Preparation method of Raman enhanced substrate applied to serum biomarker detection

Abstract

The invention discloses a preparation method of a Raman enhanced substrate applied to serum biomarker detection. Dielectric microsphere cavities are densely laid on the surface of a flexible high polymer material through a mechanical assembly method to form a micro-lens array, and a nanopore array is formed in the micro-lens array through ultrafast laser irradiation. And then gold nanoparticles are deposited in the nanopores through ion sputtering to form the novel composite structure substrate. Finally, based on excellent sensitivity and uniformity of the composite structure substrate, effective enhancement of a serum Raman spectrum and specific recognition of a biomarker are realized. The gold nanoparticle modified nanopore array composite structure substrate obtained by the invention has trace sample collection capability and excellent Raman enhancement performance, successfully realizes specific detection of serum biomarkers, and provides a new scheme for construction of a substrate with low cost, high sensitivity, high stability and accurate quantitative analysis for practical application.

Description

Technical Field

[0001] The present invention relates to the field of Raman enhancement technology, and provides a method for preparing a Raman enhancement substrate for serum marker detection. Background Art

[0002] In recent years, progress in metabolomics research has revealed the potential value of metabolic biomarkers in clinical diagnosis. By analyzing the composition and concentration changes of biomarkers in the blood (such as amino acids, lipids, carbohydrates, organic acids, etc.), it is possible to provide multi-level information, reflecting the interaction between physiological and pathological states and environmental factors, and is of great value in disease diagnosis, mechanism research and personalized treatment. Biomarker-based diagnostic methods have many advantages, such as non-invasive sample collection and the ability to detect subtle metabolic changes before clinical symptoms appear. Therefore, integrating the metabolic changes of biomarkers into clinical diagnostic strategies can significantly improve the accuracy of diagnosis, thereby achieving early intervention, improving disease management, and ultimately improving patient survival. However, the identification of blood biomarkers still faces challenges such as a wide variety, complex sample processing procedures, and low detection sensitivity.

[0003] Surface-enhanced Raman spectroscopy (SERS), an ultrasensitive biomolecular sensing tool based on molecular vibrational signatures, has attracted considerable attention due to its unique fingerprinting capabilities. This technology has been widely applied to the early diagnosis of major diseases such as cancer, neurological disorders, and cardiovascular disease through tissue biopsy, liquid biopsy, and cell identification, and has achieved significant progress. In particular, Raman spectroscopy analysis based on liquid biopsy, due to its noninvasive and real-time monitoring capabilities, plays a vital role in the process from early diagnosis to prognosis assessment. Suitable body fluids include blood, urine, sweat, saliva, and tears. Serum, a blood fraction, is rich in nucleic acids, RNA, proteins, and lipids, and can reflect the metabolic state of physiological systems. Recent studies have demonstrated that serum SERS technology can be used to diagnose a variety of diseases, such as systemic lupus erythematosus, breast cancer, and lung cancer. However, conventional SERS analysis methods have encountered bottlenecks in accurate quantification, particularly in the reliability and reproducibility of trace analysis. The fluctuations in Raman signal intensity caused by the heterogeneity of the adsorption / desorption process of the analyte molecules and the random distribution of hotspots have limited the widespread application of SERS in quantitative biosensing. The preparation of high-performance SERS substrates still faces challenges. For example, complex processes such as ultrafast laser direct writing, photolithography, and nanoimprinting result in long manufacturing times and high costs, which seriously restricts the promotion and application of new SERS substrates. Therefore, the development of highly sensitive Raman-enhanced substrates with practical application capabilities has become an urgent need.

[0004] Laser parallel processing based on the microsphere photonic nanojet effect can generate millions of focused spots in a single laser beam by modulating the light field. At the same time, the position of the focused spots can be changed to achieve efficient parallel nanomanufacturing. It has been proven to be an effective means to develop uniform SERS substrates. At the same time, digital surface-enhanced Raman scattering technology (dSERS) introduces a new analysis method that converts continuous analog Raman spectra into discrete probabilistic statistical events, thereby achieving robust statistical analysis. Unlike traditional SERS analysis methods, dSERS relies on the relative threshold of Raman shift intensity rather than the absolute value in quantitative analysis. This method eliminates the influence of fluctuations in Raman signal intensity on quantitative analysis. Therefore, the use of microsphere parallel laser processing technology and dSERS analysis is an effective solution to improve quantitative detection and processing efficiency. This solution realizes accurate quantitative analysis of Raman tests and realizes serum biomarker detection, which has extremely important scientific research significance and potential application value. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a Raman-enhanced substrate for use in serum marker detection. A mechanical assembly method is used to densely lay dielectric microsphere cavities on the surface of a flexible polymer material to form a microlens array, and ultrafast laser irradiation is used to form a nanopore array within the microlens array. Subsequently, gold nanoparticles are deposited within the nanopores by ion sputtering to form a composite structure SERS substrate. Finally, based on the high degree of independence of data acquisition of the composite structure substrate, a digital SERS analysis paradigm is introduced to analyze the data. The gold nanoparticle-modified nanopore array composite structure substrate obtained by the present invention has trace sample acquisition capability and excellent Raman enhancement performance, and realizes the detection of serum biomarkers.

[0006] Another object of the present invention is to provide a method for preparing a SERS substrate with high efficiency.

[0007] The technical solution adopted to achieve the purpose of the present invention is:

[0008] A method for preparing a Raman enhanced substrate for serum marker detection comprises the following steps:

[0009] Step 1: ultrasonically clean the glass substrate in acetone, ethanol, and deionized water in sequence, dry it, and then take it out for use;

[0010] Step 2: Mix polydimethylsiloxane (PDMS) and curing agent (diphenylurethane, DBP) in a weight ratio of 10:1 and stir evenly. Place in a degassing machine to remove bubbles.

[0011] Step 3: Drop the PDMS liquid onto a clean glass substrate and spin coat it at 500-1000 rpm / min for 50 seconds. Then, place the PDMS on a heating platform and heat it at 100°C for 2-5 minutes to form a PDMS film.

[0012] Step 4: Spread the microsphere lenses on the surface of the PDMS film and press to form a single-layer microsphere lens array;

[0013] Step 5: Use a vacuum ion sputtering coating instrument to coat gold on the surface of the microsphere lens array. The thickness of the gold film is controlled by the current and coating time, and is measured using the crystal oscillator film thickness detection device built into the instrument.

[0014] Step 6: The gold-coated microsphere array is mounted on a three-dimensional, electrically controlled displacement stage and illuminated from the backside with a femtosecond laser. A variable neutral density filter is used to control the laser energy reaching the sample surface, and a mechanical shutter controls the number of pulses.

[0015] Step 7: Deposit gold nanoparticles on top of the substrate using an ion sputtering system.

[0016] Step 8: Transfer 1-10 μL of serum stock solution to a centrifuge tube and dilute with PBS buffer;

[0017] Step 9: Take 5-10 μL of diluted serum sample and drop it onto the surface of the composite substrate. After the serum sample is captured by the composite substrate, the spectrum of the substrate is collected.

[0018] In the above technical solution, in step 4, the transparent medium microspheres have a diameter of 1-7 μm, a refractive index of 1.4682, and are made of silicon dioxide.

[0019] In the above technical solution, in step 5, the thickness of the gold film formed by ion sputtering is 5-30 nm.

[0020] In the above technical solution, in step 6, the laser energy density is controlled in the range of 55-130mJ / cm 2 800nm femtosecond pulse laser with a pulse width of 50-100fs and a pulse number of 50-300.

[0021] In the above technical solution, in step 7, the gold nanoparticle deposition time is 1-5 minutes.

[0022] In the above technical solution, in step 8, the dilution multiple of the serum is 2-5 times.

[0023] In the above technical solution, in step 9, the Raman spectrum acquisition time is 30-50 seconds, and the number of spectrum acquisition times is 30-50 times.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1) The preparation of the gold nanoparticle-modified nanopore array composite structure provided by the present invention has high preparation efficiency, simple and easy operation, and good stability and repeatability. 2) The gold nanoparticle-modified nanopore array composite structure provided by the present invention can prepare gold nanoparticle-modified nanopore array composite structures with different Raman enhancement properties by regulating the preparation parameters (microsphere cavity diameter, gold nanofilm thickness, laser energy density, and gold nanoparticle deposition time), thereby achieving controllable preparation of the composite structure. The structure has ultra-high Raman detection sensitivity and is suitable for the detection of Raman reporter molecules and serum markers. 3) The gold nanoparticle-modified nanopore array composite structure provided by the present invention has excellent comprehensive performance and has important application value in the field of enhanced Raman technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is a schematic diagram of the preparation and detection process of the gold nanoparticle-modified nanopore array composite structure;

[0026] Figure 2 Shown are the schematic diagram and scanning electron microscope image of the nanopore array obtained by femtosecond laser parallel processing;

[0027] Figure 3 Shown is a scanning electron microscope image of the nanopore cross section obtained by second laser parallel processing;

[0028] Figure 4 Shown are the Raman spectra and confusion matrix diagrams of the nanoparticle-modified nanopore array composite structure for serum markers. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] Implementation Case 1

[0031] A method for preparing a Raman enhanced substrate for serum marker detection comprises the following steps:

[0032] Step 1: ultrasonically clean a glass substrate (4 cm × 4 cm) in acetone, ethanol, and deionized water for 5 min, dry it, and then take it out for later use;

[0033] Step 2: Mix polydimethylsiloxane (PDMS) and curing agent (diphenylurethane, DBP) in a weight ratio of 10:1 and stir evenly. Place in a degassing machine to remove bubbles.

[0034] Step 3: Drop the PDMS liquid onto a clean glass substrate and spin-coat it at 1000 rpm / min for 50 seconds. Then, place the PDMS on a heating platform and heat it at 100°C for 5 minutes to form a PDMS film.

[0035] Step 4: Spread microsphere lenses with a diameter of 1 μm on the surface of the PDMS film and press them with 3M tape to form a single-layer microsphere lens array under the action of mechanical force;

[0036] Step 5: Use a vacuum ion sputtering coating device to coat the surface of the microsphere lens array with gold, with a gold film thickness of 5 nm;

[0037] Step 6: The gold-filmed microsphere array was fixed on a three-dimensional electric displacement platform and irradiated from the back of the substrate with a femtosecond laser. The laser wavelength was 800 nm, the pulse width was 50 fs, the number of pulses was 50, and the laser energy density was 55 mJ / cm 2 ;

[0038] Step 7: Deposit gold nanoparticles on top of the substrate using an ion sputtering system for 1 min.

[0039] Step 8: Transfer 5 μL of serum stock solution to a centrifuge tube and dilute it with PBS buffer at a dilution factor of 5.

[0040] Step 9: Take 10 μL of diluted serum sample and drop it onto the surface of the composite substrate. After the serum sample is captured by the composite substrate, the spectrum of the substrate is collected. The collection time is 50 seconds and the number of spectrum collections is 50 times.

[0041] Step 10: Biomarker peak identification was performed on serum spectra of rheumatoid arthritis and osteoarthritis using phenylalanine 1001 cm -1 The characteristic peak was used as the internal standard peak to obtain acetylated lysine 1301 cm -1 The characteristic peak intensity ratio was set at 75% probability of the intensity ratio being greater than 1 as the classification threshold to screen the two sera, with an accuracy of 88%, a sensitivity of 92.9%, and a specificity of 81.8%;

[0042] Implementation Case 2

[0043] A method for preparing a Raman enhanced substrate for serum marker detection comprises the following steps:

[0044] Step 1: ultrasonically clean a glass substrate (4 cm × 4 cm) in acetone, ethanol, and deionized water for 5 min, dry it, and then take it out for later use;

[0045] Step 2: Mix polydimethylsiloxane (PDMS) and curing agent (diphenylurethane, DBP) in a weight ratio of 10:1 and stir evenly. Place in a degassing machine to remove bubbles.

[0046] Step 3: Drop the PDMS liquid onto a clean glass substrate and spin-coat it at 500 rpm / min for 50 seconds. Then, place the PDMS on a heating platform and heat it at 100°C for 2 minutes to form a PDMS film.

[0047] Step 4: Spread microsphere lenses with a diameter of 4 μm on the surface of the PDMS film and press them with 3M tape to form a single-layer microsphere lens array under the action of mechanical force;

[0048] Step 5: Use a vacuum ion sputtering coating device to coat the surface of the microsphere lens array with gold, with a gold film thickness of 10 nm;

[0049] Step 6: The gold-filmed microsphere array was fixed on a three-dimensional electric displacement platform and irradiated from the back of the substrate with a femtosecond laser. The laser wavelength was 800 nm, the pulse width was 100 fs, the number of pulses was 100, and the laser energy density was 75 mJ / cm 2 ;

[0050] Step 7: Deposit gold nanoparticles on top of the substrate using an ion sputtering system for 3 min.

[0051] Step 8: Transfer 10 μL of serum stock solution to a centrifuge tube and dilute it with PBS buffer at a dilution factor of 2;

[0052] Step 9: Take 10 μL of diluted serum sample and drop it onto the surface of the composite substrate. After the serum sample is captured by the composite substrate, the spectrum of the substrate is collected. The collection time is 25 s and the number of spectrum collections is 50.

[0053] Step 9: Biomarker characteristic peak identification was performed on serum spectra of rheumatoid arthritis and osteoarthritis using phenylalanine 1001 cm -1 The characteristic peak was used as the internal standard peak to obtain tryptophan 1253cm -1 The characteristic peak intensity ratio was set at 70% probability of occurrence of the intensity ratio less than 0.8 as the classification threshold to screen the two sera, with an accuracy of 80%, a sensitivity of 85.7%, and a specificity of 72.7%;

[0054] Implementation Case 3

[0055] A method for preparing a Raman enhanced substrate for serum marker detection comprises the following steps:

[0056] Step 1: ultrasonically clean a glass substrate (4 cm × 4 cm) in acetone, ethanol, and deionized water for 5 min, dry it, and then take it out for later use;

[0057] Step 2: Mix polydimethylsiloxane (PDMS) and curing agent (diphenylurethane, DBP) in a weight ratio of 10:1 and stir evenly. Place in a degassing machine to remove bubbles.

[0058] Step 3: Drop the PDMS liquid onto a clean glass substrate and spin-coat it at 700 rpm / min for 50 seconds. Then, place the PDMS on a heating platform and heat it at 100°C for 3 minutes to form a PDMS film.

[0059] Step 4: Spread microsphere lenses with a diameter of 7 μm on the surface of the PDMS film and press them with 3M tape to form a single-layer microsphere lens array under the action of mechanical force;

[0060] Step 5: Use a vacuum ion sputtering coating device to coat the surface of the microsphere lens array with gold, with a gold film thickness of 15 nm;

[0061] Step 6: The gold-filmed microsphere array was fixed on a three-dimensional electric displacement platform and irradiated from the back of the substrate with a femtosecond laser. The laser wavelength was 800 nm, the pulse width was 100 fs, the number of pulses was 200, and the laser energy density was 130 mJ / cm 2 ;

[0062] Step 7: Deposit gold nanoparticles on top of the substrate using an ion sputtering system for 2 min.

[0063] Step 8: Transfer 8 μL of serum stock solution to a centrifuge tube and dilute it with PBS buffer at a dilution factor of 3;

[0064] Step 9: Take 15 μL of diluted serum sample and drop it onto the surface of the composite substrate. After the serum sample is captured by the composite substrate, the spectrum of the substrate is collected. The collection time is 40 seconds and the number of spectrum collections is 40 times.

[0065] Step 8: After capturing the serum samples of rheumatoid arthritis and osteoarthritis using the composite structure substrate, spectrum acquisition was performed on the substrate with an acquisition time of 50 seconds and a number of spectrum acquisitions of 50 times;

[0066] Step 9: Biomarker characteristic peak identification was performed on serum spectra of rheumatoid arthritis and osteoarthritis using phenylalanine 1001 cm -1 The characteristic peak was used as the internal standard peak to obtain citrulline 755cm -1The characteristic peak intensity ratio was set at a probability of 70% when the intensity ratio was less than 0.35 as the classification threshold to screen the two sera, with an accuracy of 76%, a sensitivity of 80%, and a specificity of 70%;

[0067] Obviously, the above-mentioned examples of implementation of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all the embodiments here. Any obvious variations or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing a Raman-enhanced substrate for serum biomarker detection, characterized in that: The following steps are involved: Step 1: ultrasonically clean the glass substrate in acetone, ethanol, and deionized water in sequence, dry it, and then take it out for use; Step 2: Mix polydimethylsiloxane (PDMS) and curing agent diphenylurethane (DBP) in a weight ratio of 10:1 and stir evenly. Place in a degassing machine to remove bubbles. Step 3: Drop the PDMS liquid onto a clean glass substrate and spin coat it at a speed of 500-1000 rpm / min for 10-50 seconds. Then, place the PDMS on a heating platform and heat it at 100°C for 2-5 minutes to form a PDMS film. Step 4: Spread the microsphere lenses on the surface of the PDMS film and press to form a single-layer microsphere lens array; Step 5: Use a vacuum ion sputtering coating instrument to coat gold on the surface of the microsphere lens array. The thickness of the gold film is controlled by the current and coating time, and is measured using the crystal oscillator film thickness detection device built into the instrument. Step 6: The gold-coated microsphere array is fixed on a three-dimensional electrically controlled displacement stage and irradiated with a femtosecond laser from the back of the substrate. A variable neutral density filter is used to control the laser energy irradiated on the sample surface, and a mechanical shutter controls the number of pulses. Step 7: Deposit gold nanoparticles on top of the substrate using an ion sputtering system. Step 8: Transfer 1-10 μL of serum stock solution to a centrifuge tube and dilute with PBS buffer; Step 9: Take 5-10 μL of diluted serum sample and drop it onto the surface of the composite substrate. After the serum sample is captured by the composite structure substrate, the spectrum of the substrate is collected.

2. The method for preparing a novel Raman enhanced substrate for serum biomarker detection according to claim 1, characterized in that: In step 4, the microspheres have a diameter of 1-7 μm, a refractive index of 1.4682, and are made of silicon dioxide.

3. The preparation method according to claim 1, wherein: In step 5, the thickness of the gold film formed by ion sputtering is 5-30 nm.

4. The preparation method according to claim 1, wherein: In step 6, the laser energy density is controlled in the range of 55-130mJ / cm 2 800nm femtosecond pulse laser with a pulse width of 50-100fs and a pulse number of 50-300.

5. The preparation method according to claim 1, wherein: In step 7, the gold nanoparticle deposition time is 1-5 minutes.

6. The preparation method according to claim 1, wherein: In step 8, the serum is diluted 2-5 times.

7. The preparation method according to claim 1, wherein: In step 9, the Raman spectrum acquisition time is 30-50 seconds, and the number of spectrum acquisition times is 30-50 times.

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