Ultra-sensitive SERS (Surface Enhanced Raman Scattering) chip detection method based on adjustable band gap porous silicon photonic crystal

Abstract

The invention belongs to the technical field of optical biological detection, and particularly discloses an ultra-sensitive SERS (Surface Enhanced Raman Scattering) chip detection method based on an adjustable band gap porous silicon photonic crystal, which comprises the following steps: cleaning a silicon wafer, preparing a PSi PhC chip with three different forbidden bands, preparing an Au NPs-X / PSi PhC SERS chip, and testing the SERS chip of a DNA (Deoxyribose Nucleic Acid) sample. According to the invention, the PSi PhCs with adjustable multi-band gaps, controllable micro-cavity aperture and high nanoparticle load and light modulation performance characteristics is designed by controlling the proportion of high and low current pulse sequences, introducing a novel zero-order sequence (an interval current strategy) and a surface strong reducibility Si-H bond mediated in-situ growth strategy. The method has the advantages that by matching with a portable Raman spectrometer, the ultrasensitive and background-free analysis of the single-stranded DNA (ss-DNA) is realized, the base composition proportion of the DNA chain is accurately revealed, and the method is successfully applied to the high-sensitivity analysis of the DNA fingerprint spectrum.

Description

Technical Field

[0001] This invention belongs to the field of optical biological detection technology, specifically relating to an ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal. Background Technology

[0002] DNA fingerprinting spectroscopy can reflect the base composition, sequence variations, and methylation status of nucleic acids. It is applicable to scenarios such as genotyping, genetic disease screening, early tumor diagnosis, and epigenetic research. Its detection accuracy and sensitivity are directly related to early disease warning, precise typing, and efficacy monitoring, making it of significant application value in clinical diagnosis and life science research. Current mainstream DNA fingerprinting spectroscopy detection methods include traditional electrophoretic separation, surface-enhanced Raman scattering (SERS), and fluorescence sequencing. While these methods can perform basic nucleic acid analysis, they generally have technical limitations: First, significant background interference and residual reducing agents easily generate interference peaks, and signal stability cannot be guaranteed even after processing; second, insufficient sensitivity, making it difficult to detect low concentrations of nucleic acids and subtle base variations; third, bulky instruments, complex operating procedures, and poor portability, making them unsuitable for grassroots and on-site testing needs. Therefore, developing a DNA fingerprinting spectroscopy detection technology that combines zero background interference, ultra-high sensitivity, and portability has become a key technical problem urgently needing to be solved in this field.

[0003] Its photonic bandgap structure, possessing wavelength-selective optical modulation capability, can trigger strong Bragg reflection and optical resonance effects when the SERS laser wavelength matches the photonic bandgap, thereby significantly enhancing the local electromagnetic field and increasing the SERS signal intensity, demonstrating technological potential in multiple scenarios. However, the research and application of existing PSi PhCs-based SERS chips still have significant limitations: First, the adaptability to multi-wavelength scenarios is lacking. There is a lack of systematic strategy support for precise tuning of mainstream SERS laser wavelengths such as 532, 638, and 785 nm, limiting the scenario coverage of the chip; Second, lateral aperture control is difficult. The traditional fabrication method relying on electrochemical high and low current cycle longitudinal etching is difficult to achieve free control of the lateral aperture. If irregular slit-like pores are formed, it will not only limit the uniform loading of nanoparticles but also weaken the microcavity effect; Third, there is a contradiction between nanoparticle loading and optical modulation performance. Although high-density nanoparticles are conducive to generating abundant SERS "hot spots," they may cover the pores and weaken the optical reflection modulation capability, thus reducing the overall performance. Traditional loading methods such as vacuum sputtering and self-assembly can easily lead to uneven hotspot density, and the preparation process that relies on reducing agents can introduce impurity interference peaks, which seriously affect the detection sensitivity and stability.

[0004] In summary, establishing a multi-wavelength precise tuning strategy, achieving controllable aperture regulation, and balancing high nanoparticle load and optical modulation performance are key to overcoming the performance bottleneck of PSi PhCs-based SERS chips and realizing precise spectral detection of DNA fingerprints. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystals. By controlling the ratio of high and low current pulse sequences, introducing novel zero-order sequences (interval current strategy), and employing an in-situ growth strategy mediated by strongly reducing Si-H bonds on the surface, PSi PhCs with tunable multiple bandgap, controllable microcavity pore size, and a balance between high nanoparticle loading and optical modulation performance are designed. This is then matched with a portable Raman spectrometer to achieve ultrasensitive, background-free analysis of single-stranded DNA (ss-DNA).

[0006] This invention systematically develops a strategy for silicon etching modulation and gold nanoparticle (Au NPs) growth. By controlling the ratio of high and low current pulse sequences, the tunability of the reflection wavelength of PSi PhCs is achieved, fabricating photonic crystals suitable for 532, 638, and 785 nm SERS excitation with a reflectivity exceeding 80%, significantly improving the light source utilization efficiency. Furthermore, a novel zero-order sequence (interval current strategy) is introduced to achieve controllable modulation of the PSi PhC pore size, making it more suitable for subsequent construction of nanostructured SERS substrates. Unlike traditional citrate-reduced Au NPs, the Au NPs in the Au NPs-785 / PSi PhC chip are grown in situ using the reducing properties of the PSi PhC's own silane-hydrogen bonds. The entire process requires no reducing agent, thus eliminating any impurity interference peaks. Detection and verification results of deoxyribonucleoside triphosphates (dNTPs) and ss-DNA show clearly distinguishable base characteristic peaks, accurately identifying changes in base composition and quantity, exhibiting zero background and ultra-high sensitivity. This invention provides a novel SERS strategy for interference-free and highly sensitive DNA fingerprint spectroscopy analysis, demonstrating its application potential in the field of DNA fingerprint spectroscopy.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a method for detecting ultrasensitive SERS chips based on tunable bandgap porous silicon photonic crystals, specifically including the following steps:

[0008] (1) Silicon wafer cleaning: P <100> The silicon wafers are cut into small pieces and soaked in aqua regia to remove the surface oxide film; then they are briefly soaked in an etching solution prepared with hydrofluoric acid and ethanol, followed by ultrasonic cleaning with acetone, ethanol and ultrapure water in sequence to remove surface organic impurities and improve hydrophilicity, finally obtaining a clean and uniform silicon wafer.

[0009] (2) Fabrication of PSi PhC chips with three different band gaps: PSi PhC chips with band gaps of 532, 638 and 785 nm were fabricated by periodically etching silicon wafers with high and low currents;

[0010] (3) Preparation of Au NPs-X / PSi PhC SERS chip: The PSi PhC chip with band gaps of 532, 638 and 785 nm obtained in step (2) was placed in chloroauric acid solution and Au NPs were reduced in situ by microwave method to obtain Au NPs-X / PSiPhC SERS chip.

[0011] (4) SERS chip test of DNA sample: DNA sample was dropped on the surface of the Au NPs-X / PSi PhC SERS chip prepared in step (3), dried, and tested with a portable Raman spectrometer. The DNA fingerprint spectrum was analyzed according to the characteristic peak positions of different dNTPs.

[0012] In a preferred embodiment of the present invention, step (1) P <100> The resistivity of the silicon wafer is 0.01-0.03Ω·cm and the thickness is 300-400μm; the size of the cut wafer is (1-2)cm×(1-2)cm.

[0013] In a preferred embodiment of the present invention, the volume ratio of hydrofluoric acid to ethanol in the corrosion solution in step (1) is 1:(1-2), and the soaking time is 1-5 min; the ultrasonic time for acetone, ethanol, and ultrapure water is 5-15 min.

[0014] In a preferred embodiment of the present invention, in step (2) the preparation of PSiPhC with band gaps of 532, 638, and 785 nm, the electrolyte is a mixture of hydrofluoric acid and ethanol in a volume ratio of 1:(1-2).

[0015] In a preferred embodiment of the present invention, in step (2), the high current is 60-80 mA and the low current is 20-40 mA; for PSi PhC with a bandgap of 532 nm, the high current etching time is 1-3 s, the low current etching time is 2-4 s, the number of cycles is 10-20, and the interval current time is 10-15 s; for PSi PhC with a bandgap of 638 nm, the high current etching time is 1.5-3.5 s, the low current etching time is 3-5 s, the number of cycles is 10-20, and the interval current time is 10-15 s; for PSi PhC with a bandgap of 785 nm, the high current etching time is 2-4 s, the low current etching time is 4-6 s, the number of cycles is 10-20, and the interval current time is 10-15 s.

[0016] By setting the high and low current etching time, the number of cycles, and the interval current time, a PSi PhC chip with accurate bandgap wavelength can be obtained.

[0017] In a preferred embodiment of the present invention, the concentration of chloroauric acid in step (3) of preparing the Au NPs-X / PSi PhC SERS chip is 0.0005-0.0015 mol / L.

[0018] In a preferred embodiment of the present invention, step (3) involves a microwave power of 500-700 W and a restoration time of 50-70 s.

[0019] In a preferred embodiment of the present invention, the volume of DNA sample solution dropped onto the chip surface in step (4) of the SERS chip test of the DNA sample is 2-5 μL.

[0020] In a preferred embodiment of the present invention, the drying temperature in step (4) is 30-50 °C.

[0021] In a preferred embodiment of the present invention, the Raman test parameters in step (4) are: the detection wavelength range is 200-2000 cm⁻¹. -1 The integration time is 1 s, the number of integrations is 1, the laser power is 100 mW, the excitation wavelength of the SERS chip with a band gap of 532 nm is 532 nm, the excitation wavelength of the SERS chip with a band gap of 638 nm is 638 nm, and the excitation wavelength of the SERS chip with a band gap of 738 nm is 738 nm.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention utilizes a high-low current pulse sequence modulation strategy to achieve precise bandgap matching of PSi PhCs with mainstream 532, 638, and 785 nm SERS lasers, with a reflectivity exceeding 80%, breaking through the limitations of traditional multi-wavelength adaptation and meeting the application requirements of multiple scenarios and diverse excitation light sources.

[0024] 2. This invention utilizes the interval current synergistic control technology to realize the construction of a three-dimensional high-reflectivity microcavity with a uniform aperture of 80-100 nm, providing high-quality sites for nanoparticle loading and ensuring the synergistic effect of PhC light modulation effect and SERS hot spot density to improve detection sensitivity;

[0025] 3. This invention utilizes an in-situ growth strategy mediated by Si-H bonds on the PSi PhC surface, without the need for external reducing agents and surfactants, to prepare Au NPs / PSi PhC chips with zero background interference, completely solving the problem of impurity interference peaks in traditional chips, achieving zero background detection, and adapting to the detection of easily interfered samples such as DNA.

[0026] 4. This invention utilizes the synergistic effect of PhC light reflection enhancement and Au NPs plasma storm to enhance the SERS signal by 5-6 orders of magnitude compared to traditional Au NPs / Si chips, and achieves an R6G detection limit as low as 10⁻¹. 4 M significantly improves the sensitivity of trace substance detection;

[0027] 5. This invention, verified by DNA and dNTP detection, achieves ultrasensitive, background-free analysis of single-stranded DNA (ss-DNA), accurately reveals the base composition ratio of the DNA strand, and has been successfully applied to highly sensitive DNA fingerprint spectral analysis.

[0028] 6. This invention utilizes the portability of SERS technology to achieve a detection mode that does not rely on large biochemical analyzers, simplifying the detection process, reducing detection costs, and adapting to diverse application scenarios such as primary healthcare and point-of-care testing. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the fabrication process of the Au NPs-X / PSi PhC chip in Embodiment 1 of the present invention;

[0030] Figure 2 The images shown are cross-sectional and surface SEM images of the PSi PhC chip with band gaps of 532, 638, and 785 nm in Embodiment 1 of the present invention: (a) cross-sectional SEM image with a band gap of 532 nm; (b) cross-sectional SEM image with a band gap of 638 nm; (c) cross-sectional SEM image with a band gap of 785 nm; (d) surface SEM image with a band gap of 532 nm; (e) surface SEM image with a band gap of 638 nm; (f) surface SEM image with a band gap of 785 nm.

[0031] Figure 3 These are the UV-Vis reflectance spectra of the PSi PhC chip with band gaps of 532, 638, and 785 nm, respectively, in Embodiment 1 of the present invention. (a) UV-Vis reflectance spectrum with a band gap of 532 nm; (b) UV-Vis reflectance spectrum with a band gap of 638 nm; (c) UV-Vis reflectance spectrum with a band gap of 785 nm;

[0032] Figure 4 This is a SEM image of the Au NPs / PSi PhC SERS chip in Embodiment 1 of the present invention;

[0033] Figure 5 The following are the DNA fingerprint spectral analysis detection diagrams of the Au NPs / PSi PhC SERS chip in Example 1 of this invention: (a) SERS spectra of T, C, G, A and their corresponding dNTPs chips; (b) ss-DNA SERS spectra obtained by matching laser with Au NPs / PSi PhC SERS chip; (c) SERS spectra of ss-DNA with the same A base and different G bases; (d) Comparison of characteristic peaks of ss-DNA with the same A base and different G bases.

[0034] Figure 6 The uniformity and detection limit of the Au NPs / PSi PhC SERS chip for detecting R6G in Embodiment 2 of the present invention are as follows: (a) uniformity of the Au NPs / PSi PhC SERS chip for detecting R6G; (b) detection limit of the Au NPs / PSi PhC SERS chip for detecting R6G.

[0035] Figure 7 The background interference and performance changes after KI treatment of different SERS enhancement substrates in Embodiment 3 of the present invention are as follows: (a) background interference of different SERS enhancement substrates; (b) performance changes of different SERS enhancement substrates after KI treatment. Detailed Implementation

[0036] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0037] A highly sensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystals specifically includes the following steps:

[0038] (1) Silicon wafer cleaning: P <100> The silicon wafers are cut into small pieces and soaked in aqua regia to remove the surface oxide film; then they are briefly soaked in an etching solution prepared with hydrofluoric acid and ethanol, followed by ultrasonic cleaning with acetone, ethanol and ultrapure water in sequence to remove surface organic impurities and improve hydrophilicity, finally obtaining a clean and uniform silicon wafer.

[0039] (2) Fabrication of PSi PhC chips with three different band gaps: PSi PhC chips with band gaps of 532, 638 and 785 nm were fabricated by periodically etching silicon wafers with high and low currents;

[0040] (3) Preparation of Au NPs-X / PSi PhC SERS chip: The PSi PhC chip with band gaps of 532, 638 and 785 nm obtained in step (2) was placed in chloroauric acid solution and Au NPs were reduced in situ by microwave method to obtain Au NPs-X / PSiPhC SERS chip.

[0041] (4) SERS chip test of DNA sample: DNA sample was dropped on the surface of the Au NPs-X / PSi PhC SERS chip prepared in step (3), dried, and tested with a portable Raman spectrometer. The DNA fingerprint spectrum was analyzed according to the characteristic peak positions of different dNTPs.

[0042] The step (1) P <100> The resistivity of the silicon wafer is 0.01-0.03Ω·cm and the thickness is 300-400μm; the size of the cut wafer is (1-2)cm×(1-2)cm.

[0043] In step (1), the volume ratio of hydrofluoric acid to ethanol in the corrosion solution is 1:(1-2), and the soaking time is 1-5 min; the ultrasonic time for acetone, ethanol, and ultrapure water is 5-15 min.

[0044] In step (2), the electrolyte used in the preparation of PSi PhC with band gaps of 532, 638, and 785 nm is a mixture of hydrofluoric acid and ethanol in a volume ratio of 1:(1-2).

[0045] In step (2), the high current is 60-80 mA and the low current is 20-40 mA. For PSi PhC with a bandgap of 532 nm, the high current etching time is 1-3 s, the low current etching time is 2-4 s, the number of cycles is 10-20, and the interval current time is 10-15 s. For PSi PhC with a bandgap of 638 nm, the high current etching time is 1.5-3.5 s, the low current etching time is 3-5 s, the number of cycles is 10-20, and the interval current time is 10-15 s. For PSi PhC with a bandgap of 785 nm, the high current etching time is 2-4 s, the low current etching time is 4-6 s, the number of cycles is 10-20, and the interval current time is 10-15 s.

[0046] In step (3) of the preparation of Au NPs-X / PSi PhC SERS chip, the concentration of chloroauric acid is 0.0005-0.0015 mol / L, the microwave power is 500-700 W, and the reduction time is 50-70 s.

[0047] In step (4), the volume of DNA sample solution dropped onto the chip surface during the SERS chip test of the DNA sample is 2-5 μL.

[0048] The drying temperature in step (4) is 30-50 ℃.

[0049] The Raman test parameters for step (4) are: the detection wavelength range is 200-2000 cm⁻¹. -1 The integration time is 1 s, the number of integrations is 1, the laser power is 100 mW, the excitation wavelength of the SERS chip with a band gap of 532 nm is 532 nm, the excitation wavelength of the SERS chip with a band gap of 638 nm is 638 nm, and the excitation wavelength of the SERS chip with a band gap of 738 nm is 738 nm.

[0050] Example 1

[0051] This example uses ultrapure water to simulate DNA fingerprint spectral detection by adding thymine (T), cytosine (C), guanine (G), and adenine (A) base standards and ss-DNA samples, respectively, to test the analytical performance of the Au NPs-X / PSi PhC chip for DNA fingerprint spectral analysis. Figure 1 As shown, before detection, the silicon wafer was pre-cleaned, PSi PhC chips with band gaps of 532, 638, and 785 nm were prepared, and an Au NPs-X / PSi PhC SERS chip was prepared. For detection, 3.5 μL of sample was added to the prepared AuNPs-785 / PSi PhC SERS chip, and a portable Raman spectrometer was used for testing. The laser wavelength was 785 nm, and the detection was performed under conditions of 1 s and 100 mW. The specific detection steps are as follows:

[0052] (1) Cleaning of silicon wafers:

[0053] P <100> Silicon wafers were cut into smaller pieces. First, the wafers were immersed in aqua regia to remove the surface oxide film; then, they were briefly immersed in an etching solution prepared with hydrofluoric acid and ethanol, followed by ultrasonic cleaning with acetone, ethanol, and ultrapure water in sequence to remove surface organic impurities and improve hydrophilicity, ultimately obtaining a clean and uniform silicon wafer surface for subsequent experiments. Among these, P... <100> The resistivity of the silicon wafer is 0.02 Ω·cm and the thickness is 340 μm; the size of the cut pieces is 1.5 cm × 1.5 cm; the volume ratio of hydrofluoric acid to ethanol in the etching solution is 1:1.5, and the soaking time is 3 min; the ultrasonic treatment time using acetone, ethanol, and ultrapure water is 10 min each.

[0054] (2) Fabrication of PSi PhC chips with band gaps of 532, 638, and 785 nm:

[0055] Three types of PSi PhC chips were fabricated by periodically etching silicon wafers with high and low currents. By setting specific high and low current etching times, cycle numbers, and interval current times, PSi PhC chips with accurate bandgap wavelengths of 532, 638, and 785 nm were obtained. Figure 2 As shown, using different high and low current etching times results in different cross-sectional etching thicknesses and apertures, thus yielding PSi PhC chips with different bandgap values. The electrolyte is a mixture of hydrofluoric acid and ethanol at a volume ratio of 1:1.5; the high current is 70 mA, and the low current is 30 mA; for PSi PhC with a bandgap of 532 nm (such as…),… Figure 2 a, Figure 2 d), the high-current etching time was 2.118 s, the low-current etching time was 3.354 s, the number of cycles was 15, and the interval current was 10-15 s; for PSi PhC with a bandgap of 638 nm (such as Figure 2 b、 Figure 2 e), the high-current etching time is 2.539 s, the low-current etching time is 4.022 s, the number of cycles is 15, and the interval current is 10-15 s; for PSi PhC with a bandgap of 785 nm (e.g. Figure 2 c. Figure 2 f), the high-current etching time was 3.126 s, the low-current etching time was 4.949 s, the number of cycles was 15, and the interval current was 10-15 s. For example... Figure 3 As shown, the bandgap positions of the prepared PSi PhCs with different bandgaps were verified by detecting them using ultraviolet-visible reflectance spectroscopy. Figure 3 As shown in Figure a, the strong absorption peak of the fabricated PSi PhC with a band gap of 532 nm is at 532 nm; Figure 3 As shown in b, the strong absorption peak of the fabricated PSi PhC with a band gap of 638 nm is at 638 nm; as Figure 3 As shown in c, the strong absorption peak of the fabricated PSi PhC with a band gap of 785 nm is at 785 nm.

[0056] (3) Fabrication of Au NPs-X / PSi PhC SERS chip:

[0057] Au NPs were reduced in situ using a microwave method on prepared PSi PhC chips with band gaps of 532, 638, and 785. The PSi PhC chips were placed in a chloroauric acid solution for microwave reduction to prepare Au NPs-X / PSi PhC SERS chips. The concentration of chloroauric acid was 0.001 mol / L, the microwave power was 600 W, and the reduction time was 60 s. Figure 4The image shown is a SEM image of the Au NPs-785 / PSi PhC SERS chip prepared by microwave method. It can be seen that the Au NPs grown in situ are very uniformly distributed.

[0058] (4) SERS microarray analysis of DNA samples:

[0059] Taking the Au NPs-785 / PSi PhC SERS chip prepared in step (3) as an example, a DNA sample was dropped onto its surface, dried, and then tested using a portable Raman spectrometer. The DNA fingerprint spectrum was analyzed based on the characteristic peak positions of different dNTPs. The volume of the DNA sample solution dropped onto the chip surface was 3.5 μL; the drying temperature was 40 ℃; the Raman test parameters were: excitation wavelength of 785 nm, detection wavelength range of 200-2000 cm-1, integration time of 1 s, integration times of 1, and laser power of 100 mW. Figure 5 The image shown is a DNA fingerprint spectral analysis result obtained using the Au NPs-785 / PSi PhC SERS chip. Figure 6 This section shows the SERS spectra of T, C, G, and A standards and their corresponding dNTP units obtained using the Au NPs-785 / PSi PhC SERS chip. The comparison shows that the main characteristic peaks of the T, C, G, and A standards and their corresponding dNTP units correspond one-to-one in the 788, 795, 652, and 729 cm⁻¹ bands, respectively, without any background interference. Figure 6 b demonstrates the SERS detection of 10 μM ordered single-stranded DNA (ss-DNA) using the prepared chip. In the obtained SERS spectrum, strong characteristic peaks for A and G bases at wavelengths of 729 and 650 cm⁻¹ are clearly observed. Figure 6 c shows the SERS spectra obtained when detecting ss-DNA with the same number of A bases but different numbers of G bases. The data show that the characteristic peak at 650 cm⁻¹ in the SERS spectra of ss-DNA with different numbers of G bases differs significantly and increases with the increase of the number of G bases, while the characteristic peak of A bases remains unchanged because the number of A bases is the same. Figure 6 The diagram clearly shows the changes in the A and G characteristic peaks with the number of bases, demonstrating the chip's advantages in sensitivity and stability in detecting ss-DNA base changes.

[0060] Example 2

[0061] This example uses a systematic SERS test with R6G aqueous solutions of 1–5–10–15 mol / L concentration gradients to verify the detection performance of the Au NPs-X / PSi PhC SERS chip. The prepared Au NPs-785 / PSi PhC SERS chip was immersed in R6G aqueous solutions of different concentrations for 10 min each, dried, and then tested using a portable Raman spectrometer with an excitation light of 785 nm. The Raman test parameters were: detection wavelength range of 200–2000 cm⁻¹, integration time of 1 s, integration count of 1, and laser power of 100 mW. Figure 6 a demonstrated the matching detection of the 785 / PSi PhC chip with a 785 nm laser device for 10⁻ 5 The spectral uniformity results of 30 random points during MR6G testing confirm that the chip has excellent stability. Figure 6 b demonstrates the high-performance Au NPs-785 / PSi PhC chip used to detect SERS spectra of different concentrations of R6G using a 785 nm laser. Based on the signal-to-noise ratio, the detection limit of R6G reached 10⁻¹² M after 10 minutes of incubation and 10⁻¹ M after 3 hours of incubation. 4 M directly verifies the chip's excellent SERS detection capability.

[0062] The specific testing method is the same as in Example 1.

[0063] Example 3

[0064] This example uses a systematic comparison of the detection performance of Au NPs-X / PSi PhC with that of chips based on traditional sodium citrate-reduced Au / Ag NPs. The results show that the detection performance of Au NPs-X / PSi PhC is significantly superior to that of chips based on traditional sodium citrate-reduced Au / Ag NPs. Besides its extremely high SERS sensitivity, its zero background signal characteristic makes it more advantageous in the detection of DNA samples that are easily interfered with by the intrinsic peaks of traditional nanoparticles. Figure 7 As shown in Figure a, compared to chips based on traditional sodium citrate reduction of Au / Ag NPs, the intrinsic SERS spectrum of the Au NPs-785 / PSi PhC chip is extremely pure, with no SERS interference peaks from the reducing agent. Currently, a more advanced step in DNA detection using SERS technology involves replacing the citrate ions on the surface of Au / Ag NPs with bromide or iodide ions to eliminate the influence of the citrate background on the DNA SERS spectrum and induce particle aggregation, allowing DNA to enter SERS hotspots for detection. However, the DNA signal obtained by this method is weak and unstable. Figure 7 b illustrates the changes in SERS detection performance of Au / Ag NPs and Au NPs-785 / PSi PhC before and after potassium iodide (KI) treatment. Data shows that the SERS performance of the chip decreased sharply after KI cleaning. The KI treatment process introduces a large amount of salt impurities onto the surface of the gold nanoparticles, leading to irregular aggregation of the nanoparticles and thus a significant attenuation of the SERS signal. Experimental data indicate that the randomness of hotspot formation and the introduction of salt impurities during the substitution of citrate ions by iodide ions severely affect the stability and sensitivity of SERS detection. The use of Au NPs-785 / PSi PhC provides better stability and sensitivity, effectively mitigating this problem.

[0065] The specific testing method is the same as in Example 1.

[0066] This invention develops an ultrasensitive SERS chip detection technology based on tunable bandgap porous silicon photonic crystal (PSi PhC), which integrates the core advantages of precise multi-wavelength adaptation, controllable three-dimensional microcavity aperture, and in-situ growth of zero-background nanoparticles to achieve ultrasensitive background-free detection of single-stranded DNA (ss-DNA) and accurate identification of base composition and ratio. By employing high- and low-current pulse sequence modulation and interleaved current synergy techniques, a PSi PhC substrate with a reflectivity exceeding 80% and precisely matched bandgap to mainstream SERS lasers at 532, 638, and 785 nm was fabricated. Utilizing the strong reducing properties of the Si-H bonds on its surface, uniformly distributed gold nanoparticles (Au NPs) were grown in situ, constructing an Au NPs / PSi PhC chip that combines high optical modulation performance with high hotspot density. This method eliminates the need for external reducing agents and surfactants, completely removing impurity interference peaks. Combining the PhC light reflection enhancement effect with the synergistic effect of Au NPs plasma, the SERS signal is enhanced by 5-6 orders of magnitude compared to traditional Au NPs / Si chips, with an R6G detection limit as low as 10⁻¹. 4 M. This method features standardized experimental procedures and a simple detection process. Detection can be completed using a portable Raman spectrometer, eliminating the need for large, precision instruments. It offers low detection costs and is suitable for various field applications. Validation with base standards, dNTP units, and ss-DNA samples with different base compositions demonstrates that this technology can clearly distinguish characteristic peaks of each base and accurately capture changes in base quantity. It effectively solves the core problems of severe background interference, insufficient sensitivity, and poor portability in traditional DNA SERS detection. This provides a novel and efficient technical solution for the further application of DNA fingerprinting spectroscopy in genotyping, genetic disease screening, and early tumor diagnosis, possessing broad potential for scientific research applications and clinical translation.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting ultrasensitive SERS chips based on tunable bandgap porous silicon photonic crystals, characterized in that, Includes the following steps: (1) Silicon wafer cleaning: P <100> The silicon wafers were cut into small pieces and soaked in aqua regia to remove the surface oxide film; then they were soaked in an etching solution prepared with hydrofluoric acid and ethanol, and then ultrasonically cleaned in sequence with acetone, ethanol and ultrapure water. (2) Fabrication of PSi PhC chips with three different band gaps: PSi PhC chips with band gaps of 532, 638 and 785 nm were fabricated by periodically etching silicon wafers with high and low currents; (3) Preparation of Au NPs-X / PSi PhC SERS chip: The PSi PhC chip with band gaps of 532, 638 and 785 nm obtained in step (2) was placed in chloroauric acid solution and Au NPs were reduced in situ by microwave method to obtain Au NPs-X / PSi PhCSERS chip. (4) SERS chip test of DNA sample: DNA sample was dropped on the surface of the Au NPs-X / PSi PhC SERS chip prepared in step (3), dried, and tested with a portable Raman spectrometer. The DNA fingerprint spectrum was analyzed according to the characteristic peak positions of different dNTPs.

2. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, The step (1) P <100> The resistivity of the silicon wafer is 0.01-0.03Ω·cm and the thickness is 300-400μm; the size of the cut wafer is (1-2)cm×(1-2)cm.

3. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, In step (1), the volume ratio of hydrofluoric acid to ethanol in the corrosion solution is 1:(1-2), and the soaking time is 1-5 min; the ultrasonic time for acetone, ethanol, and ultrapure water is 5-15 min.

4. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, In step (2), the electrolyte used in the preparation of PSi PhC with band gaps of 532, 638, and 785 nm is a mixture of hydrofluoric acid and ethanol in a volume ratio of 1:(1-2).

5. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, In step (2), the high current is 60-80 mA and the low current is 20-40 mA. For PSi PhC with a bandgap of 532 nm, the high current etching time is 1-3 s, the low current etching time is 2-4 s, the number of cycles is 10-20, and the interval current time is 10-15 s. For PSi PhC with a bandgap of 638 nm, the high current etching time is 1.5-3.5 s, the low current etching time is 3-5 s, the number of cycles is 10-20, and the interval current time is 10-15 s. For PSi PhC with a bandgap of 785 nm, the high current etching time is 2-4 s, the low current etching time is 4-6 s, the number of cycles is 10-20, and the interval current time is 10-15 s.

6. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, In step (3) of the preparation of the Au NPs-X / PSi PhC SERS chip, the concentration of chloroauric acid is 0.0005-0.0015 mol / L.

7. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, In step (3), the microwave power for the preparation of the Au NPs-X / PSi PhC SERS chip is 500-700 W, and the reduction time is 50-70 s.

8. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, In step (4), the volume of DNA sample solution dropped onto the chip surface during the SERS chip test of the DNA sample is 2-5 μL.

9. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, The drying temperature in step (4) is 30-50℃.

10. The ultrasensitive SERS chip detection method based on tunable bandgap porous silicon photonic crystal as described in claim 1, characterized in that, The Raman test parameters for step (4) are: the detection wavelength range is 200-2000 cm⁻¹. -1 The integration time is 1 s, the number of integrations is 1, the laser power is 100 mW, the excitation wavelength of the SERS chip with a band gap of 532 nm is 532 nm, the excitation wavelength of the SERS chip with a band gap of 638 nm is 638 nm, and the excitation wavelength of the SERS chip with a band gap of 738 nm is 738 nm.

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