Plasma single-molecule array chip with enhanced noble metal signal and its preparation method

Abstract

The present invention relates to the field of single-molecule immunoassay chips, specifically disclosing a precious metal signal-enhanced plasma single-molecule array chip and its preparation method. The raw materials for production include a substrate chip and a composite coating material; the composite coating material comprises a precious metal composite material and a synergistic composite metal material in a mass ratio of 1:0.03-2; the precious metal composite material comprises gold and platinum in a mass ratio of 1:0.01-0.5; and the synergistic composite metal material comprises ruthenium and palladium in a mass ratio of 1:0.2-1.5. This method has the advantage of significantly improving the signal-to-noise ratio of the single-molecule array chip, further enhancing the accuracy and reliability of the technology in single-molecule immunoassay analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection chips, in particular to the production technology of single molecule immune detection chips. Background Art

[0002] Single Molecule Array (SiMoA) technology is a method that uses single-molecule counting to detect trace biomarkers. With a detection limit of femtograms (fg / mL), this technology is currently the most representative single-molecule immunoassay technology. The system is derived from a detection system developed by David Walt and David Duffy's team. Its detection principle is similar to digital PCR, using a microreactor unit for single-molecule isolation combined with single-molecule-level signal amplification.

[0003] SiMoA technology is a single-molecule immunoassay based on a microarray chip. It involves carving (or pouring) thousands of micron-sized microwells on a millimeter-sized chip, with each microwell having a volume of approximately 40 fl (i.e., a single-molecule array chip). Immune complex magnetic beads are then distributed within the microwells. Fluorescent spots are then counted using a high-resolution fluorescence microscope. Based on Poisson distribution theory, the ratio of the number of wells containing both beads and fluorescent products to the total number of wells containing beads is calculated to determine the concentration of the analyte in the test sample. The general operating procedure is as follows:

[0004] (1) Capturing antigens in the sample using magnetic beads labeled with capture antibodies;

[0005] (2) labeling the captured antigen with a biotinylated detection antibody;

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

[0007] (4) The washed magnetic beads are mixed with the substrate and loaded into a single-molecule array chip. A magnetic field is used to make the magnetic beads fall into microwells that exactly match their size. An oil phase is added to physically isolate the microwells.

[0008] (5) The microwell containing galactosidase produces fluorescent products due to the catalysis of the substrate by the enzyme molecules;

[0009] (6) Fluorescence imaging of the microwell array is performed, and quantitative detection is achieved by comparing the number of microwells emitting fluorescent signals with a standard curve.

[0010] One of the main challenges of current SiMoA technology is the low sensitivity of its single-array signal, which directly impacts detection accuracy and reliability. 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 plasma single-molecule array chip with noble metal signal enhancement and a preparation method thereof.

[0012] The technical solution adopted by the present invention is: a method for preparing a plasma single-molecule array chip with enhanced precious metal signals, wherein the production raw materials include a substrate chip and a composite coating material; the composite coating material is composed of a precious metal composite material and a synergistic composite metal material in a mass ratio of 1:0.03 to 2; the precious metal composite material is composed of gold and platinum in a mass ratio of 1:0.01 to 0.5; 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 composite metal film on the substrate chip to obtain a coated chip. More preferably, the composite metal film of the coated chip has a thickness of 60 to 105 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 composite coating material on the substrate chip through a thermal evaporation thin film deposition technique, or by depositing the composite coating material on the substrate chip through a magnetron sputtering coating technique.

[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 composite 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 15-100 rpm, and the vacuum degree to 10 -6 ~10 -7torr, the heating temperature is 500-600℃ 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

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

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

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

[0022] The parameters of the 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 RF power to 100-500 W, and the cleaning time to 5-30 min.

[0023] The present invention also discloses a plasma single molecule array chip with enhanced noble metal signals, which is prepared by the preparation method of the plasma single molecule array chip with enhanced noble metal signals of the present invention.

[0024] The beneficial effects of the present invention are: the noble metal signal-enhanced plasma single-molecule array chip prepared by the method of the present invention can significantly improve the signal-to-noise ratio of the single-molecule array chip, further improving the accuracy and reliability of the technology in single-molecule immunoassay. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a morphology image of the single molecule array chip of Example 1 under an atomic force microscope (AFM). DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to the embodiments.

[0027] Example 1:

[0028] Prepare a single molecule array chip as follows:

[0029] (1) preparing a composite coating material in a ratio of 1:1.5 between a precious metal composite material and a synergistic composite metal material, wherein the precious metal composite material is composed of gold and platinum in a ratio of 1:0.2, and the synergistic composite metal material is composed of ruthenium and palladium in a ratio of 1:1.

[0030] (2) Ultrasonic cleaning was used to treat the surface of the substrate chip (silicon single crystal substrate chip, cut from 4 inches, 525 μm thick) to remove residual organic matter and oxides;

[0031] (3) The composite coating material was placed in the vacuum chamber of the thermal evaporation coating machine, and the substrate chip was placed on the rotary coating table. The speed was controlled to 55 rpm and the vacuum degree was 5×10 -7 torr, the heating temperature is 550℃ to start the 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 thickness of the composite metal film of the coated chip reaches 85 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample to obtain the coated chip.

[0032] (4) The coated chip was cleaned using a plasma cleaner (CPC-G / Gplus). High-purity oxygen (oxygen purity ≥ 99.995%) was used as the cleaning gas. The gas pressure was set to 0.5 mTorr, the gas flow rate was 12 sccm, the RF power was set to 200 W, and the cleaning time was 25 min. After cleaning, atmospheric pressure was gradually restored to complete the treatment, resulting in a single-molecule array chip.

[0033] The surface morphology of the single molecule array chip was observed using an atomic force microscope (AFM). Figure 1 shown.

[0034] Example 2:

[0035] Prepare a single molecule array chip as follows:

[0036] (1) preparing a composite coating material in a ratio of 1:2 between the precious metal composite material and the synergistic composite metal material, wherein the precious metal composite material is composed of gold and platinum in a ratio of 1:0.5, and the synergistic composite metal material is composed of ruthenium, palladium and platinum in a ratio of 1:0.5.

[0037] (2) Argon plasma cleaning was used to treat the surface of the substrate chip (silicon single crystal substrate substrate chip, cut from 4 inches, 525 μm thick) to remove residual organic matter and oxides;

[0038] (3) Place the composite coating material in the vacuum chamber of the thermal evaporation coating machine, place the substrate chip on the rotary coating table, control the speed to 20 rpm, and the vacuum degree to 10 -7 torr, the heating temperature is 600℃ to start the 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 thickness of the composite metal film of the coated chip reaches 105 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample to obtain the coated chip.

[0039] (4) The coated chip was cleaned using a plasma cleaner (CPC-G / Gplus). High-purity oxygen (oxygen purity ≥ 99.995%) was used as the cleaning gas. The gas pressure was set to 0.8 mTorr, the gas flow rate was 5 sccm, the RF power was set to 500 W, and the cleaning time was 30 min. After cleaning, atmospheric pressure was gradually restored to complete the treatment, resulting in a single-molecule array chip.

[0040] Example 3:

[0041] Prepare a single molecule array chip as follows:

[0042] (1) preparing a composite coating material in a ratio of 1:0.7 between a precious metal composite material and a synergistic composite metal material, wherein the precious metal composite material is composed of gold and platinum in a ratio of 1:0.1, and the synergistic composite metal material is composed of ruthenium and palladium in a ratio of 1:1.5.

[0043] (2) Ultrasonic cleaning was used to treat the surface of the substrate chip (silicon single crystal substrate chip, cut from 4 inches, 525 μm thick) to remove residual organic matter and oxides;

[0044] (3) Place the composite coating material in the vacuum chamber of the thermal evaporation coating machine, place the substrate chip on the rotary coating table, control the speed to 80 rpm, and the vacuum degree to 3×10 -7 torr, the heating temperature is 500℃ to start the 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 thickness of the composite metal film of the coated chip reaches 80 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample to obtain the coated chip.

[0045] (4) The coated chip was cleaned using a plasma cleaner (CPC-G / Gplus). High-purity oxygen (oxygen purity ≥ 99.995%) was used as the cleaning gas. The gas pressure was set to 0.7 mTorr, the gas flow rate was 17 sccm, the RF power was set to 350 W, and the cleaning time was 20 min. After cleaning, atmospheric pressure was gradually restored to complete the treatment, resulting in a single-molecule array chip.

[0046] Comparative Example 1:

[0047] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1, except that the step of coating the substrate chip is not included. The specific implementation steps are as follows:

[0048] (1) Using ultrasonic cleaning to treat the surface of the substrate chip (from the same batch as in Example 1, silicon single crystal substrate substrate chip, cut from 4 inches, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0049] (2) The chip was cleaned using a plasma cleaner (CPC-G / Gplus) using high-purity oxygen (≥99.995%) as the cleaning gas. The gas pressure was set 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, atmospheric pressure was gradually restored to complete the treatment, resulting in a single-molecule array chip.

[0050] Comparative Example 2:

[0051] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1, with the only difference being that the step of plasma cleaning the coated chip is not included. The specific implementation steps are as follows:

[0052] (1) preparing a composite coating material in a ratio of 1:1.5 between a precious metal composite material and a synergistic composite metal material, wherein the precious metal composite material is composed of gold and platinum in a ratio of 1:0.2, and the synergistic composite metal material is composed of ruthenium and palladium in a ratio of 1:1.

[0053] (2) Ultrasonic cleaning was used to treat the surface of the substrate chip (from the same batch as in Example 1, silicon single crystal substrate substrate chip, cut from 4 inches, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0054] (3) The composite coating material was placed in the vacuum chamber of the thermal evaporation coating machine, and the substrate chip was placed on the rotary coating table. The speed was controlled to 55 rpm and the vacuum degree was 5×10 -7 torr, the heating temperature is 550℃ to start the 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 thickness of the composite metal film of the coated chip reaches 85 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample, thereby obtaining a single molecule array chip.

[0055] Comparative Example 3:

[0056] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1, with the only difference being that, under the premise that the total thickness of the coating remains unchanged, the coating material is only the precious metal composite material. The specific implementation steps are as follows:

[0057] (1) The coating material is a precious metal composite material, which is composed of gold and platinum in a mass ratio of 1:0.2.

[0058] (2) Ultrasonic cleaning was used to treat the surface of the substrate chip (from the same batch as in Example 1, silicon single crystal substrate substrate chip, cut from 4 inches, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0059] (3) The above-mentioned precious metal composite material was placed in the vacuum chamber of the thermal evaporation coating machine, and the substrate chip was placed on the rotary coating table. The speed was controlled to 55 rpm and the vacuum degree was 5×10 -7 torr, the heating temperature is 550℃ to start the 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 thickness of the noble metal film on the coated chip reaches 85 nm, the gas inlet valve is opened to restore atmospheric pressure to release the sample to obtain the coated chip.

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

[0061] Comparative Example 4:

[0062] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1, with the only difference being that, under the premise that the total thickness of the coating remains unchanged, the coating material is only the synergistic composite metal 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) Ultrasonic cleaning was used to treat the surface of the substrate chip (from the same batch as in Example 1, silicon single crystal substrate substrate chip, cut from 4 inches, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0065] (3) The composite coating material was placed in the vacuum chamber of the thermal evaporation coating machine, and the substrate chip was placed on the rotary coating table. The speed was controlled to 55 rpm and the vacuum degree was 5×10 -7 torr, the heating temperature is 550℃ to start the 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 thickness of the composite metal film of the coated chip reaches 85 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample to obtain the coated chip.

[0066] (4) The coated chip was cleaned using a plasma cleaner (CPC-G / Gplus). High-purity oxygen (oxygen purity ≥ 99.995%) was used as the cleaning gas. The gas pressure was set to 0.5 mTorr, the gas flow rate was 12 sccm, the RF power was set to 200 W, and the cleaning time was 25 min. After cleaning, atmospheric pressure was gradually restored to complete the treatment, resulting in a single-molecule array chip.

[0067] Comparative Example 5:

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

[0069] (1) preparing a composite coating material in a ratio of 1:1.5 between the precious metal composite material and the synergistic composite metal material, wherein the precious metal composite material is composed of gold and platinum in a ratio of 1:0.2, and the synergistic composite metal material is ruthenium.

[0070] (2) Ultrasonic cleaning was used to treat the surface of the substrate chip (from the same batch as in Example 1, silicon single crystal substrate substrate chip, cut from 4 inches, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0071] (3) The composite coating material was placed in the vacuum chamber of the thermal evaporation coating machine, and the substrate chip was placed on the rotary coating table. The speed was controlled to 55 rpm and the vacuum degree was 5×10 -7 torr, the heating temperature is 550℃ to start the 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 thickness of the composite metal film of the coated chip reaches 85 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample to obtain the coated chip.

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

[0073] Comparative Example 6:

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

[0075] (1) preparing a composite coating material in a ratio of 1:1.5 between the precious metal composite material and the synergistic composite metal material, wherein the precious metal composite material is composed of gold and platinum in a ratio of 1:0.2, and the synergistic composite metal material is palladium;

[0076] (2) Ultrasonic cleaning was used to treat the surface of the substrate chip (from the same batch as in Example 1, silicon single crystal substrate substrate chip, cut from 4 inches, with a thickness of 525 μm) to remove residual organic matter and oxides;

[0077] (3) The composite coating material was placed in the vacuum chamber of the thermal evaporation coating machine, and the substrate chip was placed on the rotary coating table. The speed was controlled to 55 rpm and the vacuum degree was 5×10 -7 torr, the heating temperature is 550℃ to start the 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 thickness of the composite metal film of the coated chip reaches 85 nm, the gas inlet valve is opened to restore the atmospheric pressure to release the sample to obtain the coated chip.

[0078] (4) The coated chip was cleaned using a plasma cleaner (CPC-G / Gplus). High-purity oxygen (oxygen purity ≥ 99.995%) was used as the cleaning gas. The gas pressure was set to 0.5 mTorr, the gas flow rate was 12 sccm, the RF power was set to 200 W, and the cleaning time was 25 min. After cleaning, atmospheric pressure was gradually restored to complete the treatment, resulting in a single-molecule array chip.

[0079] Bioanalysis experiments of low-abundance substances:

[0080] Magnetic beads were modified with Tau217 primary antibody, and Tau-217 secondary antibody was modified with NHS-Fitc. Tau-217 antigen at different concentrations was captured using a one-step method and a sandwich method. After capture, the complex was dropped onto a single-molecule array chip for single-molecule signal statistical analysis. The specific experimental method is as follows:

[0081] 1. Experimental materials:

[0082] The 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] 2. Experimental steps:

[0089] 1. Modification of magnetic beads:

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

[0091] (2) Add Tau217 primary antibody to the magnetic beads at a reaction temperature of 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) NHS-FITC was added to the Tau217 secondary antibody to form a Tau217 secondary antibody-NHS-FITC complex. The reaction temperature was 25°C and the reaction time was 2 hours.

[0095] 3. Capture target molecules:

[0096] Using a one-step method, different concentrations of Tau217 antigen solutions (0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, and 1 ng / mL) were added to the modified magnetic beads. The reaction temperature was 4°C and the reaction time was 12 hours.

[0097] 4. Application of the compound:

[0098] The captured Tau217 antigen complex was dropped onto the single molecule array chip, using 10 μL of complex solution for each chip;

[0099] 5. Imaging analysis:

[0100] Fluorescence imaging of the single-molecule array chip was performed using a fluorescence microscope (Zeiss Axio Observer);

[0101] The fluorescence signal was detected and recorded using the FITC channel (excitation wavelength: 488 nm, emission wavelength: 515 nm); the number of single-molecule signals under capture conditions of Tau217 antigen solutions with different concentrations was counted, and the results are shown in Table 1.

[0102] Table 1 Statistics of single molecule signals under capture conditions of Tau217 antigen solutions with different concentrations

[0103]

[0104] As can be seen from the detection results of Examples 1 to 3 in Table 1, 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 solution at a concentration of 0.1 pg / mL to 1 ng / mL, and has the advantages of high detection sensitivity and stable signal intensity.

[0105] From the comparison of the detection results of Example 1 and Comparative Example 1 in Table 1, it can be seen that the single molecule array chip of the present invention has significantly higher signal intensity than the ordinary single molecule array chip without metal coating.

[0106] From the comparison between Example 1 and Comparative Example 2 in Table 1, it can be seen that the plasma cleaning step of the present invention can significantly improve the single molecule signal intensity.

[0107] From the comparison of Example 1, Comparative Example 3 and Comparative Example 4 in Table 1, it can be seen that, under the premise of completely the same coating thickness, the single-molecule signal intensity captured by the Tau217 antigen solution of different concentrations in Example 1 is significantly higher than that of Comparative Example 3 using only the precious metal coating and Comparative Example 4 using only the synergistic composite metal coating. It can be seen that the precious metal composite material and the synergistic composite metal material of the present invention have a significant synergistic effect in improving the single-molecule signal intensity.

[0108] From the comparison of Example 1, Comparative Example 5 and Comparative Example 6 in Table 1, it can be seen that under the conditions that the coating thickness is exactly the same and the mass ratio of the precious metal composite material and the synergistic metal material is also the same, the single-molecule signal intensity captured by the Tau217 antigen solution of different concentrations in Example 1 is significantly higher than that of 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 a significant synergistic effect in improving the single-molecule signal intensity.

Claims

1. A method for preparing a plasmonic single-molecule array chip with noble metal signal enhancement, characterized in that: The production raw materials include a substrate chip and a composite coating material; the composite coating material is composed of a precious metal composite material and a synergistic composite metal material in a mass ratio of 1:0.03 to 2; the precious metal composite material is composed of gold and platinum in a mass ratio of 1:0.01 to 0.5; and the synergistic composite metal material is composed of ruthenium and palladium in a mass ratio of 1:0.2 to 1.

5.

2. The method for preparing a plasmonic single-molecule array chip with noble metal signal enhancement according to claim 1, characterized in that: The method also includes the step of forming a composite metal film on the substrate chip to obtain a film-coated chip.

3. The method for preparing a plasmonic single-molecule array chip with noble metal signal enhancement according to claim 2, characterized in that: The thickness of the composite metal film of the coating chip is 60-105 nm.

4. The method for preparing a plasmonic single molecule array chip with noble metal signal enhancement according to claim 3, characterized in that: The base chip is a silicon single crystal substrate base chip.

5. The method for preparing a plasmonic single molecule array chip with noble metal signal enhancement according to claim 3, characterized in that: The specific steps of preparing the coating chip are: depositing the composite coating material on the substrate chip by thermal evaporation thin film deposition technology, thereby obtaining the coating chip.

6. The method for preparing a plasmonic single-molecule array chip with noble metal signal enhancement according to claim 3, characterized in that: The specific steps of preparing the coating chip are: depositing the composite coating material on the substrate chip by magnetron sputtering coating technology, thereby obtaining the coating chip.

7. The method for preparing a plasmonic single molecule array chip with noble metal signal enhancement according to claim 5, characterized in that: The steps for preparing the coated chip are specifically as follows: S1. Surface treatment of the substrate chip is performed by ultrasonic cleaning or argon plasma cleaning to remove residual organic matter and oxides; S2, placing the composite 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 15-100 rpm, and the vacuum degree to 10 -6 ~10 -7 torr, the heating temperature is 500-600℃ to start thermal evaporation film deposition; During the deposition process, the deposition rate was measured in real time by a quartz crystal microbalance, and the evaporation rate was adjusted by adjusting the heating power and evaporation source temperature to control the deposition rate at 1-2 Å / s. S3. Restore the atmospheric pressure by gradually increasing the ambient pressure or opening the gas inlet valve to release the sample to obtain a coated chip.

8. The method for preparing a plasmonic single molecule array chip with noble metal signal enhancement according to any one of claims 2 to 7, characterized in that: The method also includes a step of plasma cleaning the coated chip.

9. The method for preparing a plasmonic single molecule array chip with noble metal signal enhancement according to claim 8, characterized in that: The plasma cleaning parameters are as follows: using high-purity oxygen as the cleaning gas, setting 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. 10 . A plasmonic single molecule array chip with enhanced noble metal signals produced by the method for producing a plasmonic single molecule array chip with enhanced noble metal signals according to any one of claims 1 to 9 .

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