Plasmonic superlattice film functionalized sers nanocapillary, preparation method and application thereof

Abstract

The application discloses a kind of plasmonic superlattice film functionalized SERS nanocapillary, preparation method and application, it is related to nanomaterials and biochemical analysis field, the SERS nanocapillary is prepared using following method: S01 with nanoscale tip's capillary is soaked in mercaptopropyl trimethoxysilane methanol solution and is modified with surface mercapto;S02 injects perfluorodecyl mercaptan n-hexane-ethanol solution into gold nanoparticle solution, obtains gold nanoparticle superlattice film at water-oil interface, gold nanoparticle superlattice film is transferred to the surface of capillary obtained in step S01, and is prepared after washing;The above preparation step is simple and efficient, after the surface modification of the prepared plasmonic superlattice film functionalized SERS nanocapillary redox response Raman molecule, can be used for the detection of a variety of oxidation-reduction molecules and single-cell oxidation-reduction state, and detection sensitivity is high, stability and repeatability are good.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomaterials and biochemical analysis, and particularly to a plasmonic superlattice film functionalized SERS nanocapillary, a preparation method and applications thereof. BACKGROUND

[0002] The maintenance of the redox metabolism balance of the organism involves the participation of various oxidation and reduction components, and plays an important role in the regulation of cell cycle, apoptosis and immune signal transmission. However, in pathological conditions, the redox metabolism of cells is imbalanced, which promotes the occurrence of oxidative stress, causing cell oxidative damage and functional disorder of the organism. Studies have shown that the imbalance of redox metabolism is closely related to the occurrence of various diseases, such as inflammation, cardiovascular disease, neurodegenerative disease and even cancer. In addition, cells are the basic units of life composition and function, and there is heterogeneity between cells. Therefore, the development of a dynamic detection technology for the redox state of single cells has important research value and practical significance for the study of cell metabolic characteristic differences and the revelation of physiological and pathological mechanisms.

[0003] As an analysis technology that can perform specific fingerprint identification, surface-enhanced Raman spectroscopy (SERS) has the advantages of high sensitivity, strong qualitative ability, no interference from biological background, resistance to photobleaching, and non-destructive detection. SERS technology has been widely used in the detection and analysis of drugs, proteins, and pesticide residues. For example, Chinese patent document CN111678911A discloses a capillary imprint SERS sensor based on the above. The surface of the amino-modified capillary is immersed in a gold nanostar solution with a particle size of 20-50 nm for 24 hours to obtain a SERS-active glass capillary sensor. The protein and dopamine hydrochloride are dissolved in a 10 mM Tris-HCl buffer, and then the glass capillary with SERS activity is immersed. After washing, a capillary imprint SERS sensor for specific recognition of proteins is obtained. Chinese patent document CN114252429A discloses a capillary-based SERS substrate modified by gold triacontahedron. The capillary is soaked in a piranha solution to modify the hydroxyl group, and then soaked in an ethanol solution of 3-aminopropyltriethoxysilane to modify the amino group, to prepare a capillary with a positive inner wall. Then, gold triacontahedron coated with polyvinylpyrrolidone is used as an assembly unit, and the gold triacontahedron is uniformly and densely assembled on the inner wall of the capillary by electrostatic adsorption to obtain a capillary-based SERS substrate. The SERS substrate is used for the detection of fentanyl.

[0004] Although SERS technology has accumulated a certain research foundation in the detection of cell redox signals, it is still difficult to achieve precise analysis of single cells. The main reason is that most SERS nanosensors for cell detection rely on intracellular endocytosis to aggregate in cells to produce enhanced signals. To some extent, this strategy meets the needs of single cell detection, but the anti-interference ability of nanomaterials in the constantly changing intracellular environment is weak, and the uncontrollable aggregation and random distribution of nanomaterials make the reproducibility of SERS signals and the spatial resolution of imaging poor. Moreover, the incubation time of several hours of nanomaterials is difficult to meet the needs of precise monitoring of dynamic changes in cells.

[0005] Nanocapillaries with nanoscale tips (30-500 nm) have significant advantages in single-cell sensing, mainly in that they can obtain information immediately without pre-incubation with cells, can be easily removed from cells, and have less damage to cells and interference with the intracellular environment. In the prior art, noble metal nanomaterials are mainly combined with nanocapillaries by physical adsorption to construct single-cell SERS sensors, but controllability, uniformity of distribution, particle spacing and other factors directly affecting the sensitivity and reproducibility of SERS detection are still difficult to precisely control. Therefore, if the nanoparticles can be assembled into a two-dimensional film and used as a whole to explore an efficient combination method with nanocapillaries, it may be able to break through the above bottleneck and establish a reliable method for monitoring single-cell redox metabolism. SUMMARY

[0006] The application provides a preparation method of a plasmonic superlattice film functionalized SERS nanocapillary, which is simple and efficient. After the surface of the prepared plasmonic superlattice film functionalized SERS nanocapillary is modified with redox-responsive Raman molecules, it can be used for the detection of various redox molecules and the redox state of single cells, and has high detection sensitivity, good stability and good reproducibility.

[0007] The specific technical solutions adopted are as follows:

[0008] A preparation method of a plasmonic superlattice film functionalized SERS nanocapillary, comprising the following steps:

[0009] S01: soaking a capillary with a nanoscale tip in a methanol solution of mercaptopropyltrimethoxysilane for surface mercapto modification;

[0010] S02: injecting a n-hexane-ethanol solution of perfluorodecyl mercaptan into a gold nanoparticle solution to obtain a gold nanoparticle superlattice film at the water-oil interface, and transferring the gold nanoparticle superlattice film to the surface of the capillary obtained in step S01, and then washing to obtain the plasmonic superlattice film functionalized SERS nanocapillary;

[0011] The average particle size of the gold nanoparticles is 40-50 nm, which is synthesized by using seed-mediated growth method with chloroauric acid solution, trisodium citrate solution and tris(hydroxymethyl)aminomethane as raw materials.

[0012] The application constructs the SERS nanocapillary by liquid-liquid interface assembly, non-destructive film transfer and other technologies, and realizes the detection of the target by comprehensively utilizing the highly ordered superlattice film, the hotspot coupling amplification effect and the rapid response of the nanocapillary.

[0013] Preferably, in step S01, the inner diameter of the tip of the capillary with a nanoscale tip is 100-500 nm.

[0014] Preferably, in step S01, the volume fraction of mercaptopropyl trimethoxysilane in the methanol solution of mercaptopropyl trimethoxysilane is 3%-5%, and the surface mercapto-modification time is greater than or equal to 2 h.

[0015] Preferably, in step S02, the preparation method of the gold nanoparticles comprises the following steps: heating trisodium citrate solution with a mass fraction of 0.05%-0.1% to boiling, rapidly injecting a first chloroauric acid solution with a molar concentration of 20-30 mM into the above-mentioned trisodium citrate solution, keeping boiling, adding a tris(hydroxymethyl)aminomethane solution with a molar concentration of 0.05-0.2 M after 60-120 s, and keeping boiling for greater than or equal to 15 min; then, adding a second chloroauric acid solution with a molar concentration of 20-30 mM into the above-mentioned mixed solution and keeping boiling for greater than or equal to 15 min; finally, continuously adding a third chloroauric acid solution with a molar concentration of 20-30 mM into the above-mentioned mixed solution and keeping boiling for greater than or equal to 15 min, to obtain the gold nanoparticles.

[0016] Further preferably, the volume ratio of the trisodium citrate solution, the first chloroauric acid solution, the tris(hydroxymethyl)aminomethane solution, the second chloroauric acid solution and the third chloroauric acid solution is 150:0.5-2:2-8:1:1.

[0017] The gold nanoparticles prepared by the above method have a shape closer to a sphere and a more uniform particle size, which is beneficial to subsequent liquid-liquid interface assembly.

[0018] Preferably, in step S02, the concentration of perfluorodecyl mercaptan in the n-hexane-ethanol solution of perfluorodecyl mercaptan is 1-5 mM, and the volume ratio of n-hexane to ethanol is 1:1-4.

[0019] Preferably, in step S02, the gold nanoparticle superlattice film is repeatedly transferred to the capillary surface obtained in step S01 for 1-5 times, and the gold nanoparticles with weak binding force are removed by washing with deionized water and ethanol.

[0020] The application further provides a preparation method of the plasmonic superlattice film functionalized SERS nanocapillary.

[0021] The application further provides application of the plasmonic superlattice film functionalized SERS nanocapillary in detection of redox molecules, wherein the redox molecules include hydrogen peroxide, glutathione, L-cysteine or ascorbic acid.

[0022] Specifically, the method for detecting the redox molecules is as follows.

[0023] S11, a SERS active nanocapillary is prepared by modifying redox-responsive Raman molecules on the surface of the plasmonic superlattice film functionalized SERS nanocapillary; the SERS active nanocapillary is immersed in standard solutions of redox molecules with different concentrations, taken out and dried, and then spectrum collection is performed on the SERS active nanocapillary by using a Raman spectrometer; a fingerprint spectrum is constructed; and a standard curve is drawn according to the relative intensity of characteristic peaks and the concentration of redox biomolecules.

[0024] S12, after the SERS active nanocapillary is immersed in a sample solution to be detected, spectrum collection is performed thereon, the relative intensity of characteristic peaks is measured, and the content of redox molecules in the sample solution to be detected is determined according to the standard curve obtained in step S11.

[0025] Preferably, the redox-responsive Raman molecules are 2-mercapto-p-cresol or 2-mercapto-benzoquinone.

[0026] The application modifies redox-responsive Raman molecules on the surface of the plasmonic superlattice film functionalized SERS nanocapillary, which can be grafted on the surface of gold nanoparticles through gold-sulfur bonds; when 2-mercapto-p-cresol is grafted, it has sensitive response capability to some oxidized molecules; when 2-mercapto-benzoquinone is grafted, it has sensitive response capability to some reduced molecules; or after 2-mercapto-p-cresol is grafted and reacts with oxidized molecules, 2-mercapto-benzoquinone is formed, which also has sensitive response capability to reduced molecules; thus, typical redox molecules are subjected to SERS detection, an analysis model is established, and qualitative and quantitative analysis is further performed by using the analysis model and the obtained spectrum.

[0027] The sample solution to be detected contains living cells; the SERS active nanocapillary is inserted into the living cells for spectrum collection; after spectrum collection, the obtained spectrum is compared with a control group to qualitatively analyze whether the cells have redox state changes; the relative intensity of characteristic peaks is measured, and the content change of redox components in the cells is quantitatively analyzed according to the standard curve obtained in step S11.

[0028] Preferably, the sample solution to be detected contains adherent cells, which can be selected from cervical cancer Hela cells, but are not limited thereto; adherent cells are more conducive to the operation of SERS active nanocapillary spectral acquisition.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] (1) The present application uses the SERS detection substrate of the functionalized nanocapillary of the nanoparticle superlattice film, and the hot spot enhancement induced by the collective plasmon resonance coupling effect of the superlattice film effectively improves the SERS enhancement activity, and the uniform hot spot coupling provides an effective guarantee for the high reproducibility of the signal; at the same time, the redox response Raman signal molecules modified on the surface of the SERS nanocapillary can produce sensitive and clear SERS signals, and only the spectral changes before and after the reaction with the detection object need to be observed to realize qualitative and quantitative analysis, which fully utilizes the technical advantages of surface enhanced Raman spectroscopy and can realize real-time, sensitive and reproducible monitoring of the redox metabolism of single cells.

[0031] (2) When the SERS active nanocapillary is used to detect the redox molecules, the detection range of hydrogen peroxide is 0.5-200 μM, and the detection limit is 1 nM; the detection range of glutathione is 1-1000 μM, and the detection limit is 10 nM. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 1 is a transmission electron microscope image of gold nanoparticles in Example 1.

[0033] Figure 2 A in Figure 2 is a scanning electron microscope image of the gold nanoparticle superlattice film, B is a scanning electron microscope image of the capillary after surface mercapto modification, and C is a scanning electron microscope image of the SERS nanocapillary functionalized by the plasmonic superlattice film.

[0034] Figure 3 Figure 3 is a SERS spectrum of the redox response Raman molecule in Application Example 1.

[0035] Figure 4 Figure 4 is a SERS fingerprint spectrum and a standard curve of the SERS active nanocapillary in Application Example 1 after reaction with different concentrations of H2O2 and GSH, wherein A is a SERS fingerprint spectrum after reaction with different concentrations of H2O2, B is a standard curve of the relative intensity of the characteristic peak-the logarithm of H2O2, C is a SERS fingerprint spectrum after reaction with different concentrations of GSH, and D is a standard curve of the relative intensity of the characteristic peak-the logarithm of GSH.

[0036] Figure 5 Figure 5 is a surface enhanced Raman spectrum of the SERS active nanocapillary before and after reaction with cells in Application Example 1. DETAILED DESCRIPTION

[0037] The application will be further clarified by the following examples and accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the application.

[0038] Example 1

[0039] (1) Synthesis of monodisperse gold nanoparticles with SERS enhancement activity with an average particle size of 40-50 nm by seed-mediated growth method: first, 150 mL of 0.07% trisodium citrate solution was heated to boiling, 1 mL of 25 mM chloroauric acid solution was quickly added, and boiling was maintained, 5 mL of 0.1 M tris(hydroxymethyl)aminomethane solution was added after 60 s, and boiling was maintained for 15 min; then 1 mL of 25 mM chloroauric acid solution was added twice with an interval of 15 min, and boiling was maintained during the interval, after the addition was completed, boiling was maintained for 15 min, and a wine-red gold nanoparticle solution was prepared, which was left to stand and cooled to room temperature; the TEM image of the gold nanoparticles is shown in FIG. 1A, and it can be seen that the gold nanoparticles are uniform spherical particles. Figure 1

[0040] (2) Preparation of plasmonic superlattice film functionalized SERS nanocapillary: 5 mL of deionized water and 5 mL of the gold nanoparticle solution prepared in the above step were placed in a beaker and mixed uniformly by shaking; then 10 mL of 3 mM perfluorodecyl mercaptan (PTFE) mixed solution of n-hexane / ethanol (volume ratio of n-hexane to ethanol is 1:2) was quickly injected, and a cover glass was placed after shaking for 10 s, and the gold nanoparticle superlattice film was obtained at the water-oil interface after standing for 12 h or more; the scanning electron microscope image of the superlattice film is shown in FIG. 1B, and the formed superlattice film is a single-layer film, and the gold nanoparticles are arranged closely. Figure 2

[0041] Further, a nanocapillary with an inner diameter of about 200 nm at the tip was selected, and the nanocapillary was immersed in a 4% volume fraction of mercaptopropyltrimethoxysilane methanol solution for 4 h for surface mercapto-modification, then the surface mercapto-modified capillary was used as a solid substrate to adhere the superlattice film at an angle of about 30°, and the process was repeated twice, and the capillary was washed with deionized water and ethanol in sequence to remove the gold nanoparticles with weak binding force, and the plasmonic superlattice film functionalized SERS nanocapillary was prepared.

[0042] The scanning electron microscope image of the surface mercapto-modified capillary is shown in FIG. 1C, and the scanning electron microscope image of the plasmonic superlattice film functionalized SERS nanocapillary is shown in FIG. 1D. Figure 2 Figure 2 ​​​The surface of the superlattice film functionalized nanocapillary is highly uniform and has closely spaced particle distribution compared with the blank nanocapillary.

[0043] Example 2

[0044] (1) Synthesis of monodisperse gold nanoparticles with SERS enhancement activity with an average particle size of 40-50 nm by seed-mediated growth method: first, 150 mL of 0.05% mass fraction of trisodium citrate solution was heated to boiling; quickly add 0.5 mL of 25 mM chloroauric acid solution, keep boiling, after 60 s, add 4 mL of 0.1 M tris-hydroxymethyl aminomethane solution, keep boiling for 15 min; then add 1 mL of 25 mM chloroauric acid solution twice with an interval of 15 min, keep boiling during the interval, after all the addition, keep boiling for 15 min, prepare a wine red gold nanoparticle solution, stand and cool to room temperature.

[0045] (2) Preparation of plasmonic superlattice film functionalized SERS nanocapillary: 5 mL of deionized water and 5 mL of the gold nanoparticle solution prepared in the above step were placed in a beaker and mixed uniformly by shaking; then 10 mL of 1 mM perfluorodecyl mercaptan (PTFE) mixed solution of n-hexane / ethanol (volume ratio of n-hexane and ethanol is 1:3) was quickly injected, covered with a cover glass after shaking for 10 s, and stood for more than 12 h, and a gold nanoparticle superlattice film was obtained at the water-oil interface.

[0046] Further, a nanocapillary with an inner diameter of about 200 nm was selected, immersed in a 3% volume fraction of mercaptopropyl trimethoxysilane methanol solution for 3 h for surface mercapto modification, then the surface mercapto modified capillary was used as a solid substrate and attached to the superlattice film at an angle of about 30°, repeated 3 times, and washed with deionized water and ethanol in turn to remove the gold nanoparticles with weak binding force, and the plasmonic superlattice film functionalized SERS nanocapillary was prepared.

[0047] Example 3

[0048] (1) Synthesis of monodisperse gold nanoparticles with SERS enhancement activity with an average particle size of 40-50 nm by seed-mediated growth method: first, 150 mL of 0.1% mass fraction of trisodium citrate solution was heated to boiling; quickly add 2 mL of 25 mM chloroauric acid solution, keep boiling, after 60 s, add 6 mL of 0.1 M tris-hydroxymethyl aminomethane solution, keep boiling for 15 min; then add 1 mL of 25 mM chloroauric acid solution twice with an interval of 15 min, keep boiling during the interval, after all the addition, keep boiling for 15 min, prepare a wine red gold nanoparticle solution, stand and cool to room temperature.

[0049] (2) Preparation of plasmonic superlattice film functionalized SERS nanocapillary: 5 mL of deionized water and 5 mL of gold nanoparticle solution prepared in the above step were placed in a beaker and mixed uniformly by shaking; then 10 mL of 5 mM of perfluorodecanethiol (PTFE) mixed solution of n-hexane / ethanol (volume ratio of n-hexane and ethanol was 1:1) was quickly injected, and after shaking for 10 s, a cover glass was covered, and the mixture was left to stand for 12 h or more, to obtain a gold nanoparticle superlattice film at the water-oil interface.

[0050] Further, a nanocapillary with an inner diameter of about 200 nm was selected, and the nanocapillary was immersed in a 5% (volume fraction) mercaptopropyltrimethoxysilane methanol solution for 2 h for surface mercapto-modification. Subsequently, the surface mercapto-modified capillary was used as a solid substrate to adhere the superlattice film at an angle of about 30°, and the process was repeated 4 times, and the capillary was washed with deionized water and ethanol in sequence to remove gold nanoparticles with weak binding force, to obtain the plasmonic superlattice film functionalized SERS nanocapillary.

[0051] Application Example 1

[0052] The 2-mercapto-p-cresol was modified on the surface of the plasmonic superlattice film functionalized SERS nanocapillary prepared in Example 1 by gold-sulfur chemical bonding (specifically, the plasmonic superlattice film functionalized SERS nanocapillary prepared in Example 1 was immersed in a 100 μM 2-mercapto-p-cresol solution for 30 min), to construct a SERS active nanocapillary with redox response capability. The SERS spectra of redox response Raman molecules 2-mercapto-p-cresol and 2-mercaptoquinone are shown in FIG. 2. Figure 3

[0053] Taking typical biological oxidation molecules H2O2 and reduction molecules GSH as examples for SERS analysis, the SERS active nanocapillary was placed in different concentrations of H2O2 standard solution (0, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 μM) for 5 min, and after taking out, the SERS active nanocapillary was dried by nitrogen blowing and spectrum collection was performed by a micro-Raman spectrometer, and a standard curve of characteristic peak relative intensity-H2O2 concentration logarithm was established for quantitative analysis, and the results are shown in FIG. 3A and FIG. 3B; spectrum collection was performed by the same method, and a standard curve of characteristic peak relative intensity-GSH concentration logarithm was established, and the concentration of GSH standard solution was: 0, 1, 2, 5, 10, 20, 50, 100, 200, 300, 500 and 1000 μM, and the results are shown in FIG. 3C and FIG. 3D. Figure 4 Figure 4

[0054] ​​​Under the assistance of high-precision micro-operation device and microscope of micro-Raman spectrometer, the SERS active nanocapillary is used to the living cell for 5 minutes, and then the in-situ surface Raman spectrum of the SERS active nanocapillary is detected by the micro-Raman spectrometer. Figure 5 In order to obtain the surface enhanced Raman spectrum of the SERS active nanocapillary before and after the reaction with the cell, the relative intensity of the characteristic peak of the obtained surface enhanced Raman spectrum is compared with the standard curve, the redox state in the cell is analyzed quantitatively and qualitatively, the cell is preliminarily determined as the oxidation state, the content of the oxidation molecule is about 0.8 μM, and the cell is in the normal physiological range (<100 μM).

[0055] The above embodiments are used to explain the technical solutions of the present application in detail, and it should be understood that the above embodiments are only specific embodiments of the present application, and are not used to limit the present application, and any modification, supplement or similar replacement within the principle range of the present application should be included in the protection range of the present application.

Claims

1. Use of a plasmonic superlattice film functionalized SERS nanocapillary for the detection of redox molecules, characterized in that, The redox molecule is hydrogen peroxide, glutathione, L-cysteine or ascorbic acid; the method for detecting the redox molecule is: S11 functionalizing the SERS nanocapillary surface with the plasmonic superlattice film to modify the redox response Raman molecule to obtain a SERS active nanocapillary, immersing the SERS active nanocapillary in standard solutions of different concentrations of the redox molecule, drying after taking out, collecting the spectrum of the SERS active nanocapillary by using a Raman spectrometer, constructing a fingerprint spectrum, and drawing a standard curve according to the relative intensity of the characteristic peak and the concentration of the redox biomolecule; S12 inserting the SERS active nanocapillary into living cells to collect the spectrum, comparing the obtained spectrum with a control group to qualitatively analyze whether the redox state of the cells changes, measuring the relative intensity of the characteristic peak, and quantitatively analyzing the content change of the redox component in the cells according to the standard curve obtained in step S11; The redox response Raman molecule is 2-mercapto-p-cresol; The preparation method of the SERS nanocapillary functionalized with the plasmonic superlattice film comprises the following steps: S01 immersing a nanoscale-tipped capillary in a mercaptopropyltrimethoxysilane methanol solution for surface mercapto-modification; the nanoscale-tipped capillary has a tip inner diameter of 100-500 nm; S02 injecting a perfluorodecanethiol n-hexane-ethanol solution into a gold nanoparticle solution to obtain a gold nanoparticle superlattice film at the water-oil interface, and transferring the gold nanoparticle superlattice film to the surface of the capillary obtained in step S01, and then cleaning to obtain the SERS nanocapillary functionalized with the plasmonic superlattice film; The average particle size of the gold nanoparticles is 40-50 nm, and the gold nanoparticles are synthesized by using seed-mediated growth method with chloroauric acid solution, trisodium citrate solution and tris(hydroxymethyl) aminomethane as raw materials.

2. Use of the plasmonic superlattice film functionalized SERS nanocapillary according to claim 1 for the detection of redox molecules, characterized in that, The preparation method of the gold nanoparticles comprises: heating a trisodium citrate solution with a mass fraction of 0.05%-0.1% to boiling, rapidly injecting a first chloroauric acid solution with a molar concentration of 20-30 mM into the above trisodium citrate solution, keeping boiling, adding a tris(hydroxymethyl) aminomethane solution with a molar concentration of 0.05-0.2 M after 60-120 s, and keeping boiling for ≥15 min; then, adding a second chloroauric acid solution with a molar concentration of 20-30 mM into the mixed solution and keeping boiling for ≥15 min; finally, continuously adding a third chloroauric acid solution with a molar concentration of 20-30 mM into the above mixed solution and keeping boiling for ≥15 min, to obtain the gold nanoparticles.

3. Use of the plasmonic superlattice film functionalized SERS nanocapillary according to claim 2 for the detection of redox molecules, characterized in that, The volume ratio of the trisodium citrate solution, the first chloroauric acid solution, the tris(hydroxymethyl) aminomethane solution, the second chloroauric acid solution and the third chloroauric acid solution is 150:0.5-2:2-8:1:

1.

4. Use of the plasmonic superlattice film functionalized SERS nanocapillary according to claim 1 for the detection of redox molecules, characterized in that, The concentration of the perfluorodecyl mercaptan in the n-hexane-ethanol solution of the perfluorodecyl mercaptan is 1-5 mM, and the volume ratio of n-hexane and ethanol is 1:1-4.

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