Single-Molecule Array Chip Based on High-Entropy Alloy Plasmons and Preparation Method Thereof

Abstract

The present invention relates to the field of single-molecule immunoassay chips, and specifically discloses a single-molecule array chip based on high-entropy alloy plasmons and a preparation method thereof. The production raw materials include a substrate chip and a high-entropy alloy coating material. The high-entropy alloy coating material is composed of a noble metal composite material and a synergistic composite metal material in a mass ratio of 1:0.12 to 3.75. The noble metal composite material is composed of gold and silver in a mass ratio of 1:0.08 to 3. The synergistic composite metal material is composed of ruthenium, palladium, and platinum in a mass ratio of 1:0.2 to 1.5:0 to 1.2. Its advantages are: it can significantly improve the signal-to-noise ratio of the single-molecule array chip and further improve the accuracy and reliability of this technology in single-molecule immunoanalysis.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-detection chips, especially the production technology of single molecule immuno-detection chips. Background Art

[0002] Single Molecule Array (SiMoA) technology is a detection method that uses single molecule counting for trace biological markers. The detection limit of this technology can reach the femtogram (fg / mL) order of magnitude, and it is currently the most representative single molecule immuno-detection technology. The system originated from a detection system developed by the teams of David Walt and David Duffy. Its detection principle is similar to that of digital PCR, both of which are achieved through single molecule isolation in micro-reactor units combined with single molecule level signal amplification.

[0003] This technology is based on single molecule immuno-detection on a microarray chip. Thousands of micron-sized micro-wells are engraved (or cast) on a millimeter-sized chip. The volume of each micro-well is about 40 fl (i.e., the single molecule array chip). Subsequently, the immunocomplex magnetic beads are distributed in the micro-wells, and then a high-resolution fluorescence microscope is used to count the fluorescence spots. According to the Poisson distribution theory, the ratio of the number of pores containing both beads and fluorescent products to the total number of pores containing beads is calculated to determine the concentration of the analyte in the test sample. Its operation process is roughly as follows:

[0004] (1) Use magnetic beads labeled with capture antibodies on the surface to capture antigens in the sample;

[0005] (2) Use detection antibodies labeled with biotin to label the captured antigens;

[0006] (3) Add streptavidin-galactosidase complex to bind to the biotin on the detection antibody;

[0007] (4) Mix the washed magnetic beads with the substrate, load them into the single molecule array chip, use a magnetic field to make the magnetic beads fall into the micro-pores that exactly match their size, and add an oil phase to physically isolate the micro-pores;

[0008] (5) The micro-pores containing galactosidase produce fluorescent products due to the catalytic action of enzyme molecules on the substrate;

[0009] (6) Perform fluorescence imaging on the micro-well array, and achieve quantitative detection by comparing the number of micro-wells emitting fluorescence signals with the standard curve.

[0010] One of the main challenges of this technology is the low sensitivity of its single-array signal, which directly affects the accuracy and reliability of detection. In practical applications, particularly single-molecule immunoassays, extremely small amounts of molecules need to be detected and analyzed. However, existing single-molecule array chips suffer from poor signal-to-noise ratios, making it difficult to accurately extract target signals from background noise, limiting their widespread application and accuracy in biomolecule detection. Summary of the Invention

[0011] In order to improve the signal-to-noise ratio of single-molecule array chips and further enhance the accuracy and reliability of this technology in single-molecule immunoassay, the present invention provides a single-molecule array chip based on high-entropy alloy plasmons and a preparation method thereof.

[0012] The technical solution adopted by the present invention is: a method for preparing a single-molecule array chip based on high-entropy alloy plasmons, characterized in that: the production raw materials include a substrate chip and a high-entropy alloy coating material; the high-entropy alloy coating material is composed of a precious metal composite material and a synergistic composite metal material in a mass ratio of 1:0.12 to 3.75; the precious metal composite material is composed of gold and silver in a mass ratio of 1:0.08 to 3; and the synergistic composite metal material is composed of ruthenium and palladium in a mass ratio of 1:0.2 to 1.5.

[0013] As a further improvement of the present invention, the method further comprises forming a high entropy alloy film on the substrate chip to obtain a coated chip. More preferably, the thickness of the high entropy alloy film on the coated chip is 80 to 120 nm.

[0014] As a further improvement of the present invention, the base chip is selected from any one of a silicon single crystal substrate base chip, an FZ single crystal silicon wafer, a CZ single crystal silicon wafer, a semi-insulating single crystal silicon wafer, and an ultra-high purity single crystal silicon wafer.

[0015] The above-mentioned coating chip can be obtained by depositing the high entropy alloy coating material on the substrate chip through thermal evaporation thin film deposition technology, or by depositing the high entropy alloy coating material on the substrate chip through magnetron sputtering coating technology.

[0016] If thermal evaporation thin film deposition technology is used for coating, the following specific steps can be followed:

[0017] S1. Surface treatment of the substrate chip is performed by ultrasonic cleaning or argon plasma cleaning to remove residual organic matter and oxides;

[0018] S2, placing the high entropy alloy coating material in the vacuum chamber of the thermal evaporation coating machine, placing the substrate chip on the rotary coating table, controlling the rotation speed to 20-50 rpm, and the vacuum degree to 10 -6 ~10 -7Torr, start thermal evaporation thin film deposition when the heating temperature is 500 - 600 °C; during the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within

[0019] S3. Restore atmospheric pressure to release the sample by gradually increasing the ambient pressure or opening the gas inlet valve to obtain the coated chip.

[0020] The above steps can ensure uniform and high-quality deposition of the metal thin film on the substrate surface, providing a reliable basis for the subsequent preparation of the single-molecule array chip.

[0021] As a further improvement of the present invention, it further includes the step of performing plasma cleaning on the coated chip.

[0022] The parameters of the above plasma cleaning can be set as follows: use high-purity oxygen as the cleaning gas, set the gas pressure to 0.1 - 1 mTorr, the gas flow rate to 5 - 20 sccm, the radio frequency power to 100 - 500 W, and the cleaning time to 5 - 30 min.

[0023] The present invention also discloses a single-molecule array chip based on high-entropy alloy plasmons, which is prepared by the preparation method of the single-molecule array chip based on high-entropy alloy plasmons of the present invention.

[0024] The beneficial effect of the present invention is that the single-molecule array chip based on high-entropy alloy plasmons prepared by the method of the present invention can significantly improve the signal-to-noise ratio of the single-molecule array chip, and further improve the accuracy and reliability of this technology in single-molecule immunoassay. Description of the Drawings

[0025] Figure 1 It is the topographic map of the single-molecule array chip in Example 1 under an atomic force microscope (AFM). Detailed Embodiments

[0026] The present invention will be further described below in conjunction with embodiments.

[0027] Example 1:

[0028] Prepare a single-molecule array chip according to the following steps:

[0029] (1) Prepare a high-entropy alloy coating material according to the mass ratio of the noble metal composite material and the synergistic composite metal material of 1:2, wherein the noble metal composite material is composed of gold and silver according to the mass ratio of 1:1.4, and the synergistic composite metal material is composed of ruthenium and palladium according to the mass ratio of 1:1.

[0030] (2) The surface of the substrate chip (silicon single crystal substrate chip, cut from a 4-inch wafer, with a thickness of 525 μm) is treated by ultrasonic cleaning to remove residual organic substances and oxides;

[0031] (3) Place the above high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coater, and place the substrate chip on a rotating coating table. Control the rotation speed at 30 rpm and the vacuum degree at 10 -6 torr, and start thermal evaporation thin film deposition at a heating temperature of 600 °C; during the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within When the thickness of the high-entropy alloy film on the coated chip reaches 100 nm, open the gas inlet valve to restore atmospheric pressure to release the sample, and obtain the coated chip.

[0032] (4) Clean the coated chip using a plasma cleaner (CPC-G / Gplus), use high-purity oxygen (oxygen purity ≥ 99.995%) as the cleaning gas, set the gas pressure at 0.5 mTorr, the gas flow rate at 12 sccm, the radio frequency power at 200 W, and the cleaning time at 25 min. After cleaning, gradually restore atmospheric pressure to complete the treatment and obtain a single-molecule array chip.

[0033] Use an atomic force microscope (AFM) to observe the surface morphology of the above single-molecule array chip, and the results are as Figure 1 shown.

[0034] Example 2:

[0035] Prepare a single-molecule array chip according to the following steps:

[0036] (1) Prepare the high-entropy alloy coating material according to the mass ratio of the noble metal composite material to the synergistic composite metal material of 1:3.5, where the noble metal composite material is composed of gold and silver in a mass ratio of 1:1, and the synergistic composite metal material is composed of ruthenium, palladium, and platinum in a mass ratio of 1:0.5.

[0037] (2) Perform surface treatment on the substrate chip (silicon single crystal substrate chip, cut from a 4-inch wafer, with a thickness of 525 μm) by argon plasma cleaning to remove residual organic substances and oxides;

[0038] (3) Place the above high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coater, and place the substrate chip on a rotating coating table. Control the rotation speed at 35 rpm and the vacuum degree at 10 -7 torr, and start thermal evaporation thin film deposition at a heating temperature of 520 °C; during the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within When the thickness of the high-entropy alloy film of the coated chip reaches 100 nm, open the gas inlet valve to restore atmospheric pressure to release the sample, and obtain the coated chip.

[0039] (4) Use a plasma cleaner (CPC-G / Gplus) to clean the coated chip. The cleaning gas is high-purity oxygen (oxygen purity ≥ 99.995%), set the gas pressure to 0.3 mTorr, the gas flow rate to 20 sccm, the radio frequency power to 450 W, and the cleaning time to 15 min. After cleaning, gradually restore atmospheric pressure to complete the treatment, and obtain the single-molecule array chip.

[0040] Example 3:

[0041] Prepare the single-molecule array chip according to the following steps:

[0042] (1) Prepare the high-entropy alloy coating material according to the mass ratio of the noble metal composite material to the synergistic composite metal material of 1:0.5. The noble metal composite material is composed of gold and silver according to the mass ratio of 1:0.2, and the synergistic composite metal material is composed of ruthenium and palladium according to the mass ratio of 1:1.5.

[0043] (2) Use ultrasonic cleaning to perform surface treatment on the substrate chip (silicon single crystal substrate chip, cut from a 4-inch wafer, thickness 525 μm) to remove residual organic matter and oxides;

[0044] (3) Place the above high-entropy alloy coating material in the vacuum chamber of the thermal evaporation coater, place the substrate chip on the rotating coating table, control the rotation speed to 20 rpm, the vacuum degree to 10 -6 torr, and start thermal evaporation thin film deposition at a heating temperature of 500 °C; during the deposition process, measure the deposition rate in real time through a quartz crystal microbalance, and adjust the evaporation rate by adjusting the heating power and the evaporation source temperature to control the deposition rate within When the thickness of the high-entropy alloy film of the coated chip reaches 100 nm, open the gas inlet valve to restore atmospheric pressure to release the sample, and obtain the coated chip.

[0045] (4) Use a plasma cleaner (CPC-G / Gplus) to clean the coated chip. The cleaning gas is high-purity oxygen (oxygen purity ≥ 99.995%), set the gas pressure to 0.8 mTorr, the gas flow rate to 6 sccm, the radio frequency power to 250 W, and the cleaning time to 30 min. After cleaning, gradually restore atmospheric pressure to complete the treatment, and obtain the single-molecule array chip.

[0046] Comparative Example 1:

[0047] This comparative example is a control experiment for Example 1, which is implemented according to the same steps and conditions as Example 1, and the only difference is that: it does not include the step of coating the base chip. The specific implementation steps are as follows:

[0048] (1) Use ultrasonic cleaning to perform surface treatment on the base chip (the same batch as Example 1, a silicon single crystal substrate base chip, cut from a 4-inch wafer, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0049] (2) Use a plasma cleaner (CPC-G / Gplus) to clean the above chip. The cleaning gas is high-purity oxygen (oxygen purity ≥ 99.995%), set the gas pressure to 0.5 mTorr, the gas flow rate to 12 sccm, the radio frequency power to 200 W, and the cleaning time to 25 min. After cleaning, gradually restore the atmospheric pressure to complete the treatment to obtain a single-molecule array chip.

[0050] Comparative Example 2:

[0051] This comparative example is a control experiment for Example 1, which is implemented according to the same steps and conditions as Example 1, and the only difference is that: it does not include the step of plasma cleaning the coated chip. The specific implementation steps are as follows:

[0052] (1) Prepare a high-entropy alloy coating material according to the mass ratio of the noble metal composite material to the synergistic composite metal material of 1:2, where the noble metal composite material is composed of gold and silver according to the mass ratio of 1:1.4, and the synergistic composite metal material is composed of ruthenium and palladium according to the mass ratio of 1:1.

[0053] (2) Use ultrasonic cleaning to perform surface treatment on the base chip (the same batch as Example 1, a silicon single crystal substrate base chip, cut from a 4-inch wafer, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0054] (3) Place the above high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coater, place the base chip on the rotating coating table, control the rotation speed to 30 rpm, the vacuum degree to 10 -6 torr, and the heating temperature to 600 °C to start thermal evaporation film deposition; during the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate to When the thickness of the high-entropy alloy film on the coated chip reaches 100 nm, open the gas inlet valve to restore the atmospheric pressure to release the sample to obtain a single-molecule array chip.

[0055] Comparative Example 3:

[0056] This comparative example is a control experiment for Example 1, which is implemented according to the same steps and conditions as Example 1, and the only difference is that: on the premise that the total coating thickness remains unchanged, only the noble metal composite material is used as the coating material. The specific implementation steps are as follows:

[0057] (1) The coating material is a noble metal composite material, and the noble metal composite material is composed of gold and silver in a mass ratio of 1:1.4.

[0058] (2) The substrate chip (the same batch as Example 1, a silicon single crystal substrate chip, cut from a 4-inch wafer, with a thickness of 525 μm) is surface-treated by ultrasonic cleaning to remove residual organic substances and oxides;

[0059] (3) The above noble metal composite material is placed in the vacuum chamber of a thermal evaporation coater, and the substrate chip is placed on a rotating coating table. The rotation speed is controlled at 30 rpm, the vacuum degree is 10 -6 torr, and the heating temperature is 600 °C to start thermal evaporation film deposition; during the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within When the noble metal film thickness of the coated chip reaches 100 nm, the gas inlet valve is opened to restore atmospheric pressure to release the sample, and a coated chip is obtained.

[0060] (4) The coated chip is cleaned using a plasma cleaner (CPC-G / Gplus). The cleaning gas is high-purity oxygen (oxygen purity ≥ 99.995%), the gas pressure is set at 0.5 mTorr, the gas flow rate is 12 sccm, the radio frequency power is 200 W, and the cleaning time is 25 min. After cleaning, the atmospheric pressure is gradually restored to complete the treatment, and a single-molecule array chip is obtained.

[0061] Comparative Example 4:

[0062] This comparative example is a control experiment for Example 1, which is implemented according to the same steps and conditions as Example 1, and the only difference is that: on the premise that the total coating thickness remains unchanged, only the synergistic composite metal material is used as the coating material. The specific implementation steps are as follows:

[0063] (1) The coating material is a synergistic composite metal material, and the synergistic composite metal material is composed of ruthenium and palladium in a mass ratio of 1:1.

[0064] (2) The substrate chip (the same batch as Example 1, a silicon single crystal substrate chip, cut from a 4-inch wafer, with a thickness of 525 μm) is surface-treated by ultrasonic cleaning to remove residual organic substances and oxides;

[0065] (3) Place the above high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coating machine, and place the substrate chip on a rotating coating table. Control the rotation speed at 30 rpm and the vacuum degree at 10 -6 torr, and start thermal evaporation thin film deposition when the heating temperature is 600 °C. During the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within When the thickness of the high-entropy alloy film on the coated chip reaches 100 nm, open the gas inlet valve to restore atmospheric pressure to release the sample, and obtain the coated chip.

[0066] (4) Use a plasma cleaner (CPC-G / Gplus) to clean the coated chip. The cleaning gas uses high-purity oxygen (oxygen purity ≥ 99.995%), set the gas pressure at 0.5 mTorr, the gas flow rate at 12 sccm, the radio frequency power at 200 W, and the cleaning time at 25 min. After cleaning, gradually restore atmospheric pressure to complete the treatment, and obtain a single-molecule array chip.

[0067] Comparative Example 5:

[0068] This comparative example is a control experiment of Example 1, and is implemented according to the same steps and conditions as Example 1, and the only difference is that: all of the synergistic composite metal material uses ruthenium (the mass ratio of the noble metal composite material and the synergistic composite metal material remains unchanged). The specific implementation steps are as follows:

[0069] (1) Prepare a high-entropy alloy coating material according to the mass ratio of the noble metal composite material and the synergistic composite metal material of 1:2. The noble metal composite material is composed of gold and silver according to the mass ratio of 1:1.4, and the synergistic composite metal material is ruthenium.

[0070] (2) Use ultrasonic cleaning to perform surface treatment on the substrate chip (the same batch as Example 1, a silicon single crystal substrate chip, cut from a 4-inch wafer, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0071] (3) Place the above high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coating machine, and place the substrate chip on a rotating coating table. Control the rotation speed at 30 rpm and the vacuum degree at 10 -6 torr, and start thermal evaporation thin film deposition when the heating temperature is 600 °C. During the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within When the thickness of the high-entropy alloy film on the coated chip reaches 100 nm, open the gas inlet valve to restore atmospheric pressure to release the sample, and obtain the coated chip.

[0072] (4) Clean the coated chip using a plasma cleaner (CPC-G / Gplus). Use high-purity oxygen (oxygen purity ≥ 99.995%) as the cleaning gas, set the gas pressure to 0.5 mTorr, the gas flow rate to 12 sccm, the RF power to 200 W, and the cleaning time to 25 min. After cleaning, gradually restore the atmospheric pressure to complete the treatment and obtain a single-molecule array chip.

[0073] Comparative Example 6:

[0074] This comparative example is a control experiment for Example 1, which is carried out according to the same steps and conditions as Example 1, and the only difference is that: all of the synergistic composite metal material is palladium (the mass ratio of the noble metal composite material and the synergistic composite metal material remains unchanged). The specific implementation steps are as follows:

[0075] (1) Prepare a high-entropy alloy coating material according to the mass ratio of the noble metal composite material and the synergistic composite metal material of 1:2. The noble metal composite material is composed of gold and silver according to the mass ratio of 1:1.4, and the synergistic composite metal material is palladium.

[0076] (2) Perform surface treatment on the substrate chip (the same batch as Example 1, a silicon single-crystal substrate chip, cut from a 4-inch wafer, with a thickness of 525 μm) using ultrasonic cleaning to remove residual organic matter and oxides;

[0077] (3) Place the above high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coater, and place the substrate chip on a rotating coating table. Control the rotation speed to 30 rpm and the vacuum degree to 10 -6 torr, and start thermal evaporation thin film deposition at a heating temperature of 600 °C; during the deposition process, measure the deposition rate in real time through a quartz crystal microbalance, and adjust the evaporation rate by adjusting the heating power and the evaporation source temperature to control the deposition rate to When the thickness of the high-entropy alloy film on the coated chip reaches 100 nm, open the gas inlet valve to restore the atmospheric pressure to release the sample and obtain a coated chip.

[0078] (4) Clean the coated chip using a plasma cleaner (CPC-G / Gplus). Use high-purity oxygen (oxygen purity ≥ 99.995%) as the cleaning gas, set the gas pressure to 0.5 mTorr, the gas flow rate to 12 sccm, the RF power to 200 W, and the cleaning time to 25 min. After cleaning, gradually restore the atmospheric pressure to complete the treatment and obtain a single-molecule array chip. 〔

[0079] Bioanalysis experiment of low-abundance substances:

[0080] Modify magnetic beads with Tau217 primary antibody, modify Tau-217 secondary antibody with NHS-Fitc, capture Tau-217 antigens at different concentrations using one-step method and sandwich method. After capture, drop the complex on the single-molecule array chip and perform statistical analysis of single-molecule signals. The specific experimental methods are as follows:

[0081] I. Experimental materials:

[0082] Single-molecule array chips prepared in Examples 1 to 3 and Comparative Examples 1 to 6;

[0083] Tau217 primary antibody: Antibody used to modify magnetic beads;

[0084] NHS-FITC: Fluorescent dye used to modify Tau217 secondary antibody;

[0085] Tau217 antigen: Target molecule for capture;

[0086] Magnetic beads: Used to modify Tau217 primary antibody;

[0087] PBS buffer: Used for dilution and washing.

[0088] II. Experimental steps:

[0089] 1. Modification of magnetic beads:

[0090] (1) Dilute Tau217 primary antibody to a final concentration of 10 μg / mL;

[0091] (2) Add Tau217 primary antibody to magnetic beads, react at 25 °C for 1 hour;

[0092] 2. Modification of Tau217 secondary antibody:

[0093] (1) Dissolve NHS-FITC in PBS buffer at a concentration of 1 mg / mL;

[0094] (2) Add NHS-FITC to Tau217 secondary antibody to form Tau217 secondary antibody-NHS-FITC complex, react at 25 °C for 2 hours;

[0095] 3. Capture of target molecules:

[0096] Use the one-step method to add Tau217 antigen solutions at different concentrations (0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL) to the modified magnetic beads, react at 4 °C for 12 hours;

[0097] 4. Application of the complex:

[0098] Drop the captured Tau217 antigen complex on the single-molecule array chip, and use 10 μL of the complex solution for each chip;

[0099] 5. Imaging analysis:

[0100] Perform fluorescence imaging on the single-molecule array chip using a fluorescence microscope (Zeiss Axio Observer);

[0101] Use the FITC channel (excitation wavelength: 488 nm, emission wavelength: 515 nm) to detect and record the fluorescence signal; count the number of single-molecule signals under the capture conditions of Tau217 antigen solutions with different concentrations, and the results are shown in Table 1.

[0102] Table 1 Statistical table of the number of single-molecule signals under the capture conditions of Tau217 antigen solutions with different concentrations

[0103]

[0104] It can be seen from the detection results of Examples 1 to 3 in Table 1 that the single-molecule array chip prepared by the method of the present invention can generate stable single-molecule signals under the capture conditions of Tau217 antigen solutions with concentrations ranging from 0.1 pg / mL to 1 ng / mL, and has the advantages of high detection sensitivity and stable signal intensity.

[0105] It can be seen from the comparison of the detection results of Example 1 and Comparative Example 1 in Table 1 that, compared with the ordinary single-molecule array chip without metal coating, the single-molecule array chip of the present invention has significantly higher signal intensity, and the sensitivity is increased by about two orders of magnitude.

[0106] It can be seen from the comparison of Example 1 and Comparative Example 2 in Table 1 that the single-molecule signal intensity can be significantly improved through the plasma cleaning step of the present invention.

[0107] It can be seen from the comparison of Example 1, Comparative Example 3 and Comparative Example 4 in Table 1 that, on the premise that the coating thickness is exactly the same, the single-molecule signal intensities captured by Tau217 antigen solutions with different concentrations in Example 1 are significantly higher than those of Comparative Example 3 using only noble metal coating and Comparative Example 4 using only synergistic composite metal coating. It can be seen that the noble metal composite material and the synergistic composite metal material of the present invention have an obvious synergistic effect in improving the single-molecule signal intensity.

[0108] It can be seen from the comparison of Example 1, Comparative Example 5 and Comparative Example 6 in Table 1 that under the conditions of the same coating thickness and the same mass ratio of the noble metal composite material and the synergistic metal material, the single-molecule signal intensities captured by the Tau217 antigen solutions with different concentrations in Example 1 are significantly higher than those in Comparative Example 5 using ruthenium alone as the synergistic metal material and Comparative Example 6 using palladium alone as the synergistic metal material. It can be seen that the components of the synergistic composite metal material of the present invention have an obvious synergistic effect of improving the single-molecule signal intensity.

Claims

1. A method for preparing a single-molecule array chip based on high-entropy alloy plasmons, characterized in that: The production raw materials include a substrate chip and a high-entropy alloy coating material; the high-entropy alloy coating material is composed of a noble metal composite material and a synergistic composite metal material in a mass ratio of 1:0.12 to 3.75; the noble metal composite material is composed of gold and silver in a mass ratio of 1:0.08 to 3; the synergistic composite metal material is composed of ruthenium and palladium in a mass ratio of 1:0.2 to 1.

5.

2. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 1, wherein: It also includes the step of forming a high-entropy alloy film on the substrate chip to obtain a coated chip.

3. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 2, characterized in that: The thickness of the high-entropy alloy film of the coated chip is 80 to 120 nm.

4. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 3, characterized in that: The substrate chip is a single-crystal silicon substrate chip.

5. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 3, wherein: The preparation steps of the coated chip are specifically: depositing the high-entropy alloy coating material on the substrate chip by thermal evaporation thin film deposition technology to obtain a coated chip.

6. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 3, wherein: The preparation steps of the coated chip are specifically: depositing the high-entropy alloy coating material on the substrate chip by magnetron sputtering coating technology to obtain a coated chip.

7. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 5, characterized in that, The preparation steps of the coated chip are specifically: S1. Perform surface treatment on the substrate chip by ultrasonic cleaning or argon plasma cleaning to remove residual organic matter and oxides; S2. Place the high-entropy alloy coating material in the vacuum chamber of a thermal evaporation coating machine, and place the substrate chip on a rotating coating table. Control the rotation speed to be 20 - 50 rpm, the vacuum degree to be 10 -6 - 10 -7 torr, and start thermal evaporation thin film deposition when the heating temperature is 500 - 600 °C; During the deposition process, the deposition rate is measured in real time by a quartz crystal microbalance, and the evaporation rate is adjusted by adjusting the heating power and the evaporation source temperature to control the deposition rate within 1 to 2 Å / s; S3. Release the sample by gradually increasing the ambient pressure or opening the gas inlet valve to restore atmospheric pressure to obtain a coated chip.

8. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to any one of claims 2 to 7, characterized in that: It also includes the step of performing plasma cleaning on the coated chip.

9. The preparation method of the single-molecule array chip based on high-entropy alloy plasmons according to claim 8, characterized in that, The plasma cleaning parameters are: using high-purity oxygen as the cleaning gas, setting the gas pressure to 0.1 to 1 mTorr, the gas flow rate to 5 to 20 sccm, the radio frequency power to 100 to 500 W, and the cleaning time to 5 to 30 min.

10. A single-molecule array chip based on high-entropy alloy plasmon prepared by the preparation method of the single-molecule array chip based on high-entropy alloy plasmon according to any one of claims 1 to 9.

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