An optoelectrochemical sensor for detecting cortisol and a detection method thereof

Abstract

The present invention discloses a photoelectrochemical sensor for detecting cortisol and a detection method thereof. The photoelectrochemical sensor includes an ITO electrode modified with SrTiO3 semiconductor material, magnetic beads modified with cortisol aptamer, and liposomes labeled with single-stranded DNA. The present invention constructs a separated cortisol PEC aptamer sensor. Since the biological reaction is separated from the photoelectrochemical determination, it can avoid the damage of biological molecules by light and achieve high-throughput analysis.

Description

Technical Field

[0001] This invention relates to the field of cosmetic analysis and testing technology, and in particular to a photoelectrochemical sensor for detecting cortisol and a detection method thereof. Background Art

[0002] With the improvement of living standards, cosmetics have become a necessity. To cater to consumer demand, some businesses add prohibited glucocorticoids to cosmetics. Cortisol is a type of glucocorticoid and is considered a biomarker for many diseases, such as chronic fatigue syndrome, Cushing's syndrome, post-traumatic stress disorder, and fibromyalgia [Liu, Y.; Wu, B.; Tanyi, EK; Yeasmin, S.; Cheng, LJ Langmuir 2020, 36, 7781-7788]. Furthermore, quantitative analysis of cortisol has been widely used in the assessment of psychological stress [Kirschbaum, C.; Wolf, OT.; May, M.; Wippich, W.; Hellhammer, DH Life Sci. 1996, 58, 1475-1483].

[0003] Common methods for detecting cortisol include radioimmunoassay (RIA) [Franco-Martinez, L.; Tvarijonaviciute, A.; Martinez-Subiela, S.; Telesb, M.; Tort, L. Ecol. Indicat. 2019, 98, 634–640] and enzyme-linked immunosorbent assay (ELISA) [Ozgocer, T.; Yildiz, S.;]. [CJImmunoassay Immunochem. 2017, 38, 147–164.] and high-resolution chromatography [Alaejos, AR; Klaassen, SA; Fern′andez, SEClin. Chim. Acta 2019, 493, 340–341]. However, these methods all have some limitations, such as expensive equipment and time consumption. Therefore, it is necessary to develop a fast and sensitive method for the detection of cortisol.

[0004] Photoelectrochemical (PEC) sensors have attracted widespread attention due to their advantages such as simple equipment, convenient operation, low cost, and high sensitivity [Lin, Y.; Zhou, Q.; Tang, D.; Niessner, R.; Knopp, D. Anal. Chem. 2017, 89, 5637-5645], but so far there have been no reports on the application of PEC technology in the analysis and detection of cortisol. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a photoelectrochemical sensor and detection method for cortisol. This invention utilizes potassium ferrocyanide (K4[Fe(CN)6]) bound to the surface of SrTiO3 to generate metal-to-particle electron transfer (MPCT). Combined with the ferroelectric polarization characteristics of SrTiO3, a significant PEC signal can be obtained, constructing a separate cortisol PEC aptamer sensor. This separate photoelectrochemical detection, by separating the biological reaction from the photoelectrochemical measurement, avoids the damage of light to biomolecules, enabling high-throughput analysis.

[0006] The technical solution of the present invention is as follows:

[0007] The first objective of this invention is to provide a cortisol aptamer with the following nucleic acid sequence: 5'-HS-GGA ATG GAT CCA CAT CCA TGG ATG GGC AAT GCG GGG TGG AGA ATG GTT GCCGCA CTT CGG CTT CAC TGC AGA CTT GAC GAA GCT T-3'.

[0008] A second objective of this invention is to provide a photoelectrochemical sensor based on the cortisol aptamer, the photoelectrochemical sensor comprising an ITO electrode modified with SrTiO3 semiconductor material, magnetic beads modified with cortisol aptamer, and liposomes labeled with single-stranded DNA;

[0009] The method for preparing an ITO electrode modified with SrTiO3 semiconductor material is as follows: SrTiO3 semiconductor material is prepared into a suspension, drop-coated onto a pretreated ITO electrode, and then dried.

[0010] K4[Fe(CN)6] was encapsulated in liposomes labeled with single-stranded DNA. The single-stranded DNA sequence was: 5'-AAG CTT CGT CAAGTC TGC AGT GAA GCC GAA GTG CGG CA-3'.

[0011] In one embodiment of the present invention, the method for preparing liposomes labeled with single-stranded DNA encapsulating K4[Fe(CN)6] is as follows:

[0012] A mixture of soybean lecithin, cholesterol, and ethanolamine phosphate in a molar ratio of 4:1:0.15 was uniformly dispersed in a round-bottom flask containing 15-30 mL of chloroform. The mixture was then evaporated using a rotary evaporator at 25°C until a uniform lipid film formed on the flask wall. The film was dried overnight at 45°C. Subsequently, 2-5 mL of a phosphate buffer solution containing 5 mmol / L K₄[Fe(CN)₆] (0.1 mol / L, pH 7.4) was injected into the film. After sonication for 2 min, K₄[Fe(CN)₆]-encapsulated liposomes were obtained. To obtain a uniformly sized liposome suspension, the suspension was filtered through a 0.22 μm polycarbonate membrane. The resulting K₄[Fe(CN)₆]-encapsulated liposomes were stored at 4°C for later use.

[0013] Subsequently, liposomes labeled with single-stranded DNA were obtained via a glutaraldehyde cross-linking reaction. Specifically, 1-5 μL of 25 wt% glutaraldehyde was added to 100 μL of liposomes coated with K4[Fe(CN)6], and the reaction was carried out under stirring for 2 h. The solution was then dialyzed in phosphate buffer (10 mmol / L, pH 7.4) to remove excess glutaraldehyde. Next, 50-70 μL of 10 μmol / L single-stranded DNA was added to the mixture, and the mixture was gently shaken for 3-6 h. Excess single-stranded DNA was removed by centrifugation. Finally, the liposomes labeled with single-stranded DNA were dispersed in 200 μL of phosphate buffer (10 mmol / L, pH 7.4) containing 1 wt% bovine serum albumin and stored at 4°C for subsequent use.

[0014] In one embodiment of the present invention, the method for pretreating the ITO electrode is as follows: placing the unwashed ITO electrode in a boiling isopropanol solution containing 1.0 to 3.0 mol / L KOH, rinsing the electrode with deionized water and drying it after 15 to 30 minutes.

[0015] In one embodiment of the present invention, the SrTiO3 semiconductor material is prepared by mixing an aqueous SrCl2 solution with a Ti(C4H9O)4 ethylene glycol solution, adding a sodium hydroxide solution, and then heating the mixture to react.

[0016] The molar ratio of SrCl2 to Ti(C4H9O)4 is 1:1-1.2; the molar ratio of SrCl2 to sodium hydroxide is 1:3-5.

[0017] In one embodiment of the present invention, the concentration of the SrCl2 aqueous solution is 0.5-0.8 mmol / mL; the concentration of the Ti(C4H9O)4 ethylene glycol solution is 0.5-0.8 mmol / mL.

[0018] In one embodiment of the present invention, the method for preparing the ITO electrode modified with SrTiO3 semiconductor material is as follows: 5.0–8.0 mmol Ti(C4H9O)4 is dissolved in 10 mL ethylene glycol to form a homogeneous solution; then 10 mL of 0.5–0.8 mol / L SrCl2 aqueous solution is added dropwise; then 5 mL of 5.0 mol / L NaOH is added to form a white precipitate; after stirring for 30 min, the precipitate is transferred to a reaction vessel and heated at 200 °C for 20–24 h; the precipitate is then centrifuged, washed, dried, and ready for use.

[0019] The prepared SrTiO3 was prepared into a 2.0 mg / mL suspension, and 30 μL was dropped onto the pretreated ITO electrode and allowed to air dry for later use.

[0020] A second objective of this invention is to provide a method for detecting cortisol using the aforementioned photoelectrochemical sensor, the method comprising the following steps:

[0021] (1) Hybridize magnetic beads modified with cortisol aptamers with liposomes labeled with single-stranded DNA;

[0022] (2) Cortisol solution is added to the hybridization reaction solution for reaction, magnetic beads are separated and the supernatant is taken. Liposome is added to the supernatant to lyse the liposomes. Then, the liposomes are immersed in an ITO electrode modified with SrTiO3 semiconductor material. After the reaction, the electrode is taken out for photocurrent measurement to achieve qualitative or quantitative analysis of cortisol.

[0023] In one embodiment of the present invention, in step (1), the volume ratio of magnetic beads modified with cortisol aptamers to lipids labeled with single-stranded DNA is 1:2-5; the hybridization reaction conditions are: constant temperature reaction at 30-50℃ for 1-2 hours.

[0024] In one embodiment of the present invention, in step (2), the lysis solution is one or more of Triton X-100, Triton X-114, and Triton X-405;

[0025] Photocurrent measurement conditions: The 380–390 nm wavelength range was used as the excitation source. A SrTiO3-modified ITO electrode was used as the working electrode, and an Ag / AgCl electrode and a Pt wire were used as the reference and counter electrodes, respectively, forming a three-electrode system. The test solution was a 0.1 mol / L Tris-HCl buffer solution with pH = 7.5, and the test voltage was +0.2 V.

[0026] In one embodiment of the present invention, the method for preparing the standard curve for quantitative analysis in step (2) is as follows:

[0027] Different concentrations of cortisol solution were added to the hybridization reaction solution for reaction. Magnetic beads were separated and the supernatant was taken. Liposomes were lysed by adding lysis buffer to the supernatant. Then, the liposomes were immersed in an ITO electrode modified with SrTiO3 semiconductor material. After the reaction, the electrode was taken out and photocurrent was measured. The standard curve was obtained by plotting the logarithm of cortisol concentration on the x-axis and the increment of photocurrent on the y-axis.

[0028] In one embodiment of the present invention, the concentration range of the cortisol solution is 0.03-500.0 nmol / L.

[0029] The beneficial technical effects of this invention are as follows:

[0030] This invention utilizes potassium ferrocyanide (K4[Fe(CN)6]) bound to the surface of SrTiO3 to generate metal-to-particle electron transfer (MPCT). Combined with the ferroelectric polarization characteristics of SrTiO3, a significant PEC signal can be obtained, thus constructing a separate cortisol PEC aptamer sensor. This separate photoelectrochemical detection, by separating the biological reaction from the photoelectrochemical measurement, avoids the damage of light to biomolecules, enabling high-throughput analysis.

[0031] The detection principle of this invention is based on the metal-to-particle electron transfer generated by K4[Fe(CN)6] binding to the surface of SrTiO3 which has ferroelectric polarization characteristics to achieve the detection of cortisol.

[0032] This invention encapsulates K4[Fe(CN)6] in liposomes labeled with single-stranded DNA containing a sequence that hybridizes with a cortisol aptamer probe. In the presence of the target analyte, cortisol, the liposomes labeled with single-stranded DNA are released into solution due to the specific binding of cortisol to the aptamer sequence. Magnetic separation separates the solution containing the liposomes. Then, under the influence of the Triton X-ray, the liposomes are lysed, releasing free K4[Fe(CN)6] which binds to SrTiO3, thereby generating the MPCT process. The MPCT process is a direct electron transfer process; electrons excited by K4[Fe(CN)6] are directly transferred to the conduction band of SrTiO3. This rapid electron transfer results in high photoelectric conversion efficiency. Simultaneously, the ferroelectric polarization field of SrTiO3 further enhances the separation efficiency of photogenerated carriers, resulting in a strong PEC signal. The method exhibits a linear range of 0.03–500.0 nmol / L and a detection limit as low as 11.0 pmol / L, demonstrating higher sensitivity compared to conventional methods for detecting cortisol, such as radioimmunoassay (linear range 2.1–89.1 nmol / L, detection limit 1.6 nmol / L), enzyme-linked immunosorbent assay (linear range 1.0–195.0 nmol / L, detection limit 1.0 nmol / L), and chromatography (linear range 10.0–400.0 nmol / L, detection limit 1.0 nmol / L). Attached Figure Description

[0033] Figure 1 Infrared spectra of SrTiO3, K4[Fe(CN)6], and SrTiO3 / K4[Fe(CN)6];

[0034] Figure 2 (A) is a linear scan diagram of SrTiO3 semiconductor material; (B) is a cyclic voltammogram of K4[Fe(CN)6].

[0035] Figure 3 (A) Photocurrent diagrams of ITO electrodes modified with SrTiO3 semiconductor material under different concentrations of cortisol. Figure 3 (B) is a graph showing the linear relationship between cortisol concentration and ΔI;

[0036] Figure 4 To assess the (A) stability and (B) selectivity of the photoelectrochemical sensor. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0038] The method for pretreatment of ITO electrodes is as follows: place the unwashed ITO electrode in a boiling isopropanol solution containing 1.5 mol / L KOH for 20 min, rinse the electrode with deionized water and dry it.

[0039] The nucleic acid sequence of the cortisol aptamer is: 5'-HS-GGA ATG GAT CCA CAT CCA TGG ATG GGCAAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGG CTT CAC TGC AGA CTT GAC GAA GCTT-3'.

[0040] The single-stranded DNA sequence is: 5'-AAG CTT CGT CAA GTC TGC AGT GAA GCC GAA GTG CGG CA-3'.

[0041] The method for preparing liposomes labeled with K4[Fe(CN)6] and containing single-stranded DNA is as follows:

[0042] A mixture of soybean lecithin, cholesterol, and ethanolamine phosphate in a molar ratio of 4:1:0.15 was uniformly dispersed in a round-bottom flask containing 15-30 mL of chloroform. The mixture was then evaporated using a rotary evaporator at 25°C until a uniform lipid film formed on the flask wall. The film was dried overnight at 45°C. Subsequently, 2-5 mL of a phosphate buffer solution containing 5 mmol / L K₄[Fe(CN)₆] (0.1 mol / L, pH 7.4) was injected into the film. After sonication for 2 min, K₄[Fe(CN)₆]-encapsulated liposomes were obtained. To obtain a uniformly sized liposome suspension, the suspension was filtered through a 0.22 μm polycarbonate membrane. The resulting K₄[Fe(CN)₆]-encapsulated liposomes were stored at 4°C for later use.

[0043] Subsequently, liposomes labeled with single-stranded DNA were obtained via a glutaraldehyde cross-linking reaction. Specifically, 1-5 μL of 25 wt% glutaraldehyde was added to 100 μL of liposomes coated with K4[Fe(CN)6], and the reaction was carried out under stirring for 2 h. The solution was then dialyzed in phosphate buffer (10 mmol / L, pH 7.4) to remove excess glutaraldehyde. Next, 50-70 μL of 10 μmol / L single-stranded DNA was added to the mixture, and the mixture was gently shaken for 3-6 h. Excess single-stranded DNA was removed by centrifugation. Finally, the liposomes labeled with single-stranded DNA were dispersed in 200 μL of phosphate buffer (10 mmol / L, pH 7.4) containing 1 wt% bovine serum albumin and stored at 4°C for subsequent use.

[0044] Example 1:

[0045] a. Preparation of SrTiO3 semiconductor material: 5.0 mmol Ti(C4H9O)4 was dissolved in 10 mL ethylene glycol to form a homogeneous solution; then 10 mL of 0.5 mol / L SrCl2 aqueous solution was added dropwise; followed by the addition of 5 mL of 5.0 mol / L NaOH, forming a white precipitate; after stirring for 30 min, the mixture was transferred to a reaction vessel and heated at 200 °C for 22 h; then centrifuged, washed, dried, and ready for use.

[0046] b. Preparation of ITO electrode modified with SrTiO3 semiconductor material: The obtained SrTiO3 was prepared into a suspension of 2.0 mg / mL, and 30 μL was dropped onto the pretreated ITO electrode and allowed to air dry for later use.

[0047] c. Detection of cortisol: Magnetic beads modified with cortisol aptamers and liposomes labeled with single-stranded DNA were incubated at 37°C for 1 hour to ensure sufficient hybridization between the aptamer strand and the single-stranded DNA. Then, different known concentrations of cortisol were added, and the mixture was incubated at 37°C for 1.5 hours. Next, the magnetic beads were separated using a magnet, and the supernatant was collected. Triton X-114 was added to the supernatant to lyse the liposomes, releasing free K4[Fe(CN)6]. An ITO electrode modified with SrTiO3 was then used... The electrode was immersed in the above solution for 2 minutes to allow K4[Fe(CN)6] to bind to the SrTiO3-modified ITO electrode. The electrode was then removed and washed with a pH 7.5 Tris-HCl buffer. Finally, the photocurrent was measured. The 380–390 nm wavelength range was used as the excitation source. The SrTiO3-modified ITO electrode was used as the working electrode, and the Ag / AgCl electrode and Pt wire were used as the reference and counter electrodes, respectively, forming a three-electrode system. The test solution was a 0.1 mol / L Tris-HCl buffer solution with pH 7.5, and the test voltage was +0.2 V.

[0048] The infrared spectra of SrTiO3, K4[Fe(CN)6] and SrTiO3 / K4[Fe(CN)6] are as follows: Figure 1 As shown, according to SrTiO3 / K4[Fe(CN)6] at approximately 589.4 and 2041.2 cm⁻¹ -1 The presence of characteristic peaks at the point, which are attributed to the bending vibration of Fe(II)-CN and the stretching vibration of C≡N respectively, confirms that K4[Fe(CN)6] is bound to the SrTiO3 surface, thus laying the foundation for the subsequent construction of the cortisol PEC aptamer sensor;

[0049] Photocurrent diagrams generated under different concentrations of cortisol are shown below. Figure 3As shown in Figure A, the concentrations of cortisol from left to right are 0.03, 0.1, 0.5, 1.0, 10.0, 50.0, 100.0, and 500.0 nmol / L. The linear relationship between cortisol concentration and ΔI is shown in the graph below. Figure 3 As shown in Figure B; from the graph, we can see that ΔI = 176.95log[cortisol] + 331.70, R 2 =0.99; linear range is 0.03-500.0 nmol / L, detection limit is as low as 11.0 pmol / L.

[0050] The stability and selectivity test results of the photoelectrochemical sensor are as follows: Figure 4 As shown, the photocurrent response did not change significantly after repeated excitation by the light source, indicating that the photoelectrochemical sensing system has good stability. Furthermore, apart from the target compound cortisol, other interfering substances failed to enhance the photocurrent of the SrTiO3 semiconductor-modified ITO electrode, indicating that the method has good selectivity.

[0051] Example 2:

[0052] a. Preparation of SrTiO3 semiconductor material: 8.0 mmol Ti(C4H9O)4 was dissolved in 10 mL ethylene glycol to form a homogeneous solution; then 10 mL of 0.8 mol / L SrCl2 aqueous solution was added dropwise; followed by the addition of 5 mL of 5.0 mol / L NaOH, forming a white precipitate; after stirring for 30 min, the mixture was transferred to a reaction vessel and heated at 200 °C for 24 h; then centrifuged, washed, dried, and ready for use.

[0053] b. ITO electrode modified with SrTiO3 semiconductor material: Prepare a 2.0 mg / mL suspension of the obtained SrTiO3, take 30 μL and drop it onto the pretreated ITO electrode, let it air dry, and set aside for later use;

[0054] c. Detection of cortisol: Magnetic beads modified with cortisol aptamers and liposomes labeled with single-stranded DNA were incubated at 37°C for 2 hours to ensure sufficient hybridization between the aptamer strand and the single-stranded DNA. Then, different concentrations of cortisol were added, and the reaction was incubated at 37°C for 1.5 hours. Next, the magnetic beads were separated using a magnet, and the supernatant was collected. Triton X-114 was added to the supernatant to lyse the liposomes, releasing free K4[Fe(CN)6]. An ITO electrode modified with SrTiO3 was then used... The electrode was immersed in the above solution for 2 minutes to allow K4[Fe(CN)6] to bind to the SrTiO3-modified ITO electrode. The electrode was then removed and washed with a pH 7.5 Tris-HCl buffer. Finally, the photocurrent was measured. The 380–390 nm wavelength range was used as the excitation source. The SrTiO3-modified ITO electrode was used as the working electrode, and the Ag / AgCl electrode and Pt wire were used as the reference and counter electrodes, respectively, forming a three-electrode system. The test solution was a 0.1 mol / L Tris-HCl buffer solution with pH 7.5, and the test voltage was +0.2 V.

[0055] Example 3

[0056] a. Preparation of SrTiO3 semiconductor material: 5.0 mmol Ti(C4H9O)4 was dissolved in 10 mL ethylene glycol to form a homogeneous solution; then 10 mL of 0.6 mol / L SrCl2 aqueous solution was added dropwise; followed by the addition of 6 mL of 5.0 mol / L NaOH, forming a white precipitate; after stirring for 30 min, the mixture was transferred to a reaction vessel and heated at 200 °C for 24 h; then centrifuged, washed, dried, and ready for use.

[0057] b. ITO electrode modified with SrTiO3 semiconductor material: Prepare a 2.0 mg / mL suspension of the obtained SrTiO3, take 30 μL and drop it onto the pretreated ITO electrode, let it air dry, and set aside for later use;

[0058] c. Detection of cortisol: Magnetic beads modified with cortisol aptamers and liposomes labeled with single-stranded DNA were incubated at 37°C for 2 hours to ensure sufficient hybridization between the aptamer strand and the single-stranded DNA. Then, different concentrations of cortisol were added, and the reaction was incubated at 37°C for 1.5 hours. Next, the magnetic beads were separated using a magnet, and the supernatant was collected. Triton X-405 was added to the supernatant to lyse the liposomes, releasing free K4[Fe(CN)6]. An ITO electrode modified with SrTiO3 was then used… The electrode was immersed in the above solution for 2 minutes to allow K4[Fe(CN)6] to bind to the SrTiO3-modified ITO electrode. The electrode was then removed and washed with a pH 7.5 Tris-HCl buffer. Finally, the photocurrent was measured. The 380–390 nm wavelength range was used as the excitation source. The SrTiO3-modified ITO electrode was used as the working electrode, and the Ag / AgCl electrode and Pt wire were used as the reference and counter electrodes, respectively, forming a three-electrode system. The test solution was a 0.1 mol / L Tris-HCl buffer solution with pH 7.5, and the test voltage was +0.2 V.

Claims

1. A photoelectrochemical sensor based on a cortisol aptamer, characterized in that: The photoelectrochemical sensor includes an ITO electrode modified with a SrTiO3 semiconductor material, magnetic beads modified with a cortisol aptamer, and liposomes labeled with single-stranded DNA; The preparation method of the ITO electrode modified with the SrTiO3 semiconductor material is as follows: the SrTiO3 semiconductor material is prepared into a suspension, drop-coated on the pre-treated ITO electrode, and dried; The nucleic acid sequence of the cortisol aptamer is: 5'-HS-GGA ATG GAT CCA CAT CCA TGG ATG GGCAAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGG CTT CAC TGC AGA CTT GAC GAA GCTT-3'; K4[Fe(CN)6] was encapsulated by liposomes labeled with single-stranded DNA, and the single-stranded DNA sequence was: 5'-AAG CTT CGT CAA GTCTGC AGT GAA GCC GAA GTG CGG CA-3'.

2. The photoelectrochemical sensor according to claim 1, wherein The method for pretreatment of ITO electrodes is as follows: place the unwashed ITO electrode in a boiling isopropanol solution containing 1.0-3.0 mol / L KOH, rinse the electrode with deionized water after 15-30 minutes, and dry it.

3. The photoelectrochemical sensor according to claim 1, wherein The preparation method of SrTiO3 semiconductor material is as follows: after mixing SrCl2 aqueous solution and Ti(C4H9O)4 ethylene glycol solution, adding sodium hydroxide solution, and then heating the mixture for reaction; The molar ratio of SrCl2 to Ti(C4H9O)4 is 1:1-1.2; the molar ratio of SrCl2 to sodium hydroxide is 1:3-5.

4. The photoelectrochemical sensor according to claim 3, characterized in that The concentration of SrCl2 aqueous solution is 0.5~0.8 mmol / mL; the concentration of Ti(C4H9O)4 ethylene glycol solution is 0.5~0.8 mmol / mL.

5. A method for detecting cortisol using the photoelectric chemical sensor according to claim 1, characterized in that: The method comprises the following steps: (1) Hybridization reaction between magnetic beads modified with cortisol aptamers and liposomes labeled with single-stranded DNA; (2) Add cortisol solution to the hybridization reaction solution for reaction, separate the magnetic beads and take the supernatant, add lysis solution to the supernatant to lyse the liposomes, and then immerse them in an ITO electrode modified with SrTiO3 semiconductor material. After the reaction, take out the electrode and measure the photocurrent to achieve qualitative or quantitative analysis of cortisol.

6. The method according to claim 5, characterized in that In step (1), the hybridization reaction conditions are: 30-50°C for 1-2 h.

7. The method according to claim 5, characterized in that In step (2), the lysis solution is one or more of Triton X-100, Triton X-114, and Triton X-405; Photocurrent measurement conditions: 380-390 nm wavelength was used as the excitation light source, SrTiO3-modified ITO electrode was used as the working electrode, Ag / AgCl electrode and Pt wire were used as the reference electrode and counter electrode, respectively, to form a three-electrode system.

8. The method according to claim 5, characterized in that In step (2), the method for preparing the standard curve for quantitative analysis is as follows: Cortisol solutions of different concentrations are added to the hybridization reaction solution for reaction, the magnetic beads are separated and the supernatant is taken, a lysis solution is added to the supernatant to lyse the liposomes, and then the liposomes are immersed in an ITO electrode modified with a SrTiO3 semiconductor material. After the reaction, the electrode is taken out for photocurrent measurement, and the logarithm of the cortisol concentration is used as the horizontal axis and the increment of the photocurrent is used as the vertical axis to obtain the standard curve.

9. The method according to claim 8, characterized in that The concentration range of the cortisol solution was 0.03-500.0 nmol / L.

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