Construction and application of a membrane filter-based photothermal immunoassay method

Abstract

The present invention discloses a membrane-type photothermal immunoassay method, comprising the steps of: (1) synthesizing Cu 2‑x Se nanomaterials; (2) Prepare the signal probe by first preparing liposomes to embed the synthesized Cu 2‑ x Se complex, and then prepare an aptamer-modified liposome-embedded complex signal probe; (3) combine the prepared signal probe with a syringe with a filter membrane, that is, construct a filter-type photothermal immunoassay method. The signal probe prepared by the present invention constructs a filter-type photothermal immunoassay method with high sensitivity. The method is not only easy to operate but also has a fast response speed. The entire detection process can be easily performed within 10 minutes, which is much shorter than traditional clinical methods. The present invention also proves the feasibility and reliability of using this method to detect tumor marker levels in patient serum samples, thereby revealing the great prospects of the proposed strategy in early POCT of cancer.

Description

Technical Field

[0001] The present invention relates to the field of detection of tumor markers in human serum, and specifically to the preparation of a signal probe by utilizing its photothermal effect and combining it with a syringe with a filter membrane to construct a filter-based photothermal immunoassay method. Background Art

[0002] Cancer is one of the leading causes of death worldwide. Over the past few decades, tremendous efforts have been made to diagnose and treat cancer. Early, accurate, and sensitive detection of tumor markers is crucial to reducing cancer mortality. Currently, human serum is the most common and intensively studied diagnostic sample for the detection of tumor markers. Currently, enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence immunoassay, reverse transcriptase-polymerase chain reaction, etc. are commonly used methods for clinical tumor marker detection. However, most bioassays require expensive instruments and well-trained personnel to be performed in homogeneous or heterogeneous conditions under laboratory conditions. In order to overcome the limitations of traditional bioassays, the development of simple assays that are portable and easy to operate is of great significance for the rapid screening and detection of tumor markers in the early stages of cancer.

[0003] Nanomaterial-mediated photothermal effects have attracted widespread attention due to their unique photothermal conversion properties and good biocompatibility. The photothermal effect has been widely studied in photothermal cancer therapy for more than 20 years, but has only recently been explored for the construction of biosensors. The signal output of traditional enzyme-linked immunosorbent assays is limited by the measurement volume and precision instruments. The photothermal effect is a new visual biosensing signal transduction principle. When irradiated with a laser of the required wavelength, the photothermal agent can absorb light energy and continuously convert it into heat, breaking through the volume limitation. Using photothermal agents as specific biosensor probes, the photothermal properties of nanomaterials are used to establish a linear relationship between temperature changes and biomarker concentrations, providing an environmentally friendly analytical method for portable signal output analysis of biomarkers.

[0004] However, the photothermal immunoassay platforms that have been developed for detecting tumor markers are all accompanied by tedious separation and washing procedures. Therefore, it is particularly urgent to develop a sensitive and rapid photothermal immunoassay sensor that integrates separation and detection based on temperature as the output signal for POCT detection.

[0005] Among the previously published patents related to tumor marker detection, CN110133266A describes a method for preparing a conjugate of oncofetal antibodies and polyaniline@gold and constructing a photothermal immunosensor for detecting carcinoembryonic antigen. This method involves preheating an aqueous solution of chloroauric acid in a 45°C water bath for 10 minutes. A solution of aniline dissolved in hexane is then slowly added to the chloroauric acid solution. The mixture is incubated at 45°C overnight. Once the polyaniline@gold nanoparticles have grown, the hexane on the surface of the aqueous phase is discarded, the polyaniline@gold nanoparticles are collected by centrifugation, and rinsed several times with water to obtain a polyaniline@gold complex, designated PANi@Au. The prepared polyaniline@gold complex is then added with anti-CEA, shaken overnight, and finally centrifuged to obtain a conjugate of anti-CEA and polyaniline@gold, designated anti-CEA-PANi@Au. This invention also relates to a method for constructing a photothermal immunosensor with the polyaniline@gold complex to detect carcinoembryonic antigen. However, the main shortcoming of this method is that it cannot achieve separation-detection integration. Summary of the Invention

[0006] Immunoassay methods primarily consist of two components: an immunoassay substrate and an immunoprobe. The present invention prepares a signal probe and constructs a filter-based photothermal immunoassay method for tumor marker detection. The signal probe preparation method is simple, and quantitative detection of tumor markers can be achieved using a common thermometer. Compared with traditional immunoassay methods, this method is highly selective, simple, and effective, and can be conveniently read visually without the use of complex instrumentation, facilitating on-site, real-time testing. Furthermore, no filter-based photothermal immunoassay methods have been reported.

[0007] The technical solution adopted in the present invention is:

[0008] A membrane filter-type photothermal immunoassay method is constructed, comprising the following steps:

[0009] Step 1: Synthesis of Cu 2-x Se;

[0010] Step 1.1: Add cetyltrimethylammonium bromide (CTAB) to the round-bottom flask and spin at 1200 rpm. -1 Selenium dioxide (SeO2) and ascorbic acid (AA) solution were added sequentially under vigorous stirring and mixed for 10-20 min;

[0011] Step 1.2: After the reaction, add CuSO4∙5H2O and appropriate amount of AA solution, where AA converts Cu 2+ Reduction to Cu + , the resulting mixture was vigorously stirred at 30°C-40°C until a green solution was obtained;

[0012] Step 1.3: The green solution was placed in a dialysis bag for purification. 2-x Se was stored at 0-4°C until use;

[0013] Step 2: Prepare signal probe;

[0014] Step 2.1: Distearoylphosphatidylcholine (DSPC), DSPE-PEG(2000)-Biotin, and cholesterol were dissolved in a round-bottom flask containing chloroform to obtain a mixed solution. The mixed solution was rotary evaporated at 30-40°C to remove the organic solvent to obtain a liposome film;

[0015] Step 2.2: Adding Cu to the liposome film 2-x Se solution and PBS buffer, the mixture was stirred in a water bath at 30-45°C for 10-30 min, and then ultrasonicated. The obtained solution was placed in a dialysis bag for purification overnight to obtain liposome embedding material, which was stored at 0-4°C until use;

[0016] Step 2.3: Mix the prepared liposome-embedded material with streptavidin and shake for 10-20 minutes. Then add the biotin-modified aptamer and incubate at 20-40°C with shaking for 10-30 minutes. Then, store at 0-4°C until use.

[0017] Step 3: Combine the prepared signal probe with a syringe with a filter membrane to construct a filter membrane photothermal immunoassay method.

[0018] Furthermore, in the construction method of the present invention, the concentration of hexadecyltrimethylammonium bromide in step 1.1 is 0.0001-0.05 mol L -1 , the volume is 1-20 mL; the concentration of selenium dioxide is 0.01-2 mol L -1 , the volume is 0.001-1 mL; the concentration of ascorbic acid is 0.01-2 mol L -1 , volume 0.001-1 mL;

[0019] The concentration of CuSO4∙5H2O in step 1.2 is 0.01-4 mol L -1 , with a volume of 0.001-1 mL.

[0020] The mass of distearoylphosphatidylcholine in step 2.1 is 0.00001-2 g, and the volume is 0.01-10 mL; the mass of DSPE-PEG(2000)-Biotin is 0.00001-1 g, and the volume is 0.01-10 mL; the mass of cholesterol is 0.00001-1 g, and the volume is 0.01-10 mL; the volume of chloroform is 1-20 mL;

[0021] Cu in step 2.2 2-x The volume of Se solution is 1-20 mL; the concentration of PBS buffer is 0.0001-10 mol L -1 , volume 1-20 mL;

[0022] The volume of the liposome embedding in step 2.3 is 0.0001-1 mL; the concentration of streptavidin is 0.000001-1 ng mL -1 ; Volume 0.0001-1 mL; Concentration of biotin-modified aptamer 0.00001-1 ng mL -1 , with a volume of 0.0001-1 mL.

[0023] Another object of the present invention is to utilize the prepared signal probe's photothermal effect and combine it with a syringe with a filter membrane to construct a filter-type photothermal immunoassay method for detecting tumor markers. The detection method is carried out according to the following steps:

[0024] Step A: Dilute the tumor marker standard solution to different concentrations with PBS buffer, then quickly aspirate the tumor marker standard solution and inject it into the filter with a syringe. Incubate and rinse for 2 minutes, and rinse several times with PBST solution to remove excess tumor markers.

[0025] Step B: The signal probe solution was quickly introduced into the detachable filter through a syringe and incubated for 2 minutes. The filter was then rinsed several times with PBST solution. After washing, the fiber membrane was taken out and vacuum-dried at room temperature. It was then irradiated with a laser for 2 minutes. The temperature of the fiber membrane was recorded with a handheld thermal imager. The temperature change (ΔT) of tumor markers with different concentrations was plotted to construct a calibration curve.

[0026] Furthermore, in the detection method:

[0027] The concentration of the PBS buffer solution in step A is 0.0001-10 mol L -1 The volume is 1-20 mL; the concentration of tumor marker dilution is 0.00001-10 mol L -1 , with a volume of 0.001-10 mL.

[0028] The concentration of PBST solution in step A and step B is 0.0001-10 mol L -1 , with a volume of 1-20 mL.

[0029] In step B, the quantification of tumor marker content by monitoring the temperature change (ΔT) of the fiber membrane means that the concentration of the tumor marker is linearly correlated with the temperature change. The corresponding linear equation is y = 1.47 x + 0.23, where x is the concentration of the tumor marker in ng mL -1 , y is the detected temperature change.

[0030] The present invention has the following beneficial effects: the signal probe prepared in this invention is used to construct a filter-based photothermal immunoassay method with high sensitivity. The method is not only convenient to operate but also has a fast response speed. The entire detection process can be easily performed within 10 minutes, which is much shorter than traditional clinical methods. In addition, the present invention demonstrates the feasibility and reliability of using this method to detect tumor marker levels in patient serum samples, revealing the great potential of the proposed strategy in early-stage cancer point-of-care testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the principle of detecting tumor markers using a filter-type photothermal immunoassay in an embodiment;

[0032] in, Figure 1 A is a schematic diagram of preparing a signal probe;

[0033] Figure 1 B is a schematic diagram of the modification of monoclonal antibodies on nitrocellulose membrane;

[0034] Figure 1 C is a schematic diagram of photothermal immunoassay for detecting tumor markers;

[0035] Figure 2 For example, plasma Cu 2-x UV-visible-near-infrared absorption spectra of Se, ptSNBs and liposomes;

[0036] Figure 3 This is a response curve diagram of the photothermal immunoassay for determining tumor markers in the embodiment;

[0037] Figure 4 This is a linear response curve diagram of the photothermal immunoassay method for determining tumor markers in the embodiment. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below with reference to the embodiments and drawings, but the present invention is not limited thereto. Example

[0039] A filter-type photothermal immunoassay method was constructed with reference to Figure 1 , including the following steps:

[0040] Step 1: Synthesis of Cu 2-x Se;

[0041] Add 1.6 mL of cetyltrimethylammonium bromide (CTAB) to the round-bottom flask and spin the mixture at 1200 rpm min. -1 0.1 mL of selenium dioxide (SeO2) and 0.6 mL of ascorbic acid (AA) solution were added successively under vigorous stirring at a high speed and reacted for 10 min. Then, 0.1 mL of CuSO4∙5H2O and 0.8 mL of AA solution were added respectively. The resulting mixture was vigorously stirred at 30 °C until a green solution was obtained. The obtained green solution was placed in a dialysis bag for purification. The synthesized plasma Cu 2-x Se was stored at 4°C until use;

[0042] Step 2: Prepare signal probe;

[0043] 0.5 mL of distearoylphosphatidylcholine, 0.1 mL of DSPE-PEG (2000)-Biotin and 0.5 mL of cholesterol were mixed evenly, and 3 mL of chloroform was added. The organic solvent was removed by rotary evaporation at 40 °C to obtain a liposome film. Then 2 mL of Cu was added. 2-x Se solution and 3 mL PBS buffer solution, the mixture was stirred in a 45 ℃ water bath for 30 min, ultrasonicated for 5 min, and then placed in a dialysis bag for purification overnight. The obtained liposome embedding material was stored at 4 ℃ for use; 0.3 mL liposome embedding material was mixed with 0.2 mL streptavidin shaking solution for 10 min, and then 0.1 mL nucleic acid aptamer was added and incubated at 37 ℃ for 30 min and then stored at 4 ℃ for use, thus synthesizing the signal probe, such as Figure 1 As shown in A;

[0044] Step 3: Combine the prepared signal probe with a syringe with a filter membrane to construct a filter membrane photothermal immunoassay method.

[0045] The filter-type photothermal immunoassay method constructed in the embodiment is used to detect tumor markers. The detection method includes the following steps:

[0046] Step A: The stationary phase, a 2 cm diameter nitrocellulose filter membrane (NC membrane) with a pore size of 1.2 µm, was washed several times with a pH 9.6 Na2CO3-NaHCO3 buffer solution and then incubated with 12.5 µg mL -1The monoclonal antibody was incubated at 4 °C overnight. After washing several times with 3 mL of PBST solution, the stationary phase was blocked in 2% BSA solution overnight, dried under vacuum at 25 °C, and stored in N2 until use. Figure 1 As shown in B;

[0047] Step B: Use a disposable syringe to quickly draw an appropriate amount of tumor marker standard solution, inject it into the filter with a 1 mL syringe, incubate and rinse for 2 minutes, and rinse several times with 3 mL of PBST solution to remove excess tumor markers. After washing, remove the fiber membrane and vacuum dry it at room temperature. Then, irradiate it with a 1064 nm laser for 2 minutes and record the fiber membrane temperature with a handheld thermal imager. Complete the immunoassay process and record the temperature with a handheld infrared temperature detector, as shown in Figure 2. Figure 1 As shown in C.

[0048] UV-visible-near-infrared absorption spectra, such as Figure 2 As shown, Cu 2-x Se has strong UV absorption at 987 nm; in comparison, blank liposomes have no obvious absorption peak and Cu 2-x The UV absorption peak of ptSNBs formed by Se encapsulation in liposomes showed an obvious red shift, indicating that Cu 2-x Se was successfully encapsulated in liposomes.

[0049] The photothermal immunoassay method constructed using the prepared signal probes is used to detect tumor markers, such as Figure 3 As shown in Figure 2, the content of tumor markers was determined by monitoring the temperature change (ΔT) of the fiber membrane. -1 Within this range, with the increase of tumor marker concentration, the fiber membrane temperature change (ΔT) gradually increased and reached a platform.

[0050] The concentration of tumor markers is quantitatively analyzed by monitoring the temperature change of the fiber membrane. This is done by using a thermometer to detect the temperature change after irradiation with a near-infrared laser. Figure 4 As shown, in the range of 0-20 ng mL -1 Within the range, after irradiation with 1064 nm laser for 2 min, the temperature change of the fiber membrane (ΔT) has a strong linear relationship with the tumor marker concentration, and the linear equation is: y = 1.47 x + 0.23 (R 2 = 0.9963, n = 3), and the LOD value was 0.097 ng mL -1 (n = 15, 3σ 0，n=15 / K). Where x is the concentration of tumor markers, in ng mL -1, y is the temperature change. Compared with other detection methods, the photothermal immunosensor constructed in the present invention has a lower detection limit and a wider detection range (R is the linear correlation coefficient, R 2 is the square of the linear correlation coefficient, and n represents the number of experiments).

Claims

1. Construction of a membrane filter photothermal immunoassay method, characterized in that: The steps include: Step 1: Synthesis of Cu 2-x Se; Step 1.1: Add cetyltrimethylammonium bromide to the round-bottom flask and spin at 1200 rpm min. -1 Selenium dioxide and ascorbic acid solution were added sequentially under vigorous stirring and mixed for 10-20 min; Step 1.2: After the reaction, add CuSO₄∙5H₂O and an appropriate amount of ascorbic acid solution, and stir the resulting mixture vigorously at 30°C-40°C until a green solution is obtained. Step 1.3: The green solution was placed in a dialysis bag for purification. 2-x Se was stored at 0-4°C until use; Step 2: Prepare signal probe; Step 2.1: Distearoylphosphatidylcholine, DSPE-PEG(2000)-Biotin, and cholesterol were dissolved in a round-bottom flask containing chloroform to obtain a mixed solution of the three. The mixed solution was rotary evaporated at 30-40°C to remove the organic solvent to obtain a liposome film; Step 2.2: Adding Cu to the liposome film 2-x Se solution and PBS buffer, the mixture was stirred in a water bath at 30-45°C for 10-30 min, and then ultrasonicated. The obtained solution was placed in a dialysis bag for purification overnight to obtain liposome embedding material, which was stored at 0-4°C until use; Step 2.3: Mix the prepared liposome-embedded material with streptavidin and shake for 10-20 minutes. Then add the biotin-modified aptamer and incubate at 20-40°C with shaking for 10-30 minutes. Then, store at 0-4°C until use. Step 3: Combine the prepared signal probe with a syringe with a filter membrane to construct a filter membrane photothermal immunoassay method.

2. The construction of the filter membrane photothermal immunoassay method according to claim 1, characterized in that: The concentration of hexadecyltrimethylammonium bromide in step 1.1 is 0.0001-0.05 mol L -1 , volume 1-20 mL; The concentration of selenium dioxide is 0.01-2 mol L -1 , volume 0.001-1 mL; The concentration of ascorbic acid is 0.01-2 mol L -1 , volume 0.001-1 mL; The concentration of CuSO4∙5H2O in step 1.2 is 0.01-4 mol L -1 , with a volume of 0.001-1 mL.

3. The construction of the filter membrane photothermal immunoassay method according to claim 1, characterized in that: The mass of distearoylphosphatidylcholine in step 2.1 is 0.00001-2 g, and the volume is 0.01-10 mL; the mass of DSPE-PEG(2000)-Biotin is 0.00001-1 g, and the volume is 0.01-10 mL; The mass of cholesterol is 0.00001-1 g, and the volume is 0.01-10 mL; the volume of chloroform is 1-20 mL; Cu in step 2.2 2-x The volume of Se solution was 1–20 mL; PBS buffer concentration is 0.0001-10 mol L -1 , volume 1-20 mL; The volume of the liposome embedding in step 2.3 is 1-20 mL; Streptavidin concentration is 0.000001-1 ng mL -1 ; Volume 0.0001-1 mL; The concentration of biotin-modified aptamer was 0.00001-1 ng mL -1 , with a volume of 0.001-1 mL.

4. Application of the filter membrane photothermal immunoassay method constructed according to any one of claims 1 to 3, characterized in that: Used to detect tumor markers.

5. The use according to claim 4, characterized in that The method for detecting tumor markers comprises the following steps: Step A: Dilute the tumor marker standard solution to different concentrations with PBS buffer, then quickly aspirate the tumor marker standard solution and inject it into the filter with a syringe. Incubate and rinse for 2 minutes, and rinse several times with PBST solution to remove excess tumor markers. Step B: The signal probe solution was quickly introduced into the detachable filter head through a syringe and incubated for 2 minutes. It was then rinsed several times with PBST solution. After washing, the fiber membrane was taken out and vacuum-dried at room temperature. It was then irradiated with a laser for 2 minutes. The fiber membrane temperature was recorded with a handheld thermal imager. The temperature change ΔT of tumor markers with different concentrations was plotted to construct a calibration curve.

6. The use according to claim 5, characterized in that: The concentration of the PBS buffer solution in step A is 0.0001-10 mol L -1 , volume 1-20 mL; The concentration of tumor marker dilution is 0.00001-10 mol L -1 , volume 0.001-10 mL; The concentration of PBST solution in step A and step B is 0.0001-10 mol L -1 , with a volume of 1-20 mL.

7. The use according to claim 5, characterized in that: The concentration of the tumor marker in step B is linearly correlated with the temperature change, and the corresponding linear equation is y = 1.47 x + 0.23, where x is the concentration of the tumor marker in ng mL -1 , y is the detected temperature change.

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