanced Raman C) spectra of p-aminothiophenol adsorbed on a silver mirror transmitted through silica-coated silver nanoparticles and D) the intensity of the C-S stretch band as a function of distance.

[0127] Figure 19 shows remote detection of analytes adsorbed on oxygen-removed and aggregated silver nanoparticles on a 96-well microtiter plate. Specifically, Figure 19 shows surface enhanced Raman spectra measured using a glass capillary pre-filled with fresh oxygen-removed and aggregated silver nanoparticles (the incident laser was 6 cm from the end of the capillary). Figure 19 shows the spectra of A) p-aminothiophenol and B) p-nitrothiophenol as a function of integration time.

[0128] Figure 20 shows analyte remote detection at distances greater than 1 m by integrated plasmonic coupling. Specifically, Figure 20 shows a schematic of A) remote detection of analytes at the distal end of a 1 m long PEEK capillary using a probe of the present application. In addition, Figure 20 shows detection of B) p-aminophenylthiol and C) p-nitrothiophenol with the device in A). DETAILED DESCRIPTION

[0129] Methods of altering a liquid sample containing an analyte to increase the SERS signal intensity of the analyte in the sample, and probes for remote detection of analytes in a liquid sample using SERS are provided.

[0130] The inventors have found that dissolved oxygen (DO) in a liquid sample is the main limiting factor for the transport of the plasmonic field throughout the liquid sample, and thus the sensitivity of SERS. In particular, the inventors have found that by removing DO using a chemical oxygen scavenger, an improved SERS sensitivity can be achieved, for example detecting analytes at zM concentration levels in water. Crucially, such good results are not achieved with other methods of oxygen removal, for example with an oxygen-free gas. Sufficient DO removal cannot be achieved with sparging. Furthermore, when using an oxygen scavenger, the inventors detected analytes outside the field-of-view of the detector, which indicates an unexpectedly long-range signal propagation along the plasmonic material, which allows for example long-range detection of analytes with the probes described herein.

[0131] Method of modifying a liquid sample

[0132] A first aspect of the present invention provides a method of modifying a liquid sample containing an analyte to increase the SERS signal intensity of said analyte. The method of the present invention comprises the steps of:

[0133] (a) providing said liquid sample to be analysed using SERS; and

[0134] (b) adding an oxygen scavenger to said liquid sample to remove dissolved oxygen from said liquid sample.

[0135] Liquid sample

[0136] The analyte in the sample to be analysed can be any analyte commonly measured by SERS, i.e. molecules and ions comprising two or more atoms, organic or inorganic. It is also known that single atom ions can be indirectly detected using SERS, for example by forming a vibrationally active complex. In this case, the substance under investigation is strictly speaking one atom, but the analyte detected by SERS (i.e. the vibrationally active complex) is polyatomic. In embodiments, the analyte is a thiol-containing compound, an amine, a pesticide, a persistent organic pollutant, a transition metal complex, a peptide, a protein, a nucleic acid, a polysaccharide or a hormone. In preferred embodiments, the analyte is a transition metal complex, a peptide, a protein, a nucleic acid, a persistent organic pollutant, a pesticide or a hormone. For example, the analyte can be p-aminothiophenol (pATP) or p-nitrothiophenol (pNTP).

[0137] The liquid in the sample is preferably water or an organic solvent, for example methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, a hydrocarbon or a compound thereof. In preferred embodiments, the liquid is water.

[0138] In embodiments, the method of the present application further comprises a step of modifying the analyte as necessary to enable the analyte to interact with the plasmonic material surface for SERS detection. Such modifications are well known to those skilled in the art of SERS spectroscopy. One common method is to attach a thiol group to the analyte, whereby the analyte can be chemisorbed to a silver or gold plasmonic material surface through the thiol group. Other methods include physical adsorption through hydrophobic / hydrophilic interactions or van der Waals forces; chemical adsorption through other binding atoms; indirect detection through chemical reaction with a pre-adsorbed reporter molecule; chelation of metal ions through a pre-adsorbed chelator; indirect detection through affinity interactions, such as antigen-antibody binding of biomolecules; and indirect detection through retention of plasmonic nanoparticles with SERS-active reporter molecules using specific interactions with the analyte, such as antibody-antigen interactions.

[0139] The liquid sample to be analyzed using SERS will be modified using the method of the present application. Since the method of the present application 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 will be understood that if the liquid sample does not contain the analyte (e.g., a blank control), then naturally the desired increase in SERS signal intensity will not occur.

[0140] Oxygen scavenger

[0141] As previously described, the inventors have found that the use of an oxygen scavenger to remove DO results in an increase in the sensitivity of SERS. Accordingly, the oxygen scavenger can be any oxygen scavenger known in the art to reduce DO in a liquid. In embodiments, the oxygen scavenger is sodium sulfite, hydrazine, ascorbic acid, or ascorbyl palmitate.

[0142] Preferably, as much of the dissolved oxygen in the liquid sample is removed. Accordingly, it is preferred that an excess of the oxygen scavenger is added to ensure that as much DO as possible is removed. In embodiments, a sufficient amount of the oxygen scavenger is added to remove at least 98%, 99%, 99.5%, or 99.9% of the DO in the liquid sample, preferably at least 99.5% or 99.9%. In embodiments, a sufficient amount of the oxygen scavenger is added such that the remaining DO concentration 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.

[0143] Other optional steps

[0144] As mentioned above, the liquid sample will be subjected to SERS analysis. This spectroscopic technique relies on localized surface plasmon resonance (LSPR). LSPR occurs when the free conducting electrons of a plasmonic material resonate with the incident light (typically a laser). Both the position and intensity of the LSPR band are dependent on the properties of the plasmonic material, and they are highly sensitive to the dielectric properties of the surrounding medium. In particular, when different groups are adsorbed to (or in close proximity to) the plasmonic material, the position and intensity of the LSPR band change. Indeed, when light is shone on the plasmonic material, the electromagnetic field is locally amplified (due to the excitation of localized surface plasmons), leading to what we know as "hot spots", where the electromagnetic field is even more enhanced. When analyte molecules come into contact (or proximity) with the plasmonic material (e.g. adsorbed on the material surface), these molecules interact with these hot spots, leading to a change in the surface plasmon resonance conditions. This change in resonance conditions leads to a SERS signal that can be detected by the SERS spectrometer, ultimately allowing the analyte to be detected and quantified.

[0145] Contacting the liquid sample with the plasmonic material

[0146] As the liquid sample will be subjected to SERS analysis, in embodiments the method further comprises the step of contacting the liquid sample (as modified above, or to be modified as described above) with a plasmonic material (also referred to as a SERS substrate). This is done so that the analyte can come into close proximity with the surface of the plasmonic material (so that the analyte to be analysed can interact with the amplified electromagnetic field generated by the plasmonic material upon SERS laser irradiation, thereby benefiting from the SERS signal amplification factor inherent to the SERS technique). Various ways of increasing this SERS enhancement factor (and the corresponding measured signal) are well known to the skilled person.

[0147] In embodiments, at least a portion of the analyte is (or becomes) adsorbed on the surface of the plasmonic material. As mentioned above, one common technique to achieve this is to chemisorb the analyte onto the plasmonic material, for example, to chemisorb analytes bearing a thiol group on the surface of Ag or Au plasmonic materials.

[0148] By modifying the liquid sample using the method of the present application, the limit of detection (LOD) of the analyte is unexpectedly and substantially reduced compared to the unmodified liquid sample. In embodiments, for a given analyte / plasmonic combination, the LOD is typically reduced by a factor of about 10 8 , preferably by a factor of about 10 9 , and even by a factor of about 10 10In preferred embodiments, the LOD of the analyte using SERS is increased by at least about 10 9 or about 10 10 times using the methods of the present application. In embodiments, the LOD of 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. In preferred embodiments, the LOD of the analyte is at most about 10 zM. Of course, the particular LOD in the modified liquid sample will depend on the plasmonic material and the analyte used as described above.

[0149] Based on the experimental results discussed below, the removal of oxygen results in the efficient coupling of the SERS field, and this plasmonic coupling enables the propagation of the electromagnetic (EM) field throughout the sample volume with no significant loss of signal energy (i.e., limited quenching) during the propagation. The plasmonic field generated by the photoexcitation of the plasmonic material propagates throughout the sample to reach the plasmonic material (which is in contact with or in close proximity to the analyte) and back, thereby returning the Raman scattered signal of the analyte to the detector. It has been discovered herein that experiments and computer simulations as described below indicate that in the absence of the removal of DO, oxygen, due to its high electron affinity, can trap electrons and prevent the efficient generation, coupling, propagation, and integration of the SERS field along the plasmonic material. Conversely, the removal of DO enables the plasmonic coupling and the propagation of the SERS field. This is the reason for the increase in the SERS signal intensity of the analyte.

[0150] In general, the presence of oxygen molecules hovering in the nanogap or adsorbed on the surface of the plasmonic material results in a charge redistribution and quenching of the plasmonic field and coupling. However, the following experiments demonstrate that this can be avoided using the methods of the present application. Even for Ag2O-coated nanoparticles, despite the fact that their plasmonic field is somewhat attenuated compared to uncoated silver nanoparticles, the introduction of DO can dramatically quench the plasmonic field and prevent coupling. In the following experiments using plasmonic nanoparticles, the quenching power of DO is independent of typical parameters of the nanogap, such as the gap distance, the number of nanoparticles in the nanocluster (2, 3, 4 nanoparticles), and the characteristics of the plasmonic nanoparticles (size, shape, position, orientation, and surface chemistry). The results of the simulation calculations show that DO can quench the plasmonic field; in the absence of DO, the plasmonic field is able to propagate throughout the sample by the adjacent plasmonic nanostructures and their clusters through plasmonic coupling.

[0151] As mentioned above, the traditional understanding of SERS is based on signal amplification of a single nanostructure or coupling of adjacent nanoparticles in the plasmonic field (i.e. at the nanogap) - this is the "hotspot" theory. However, in the present experiments (see below) it was demonstrated that after removal of DO with an oxygen scavenger, large scale long range plasmonic field coupling (i.e. integrated surface plasmon resonance) becomes possible. This integrated field enables efficient transmission of SERS signals of a single molecule located in close proximity or in contact with any individual plasmonic material (e.g. plasmonic nanoparticles) (preferably adsorbed on their surface), e.g. each nanoparticle becomes SERS active and can act as a "hotspot" or propagate SERS signals of adsorbed analytes, thus enabling true single molecule detection.

[0152] Signal enhancement due to oxygen removal in the method of the present application has been observed in different plasmonic nano-materials (see examples below), including silver nanoparticles, and even silica-coated plasmonic nanoparticles, e.g. AgNPs (i.e. SHINERS technology), where LOD was observed to drop from 500 nM to 50 aM (10 10 fold). It should be mentioned that while the overall SERS sensitivity of silica-coated plasmonic nanoparticles is typically lower than that of bare (uncoated) plasmonic nanoparticles (10 4 fold lower when comparing silica-coated AgNPs to uncoated AgNPs), it appears that DO and not the surface oxide layer is the main factor quenching the SERS signal, i.e. the relative signal enhancement after removal of DO is always 10 9 -10 10 fold, although the absolute sensitivity depends on the nano-plasmonic particles used. For example, by using AuNRs (gold nanorods, which are not easily oxidized on the surface) and silica-coated AgNPs, it is possible to separate the effect of DO from the effect of surface oxidation on SERS sensitivity.

[0153] Thus, in the method of the present application, the plasmonic material is any plasmonic material suitable for use in SERS. Such materials are well known to those skilled in the art and are extensively described in the literature. As is well known to those skilled in the art, plasmonic materials for use in SERS are made of substances that can produce localized surface plasmon resonance when irradiated with a SERS laser, which substances can be electrically conductive, semiconductive, and / or dielectric materials, combinations thereof, or composites thereof. Non-limiting examples of preferred substances that can exhibit localized surface plasmon resonance when irradiated with a SERS laser include gold, silver, and copper, and gold, silver, and copper whose surfaces are covered with a thin dielectric oxide layer (such as a thin layer of silicon dioxide or aluminum oxide as reported in the prior art). Preferred plasmonic materials are gold, silver, and copper, preferably gold and silver, and more preferably silver.

[0154] Furthermore, it is well known that plasmonic materials either have a rough surface (referred to herein as a "rough plasmonic surface") or are composed of nanoparticles (referred to herein as "plasmonic nanoparticles"), or combinations thereof. In preferred embodiments, the plasmonic material is a plasmonic nanoparticle.

[0155] For the sake of clarity, when referring to a "rough plasmonic surface", the roughness should be sufficient to produce the desired localized surface plasmon resonance. Those skilled in the art will understand the meaning of the expression "rough plasmonic surface" in the context of SERS. For the sake of clarity, in specific embodiments, the rough plasmonic surface has a roughness (Ra) of between about 5 nm and about 50 nm, preferably between about 10 nm and about 20 nm. In embodiments, the rough plasmonic surface has a roughness of at least about 5 nm, at least about 10 nm, or at least about 20 nm; and / or at most about 200 nm, at most about 100 nm, or at most about 50 nm. For the sake of clarity, the rough plasmonic surface does not necessarily need to be subjected to a separate "roughening" process; the surface can already have the above-defined roughness when it is prepared.

[0156] Those skilled in the art will appreciate that, although the above roughness parameters relate to rough plasmonic surfaces, other plasmonic materials (such as plasmonic nanoparticles) can have high-curvature regions (such as edges and vertices) where the electromagnetic field strength is typically highest.

[0157] Common examples of plasmonic nanoparticles include gold, silver, or copper nanoparticles (including silica-coated gold, silver, or copper nanoparticles). In preferred embodiments, the plasmonic nanoparticles are silver or gold nanoparticles (uncoated), more preferably silver nanoparticles (uncoated).

[0158] It is well known to those skilled in the art that plasmonic nanoparticles can have different (nano) shapes, sizes, morphologies and size distributions. In particular, the size and shape of the nanoparticles can be adjusted to improve the SERS enhancement of a given analyte. In preferred embodiments, the plasmonic microparticles are pyramidal, spheroidal (including spherical), platelet-like or rod-like. In embodiments, the D50 size of the plasmonic nanoparticles is between about 2 nm and about 500 nm. In embodiments, the D50 size 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 at most about 500 nm, at most about 250 nm, at most about 200 nm, at most about 100 nm, at most about 75 nm, or at most about 50 nm.

[0159] In preferred embodiments of the method of the application, the plasmonic nanoparticles are agglomerated, such that they are in close proximity (and / or contact) with each other. Indeed, as shown in the examples below, the use of an oxygen scavenger can enable long-range signal propagation along the plasmonic material, in turn enabling long-range detection of an analyte. The plasmonic nanoparticles can be agglomerated using any known technique in the art. It is well known to those skilled in the art that there are many ways in which nanoparticles can be agglomerated, for example by adding an agglomerating agent, such as an agglomerating molecule (i.e. a molecule capable of bridging nanoparticles and inducing agglomeration) or an electrolyte (e.g. a salt), adjusting the pH, adding an organic solvent such as an organic alcohol, or a combination of these methods. In preferred embodiments of the application, the nanoparticles are agglomerated by adding a dissolved salt and / or adjusting the pH.

[0160] In embodiments, the plasmonic nanoparticles are agglomerated by adding an agglomerating agent such as a sulphate or a sulphite salt. In embodiments, the agglomerating agent and the oxygen scavenger are different materials. In embodiments, the agglomerating agent is the oxygen scavenger, preferably a sulphite salt, preferably sodium sulphite. In some embodiments, the pH of the liquid sample is adjusted to about 4 prior to agglomeration. For example, SiO2-coated AgNPs can have the pH adjusted to about 4 prior to agglomeration by the addition of a sulphate or sulphite salt.

[0161] Furthermore, in preferred embodiments, the plasmonic nanoparticles can be stabilised by small, loosely bound coating molecules, preferably citrate, ethanol, ethylene glycol and / or polyethylene glycol, more preferably citrate and / or ethanol. These molecules can act as reducing agents and structure-directing agents, meaning that they can dictate the shape of the particles during synthesis, and stabilise the resulting nanoparticles. Such coating molecules (coating ligands) can also improve the optical properties of the plasmonic material (as a SERS substrate), while being easily replaced by a mercaptanated analyte.

[0162] In preferred embodiments, the plasmonic nanoparticles are added to the liquid sample prior to aggregation of the nanoparticles and prior to removal of dissolved oxygen using an oxygen scavenger.

[0163] In embodiments, molecules that interact with a particular analyte to produce a unique signal are pre-adsorbed to the plasmonic nanoparticles or roughened plasmonic surface prior to contact with the analyte, the molecules being, for example, chelators of metal ions.

[0164] Measuring SERS spectra

[0165] Once a liquid sample has been modified according to the methods described above, and then contacted with plasmonic material, the methods described herein can further comprise the step of measuring the SERS spectrum of the liquid sample.

[0166] This step can be performed using any known SERS technique in the art, using any known SERS system in the art, and in general terms, this step comprises irradiating the plasmonic material with light, typically from a laser, and detecting the Raman signal scattered by the plasmonic material.

[0167] In preferred embodiments, the measuring step is performed using a probe of the application, which is described below.

[0168] In preferred embodiments, the SERS system used is defined in the next section.

[0169] Probes for remote detection using SERS

[0170] In a second aspect of the application, there is provided a probe for remote detection of an analyte in a liquid sample using SERS. The probe of the application comprises:

[0171] a detection chamber having a window transparent to SERS excitation light and Raman scattered signals; and

[0172] a conduit having a first end and a second end, the first end of the conduit being flowably connected to the detection chamber, and the second end of the conduit being configured to be in contact with a liquid sample,

[0173] wherein:

[0174] the detection chamber and the conduit between the first and second ends comprise plasmonic nanoparticles immersed in an oxygen scavenging solvent, wherein the plasmonic nanoparticles are in close proximity to one another, thereby allowing unimpeded propagation of plasmonic fields from the detection chamber to the second end of the conduit and back, and / or

[0175] the interior walls of the detection chamber and the interior walls of the conduit between the first and second ends are coated with a plasma nano-layer, wherein the plasma nano-layer is continuous from the detection chamber to the second end of the conduit, thereby allowing uninterrupted propagation and return of a plasma field from the detection chamber to the second end of the conduit, and wherein the conduit and the detection chamber are filled with an oxygen-removing solvent; and

[0176] wherein the oxygen-removing solvent comprises an oxygen scavenger that removes dissolved oxygen from the solvent.

[0177] A plasma layer is a layer of plasma material that contains a rough plasma surface. In this section, the plasma material (including the plasma nanoparticles and the rough plasma surface) is as described in the previous section (e.g., the material, roughness, shape, etc. are the same).

[0178] During use of the probe, SERS excitation light will enter the detection chamber through the transparent window. The SERS excitation light will impinge on the nanoparticles in the detection chamber, thereby creating a plasma field. Because the plasma nanoparticles are in close proximity to each other throughout the probe (the conduit and the detection chamber) (or the plasma layer is continuous), and because they are in an oxygen-removing solvent that contains an oxygen scavenger, the plasma field will propagate from the detection chamber to the second end of the conduit in an uninterrupted manner. There, the plasma field will interact with the analyte, creating a signal plasma field (i.e., a SERS signal) that will propagate back along the conduit to the detection chamber. Finally, the signal plasma field will exit the detection chamber through the transparent window of the detection chamber and will ultimately be detected by the light detector.

[0179] As mentioned, the probe of the present application utilizes SERS for remote detection. Specifically, by placing the second end of the conduit in direct contact with a liquid sample, a SERS spectrum of the sample can be obtained despite the distance (i.e., the length of the conduit) between the sample and the spectrometer. This allows for direct measurement of the spectrum of the sample without having to bring the sample close to the spectrometer, and also allows for measurement of the sample in spaces that would otherwise be too small for the spectrometer (as only the conduit needs to enter the space where the liquid sample is located).

[0180] The dimensions and length of the probe depend on the dimensions of the conduit and the detection chamber, which will be defined in detail below. For example, using a longer, narrower tube will naturally make the probe longer and narrower. However, one skilled in the art will appreciate that the dimensions of the probe can be selected and adjusted depending on the intended use of the probe. For example, if the probe is intended to be used to measure a liquid sample in a nuclear reactor, the dimensions of the conduit (and thus the probe) will be adjusted so that the conduit is able to safely reach and detect the liquid sample within the nuclear reactor.

[0181] In a preferred embodiment, the detection chamber and the conduit between the first end and the second end comprise plasmonic nanoparticles immersed in an oxygen depleted solvent. In an embodiment, the plasmonic nanoparticles are condensed along the longitudinal direction of the conduit using any of the techniques discussed in the previous section as defined in the previous section.

[0182] The oxygen depleted solvent used can be any solvent known in the art. In an embodiment, 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. In a preferred embodiment, the solvent is chosen to be water. As mentioned before, the solvent is oxygen depleted and the solvent comprises an oxygen scavenger that removes dissolved oxygen from the solvent, which means that its dissolved oxygen has been removed by the oxygen scavenger. The oxygen scavenger is as defined in the previous section. Furthermore, the concentration of the oxygen scavenger added as well as the concentration of the remaining DO is as defined in the previous section.

[0183] As mentioned in the previous section, the removal of dissolved oxygen results in an efficient coupling of the SERS field, which plasmonic coupling enables the electromagnetic field to propagate over long distances without significant loss of signal energy. Therefore, by using an oxygen depleted solvent, the electromagnetic field propagates along the longitudinal direction of the conduit.

[0184] Conduit

[0185] The conduit of the probe of the present invention allows the plasmonic field to propagate from the detection chamber to the second end of the conduit and back. The signal plasmonic field can be generated at the second end in a number of ways. One example is if the analyte comprised in the liquid sample is in contact (preferably adsorbed) with plasmonic material at the second end of the conduit, such as nanoparticles immersed in an oxygen depleted solvent. Another example is if the liquid sample itself generates a plasmonic field (for example if the liquid sample has been modified using the method defined in the previous section and comprises condensed plasmonic nanoparticles). The latter example means that both the liquid sample and the oxygen depleted solvent comprised in the conduit have been treated with an oxygen scavenger.

[0186] Figure 1A Longitudinal cross-sectional view of the second end of the conduit (g) of an embodiment of the probe according to the present invention. The figure shows the inner wall (12) and the outer wall (16) of the conduit g as well as the end wall (14) of the end of the conduit g.

[0187] The second end of the conduit can be open or closed (preferably open). Figure 1B and 1C An embodiment is shown in which the second end of the conduit g is open. Figure 1B Longitudinal cross-sectional view of the second end of the conduit g of an embodiment of the probe according to the present invention, said conduit g comprising plasmonic nanoparticles (18) immersed in an oxygen depleted solvent. Figure 1CLongitudinal cross-sectional view of the second end of a conduit g for another embodiment of a probe according to the present application, wherein the inner wall 12 of the conduit g is coated with a plasma layer (20) and the conduit g is filled with deoxygenated solvent (23).

[0188] When the conduit is open, the deoxygenated solvent (and the plasma nanoparticles, if used) can in some cases flow out of the second end of the conduit. Accordingly, in embodiments where the second end of the conduit is open, the detection chamber and conduit are refilled with deoxygenated solvent (and plasma nanoparticles, if they are used) from, for example, a pump or reservoir device, so that the tube and detection chamber remain filled with deoxygenated solvent (and plasma nanoparticles, if they are used, so that the plasma nanoparticles can remain in close proximity to one another). The pump or reservoir device can be directly connected to the first end of the conduit, or it can be connected to the detection chamber so that any deoxygenated solvent / plasma nanoparticles that flow out of the second end of the conduit are replaced. In other cases, particularly when the conduit is small in diameter, capillary forces can keep the deoxygenated solvent (and plasma nanoparticles, if they are used) within the conduit even though the second end of the conduit is open. In yet other cases, it can be sufficient to ensure that the detection chamber and conduit are airtight even if the second end of the conduit is open, so long as the deoxygenated solvent (and plasma nanoparticles, if they are used) remain in the conduit (this is analogous to removing a straw from a cup of water with the top end of the straw blocked: as long as the top end of the straw is blocked, the water will remain in the straw).

[0189] In preferred embodiments where the second end of the conduit is open, the second end of the conduit is at least partially coated with a plasma layer. Figure 1D and 1E An embodiment of such a coating is shown. Figure 1D Longitudinal cross-sectional view of the second end of a conduit g containing plasma nanoparticles 18, wherein the end wall 14, the inner wall (22) facing the second end of the conduit g, and the outer wall (24) facing the second end of the conduit g are coated with a plasma layer (26). This plasma layer 26 is in close proximity to (preferably in contact with) the plasma nanoparticles and the deoxygenated solvent, thereby ensuring the propagation of the plasma field between the nanoparticles and the layer. Figure 1E is a longitudinal cross-sectional view of the second end of a conduit g, wherein the inner wall 12 is coated with a plasma layer (28) (similar to Figure 1C shown), but wherein the plasma layer (28) also covers the end wall 14 and the outer wall (24) facing the second end of the conduit g. These plasma layers (26, 28) are also in contact with the liquid sample, thereby allowing the desired interaction between the analyte and the plasma field.

[0190] In alternative embodiments, the second end of the conduit can also be sealed by a plasma layer. Figure 1F and 1GAn embodiment of the seal is shown. Figure 1F is a longitudinal cross-sectional view of the second end of the conduit g, which contains the plasmonic nanoparticles 18, the tip being sealed by a plasmonic layer (30). The inner side of the plasmonic layer (30) is in close proximity (preferably in contact) with the plasmonic nanoparticles and the oxygen-removing solvent, thereby ensuring the propagation of the plasmonic field between the nanoparticles and the layer. Figure 1G is a longitudinal cross-sectional view of the second end of the conduit g, wherein the inner wall 12 and the tip are coated with a plasmonic layer (32) (similarly to Figure 1C shown), but wherein the plasmonic layer 32 also seals the second end of the conduit g. In use, the outer side of these plasmonic layers (30, 32) is in contact with the liquid sample, thereby allowing the desired interaction between the analyte and the plasmonic field to occur. Indeed, any analyte in contact or in close proximity with the plasmonic layer will generate a signal plasmonic field, which will be transmitted to the detection chamber, where the SERS signal can be detected by a spectrometer. With such a configuration, there is no need to constantly replenish the plasmonic nanoparticles immersed in the oxygen-removing solvent, as no plasmonic nanoparticles will escape from the second end (unlike the configuration with the second end open).

[0191] The conduit can be made of any conduit material known in the art and which does not adversely affect the generation and transmission of the plasmonic field. It is generally understood that the intended use of the probe can influence the choice of conduit material. For example, when the probe is used in situations requiring a curved conduit, a softer material can be preferred. In embodiments, the conduit is made of metal, quartz, glass or a polymeric plastic, such as polyethylene, polypropylene, polystyrene, polyether ether ketone, polyvinyl chloride, polytetrafluoroethylene or polydimethylsiloxane. In preferred embodiments, the conduit is a polyether ether ketone (PEEK) polymer tube or a glass capillary tube.

[0192] In general, it is more advantageous for the conduit to be thinner. By using a conduit with a smaller inner diameter, less plasma nanoparticles immersed in the oxygen-removing solvent will be needed to fill the conduit, the capillary action within the conduit will increase; the transport of the plasma field along the length of the conduit will be improved as it will be easier to pack the plasma nanoparticles together sufficiently so that they are in close proximity to each other. Furthermore, a conduit with a smaller outer diameter is also smaller in size (meaning that the conduit can access smaller spaces). Moreover, the inner diameter of the conduit should be suitable for scatter detection. In embodiments, the inner diameter of the conduit is between about 0.08 mm and about 1 cm, preferably between about 0.3 mm and about 0.5 mm. In embodiments, the inner diameter of the conduit 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 at most about 1 cm; at most about 5 mm; at most about 2 mm; at most about 1 mm; at most about 0.75 mm; or at most about 0.5 mm. In preferred embodiments, the inner diameter of the conduit is about 0.3 mm or about 0.5 mm.

[0193] As mentioned previously, increasing the length of the conduit will increase the length of the probe, thereby allowing the SERS spectrum of the sample to be measured at a greater distance from the SERS spectrometer. In embodiments, the length of the conduit 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 at most about 50 m; at most about 20 m; at most about 10 m; at most about 5 m; at most about 2 m; or at most about 1 m. In preferred embodiments, the length of the conduit is about 1 m or about 2 m.

[0194] It will be appreciated by those of ordinary skill in the art that the conduit need not necessarily have a circular cross-section. In fact, its cross-section can be of any shape (elliptical, rectangular, etc.).

[0195] “Close proximity”

[0196] It will be well appreciated by those of ordinary skill in the art of spectroscopy that the strength of the plasma field generated at the surface of a plasmonic material decreases (more or less exponentially) as the distance from the surface of the plasmonic material increases.

[0197] In the context of an analyte being in close proximity to a plasmonic nanoparticle or plasmonic layer, “close proximity” means that the analyte is close enough to the layer / nanoparticle so as to interact with the plasma field of the nanoparticle / layer, which in turn generates a plasmonic signal field.

[0198] In the context of plasmonic nanoparticles herein, "close proximity" means that the nanoparticles are close enough that the plasmonic field of one nanoparticle can excite the plasmonic field of the next adjacent nanoparticle, such that the plasmonic field propagates in an unbroken fashion from the detection chamber to the second end of the conduit and back. Of course, "close proximity" includes the case where the particles are in contact with each other. Similarly, in the case of a plasmonic layer in close proximity to a plasmonic nanoparticle, "close proximity" means that the nanoparticle and the layer are close enough that the plasmonic field of the nanoparticle can excite the plasmonic field of the layer, and vice versa.

[0199] It is noted that the exact propagation distance of the plasmonic field depends on the plasmonic material used. For example, as discussed further below, computational models suggest that for silver nanoparticles that are brand new (uncoated) and coated with silver oxide, the propagation of the plasmonic field appears to end at a distance of 17 nm and 9.8 nm, respectively. The closer distances have a stronger resonance, and thus they can further propagate the plasmonic field. Thus, in embodiments, "close proximity" means a distance of at most about 15 nm, preferably a distance of at most about 10 nm, and most preferably a distance of at most about 5 nm (between plasmonic nanoparticles, between a plasmonic nanoparticle and a plasmonic layer, or between one of the foregoing and an analyte).

[0200] It should be clear that the plasmonic nanoparticles can be in close proximity in such a way that the density of these plasmonic nanoparticles varies along the longitudinal direction of the conduit; what is important is that the SERS signal can be propagated along the longitudinal direction of the conduit.

[0201] Because the plasmonic nanoparticles are in close proximity to each other (or because the plasmonic layer is continuous), the signal plasmonic field that emerges at the second end of the conduit is able to propagate along the conduit to the first end of the conduit, where the SERS signal can be measured with a spectrometer. It is important to emphasize that the above propagation of the plasmonic field (between plasmonic nanoparticles or along a plasmonic layer) does not occur if no oxygen scavenger is used to remove dissolved oxygen. In this case, the plasmonic field is quenched within a very short distance, even when other oxygen removal techniques are used (such as the injection of an inert gas into the sample).

[0202] Detection chamber

[0203] The detection chamber is a component of the probe that is fluidly connected to the first end of the conduit. The detection chamber of the probe is configured to be used with a surface enhanced Raman spectrometer such that the incident light of the surface enhanced Raman spectrometer can enter the detection chamber. The detection chamber is also where the SERS signal returned from the conduit is measured by the field of view of the spectrometer detector. This is why the detection chamber is transparent to the SERS laser and Raman scattered light because this allows the SERS laser to act on the detection chamber and because this allows the field of view of the detector to measure the analyte SERS signal returned from the sample.

[0204] The detection chamber is fluidly connected to the first end of the conduit, preferably through a hole in the detection chamber, such that the oxygen scavenging solvent (and the plasmonic nanoparticles if used) can flow between the detection chamber and the conduit, or such that the plasmonic layer is continuous (i.e. unbroken) from the detection chamber to the conduit. The detection chamber also comprises plasmonic nanoparticles immersed in the oxygen scavenging solvent or an inner wall coated with a plasmonic layer because this will propagate the SERS signal that has been transmitted to the first open end of the conduit to the entire detection chamber where it can be measured by the field of view of the detection chamber.

[0205] It will be appreciated by the person of ordinary skill in the art that the detection chamber can have any shape and structure suitable for SERS detection and can be made of any material suitable for SERS detection, in preferred embodiments the detection chamber is made of glass, plastic or quartz, more preferably glass or quartz.

[0206] Liquid sample

[0207] The liquid sample can be any liquid sample to be measured by the probe. In embodiments, the analyte contained in the liquid can be placed in contact with the plasmonic material contained in the probe (defined in the previous section) (e.g. fully coated with a plasmonic layer in the case of a closed second end; or coated with a plasmonic layer or plasmonic nanoparticles immersed in an oxygen scavenging solvent in the case of an open second end). In embodiments, the liquid sample can be placed in contact with the plasmonic material defined in the previous section. In such embodiments, the plasmonic material (and hence the liquid sample) can be positioned on a surface in contact with the second end of the conduit and / or a container. In preferred embodiments, the analyte is adsorbed onto the plasmonic material.

[0208] In preferred embodiments, the liquid sample has been deoxygenated, preferably using an oxygen scavenger as defined in the previous section. The amount of oxygen scavenger and the concentration of DO remaining in the liquid sample can be as defined in the previous section.

[0209] In embodiments, the liquid sample is a liquid sample as defined in the previous section. In preferred embodiments, the liquid sample is modified using the method of the present application. In preferred embodiments, the liquid sample is a liquid sample as defined in the previous section. In preferred embodiments, the liquid sample is modified using the method of the present application.

[0210] Those skilled in the art will understand that, since the probe is designed for SERS measurements, the liquid sample can be any liquid sample capable of being measured for SERS.

[0211] As described above, the probe of the present invention is configured to be placed in 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 placed on the surface of the liquid sample. For clarity, it is the end of the second end of the conduit (whether closed or not) that is placed in contact with the liquid sample.

[0212] Probes that work with SERS spectrometers

[0213] This section describes how to configure the probe to work with the SERS spectrometer. Figure 2 This is a schematic diagram of a conventional surface-enhanced Raman spectrometer. Figure 2 The system consists of a transparent sample container, a laser source, a detector, optical cables connecting the laser source and detector to the sample container, and a computer for interpreting and displaying the data acquired by the detector. Figure 3 A schematic diagram of a surface-enhanced Raman spectrometer for long-range detection is depicted, which includes... Figure 2 The surface-enhanced Raman spectrometer described herein uses most of the same components. However, instead of a sample container, a detection chamber is used instead of a probe, in which incident light is introduced and scattered light (i.e., the SERS signal) propagating in a conduit connected to a tip (the second end of the conduit) is in contact with the liquid sample (e.g., immersed in the sample or in contact with its surface). Figure 3 The detection chamber shown is connected to a pump and a liquid storage device for filling the conduit with liquid.

[0214] It should also be noted that the probe of the present invention can be used in SERS spectrometer systems, such as those used in the remote testing systems described in the experimental section below.

[0215] In use, the probe of the present invention comes into contact with the analyte, i.e., the second end of the conduit comes into contact with the analyte. In a preferred embodiment, the analyte is preferably adsorbed onto the surface of a plasma material, which may be the surface of nanoparticles in the conduit, a plasma layer coated on the second end of the conduit, and / or a plasma layer capping on the second end of the conduit. In an alternative embodiment, the analyte is in a deoxygenated liquid sample produced by the method according to the present invention, the sample comprising aggregated plasma nanoparticles or a rough plasma surface, one of which is in contact with nanoparticles in the conduit; in contact with a plasma layer coated on the second end of the conduit; and / or in contact with a plasma layer capping on the second end of the conduit.

[0216] In the embodiments, molecules that interact with a specific analyte to produce different signals (e.g., chelators of metal ions or antibodies, peptides or aptamers that interact with biomolecules) may be pre-adsorbed onto plasma nanoparticles in the catheter, onto plasma nanoparticles in a liquid sample, onto a plasma layer coated on the second end of the catheter, and / or adsorbed onto a plasma layer on the second end of the catheter.

[0217] Figure 2 Surface-enhanced Raman spectroscopy (when using liquid samples modified according to the method of the present invention) and Figure 3 The surface-enhanced Raman spectrometer (including the probe of this invention) shown for long-distance detection detects analytes by illuminating an incident laser onto a sample container or detection chamber and measuring the scattered light using a detector. In a preferred embodiment, backscattered light (light scattered 180° relative to the incident light) is detected, but light scattered at any angle relative to the incident light may also be detected.

[0218] When using chemical oxygen scavengers to remove oxygen, Figure 2 Surface-enhanced Raman spectroscopy (when using liquid samples modified according to the method of the present invention) and Figure 3 Significant signal amplification was observed in the surface-enhanced Raman spectrometer (including the probe of the present invention) used for long-distance detection. Deoxygenation also enables the long-distance detection capability of the surface-enhanced Raman spectrometer using the probe of the present invention.

[0219] Advantages of the present invention

[0220] In studying the method of the present invention, the inventors discovered a surprising result: the SERS spectral signal intensity of the analyte increased significantly after removing DO using an oxygen scavenger.

[0221] In the embodiments, in addition to the advantages previously discussed, the method and probe of the present invention may exhibit one or more of the following advantages:

[0222] • The limit of detection (LOD) of the analyte can reach 10 μm (compared to 10 μm for aerobic (non-deoxygenated) samples, which improves sensitivity by 10 μm). 9 times).

[0223] • When using silica-coated nanoparticles (such as AgNPs) (i.e., SHINERS technology), signal enhancement can also be observed, with the LOD decreasing from 500 nM to 50 aM.

[0224] (10 10 times).

[0225] Although the absolute sensitivity depends on the plasma particles themselves, the relative signal enhancement (compared to the unmodified liquid sample) upon DO removal can be maintained at 10.9 -10 10 between 1 and 10 times.

[0226] • The analyte no longer needs to be illuminated (meaning it no longer has to be within the beam diameter of the incident light source), nor does it have to be within the field of view of the detector, which allows for a wider potential application of the method and probe of the present invention, such as environmental monitoring.

[0227] • The detection time is not determined by the diffusion of the analyte into the field of view of the detector, but rather by the transport of the plasma field. The SERS signal can be obtained immediately after illumination at the other end of the conduit. This fast response, in addition to the high sensitivity, results in a high temporal resolution, which allows for the monitoring of fast chemical events, such as studying chemical reactions or detecting short-lived species at the single molecule level.

[0228] • The method of the present invention is cost-effective, requiring only a conventional Raman spectrometer and not an expensive SERS detector. This greatly reduces the investment in performing SERS experiments and will promote research in this field globally, especially in developing countries and resource-limited areas. It is believed that the unprecedented detection sensitivity, fast response time, and remote detection capability of the technology can result in many applications in medical diagnostics, environmental monitoring, and national security, among others.

[0229] Definitions

[0230] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0231] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0232] Unless otherwise stated in this document, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All sub-ranges between the values recited in the range are also incorporated into the specification as if they were individually recited herein.

[0233] All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context.

[0234] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed.

[0235] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0236] Here the term "about" has its ordinary meaning. In embodiments it can refer to plus or minus 10% or plus or minus 5% of a defined numerical value.

[0237] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0238] Other objects, advantages and features of the present application will become more apparent from the following non-limiting description of specific embodiments thereof, merely by way of example, with reference to the annexed drawings.

[0239] Description of illustrative embodiments

[0240] The present application is more particularly described in the following non-limiting examples.

[0241] Experimental procedures

[0242] Surface enhanced Raman spectra were recorded with a 785 nm solid state Nd-YAG laser (line width < 3.2 cm -1 , 500 mW) using a standard sunlight TG-Raman fiber optic spectrometer (Changchun Xin Industrial Optoelectronics Technology Co., Ltd.) to detect the backscattered light. The detailed experimental procedures, including sample preparation, analysis and computer simulation, are as follows.

[0243] Materials and reagents

[0244] All reagents were purchased and used without further purification. Silver nitrate (AgN03, 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). Absolute ethanol (EtOH) and hydrogen peroxide (H202, 30 wt%) were purchased from VWR (Mississauga, ON, Canada). Single-stranded thiolated DNA 5-mers, 5A, 5C, 5G, and 5T were purchased from Eurofins MWG Operon (Huntsville, AL, USA). Pure water (18 MΩ cm) was obtained from a Barnstead Nanopure system (Thermo Fisher Scientific, Waltham, MA, USA).

[0245] Silver nanoparticle synthesis

[0246] Silver nanoparticles (AgNPs) were synthesized using the method reported in "Q. Zhang, N. Li, J. Goebl, Z. Lu, Y. Yin, A Systematic Study of the Synthesis of Silver Nanoplates: Is Citrate a "Magic" Reagent? J. Am. Chem. Soc. 133, 18931-18939 (2011)", the entire contents of which are incorporated herein by reference. The resulting AgNPs had a maximum absorbance at 590 nm. TEM imaging showed triangular nanoparticles with an average diameter of 17 ± 2 nm (Figure 4 shows transmission electron micrographs of silver nanoparticles and aggregates used in the following experiments). As prepared, AgNPs at a concentration of 0.3 nM were concentrated to 7 nM by centrifugation at 12,000 rpm for 20 minutes. Unless otherwise stated, all references to AgNPs in the following experiments refer to the AgNPs prepared as described above.

[0247] Preparation of silica-coated silver nanoparticles

[0248] An equal volume of AgNPs (7 nM) and TEOS (1 pm) were sonicated in 1.5 mL EtOH for 10 minutes in a polypropylene microfuge tube. No aggregation was observed after centrifugation at 12500 rpm for 20 minutes. The pH was adjusted to 4 before adding the aggregating agent for SERS measurements. Unless otherwise stated, all references to silica-coated AgNPs in the following experiments refer to the above preparation. Unless otherwise stated, all references to AgNPs in the following experiments refer to the above preparation of AgNPs.

[0249] Electrochemical measurements

[0250] Current analysis was performed using a bipotentiostat (CS2350, Wuhan Crest, Wuhan, China). Screen-printed honeycomb gold working electrodes and counter electrodes were used with an external Ag / AgCl reference electrode (Pine Research instrument, Durham, NC, USA). The honeycomb electrodes have 19 holes of 0.50 mm diameter, 1.5 mm deep, with 0.75 mm spacing between the holes, which allowed the 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, the low potentials were -0.9 V, -1 V and -1.7 V, and the high potentials were set to +0.9 V, +1 V and +1.7 V. The current enhancement was independent of the applied voltage, and no effects of electrode instability were observed. The pulse width was 10 s, and the frequency was 10 Hz. A quartz cuvette was used as the sample container (no background interference was observed). The sample was irradiated with a 785 nm laser outside the electrode area. Figure 5A An experimental setup schematic of the above system for electrochemical measurements is shown. For clarity, these electrochemical experiments were designed to show the transmission of the plasmonic field enhanced by laser excitation of AgNPs. No analyte was present or a spectrum was generated.

[0251] Surface-enhanced Raman spectroscopy

[0252] To evaluate the detection sensitivity of different analytes adsorbed by AgNPs, SERS signals were measured in 5 x 100 mm quartz NMR tubes (0.5 mm quartz thickness). The samples were aggregated by adding sulfite or sulfate salts and any other additives as presented in the experiments. It is worth noting that silica-coated AgNPs require the pH to be adjusted to ~4 before adding the sulfite or sulfate salts to aggregate them. The samples were excited with a solid Nd-YAG laser (785 nm, line width <3.2 cm -1 -1 ​A 785 nm fiber-optic laser probe was connected to a laser source with an output power of 450 mW and a beam diameter of 2.5 μm. The power of the sample (~5 mw) was recorded with a Thorlabs PM100 optical power meter. Backscattered light (180°) was collected by a standard sunlight TG-Raman fiber-optic spectrometer (Changchun New Industrial Optoelectronics Technology Co., Ltd, Changchun, China). Due to strong spectral interference from the coating molecules (i.e., citrate and ethanol), the SERS spectra of the analyte were analyzed between 1000 cm -1 above collection, this is. However, the SERS spectra of the sample between 200-1650 cm -1 are provided herein to support the following conclusions. All spectra are represented as the average of three measurements, each consisting of 1505 data points. Sample preparation and single molecule detection probability analysis

[0253] A careful sample preparation was performed using serial dilutions to accomplish accurate and precise measurements of samples containing very low concentrations of the analyte (pNTP). All pre-cleaned glass containers (e.g., 100-mL volumetric flasks and quartz cuvettes) were soaked in freshly prepared aqua regia for 2 hours, then rinsed with nanopure water until neutral, and then with absolute ethanol. The analyst preparing the sample tested the accuracy and precision of the micropipette by weighing the mass of a specific volume (20 μL) of water delivered by the micropipette using an analytical balance. The resulting value was 20.00 ± 0.09 μL (n = 10). The working stock was carefully prepared by a series of 5000-fold serial dilutions (20 μL into 100 mL) of the initial stock (which was prepared from greater than 100.0 mg of pNTP to obtain a 4-significant figure measurement); i.e.: 102.7 mg of pNTP was dissolved in 50 mL of absolute ethanol to prepare the initial stock (13.22 mM), ensuring complete dissolution of the pNTP particles by vortex mixing and sonication. The number of dilution steps was minimized to reduce error propagation of the final concentrations obtained. The first of the serial dilutions (8 μM) was made by diluting 60.5 μL of the initial stock into a 100-ml Class A volumetric flask (100.00 ± 0.08 mL at 25 degrees Celsius). The flask was inverted several times to allow the liquid to mix thoroughly. This process was repeated three times, each time by pipetting 20 μL of the solution made in the previous step into a new 100-mL volumetric flask, to make solutions with nominal concentrations of 1.6 nM, 320 fM, and 64 aM. The 160 zM and 32 zM stocks were prepared by diluting 37.5 μL or 7.5 μL, respectively, of the 64 aM solution in new 15-ml centrifuge tubes prior to use.

[0254] Two batches of samples (100 samples each) were prepared at two concentrations such that the estimated probability of containing one analyte molecule per sample was 60% or 12%, respectively. The required analyte concentrations to contain a mean of 0.60 and 0.12 molecules in 25 μL were 40 zM and 8 zM, respectively. The samples were prepared by adding 10 μL of pNTP stock solution (160 zM or 32 zM), 10 μL of 7 nM AgNP solution, and 20 μL of nanopure water to a 600 μL microfuge tube. For rapid screening, each 40 μL sample was deoxygenated and aggregated by adding Na2SO3 powder to the sample, and 25 μL was completely aspirated into a standard melting point capillary (1.5-1.8 x 100 mm 9530 Corning-1 Pyrex capillary) for SERS measurement.

[0255] Similar sample preparation procedures were used to prepare other analytes, including pATP and a multimeric oligo DNA sequence at four different concentrations, as well as pNTP at different concentrations. However, while the above pNTP samples were prepared by 5000-fold serial dilution, the samples defined in this paragraph were prepared by 100-fold serial dilution. Subsequently, the detector (e.g., pATP) was mixed with the AgNP solution to obtain the target analyte concentration; for example, 75 μL each of pATP and AgNP solution were mixed with 150 μL of nanopure water. Each concentration was prepared in three independent replicates to obtain the standard deviation. For each solution (e.g., pATP + AgNP solution) (300 μL), 10 mg of Na2SO3 or Na2SO4 powder was added to obtain solutions of the same ionic strength, but each without (Na2SO3 addition) or with dissolved oxygen (Na2SO4 addition). Unless otherwise noted, the analyte-containing solutions used in the experiments discussed below were prepared according to the above method, with necessary adjustments to achieve the target concentration. Error propagation in serial dilution sample preparation

[0256] Considering the ±0.1 mg error of the analytical balance and assuming a ±0.1 mL error for the 50 mL volumetric flask, there is a potential maximum error of 0.22% in the concentration of the initial stock solution of pNTP. For each dilution, the relative standard deviation of the concentration is determined by the root sum square of the relative standard deviation of the pipetting (0.47%) and the relative standard deviation of the volumetric flask (0.08%), which is ~0.48%. This error is compounded over the initial and three consecutive dilution steps and is considered an independent event, i.e., the bias is random, not systematic, resulting in a concentration error of ~1.9%. After this, there is an additional dilution step to form the 160 zM and 32 zM working stocks for analysis. Assuming a ±0.1 mL error in the 15 mL volumetric flask and the same relative bias as pipetting, this results in an additional 0.81% error. The nanoparticle adsorption step includes three pipetting events, which all contribute to the error in the final concentration of the analyte solution. Assuming the same error occurs in the 10 μL and 20 μL additions, an additional ~0.43% concentration error also occurs. Each dilution step is an independent event, so the overall relative error in the concentration is expected to be ~2.1%. Therefore, the concentration in the analyte solution should be 40 ± 0.85 zM or 8 ± 0.17 zM, which corresponds to a detection probability of 58.7% - 61.3% or 11.7% - 12.3% in 25 μL samples, respectively.

[0257] Results and implications of the probability analysis

[0258] After nanoparticle aggregation and deoxygenation by the addition of Na2SO3to the standard melting point capillary, samples of 40 zM and 8 zM (25 μL each, 100 each) were analyzed; the nominal probability of a single pNTP molecule in the samples was 60% and 12%, respectively. In 100 samples at the 40 zM and 8 zM concentration levels, pNTP was detected in 55 and 8 samples, respectively. Fisher's exact test was used to compare the measured frequency to the expected frequency to determine if there was a non-random association between the expected frequency and the measured frequency. The probabilities of 60% and 12% were p = 0.57 and 0.48, respectively, which were not statistically significantly different. Therefore, the measured detection frequencies at both concentrations were within the expected values for single molecule detection. For clarity, the probability analysis did not generate analyte SERS spectra.

[0259] Long range detection

[0260] To test the efficacy of the probes in the present application and to determine the degree of plasmonic coupling within and between the plasmonic nanoparticle aggregates, two experiments were designed. These experiments attempt to isolate the analyte molecules from the field of view of the incident light source and detector by adsorbing them onto the silver mirror surface and to completely remove any unadsorbed or loosely bound analyte from the silver mirror surface by extensive rinsing (these experiments are related to Figures 18-20 and their results are discussed below). This was accomplished using either a 10 cm long quartz NMR tube or a 1 m long PEEK capillary tube with a short section of quartz NMR tube attached to each end.

[0261] First, to prepare the silver mirror surface at the end of the tube, Tollen's reagent was prepared from 0.1 M AgNO3; the AgNO3 was first precipitated by the addition of dilute NaOH to form Ag2O and the Ag2O was then redissolved by the dropwise addition of concentrated aqueous NH3 to form the Ag(NH3)2 complex. The Tollen's reagent was added to a polypropylene microfuge tube and the end of the quartz NMR tube or PEEK tube was placed in the microfuge tube so that the Tollen's reagent covered half of the desired height of the silver mirror to be coated (~5 mm) and an equal volume of 0.1 M glucose was added to form the silver mirror on the end surface of the quartz NMR tube or PEEK tube. After the silver mirror was formed on the surface, it was rinsed with nanopure water and ethanol ~30 times before drying at room temperature to remove any excess reagents and other materials adsorbed on the silver mirror surface. Note that the silver mirror adheres weakly to the polymer surface and can be easily wiped off, so it should be handled with care.

[0262] Next, 100 nM of the analyte (e.g., pATP) was added to cover the silver mirror surface and the NMR tube and PEEK tube were then rinsed with EtOH and nanopure water 30 times each to remove any unbound analyte from the silver mirror surface (the PEEK tube was rinsed from the end opposite the silver mirror to prevent the analyte from entering the tube). To detect the analyte adsorbed on the distal (second) end of the glass NMR tube or PEEK tube, the completely rinsed device was filled with an AgNP solution that was immediately aggregated and deoxygenated by the addition of excess Na2SO3. The end of the cuvette or tube (the end opposite the adsorbed analyte) was aligned with the optical cable connecting the incident laser light source and detector and the Raman spectrum was recorded. As discussed in detail below, the analyte was detected when the AgNPs were aggregated and deoxygenated with Na2SO3, but not when they were aggregated with Na2SO4 alone.

[0263] Long-range blind detection by nanoparticle aggregates

[0264] A sample for remote detection using the probes of the present application was prepared for a blind test. The stock solution of the sample was 50% volume ethanol (400 μί) with 0 or 2 μΜ pNTP and 3.5 nM AgNPs. The solution was sonicated for 30 minutes to ensure good mixing of the pNTP and AgNPs and to allow the pNTP to adsorb to the AgNP surface. To remove any residual unadsorbed pNTP (which could diffuse through the solution), the sample was centrifuged at 12,700 rpm for 30 minutes to pellet the NPs; everything but 20 μί of the solution was removed (to avoid disturbing the pelleted NPs). The sample was diluted with ethanol to 1500 μί and sonicated for another 30 min. This process was repeated a total of two times (to reduce the maximum possible free pNTP to ~356 pM), solvent removal (to 20 μί), and resuspension in 50 wt% ethanol, with the final maximum possible free pNTP concentration of ~18 pM. The sample preparer randomly assigned 20 μί of the sample to 20 microfuge tubes, then diluted with 20 μί of water (with a final maximum free pNTP concentration of ~9 pM if the pNTP was not adsorbed, and a positive control AgNP adsorption concentration of 1 μΜ), and documented (without telling the analyst). In the blind test, the number and identity of the positive and negative controls were unknown to the analyst, who deoxygenated the AgNPs and sample by adding Na2S03 powder prior to measurement. A glass capillary tube preloaded with the deoxygenated and AgNP-adsorbed solution was aligned with the Raman laser, and the glass capillary tube was immersed in the deoxygenated sample solution (with the sample solution surface 6 cm from the incident light position), and the Raman spectrum was recorded, and the analyst identified the positive and negative controls and reported the results to the sample preparer. The sample attendant compared the test results with the sample preparation documentation, and the results were that all samples (positive controls n = 9, negative controls n = 11) were correctly identified. For clarity, this blind test did not produce spectra.

[0265] Theoretical explanation of the computational modeling

[0266] To understand how Ag20 and DO affect the signal response of SERS, the electromagnetic-field (EM-field) enhancement of a surface plasmonic nanogap was simulated by using the boundary element method (BEM). The surface integrals depend on scalar and vector potentials of the interface charge and current, which are related to the frequency-dependent local dielectric function. The surface charge was assumed to be located at the center of a single infinitesimal triangle (indexed by j).

[0267] The E-field is expressed in terms of scalar and vector potentials, Φ (electric scalar potential) and A (magnetic vector potential), respectively.

[0268]

[0269] After applying the boundary conditions, the electric displacement (D), 1 and 2, at the interface (s) of the two media are suggested as (n s Normal vector, ε a Dielectric function of the medium a) :

[0270]

[0271] The spatial convolution is done using compact matrix representation. The electric displacement of the oxygen-free or oxidized layer system will be the sum of the electric displacement at each interface of each AgNP, resulting in:

[0272]

[0273] In the equation, Ag represents the AgNPs, Ag x O represents the oxidized layer. In the system with an oxidized layer, the electric displacement of each AgNP will be:

[0274]

[0275] When molecular oxygen (O2) is present in the medium, each AgNP will have the following electric displacement:

[0276] The skilled person will understand that a detailed exposition of the BEM method and its application in NP surface plasmons can be found in the literature. AgNPs, Ag x The dielectric coefficients of O, oxygen, and water can also be found in the literature, the results of the model are shown in Figure 6 and 7, which are described in the above and below.

[0277] In figure 7 it is notable that the electromagnetic field transmission ends at 17 and 9.8 nm, respectively, in the case of silver nanoparticles without and with silver oxide coating.

[0278] Results and discussion

[0279] Results and discussion of the surface-enhanced Raman spectroscopy of the prepared samples and the probability analysis of single molecule detection

[0280] For the first series of measurements, solutions of p-aminothiophenol and p-nitrothiophenol of different concentrations were prepared with and without dissolved oxygen using the method described above. AgNPs were used as plasmonic material (SERS substrate) and sodium sulfite as oxygen scavenger.

[0281] In Figure 8, the spectrum labeled "Blank" was obtained from a sample of 7 nM AgNPs (as described above, they were prismatic with an average size of 17 nm) containing Na2SO3aggregation. The spectra labeled "Na2SO3" (oxygen removal with 10 mg of sodium sulfite) and Na2SO4(10 mg of sodium sulfate was added, so the sample contained dissolved oxygen) were obtained from liquid samples containing 1 μM of analyte, respectively, with Na2SO3or Na2SO4as AgNPs aggregation agent. As described above, the spectra were recorded while irradiating a 5 x 100 mm quartz NMR tube with a solid-state Nd-YAG laser (785 nm, line width < 3.2 cm -1 500 mW, beam diameter 2.5 μm). The backscattered light (180°) was detected using a standard sunlight TG-Raman fiber-optics spectrometer. All spectra are represented as the average of three measurements, each consisting of 1505 points (integration time = 100 ms). The analytes were p-aminothiophenol (pATP) and p-nitrothiophenol (pNTP). Figure 8A ) and p-nitrothiophenol ( Figure 8B ).

[0282] In Figure 9, the same measurement parameters as in Figure 8 were used, but the analyte concentration of p-aminothiophenol was different. The other measurement parameters of Figure 9 were the same as in Figure 8. Figure 9A The spectrum in -C was obtained from a sample containing dissolved oxygen (10 mg of sodium sulfate), while the spectrum of the sample that was deoxygenated with 10 mg of sodium sulfite is in -F. Note that Figure 9D and Figure 9B only represent the individual spectra of 9E and Figure 9A respectively. 9D

[0283] By using thiophenols (i.e. p-aminothiophenol (pATP) and p-nitrothiophenol (pNTP)) as the main analytes and pyramidal AgNPs as the plasmonic species (SERS substrate) for proof-of-principle studies, a significant increase in the SERS spectra of water pATP and pNTP was observed after the removal of DO with sodium sulfite (Na2SO3, see Figures 8 and 9) (described in detail above), it should be mentioned that the peaks in Figure 8 are related to in-plane distortions of the aromatic ring, which at higher concentrations overlap with the N=N band of the azo-dimer.

[0284] ​As mentioned previously, all experiments were performed using triangular pyramidal AgNPs, unless otherwise stated. In all experiments performed, unless otherwise mentioned, sodium sulfite was used as the DO scavenger, which coagulates the AgNPs by screening the surface charge of the citrate capping agent, while simultaneously condensing the AgNPs (sodium sulfate Na2S04was used as a control to sodium sulfite Na2S03for coagulating the AgNPs in aerobic (i.e. not deoxygenated) experiments); due to the strong signal from the citrate (see Figure 4 and Figure 10, described in more detail above), the spectral window was limited to > 1000 cm -1 .

[0285] As mentioned previously, Figure 4 shows transmission electron micrographs of AgNPs deposited on a copper grid; Figure 4 a) and b) are photographs before coagulation, c) and d) are photographs after coagulation with 10 mg of sodium sulfite and thorough rinsing to remove salt crystals.

[0286] Figure 10A AgNP size distribution was shown by dynamic light scattering, while Figure 10B D shows the Raman spectra of B) AgNPs, C) p-aminothiophenol and D) p-nitrothiophenol. Specifically, Figure 10B is a blank spectrum of the AgNPs prepared, where the large peak is associated with the citrate capping agent. Figure 10C and D are Raman spectra of high concentrations (100 mM) of pATP and pNTP to show which peaks can be used for detection (it should be noted that 1000 cm -1 The peaks below are masked by the strong signal of surface-bound citrate).

[0287] Based on the C-H bend (1135 - 1142 cm -1 ) and C-S (1070 - 1080 cm -1 ) stretch peaks (although not all vibrational modes are equally sensitive, and some modes become undetectable as the concentration of the analyte decreases), the limit of detection (LOD) for pATP reached 10 zm (a 10 9 fold increase in sensitivity compared to the 10 pm for aerobic samples).

[0288] Similar increases in SERS enhancement factor (EF) were observed for other analytes. The other analytes were four multimeric oligonucleotide sequences prepared using the method described above (see Figure 11A and B, described in detail above). Specifically, Figure 11A and B show the SERS spectra of the oligonucleotides for an aerobic sample (10 pm analyte) and a deoxygenated sample (8 zm analyte) using the same measurement parameters as used in Figure 8.

[0289] Similar results were observed when spherical rather than pyramidal AgNPs were used. Regardless of whether chemical deoxygenation (e.g., sodium sulfite, hydrazine, or ascorbate) or physical removal with inert gases (e.g., N2 or Ar injection) was used, DO concentration (electrochemically measured) was negatively correlated with SERS signal intensity (see Figures 12, 13, and 14 above for a more detailed description). Figure 12B middle( Figure 12A The TEM of spherical AgNPs is shown, and the SERS spectrum was obtained using the same measurement parameters as in Figure 8, except that spherical AgNPs were used instead of pyramidal AgNPs.

[0290] exist Figure 13A In Figures B and B, using the same measurement parameters as in Figure 8, but with various analyte concentrations of p-nitrothiophenol and different oxygen scavengers, various SERS spectra were obtained. Specifically, Figure 13A The SERS spectrum of pNTP deoxygenation using 10 mg of ascorbic acid is shown, while Figure 13B The SERS spectrum of pNTPs after deoxygenation by hydrazine (50 mM) is shown.

[0291] exist Figure 14A In Figure B, SERS spectra (analyte pATP concentration of 100 fM) were obtained using the same measurement parameters as those used in Figure 8. The difference was that the measurements were performed in an electrochemical system to monitor oxygen concentration, and different amounts of oxygen scavenger (Na2SO3) were added to test different DO concentrations.

[0292] Similarly, in Figure 14C In Figures D and D, SERS spectra were obtained using the same measurement parameters as those used in Figure 8 (analyte pATP concentration of 100 fM), the difference being that the measurements were performed in an electrochemical system to monitor oxygen concentration, and argon gas was injected into the sample instead of adding an oxygen scavenger. To test different DO concentrations, the argon injection time was gradually increased.

[0293] As shown in Figure 13, similar to Na2SO3, detection limits in the zM range can also be obtained for pNTPs, hydrazine, and ascorbic acid deoxygenation. As shown in Figure 14, argon injection does reduce DO and increase SERS signal intensity, but the DO removal level is still significantly lower than the minimum level achieved using oxygen scavengers. It should also be mentioned that... Figure 14C The lowest oxygen concentration achieved (0.013 mM, almost an order of magnitude higher than the 0.0019 mM DO concentration obtained using Na₂SO₃) required 20 minutes of argon injection and simultaneous sonication to obtain the spectrum. As previously mentioned, the dissolved oxygen concentration was determined using an electrochemical method, as shown in Figure 14. Furthermore, Figure 14 presents the average values ​​of the three spectra; the standard deviations of the data in B) and D) are less than the data markers.

[0294] The effect of AgNP surface oxidation on SERS enhancement was investigated (Figure 15, described in detail above), since the removal of DO also reduces the formation of Ag20 on the AgNP surface compared to aerobic (i.e. non-deaerated) samples. Figure 15A The UV-Vis spectra of Ag + NPs are shown (obtained by addition of Ag20s), while Figure 15B and C show the voltammograms of AgNPs in samples containing DO and samples deaerated using 10 mg of sodium sulfite, respectively.

[0295] Similarly, Figure 16A The UV-Vis spectra of gold nanorods (AuNRs) are shown, while Figure 16B The SERS spectra of cetyltrimethylammonium bromide (CTAB) on AuNRs with and without removal of oxygen and the Raman spectra of a higher concentration of CTAB (100 mM) are shown. For clarity, the spectra labelled "Na2S03" are from samples deaerated using 10 mg of sodium sulfite and the spectra labelled "Na2S04" are from samples containing 10 mg of sodium sulfate and therefore containing dissolved oxygen. Furthermore, the amount of CTAB in the samples of the spectra labelled "Na2S03" and "Na2S04" is comparable to the amount of CTAB remaining on the surface of the AuNRs as supplied commercially.

[0296] As described, Figure 17A The transmission electron microscopy TEM of silica-coated AgNPs is shown, while Figure 17B The SERS spectra of pATP on deaerated aggregated silica-coated AgNPs (at pH 4) are shown, Figure 17C The SERS spectra of pATP (50 aM) and pNTP (50 aM) on deaerated aggregated silica-coated AgNPs (at pH 4) are shown.

[0297] Based on these results, the formation of a dielectric oxide layer (in this case, the formation of Ag20) appears to be a secondary factor compared to the presence of DO. When silica-coated AgNPs were used, signal enhancement was also observed (i.e. SHINERS technique, LOD from 500 nM to 50 aM (10 10 Although the overall sensitivity of silica-coated AgNPs was 10 4 times lower than bare AgNPs, DO (rather than the surface oxide layer) appears to be the main factor inhibiting the SERS signal (10 10 times vs 10 4 times), i.e.: the relative signal enhancement when DO is removed is always 10 9 - 10 10fold, although the absolute sensitivity of SERS is particle dependent. By using AuNRs and silica-coated AgNPs, the role of DO was resolved separately from the role of plasmonic nanoparticle surface oxidation.

[0298] This finding challenges the conventional understanding of SERS. Based on the conventional understanding, the analyte must be illuminated by the excitation light and simultaneously present in the detector’s field of view. Since the laser beam is quite narrow (2.5 pm in diameter when collimated), the probability of the analyte being illuminated is 5.4 x 10 -8 (close to zero) based on the ratio of the volume illuminated by the laser to the volume of the sample. Since this probability is almost zero, the analyte is undetectable according to the conventional SERS theory. However, consistent and reproducible results were obtained from numerous independent analyses of zM analyte levels in samples (n > 300, performed by different analysts on different days with different batches of AgNPs, including blind tests, as described above). To verify whether single-molecule detection was real, two groups of 100 samples each (where the probability of each sample containing a single analyte molecule was < 1) were subjected to statistical analysis. By comparing the expected and actual detection frequencies using Fisher’s exact test, it was determined that there was no statistically significant difference between the detected and expected detection frequencies (p = 0.57 and p = 00.48 for 60% and 12% detection probabilities, respectively).

[0299] Based on these observations, the removal of DO from the sample leads to the effective coupling of the SERS field, which enables the electromagnetic (EM) field to propagate throughout the volume of the liquid sample without significant loss of signal energy. The plasmonic field generated by the photoexcitation of AgNPs propagates throughout the sample, reaching AgNPs with adsorbed analytes and returning the Raman scattered signal of the analyte to the detector. Without the removal of DO, the high electron affinity of oxygen would trap electrons and prevent the effective generation, coupling, propagation, and integration of the SERS field between AgNPs and their aggregates, which was verified experimentally and through computational simulations. However, DO removal enables plasmonic coupling and SERS field transport by plasmonic species (AgNP aggregates in this case).

[0300] Computational results for the plasmonic field generated by an AgNP aggregate show the expected plasmonic coupling between nanoparticles and the enhancement of the plasmonic field in the nanogap (see Figure 6and 7, described in detail above). Molecular oxygen suspended in the nanogap or adsorbed on the surface of AgNPs causes charge redistribution and quenching of plasmonic coupling. When simulating silver oxide-coated AgNPs, the plasmonic field is reduced, but the introduction of DO significantly suppresses the EM field, preventing coupling. The DO quenching effect is independent of representative parameters of the nanogap, including the distance of the nanogap, the number of nanoparticles in the nanocluster (2, 3, and 4 AgNPs), and the characteristics of the nanoparticles (size, shape, position, orientation, and surface chemistry). This model suggests that DO quenches the SERS field, and removing DO allows the EM field to propagate throughout the sample in a plasmonic coupling manner through adjacent AgNPs and clusters.

[0301] Electrochemical studies were also performed on the propagation of SERS signals through liquid samples (e.g., aqueous solutions). DO removal significantly enhanced the photocurrent induced by laser irradiation of those AgNPs suspended (i.e., not physically attached to the gold electrode surface) (see Figure 5, described in detail above). As described in the methodology section, Figure 5A A schematic of the experimental setup used is shown, in which the photocurrent generated by AgNPs solutions coagulated by Na2SO3(with DO removal) and Na2SO4(without DO removal), respectively, was measured on a honeycomb gold electrode by irradiating the AgNPs solution (not the electrode) with light. At the same time, Figure 5B The photocurrent generated by NIR (λ = 785 nm) irradiation at a distance of 1 cm from the electrode surface in the presence and absence of DO at constant ionic strength under the conditions listed is shown. As Figure 5B shown, the increase in the current generated by irradiation is related to the addition of salt (sodium sulfate or sulfite) and the increase in conductivity. However, sodium sulfite also scavenges dissolved oxygen and significantly enhances the generation of photocurrent.

[0302] Results and discussion of long-range detection and long-range blind detection

[0303] Additional evidence supporting long-range SERS field propagation in liquid samples (e.g., aqueous solutions) comes from the analysis of analytes by oxygen-free SERS long-range detection using the probes of the present application.

[0304] First, when the measurement was taken at a distance of 10 cm from the silver mirror (the silver mirror was located at the distal end of an NMR tube filled with a solution of AgNPs (the surface of the AgNPs was not coated) that was free of oxygen), the signal intensity of the stretching band of the C-S bond (1070-1080 cm -1 ) in the analyte molecules adsorbed on the silver mirror, which was close to and perpendicular to the Ag surface, remained about 70%, although the vibrational bands of other functional groups that were farther away or in other orientations (not perpendicular to the surface of the silver mirror) rapidly decreased (see Figure 18, described in detail above).

[0305] For clarity, in Figure 18, the same measurement parameters as in Figure 8 were used to record the spectra. However, the analyte was adsorbed on a silver mirror at the bottom of the NMR tube instead of being dispersed in the liquid sample. The AgNPs solution was added to the NMR tube and Na2SO3 was added to coagulate the AgNPs and remove oxygen as in Figure 8; however, the AgNPs were coagulated along the tube and in the detection chamber.

[0306] As previously described, to prepare the silver mirror at the end of the tube, Tollen's reagent was first prepared from 0.1 M AgNO3, which was first precipitated by the addition of dilute NaOH to form Ag2O. The Ag2O obtained was redissolved by the addition of concentrated aqueous NH3 to form the Ag(NH3)2 complex. The Tollen's reagent solution was added to the bottom of a quartz NMR tube to cover half of the desired height (~5 mm) to be silvered. Then, an equal volume of 0.1 M glucose was added to allow the silver mirror to grow. After the surface was silvered, it was rinsed 30 times with nanopure water and EtOH before drying at room temperature to remove excess reagents or other adsorbed materials.

[0307] As shown in Figure 18, using the above setup and parameters, the following SERS spectra were obtained:

[0308] Figure 18A : p-Aminothiophenol adsorbed at 0 and 10 cm from the silver mirror.

[0309] Figure 18B : p-Nitrothiophenol and blank adsorbed at 10 cm from the silver mirror.

[0310] Figure 18C : Spectra of p-aminothiophenol adsorbed on the silver mirror at different distances from the silver mirror.

[0311] Similarly, Figure 18D The signal intensity of the C-S stretch band of p-aminothiophenol as a function of the distance from the silver mirror is shown.

[0312] As shown in Figure 18, the propagation efficiency of the silica-coated AgNPs was lower. Notably, in Figure 18, the blanks of all experiments showed no significant signal above the noise level. The spectra in the figure are the average of three separate measurements, and the error bars (representing the standard deviation) in D) are smaller than the size of the data markers.

[0313] Second, the remote SERS was used for high-throughput detection in a 96-well plate, where the analyte was pre-adsorbed in the AgNPs, and then the DO was removed and the AgNPs were coagulated with sodium sulfite (see Figure 19, described in detail above). The measurement parameters used in Figure 19 were similar to those in Figure 18, however, in the wells of the microtiter plate, the analyte was pre-adsorbed on the AgNPs that were deoxygenated and coagulated. A capillary melting point tube pre-filled with deoxygenated and coagulated AgNPs was immersed in the well. The SERS spectra were measured through the distal end of the capillary tube, 6 cm away from the well of the plate. These spectra are presented as a function of integration time for p-aminothiophenol Figure 19A ) and p-nitrothiophenol Figure 19B ).

[0314] For clarity, a 6 cm glass capillary was filled with an aqueous solution of freshly deoxygenated AgNP aggregates, and the capillary was placed vertically in each well to transmit the SERS signal upward, with the incident light shining perpendicular to the capillary (parallel to the microtiter plate). This was verified by the blind method described above, where 20 wells were randomly filled with either analyte or blank, and the blind results gave 100% accuracy. It should be noted that in Figure 19, all of the blanks for the experiments showed no significant signal (above the noise level).

[0315] Finally, the remote SERS technique was verified by using a 1 m long PEEK capillary tube with a silver mirror coating on the distal end (see Figure 20, described in detail above). The measurement parameters used in Figure 20 were similar to those in Figure 18. However, a silver mirror was grown on one end of a 1 m long PEEK capillary tube. The other end was connected to a piece of NMR tubing Figure 20A ), and the system was flushed with solvent. First, a blank measurement was made, where the conduit was filled with deoxygenated and coagulated AgNPs, and no analyte was adsorbed on the silver mirror. Then, the analyte was adsorbed onto the silver mirror, and after repeated flushing of the probe, the conduit was filled with deoxygenated and coagulated AgNPs, and the SERS detection was performed. Figure 20A A schematic of the probe setup described above is shown in Figure 21, while Figure 20B SERS spectra using p-aminothiophenol as the analyte are shown in Figure 22, including deoxygenated and non-deoxygenated, Figure 20C SERS spectra using p-nitrothiophenol as the analyte are shown in Figure 23, where the samples were deoxygenated with 10 mg of sodium sulfite. For clarity, in Figure 23, the spectra labeled "Na2SO3" were deoxygenated with 10 mg of sodium sulfite, and the spectra labeled "Na2SO4" contained 10 mg of sodium sulfate, and therefore contained dissolved oxygen. Figure 20B It should be noted that in Figures 22 and 23, no analyte signal was detected in the blanks or samples that were not deoxygenated.

[0316] Figure 20B

[0317] ​​The above experiments demonstrate the long-range propagation of surface plasmon field in liquid samples (e.g. aqueous solutions), although the length of signal transmission depends on the nature of the plasmonic nanoparticles themselves, i.e. fresh AgNPs can propagate SERS signal at least 1 m, while silica-coated AgNPs decrease after ~6 cm. This is in agreement with the results of computational simulations Figure 6 ), the weaker SERS field and poor propagation ability produced using silica-coated AgNPs results in a 10 4 times higher detection limit LOD (50 aM) than using fresh AgNPs (10 zM).

[0318] The conventional understanding of SERS is based on signal amplification produced by individual nanostructures or plasmonic field coupling between adjacent plasmonic nanoparticles (i.e. nanogap). The "hot spot" theory is sufficient to explain all experimental observations, where SERS signal amplification is based on the principle of localized surface plasmon resonance, which is a discrete EM field around a single or small cluster of plasmonic nanoparticles. However, in the present experiments it has been demonstrated that long-range large-scale plasmonic field coupling (or integrated surface plasmon resonance) is possible after DO removal. This integrated plasmonic field enables the efficient transmission of SERS signal of a single molecule, even if this single molecule is adsorbed on any of the plasmonic nanoparticles in the sample. Therefore, each plasmonic nanoparticle becomes SERS active, either acting as a "hot spot" adsorbing analyte or transmitting SERS signal, thus enabling true single molecule detection in the sample. The experimental results also show that this plasmonic field can also be transmitted over long distances in deoxygenated aqueous solutions. Fresh uncoated AgNP aggregates are still the preferred choice for oxygen-free SERS, with the advantage of using it for plasmonic field transmission (see Figures 7A-F, described in more detail above, where it can be observed that for fresh uncoated and coated with silver oxide silver nanoparticles, the electromagnetic field transmission is terminated at 17 nm and 9.8 nm, respectively). In the SHINERS experiments, it was found that higher SERS sensitivity (50 aM) was achieved only when sodium sulfite was added and the pH of the solution was lowered to agglomerate the silica-coated AgNPs, where sodium sulfite alone is sufficient to remove DO, but cannot agglomerate silica-coated AgNPs.

[0319] A significant advantage of the present oxygen-free (i.e. deoxygenated) SERS measurements is that the detection time is not determined by the time it takes for the analyte to diffuse into the field of view of the detector (especially considering the analyte binding to the surface of a stationary silver mirror or a slowly diffusing nanoparticle aggregate), but rather by the speed of propagation of the plasmonic field. In 1 m standoff SERS detection using the probe of the present application, the analyte is immobilized on a silver mirror at one end of a PEEK tube and cannot migrate, and SERS signals are obtained immediately upon turning on the laser source to illuminate the detection chamber at the other end of the conduit. This ultrafast response, in addition to the high sensitivity, results in high temporal resolution, which can be used to monitor fast chemical events, for example, to study chemical reactions or to detect short-lived species at the single molecule level. This technology is also cost effective, requiring only a common Raman spectrometer, and not an expensive SERS detector. This greatly reduces the expense of performing SERS experiments and will promote single molecule studies worldwide, but especially in developing countries and resource-limited areas. The unprecedented detection sensitivity, fast response time, and standoff detection capability can potentially lead to many applications in medical diagnostics, environmental monitoring, and national security.

[0320] The scope of the claims should not be limited to the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the entire description.

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Claims

1. A method of modifying a liquid sample containing an analyte to increase the SERS signal intensity of the analyte, the method comprising the steps of: providing said liquid sample to be analyzed using SERS; and adding an oxygen scavenger to the liquid sample to remove dissolved oxygen from the liquid sample; wherein, the oxygen scavenger is added such that the concentration of the remaining dissolved oxygen is at most 0.020 mM; wherein, the method further comprises the step of contacting the liquid sample with a plasmonic material; wherein, the plasmonic material has a rough plasmonic surface, is composed of plasmonic nanoparticles, or both; the plasmonic nanoparticles are aggregated, such that the plasmonic nanoparticles are in close proximity and / or contact with each other; and wherein, at least a portion of the analyte is adsorbed on or becomes adsorbed on the surface of the plasmonic material; or, the analyte is modified to enable interaction with the surface of the plasmonic material.

2. The method of claim 1, wherein the analyte is a thiol compound, an amine, a pesticide, a persistent organic pollutant, a transition metal complex, a peptide, a protein, a nucleic acid, a polysaccharide, or a hormone.

3. The method of claim 2, wherein the analyte is a transition metal complex, a peptide, a protein, a nucleic acid, a persistent organic pollutant, a pesticide, or a hormone.

4. The method of claim 1, wherein the analyte is p-aminothiophenol (pATP) or p-nitrothiophenol (pNTP).

5. The method of claim 1, wherein the liquid in the liquid sample is water or an organic solvent.

6. The method of claim 5, wherein the liquid in the liquid sample is an organic solvent comprising methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, a hydrocarbon, or a mixture thereof.

7. The method of claim 5, wherein the liquid is water.

8. The method of claim 1, wherein the modification of the analyte is accomplished by binding a thiol group to the analyte, whereby the analyte will chemisorb to an Ag or Au plasmonic surface through the thiol group.

9. The method of claim 1, wherein the modification of the analyte is accomplished by physical adsorption through hydrophobic / hydrophilic interactions or van der Waals forces, chemical adsorption through other binding atoms, indirect detection by chemical reaction with a pre-adsorbed reporter molecule, chelation of metal ions by a pre-adsorbed chelator, indirect detection by affinity, indirect detection by retaining plasmonic nanoparticles with SERS-active reporter molecules through specific interaction with the analyte.

10. The method of claim 9, wherein the affinity comprises antibody-antigen binding of biomolecules; and / or, the specific interaction with the analyte comprises an antigen antibody reaction.

11. The method of any one of claims 1 to 10, wherein the oxygen scavenger is sodium sulfite, hydrazine, ascorbic acid, or ascorbyl palmitate.

12. The method of any one of claims 1 to 10, wherein an excess of the oxygen scavenger is added.

13. The method of any one of claims 1 to 10, wherein sufficient oxygen scavenger is added to remove at least 98% of dissolved oxygen in the liquid sample.

14. The method of claim 13, wherein sufficient oxygen scavenger is added to remove at least 99% of dissolved oxygen in the liquid sample.

15. The method of claim 13, wherein sufficient oxygen scavenger is added to remove at least 99.5% of dissolved oxygen in the liquid sample.

16. The method of claim 13, wherein sufficient oxygen scavenger is added to remove at least 99.9% of dissolved oxygen in the liquid sample.

17. The method of any one of claims 1 to 10, wherein sufficient oxygen scavenger is added such that the concentration of remaining dissolved oxygen is at most 0.010 mM.

18. The method of claim 17, wherein sufficient oxygen scavenger is added such that the concentration of remaining dissolved oxygen is at most 0.005 mM.

19. The method of claim 17, wherein sufficient oxygen scavenger is added such that the concentration of remaining dissolved oxygen is at most 0.002 mM.

20. The method of any one of claims 1 to 10, wherein the limit of detection (LOD) of a given analyte is reduced by at least 10 8 fold after the liquid sample is modified.

21. The method of claim 20, wherein the LOD of the analyte using SERS is reduced by at least 10 9 fold after the liquid sample is modified.

22. The method of claim 20, wherein the LOD of the analyte using SERS is reduced by at least 10 10 fold after the liquid sample is modified.

23. The method of any one of claims 1 to 10, wherein the LOD of the analyte using SERS is at most 1 pM.

24. The method of claim 23, wherein the LOD of the analyte using SERS is at most 10 fm.

25. The method of claim 23, wherein the LOD of the analyte using SERS is at most 100 aM.

26. The method of claim 23, wherein the LOD of the analyte using SERS is at most 1 aM.

27. The method of claim 23, wherein the LOD of the analyte is at most 10 zM.

28. The method of any one of claims 1 to 10, wherein the plasmonic material is a substance made of electrically conductive, semiconductive and / or dielectric materials, combinations thereof, or composites thereof.

29. The method of claim 28, wherein the plasmonic material is gold, silver, copper, or mixtures thereof.

30. The method of claim 29, wherein the plasmonic material is gold or silver.

31. The method of claim 30, wherein the plasmonic material is silver.

32. The method of any one of claims 1 to 10, wherein the roughness of the rough plasmonic surface is between 5 nm and 50 nm.

33. The method of claim 32, wherein the roughness of the rough plasmonic surface is between 10 nm and 20 nm.

34. The method of any one of claims 1 to 10, wherein the roughness of the rough plasmonic surface is at least 5 nm; and / or at most 200 nm.

35. The method of claim 34, wherein the roughness of the rough plasmonic surface is at least 10 nm; and / or at most 100 nm.

36. The method of claim 34, wherein the roughened plasma surface has a roughness of at least 20 nm; and / or at most 50 nm.

37. The method of any one of claims 1 to 10, wherein the plasma material comprises plasma nanoparticles.

38. The method of claim 37, wherein the plasma nanoparticles comprise gold, silver or copper nanoparticles; the nanoparticles having a coating or no coating on their surface.

39. The method of claim 38, wherein the plasma nanoparticles are uncoated silver nanoparticles or uncoated gold nanoparticles.

40. The method of claim 38, wherein the coating comprises silicon dioxide.

41. The method of claim 37, wherein the plasma nanoparticles are pyramidal, spheroid-like, spherical, platelet-like or rod-like.

42. The method of claim 37, wherein the plasma nanoparticles have a size of D50 between 2 nm and 500 nm in diameter.

43. The method of claim 37, wherein the plasma nanoparticles have a size of D50 of at least 2 nm; and / or at most 500 nm in diameter.

44. The method of claim 43, wherein the plasma nanoparticles have a size of D50 of at least 2 nm; and / or at most 250 nm in diameter.

45. The method of claim 44, wherein the plasma nanoparticles have a size of D50 of at least 10 nm; and / or at most 200 nm in diameter.

46. The method of claim 45, wherein the plasma nanoparticles have a size of D50 of at least 25 nm; and / or at most 100 nm in diameter.

47. The method of claim 46, wherein the plasma nanoparticles have a size of D50 of at least 50 nm; and / or at most 75 nm in diameter.

48. The method of claim 43, wherein the plasma nanoparticles have a size of D50 of at least 10 nm; and / or at most 50 nm in diameter.

49. The method of claim 37, wherein the plasma nanoparticles are agglomerated by adding an agglomerating agent, adjusting the pH, adding an organic solvent or a combination of these methods.

50. The method of claim 49, wherein the organic solvent is an alcohol; and / or the agglomerating agent is a molecule or an electrolyte capable of bridging the plasma nanoparticles and inducing agglomeration.

51. The method of claim 50, wherein the electrolyte is a salt.

52. The method of claim 37, wherein the plasma nanoparticles are agglomerated by adding a sulfate or a sulfite salt as an agglomerating agent.

53. The method of any one of claims 49 to 52, wherein the agglomerating agent and the oxygen scavenger are different materials.

54. The method of any one of claims 49 to 52, wherein the agglomerating agent is the oxygen scavenger.

55. The method of claim 54, wherein the agglomerating agent is a sulfite salt.

56. The method of claim 55, wherein the sulfite is sodium sulfite.

57. The method of claim 37, wherein the plasmonic nanoparticles are aggregated by the addition of a dissolved salt and / or adjustment of pH.

58. The method of claim 37, wherein the pH of the liquid sample is adjusted to 4 prior to aggregation.

59. The method of claim 37, wherein the plasmonic nanoparticles are added to the liquid sample prior to aggregation of the nanoparticles and prior to removal of dissolved oxygen using an oxygen scavenger.

60. The method of claim 37, wherein the plasmonic nanoparticles are stabilized by small, loosely bound coating molecules.

61. The method of claim 60, wherein the coating molecules are citrate, ethanol, ethylene glycol, and / or polyethylene glycol.

62. The method of claim 61, wherein the coating molecules are citrate and / or ethanol.

63. The method of any one of claims 1-10, wherein molecules that interact with an analyte to produce a unique signal are pre-adsorbed to the plasmonic nanoparticles or rough plasmonic surface prior to contact with the analyte.

64. The method of claim 63, wherein the molecules that interact with an analyte to produce a unique signal are chelators of metal ions.

65. The method of any one of claims 1-10, wherein the method further comprises the step of measuring a SERS spectrum of the liquid sample.

66. The method of claim 65, wherein the step of measuring comprises illuminating the plasmonic material with light from a laser and detecting Raman signals scattered by the plasmonic material.

67. A probe for remote detection of an analyte in a liquid sample using SERS, the probe comprising: a detection chamber having a window transparent to SERS excitation light and Raman scattered signals; and a conduit having a first end and a second end, the first end of the conduit being flowably connected to the detection chamber, and the second end of the conduit being configured to be in contact with a liquid sample, wherein: the detection chamber and the conduit between the first and second ends contain a plasmonic material immersed in an oxygen-free solvent, the plasmonic material being plasmonic nanoparticles, wherein the plasmonic nanoparticles are aggregated such that they are in close proximity to and / or in contact with each other, thereby allowing unimpeded propagation and return of a plasmonic field from the detection chamber to the second end of the conduit, and / or the inner walls of the detection chamber and the conduit between the first and second ends are coated with a plasmonic material, the plasmonic material being a plasmonic layer, wherein the plasmonic layer is continuous from the detection chamber to the second end of the conduit, thereby allowing uninterrupted propagation and return of a plasmonic field from the detection chamber to the second end of the conduit, and wherein the conduit and the detection chamber are filled with an oxygen-free solvent; and wherein the oxygen scavenging solvent comprises an oxygen scavenger that removes dissolved oxygen from the oxygen scavenging solvent; the oxygen scavenger is added in an amount such that the concentration of the remaining dissolved oxygen is at most 0.020 mM.

68. The probe of claim 67, wherein the plasmonic nanoparticles satisfy one or more of (1)-(5): (1) the plasmonic nanoparticles comprise gold, silver, or copper nanoparticles; the nanoparticles have a surface that is coated or uncoated; (2) the plasmonic nanoparticles are pyramidal, spheroidal, spherical, platelet-like, or rod-like; (3) the plasmonic nanoparticles have a size of at least 2 nm; and / or at most 500 nm in diameter D50; (4) the plasmonic nanoparticles are stabilized by small, loosely bound coating molecules; (5) molecules that interact with an analyte to produce a unique signal are pre-adsorbed to the surface of the plasmonic nanoparticles prior to contact with the analyte; and / or the plasmonic layer is a layer of plasmonic material that includes a rough plasmonic surface having a roughness of at least 5 nm; and / or at most 200 nm.

69. The probe of claim 67 or 68, wherein the detection chamber and the conduit between the first end and the second end comprise plasmonic nanoparticles immersed in an oxygen scavenging solvent.

70. The probe of claim 67 or 68, wherein the plasmonic nanoparticles are agglomerated within the conduit, the agglomeration satisfying one or more of (11)-(14): (11) the plasmonic nanoparticles are agglomerated by adding an agglomeration agent, adjusting the pH, adding an organic solvent, or a combination of these methods; (12) the substance that agglomerates the plasmonic nanoparticles comprises an agglomeration agent that is a different material than the oxygen scavenger; (13) the substance that agglomerates the plasmonic nanoparticles comprises an agglomeration agent that is the same material as the oxygen scavenger; (14) the plasmonic nanoparticles are agglomerated by adding a dissolved salt and / or adjusting the pH.

71. The probe of claim 67 or 68, wherein the oxygen scavenging solvent is water or an organic solvent.

72. The probe of claim 71, wherein the organic solvent that is the oxygen scavenging solvent is methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, a hydrocarbon, or a mixture thereof.

73. The probe of claim 71, wherein the oxygen scavenging solvent is water.

74. The probe of claim 67 or 68, wherein the oxygen scavenger is a sulfite, a hydrazine, ascorbic acid, or ascorbyl palmitate.

75. The probe of claim 67 or 68, wherein, further satisfying one or more of (21)-(23): (21) an excess amount of the oxygen scavenger is added; (22) sufficient oxygen scavenger is added to remove at least 98% of the dissolved oxygen in the liquid sample; (23) wherein sufficient oxygen scavenger is added such that the concentration of the remaining dissolved oxygen is at most 0.010 mM.

76. The probe of claim 67 or 68, wherein the second end of the conduit is open or closed.

77. The probe of claim 76, wherein the second end of the conduit is open.

78. The probe of claim 67 or 68, wherein the inner wall of the conduit is coated with a plasma layer and the conduit is filled with oxygen-removing solvent.

79. The probe of claim 67 or 68, wherein the second end of the conduit is open and the detection chamber and the conduit are refilled with oxygen-removing solvent such that the conduit and the detection chamber remain filled with oxygen-removing solvent.

80. The probe of claim 67 or 68, wherein, Plasma material comprises plasma nanoparticles; the second end of the conduit is open and the detection chamber and the conduit are refilled with oxygen-removing solvent and plasma nanoparticles such that the conduit and the detection chamber remain filled with oxygen-removing solvent and plasma nanoparticles and the plasma nanoparticles are held in close proximity to each other.

81. The probe of claim 79, wherein the oxygen-removing solvent is from a pump or a reservoir device.

82. The probe of claim 81, wherein the pump or the reservoir device is directly connected to the first end of the conduit or connected to the detection chamber in such a way that any oxygen-removing solvent / plasma nanoparticles flowing out of the second end of the conduit are replaced.

83. The probe of claim 67 or 68, wherein capillary forces hold oxygen-removing solvent within the conduit despite the second end of the conduit being open.

84. The probe of claim 67 or 68, wherein plasma material comprises plasma nanoparticles; and capillary forces hold oxygen-removing solvent and plasma nanoparticles within the conduit despite the second end of the conduit being open.

85. The probe of claim 67 or 68, wherein the second end of the conduit is open and the second end of the conduit is at least partially coated with a plasma layer.

86. The probe of claim 85, wherein the second end of the conduit comprises plasma nanoparticles and an end wall, the inner wall towards the second end of the conduit, and an outer wall towards the second end is coated with a plasma layer.

87. The probe of claim 86, wherein the inner wall of the conduit is coated with a plasma layer and the plasma layer also covers the end wall and the outer wall towards the second end of the conduit.

88. The probe of claim 67 or 68, wherein the second end of the conduit is also sealed by a plasma layer.

89. The probe of claim 67 or 68, wherein the conduit is made of metal, quartz, glass, or polymeric plastic.

90. The probe of claim 89, wherein the polymeric plastic comprises polyethylene, polypropylene, polystyrene, polyether ether ketone, polyvinyl chloride, polytetrafluoroethylene, or polydimethylsiloxane.

91. The probe of claim 89, wherein the conduit is a polyether ether ketone (PEEK) polymeric tube or a glass capillary tube.

92. The probe of claim 67 or 68, wherein the inner diameter of the conduit is between 0.08 millimeters and 1 centimeter.

93. The probe of claim 92, wherein the inner diameter of the conduit is between 0.3 millimeters and 0.5 millimeters.

94. The probe of claim 67 or 68, wherein the inner diameter of the conduit is at least 0.08 mm; and / or at most 5 mm.

95. The probe of claim 94, wherein the inner diameter of the conduit is at least 0.1 mm; and / or at most 2 mm.

96. The probe of claim 95, wherein the inner diameter of the conduit is at least 0.1 mm; and / or at most 1 mm.

97. The probe of claim 96, wherein the inner diameter of the conduit is at least 0.2 mm; and / or at most 0.75 mm.

98. The probe of claim 92, wherein the inner diameter of the conduit is 0.3 millimeters or 0.5 millimeters.

99. The probe of claim 67 or 68, wherein the length of the conduit is at least 5 cm; and / or at most 50 m.

100. The probe of claim 99, wherein the length of the conduit is at least 5 cm; and / or at most 20 m.

101. The probe of claim 99, wherein the length is at least 10 cm; and / or at most 10 m.

102. The probe of claim 99, wherein the length of the conduit is at least 20 cm; and / or at most 5 m.

103. The probe of claim 99, wherein the length of the conduit is at least 50 cm; and / or at most 2 m.

104. The probe of claim 99, wherein the length of the conduit is at least 75 cm; and / or at most 1 m.

105. The probe of claim 99, wherein the length of the conduit is 1 m or 2 m.

106. The probe of claim 67 or 68, wherein the cross-section of the conduit is circular, elliptical, or rectangular.

107. The probe of claim 67 or 68, wherein "in close proximity" means a distance of at most 15 nm; the distance is between plasmonic nanoparticles, between a plasmonic nanoparticle and a plasmonic layer, or between a plasmonic nanoparticle or plasmonic layer and an analyte.

108. The probe of claim 107, wherein "in close proximity" means a distance of at most 10 nm.

109. The probe of claim 107, wherein "in close proximity" means a distance of at most 5 nm.

110. The probe of claim 67 or 68, wherein the density of the plasmonic nanoparticles varies along the longitudinal direction of the conduit.

111. The probe of any one of claims 67 or 68, wherein the detection chamber and the first end of the conduit are fluidically connectable to each other through a hole defined by the detection chamber.

112. The probe of claim 67 or 68, wherein the detection chamber is made of glass, plastic or quartz.

113. The probe of claim 112, wherein the detection chamber is made of glass or quartz.

114. The probe of claim 67 or 68, wherein the analyte contained in the liquid is contacted with a plasmonic material contained in the probe, the plasmonic material being a substance made of an electrically conductive, semi-conductive and / or dielectric material, a combination thereof or a composite thereof.

115. The probe of claim 114, wherein the plasmonic material in contact with the analyte is a plasmonic layer that completely covers the second end in the case of a closed second end; or a plasmonic nanoparticle coated on the second end in the case of an open second end or immersed in the oxygen scavenging solvent. the contact of the liquid sample with the plasmonic material satisfies one or more of (31)-(32); 116. The probe of any one of claims 67 or 68, wherein, (31) the pH of the liquid sample is adjusted to 4 before the plasmonic nanoparticle is aggregated; (32) the plasmonic nanoparticle is added to the liquid sample before the plasmonic nanoparticle is aggregated and before the dissolved oxygen is removed using an oxygen scavenger.

117. The probe of claim 116, wherein the liquid sample is adsorbed on the plasmonic material.

118. The probe of claim 67 or 68, wherein the oxygen scavenger is sodium sulfite. the residual dissolved oxygen concentration satisfies one or more of (41)-(42); 119. The probe of claim 67 or 68, wherein, (41) sufficient oxygen scavenger is added to remove at least 99% of the dissolved oxygen in the liquid sample; (42) wherein sufficient oxygen scavenger is added such that the concentration of the residual dissolved oxygen is at most 0.005 mM. for the liquid sample, one or more of (51)-(52) is satisfied:

120. The probe of claim 67 or 68, wherein, (51) the analyte in the liquid sample is a thiol compound, an amine, a pesticide, a persistent organic pollutant, a transition metal complex, a peptide, a protein, a nucleic acid, a polysaccharide or a hormone; (52) the liquid in the liquid sample is water or an organic solvent. the analyte is modified to enable it to interact with the surface of the plasmonic material, the modification satisfying one or more of (61)-(62):

121. The probe of claim 67 or 68, wherein, (61) the modification to the analyte is done by binding a thiol to the analyte, whereby the analyte will be chemisorbed on the Ag or Au plasmonic surface through this thiol; (62) the modification to the analyte is done by: physisorption through hydrophobic / hydrophilic interactions or van der Waals forces, chemisorption through other binding atoms, indirect detection by chemical reaction with a pre-adsorbed reporter molecule, chelation of metal ions by a pre-adsorbed chelator, indirect detection by affinity, indirect detection by retaining plasmonic nanoparticles with SERS-active reporter molecules through specific interactions with the analyte. ​ 122. The probe of claim 67 or 68, wherein the analyte becomes adsorbed to a surface of a plasmonic material, the surface of the plasmonic material being a surface of the nanoparticle in the conduit, a plasmonic layer coated on the second end of the conduit, and / or a plasmonic layer adsorbed on the second end of the conduit.

123. The probe of claim 67 or 68, wherein, the analyte in the liquid sample is in contact with the plasmonic nanoparticle in the conduit, and / or a plasmonic layer coated on the second end of the conduit, and / or a plasmonic layer adsorbed on the second end of the conduit; the liquid sample satisfies one or more of (71)-(75): (71) the analyte in the liquid sample is p-aminothiophenol (pATP) or p-nitrothiophenol (pNTP); (72) the liquid in the liquid sample is an organic solvent, including methanol, ethanol, isopropanol, acetone, acetonitrile, diethyl ether, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, xylene, a hydrocarbon, or a mixture thereof; or the liquid in the liquid sample is water; (73) the analyte in the liquid sample is modified to enable it to interact with a surface of the plasmonic material, the modification being done through an affinity and / or a specific interaction with the analyte; the affinity including an antibody-antigen binding of biomolecules; the specific interaction with the analyte including an antigen antibody reaction; (74) sufficient oxygen scavenger is added to remove at least 99.5% of dissolved oxygen in the liquid sample; (75) sufficient oxygen scavenger is added such that the concentration of dissolved oxygen remaining in the liquid sample is at most 0.002 mM.

124. The probe of claim 67 or 68, wherein a molecule that produces a unique signal upon interaction with an analyte is pre-adsorbed on the plasmonic nanoparticle within the conduit, on the plasmonic nanoparticle in the liquid sample, on a plasmonic layer coated on the second end of the conduit, and / or on a plasmonic layer adsorbed on the second end of the conduit.

125. The probe of claim 124, wherein the molecule that produces a unique signal upon interaction with an analyte includes a chelator of a metal ion or an antibody, a peptide, or an aptamer that interacts with a biomolecule.

126. The probe of claim 67 or 68, wherein the probe is configured to work with a surface enhanced Raman spectrometer for remote detection, the surface enhanced Raman spectrometer being operable to detect an analyte by shining an incident laser light to a sample container or a detection chamber and measuring scattered light using a measurer.

127. The probe of claim 126, wherein backscattered light is detected, i.e. scattered 180 o relative to the incident light.

128. The method of any one of claims 1 to 10, wherein the method is performed using the probe as defined in claim 67 or 68.

129. The method of any one of claims 1 to 10, wherein the probe of claim 67 or 68 is used.

130. The probe of claim 68, wherein the plasmonic nanoparticle and / or the plasmonic layer satisfies one or more of (81)-(87): (81) the plasmonic nanoparticle has a diameter of 1 nm to 100 nm; (82) the plasmonic nanoparticle has a diameter of 1 nm to 50 nm; (83) the plasmonic nanoparticle has a diameter of 1 nm to 20 nm; (84) the plasmonic nanoparticle has a diameter of 1 nm to 10 nm; (85) the plasmonic nanoparticle has a diameter of 1 nm to 5 nm; (86) the plasmonic nanoparticle has a diameter of 1 nm to 2 nm; (87) the plasmonic nanoparticle has a diameter of 1 nm. (81) the material of the plasmonic nanoparticles and / or plasmonic layer is gold, silver, copper or a mixture thereof; (82) the roughness of the rough plasmonic surface of the plasmonic layer is at least 10 nm; and / or at most 100 nm; (83) the plasmonic nanoparticles are uncoated silver nanoparticles or uncoated gold nanoparticles; (84) the plasmonic nanoparticles have a coating, the coating comprising silicon dioxide; (85) the size of the diameter D50 of the plasmonic nanoparticles is at least 10 nm; and / or at most 200 nm; (86) the plasmonic nanoparticles are agglomerated by adding a sulfate or a sulfite salt as agglomerating agent; (87) the capping molecule stabilizing the plasmonic nanoparticles is citrate, ethanol, ethylene glycol and / or polyethylene glycol.

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