Metal heterogeneous material, chip and application in drug mass spectrum analysis

Abstract

The invention relates to the technical field of mass spectrum detection, in particular to a metal heterogeneous material, a chip and application of the metal heterogeneous material and the chip in drug mass spectrum analysis. According to the metal heterogeneous material, tight combination of TiO2 and gold is achieved through lattice matching design, and a plasma resonance enhanced heterogeneous interface is constructed. The chip comprises a titanium dioxide nanorod array formed on the substrate, a gold array deposited on the surface of the semiconductor layer and formed by gold nanoparticles, and a surface modification layer. Through the heterostructure design of a semiconductor and gold nanoparticles and a surface modification technology, the laser desorption ionization efficiency and the specific adsorption capacity to drug small molecules are remarkably improved, high-flux, high-sensitivity and high-resolution detection of complex components of traditional drugs is achieved, the gold nanoparticles are applied to drug screening and quality control, and the application prospect is wide. And an efficient analysis tool is provided for traditional drug research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometry, and particularly relates to a metal hetero-material, a chip and application thereof in drug mass spectrometry analysis. BACKGROUND

[0002] In the field of mass spectrometry, the traditional matrix-assisted laser desorption ionization (MALDI) technology relies on organic matrix to assist the desorption and ionization of samples. However, such organic matrix can generate strong background signal, which seriously interferes with the detection of low molecular weight substances such as small molecule metabolites, resulting in low sensitivity of small molecule detection. At the same time, although single nanomaterials such as graphene and gold nanoparticles have been tried to replace organic matrix, due to their insufficient adsorption capacity for polar compounds, they cannot effectively promote the ionization of sample molecules, so that the ionization efficiency is limited, and it is difficult to meet the needs of complex sample analysis.

[0003] Traditional drugs include traditional Chinese medicine compound, natural drug extract and the like, and the components thereof are extremely complex, containing not only a large amount of small molecule metabolites, lipids, proteins and the like, but also a large number of low-abundance active ingredients. These low-abundance active ingredients are easily covered by high-abundance components, and the existing technology is difficult to realize the synchronous high-sensitivity detection of multiple components in traditional drugs, which greatly hinders the development of related researches such as screening of active ingredients of traditional drugs, research of action mechanism and quality control. Therefore, it is urgent to develop new detection technology and tools to break through the technical bottleneck of traditional drug analysis. SUMMARY

[0004] Therefore, the present application provides a metal hetero-material, a chip and application thereof in drug mass spectrometry analysis. Through the heterostructure design of semiconductor and gold nanoparticles and surface modification technology, the present application significantly improves the laser desorption ionization efficiency and the specific adsorption capacity for small molecules of drugs, realizes the high-throughput, high-sensitivity and high-resolution detection of complex components of traditional drugs, and applies it to drug screening and quality control, thereby providing an efficient analysis tool for traditional drug research.

[0005] One of the purposes of the present application is to provide a metal hetero-material, which comprises a substrate formed of semiconductor material and metal nanoparticles loaded on the substrate. The semiconductor material is TiO2 with a band gap of 3.2 eV, and the metal nanoparticles are gold nanoparticles with a particle size of 1-20 nm.

[0006] The metal hetero-material realizes the close combination of semiconductor and gold through lattice matching design (for example, the lattice mismatch rate of TiO2 (101) crystal surface and Au (111) crystal surface is less than 5%), and constructs a hetero-interface with enhanced plasmonic resonance. A Schottky barrier is formed at the interface, which promotes the separation of photo-generated electron-hole pairs. A thiol (for example, HS-CH2-COOH) functional group is introduced to the hetero-interface of the metal hetero-material, and the specific adsorption of a polar drug molecule (for example, alkaloids, flavones) is enhanced through coordination or hydrogen bonding.

[0007] The second object of the present application provides a mass spectrometry detection chip based on a metal hetero-material. The chip comprises a substrate, a semiconductor layer, a metal nano-array and a surface modification layer. The semiconductor layer is a titanium dioxide nanocolumn array formed on the substrate. The metal nano-array is a gold array formed by gold nanoparticles deposited on the surface of the semiconductor layer. The surface modification layer is a hetero-interface formed on the surface of the chip by grafting a thiol functional group and covering the semiconductor layer and the gold nanoparticles.

[0008] Specifically, the substrate is a hydroxylated silicon wafer.

[0009] Specifically, the thickness of the semiconductor layer is 200 nm.

[0010] Specifically, the particle size of the gold nanoparticles is 10 nm.

[0011] The third object of the present application provides a preparation method of a mass spectrometry detection chip. The method comprises: providing a substrate; forming a titanium dioxide nanocolumn array on the substrate as a semiconductor layer; forming a gold array formed by gold nanoparticles on the surface of the semiconductor layer; immersing a preliminary chip comprising the substrate, the semiconductor layer and the gold array into a mercaptoacetic acid-containing ethanol solution for thiol functional group grafting to obtain the mass spectrometry detection chip.

[0012] Specifically, the step of forming a titanium dioxide nanocolumn array on the substrate as a semiconductor layer comprises: forming a titanium dioxide thin film on the surface of the substrate by magnetron sputtering of titanium dioxide nanoparticles; and preparing the titanium dioxide nanocolumn array from the titanium dioxide thin film by using a nano-imprint lithography process.

[0013] Specifically, the step of forming a gold array formed by gold nanoparticles on the surface of the semiconductor layer comprises: forming a layer of patterned photoresist mask on the surface of the titanium dioxide nanocolumn array silicon wafer with the semiconductor layer; and placing the chip with the photoresist mask as a working electrode into a three-electrode system containing a HAuCl4 electrolyte for electrochemical deposition to form the gold array.

[0014] Specifically, the photoresist mask forming step comprises: A positive photoresist AZ5214E is provided; After the titanium dioxide nanorod array silicon wafer is ultrasonically cleaned and dried, it is treated in a hexamethyldisilazane gas phase environment for 30 seconds, with the temperature controlled at 200-250 DEG C; The positive photoresist AZ5214E is dynamically spin-coated on the treated titanium dioxide nanorod array silicon wafer; The photoresist-coated silicon wafer is placed on a vacuum hot plate and baked at 85-120 DEG C for 30-60 seconds; A chromium plate mask is provided, which has a pattern of an array of holes with a period of 50-200 nm and a line width error controlled at ±2 nm; The chromium plate mask is aligned with the treated photoresist-coated silicon wafer, and exposed to ultraviolet light with a wavelength of 365 nm, with the energy set at 200 mJ / cm²; The exposed silicon wafer is immediately post-baked on a vacuum hot plate at 95-110 DEG C for 2-3 minutes; The post-baked silicon wafer is immersed in a developer AZ300MIF, and the developing time is accurately controlled at 60 seconds; After the developing is completed, the silicon wafer surface is immediately rinsed with deionized water to remove the residual developer, and after nitrogen blowing, the photoresist pattern integrity is preliminarily checked by an optical microscope to ensure that there is no pattern missing, breaking or sticking The fourth object of the present application provides a metal hetero-material or chip and its application in drug mass spectrum analysis.

[0015] The present application has the following beneficial effects: By adopting the synergistic effect of nanoimprint and RIE etching technology, the present application successfully realizes the controllable growth of the three-dimensional structure of the semiconductor layer, so that the specific surface area is increased by 3-5 times, from the original 5 m² / g to 20 m² / g. This technology significantly increases the laser energy absorption sites, thereby greatly improving the energy absorption efficiency. In addition, by integrating an electrochemical workstation (CHI660E) with SEM real-time monitoring, a mathematical model (R²>0.98) between deposition time and particle size / pitch is established, realizing the parameterized customization of metal nanometer arrays.

[0016] In the modification layer oriented bonding technology, the application utilizes the strong covalent bond (bond energy 250 kJ / mol) of gold-thiol and the condensation reaction (activation energy 45 kJ / mol) of silane-hydroxyl, ensures the vertical orientation arrangement of the modification layer, and makes the adsorption efficiency increase by 200% than random adsorption. Through these fine preparation processes, we can control the preparation period of a single chip within 4±0.5 hours, and ensure the consistency of the performance of the chips in batches, RSD<8% (taking the berberine detection signal strength as an index), which lays a solid technical foundation for large-scale production and commercial application.

[0017] The core technical advantage of the application lies in the synergistic effect of "three-dimensional structure enhanced absorption-heterojunction efficient ionization-surface modification specific capture-step process precise control". At the material level, the semiconductor-metal heterojunction solves the problem of low ionization efficiency of single material, and the surface modification layer overcomes the difficulty of adsorption of polar / weak polar molecules. At the structural level, the nanocolumn array increases the specific surface area by 4 times, increases the laser energy action site, and maximizes the plasmonic resonance effect through the precise regulation of the spacing between metal nanoparticles (error <5%). At the process level, the step-by-step assembly realizes the parameterization control from the substrate to the modification layer, ensuring the consistency of the chip performance (batch-to-batch RSD<10%), laying a foundation for industrial production and clinical standardized detection.

[0018] These advantages enable the chip of the application to realize the breakthrough from "low sensitivity and missed detection" to "precision and high throughput" in traditional drug multi-omics analysis, and it is especially suitable for screening and quality control of low-abundance active ingredients in complex systems, which is significantly superior to the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 SEM image of a titanium dioxide nanocolumn array silicon wafer with deposited nanogold particles.

[0020] Figure 2 Gold element face scanning transmission electron micrograph of a titanium dioxide nanocolumn array silicon wafer with deposited nanogold particles.

[0021] Figure 3 Mass spectrum of a chip provided in Example 1 for detecting Angelica processed sample.

[0022] Figure 4 Mass spectrum of a chip provided in Comparative Example 1 for detecting Angelica processed sample.

[0023] Figure 5 Mass spectrum of a chip provided in Comparative Example 2 for detecting Angelica processed sample.

[0024] Figure 6 Mass spectrum of a chip provided in Comparative Example 3 for detecting Angelica processed sample.

[0025] Figure 7 The chip provided for Comparative Example 4 detects the mass spectrum of the angelica processing sample. DETAILED DESCRIPTION

[0026] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below. It should be understood that the specific groups described herein are only used to explain the present application and are not used to limit the present application. The reagents not described in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be known from the prior art.

[0027] Example 1: Mass spectrometry detection chip and preparation method The preparation of the mass spectrum detection chip of the present application adopts a four-step precise control process, realizes the controllability and stability of the chip performance through a systematic process of material deposition, structure processing, interface modification and solidification enhancement, and the specific technical solutions are as follows: 1. Semiconductor layer preparation (1) Substrate pretreatment High-purity silicon wafer (100 crystal surface, thickness 500±10 μm, roughness <1 nm) is selected as the substrate, and is sequentially subjected to acetone ultrasonic degreasing (power 200 W, time 15 min), ethanol ultrasonic organic matter removal (power 200 W, time 10 min), deionized water ultrasonic washing (power 200 W, time 5 min), nitrogen blowing and drying in a 120°C oven for 30 min, then the dried substrate is immersed in a 5% (v / v) hydrogen peroxide solution, treated at room temperature for 10 min, and a hydroxylated surface is formed (water contact angle measurement shows that the water contact angle decreases from 75° to 30°).

[0028] (2) Magnetron sputtering deposition of semiconductor thin film This step uses a high vacuum magnetron sputtering instrument equipped with a radio frequency power source to prepare the thin film. The target material used is a high-purity (≥99.95%) TiO2 ceramic target with a diameter of 50 mm. During the preparation process, the base vacuum is maintained at 1×10 -4 Pa or below. The sputtering gas is high-purity argon, and the flow rate is controlled at 50 sccm. The sputtering power is set between 50-100 W, preferably 80 W to obtain a deposition rate of 1.2 nm / min. The deposition time is controlled between 30-60 min, which is monitored in real time by a quartz crystal microbalance to ensure that the film thickness is about 200 nm. In order to control the crystallinity of the film, the deposited film is subjected to an annealing treatment at 450°C in air for 2 hours, and the heating rate of the muffle furnace is 5°C / min. Through XRD detection, the anatase phase characteristic peak (101 crystal plane diffraction angle 25.3°) is obvious, and the crystallinity calculated by Rietveld refinement is more than 80%.

[0029] (3) Nanopillar array fabrication The deposited titanium dioxide film layer is subjected to a nanoimprint lithography process to form a nanopillar array using the following steps.

[0030] First, a PDMS nanopillar array template is prepared by electron beam lithography and silicon etching. Next, a negative photoresist (SU-82005, Microchem) is spin-coated on the titanium dioxide film layer chip at a speed of 3000 rpm to obtain a thickness of 5 μm, and is pre-baked at 95°C for 10 minutes to solidify. Then, the template is aligned with the photoresist-coated titanium dioxide film layer chip and hot-pressed using an 80°C hot press, with a pressure of 500 mbar and a holding time of 5 minutes, and after demolding, a photoresist nanopillar array is formed. Next, further processing is carried out by reactive ion etching (RIE) using CF4 / Ar mixed gas (flow ratio 10:5 sccm), power 100 W, pressure 10 Pa, and etching time 8-12 minutes to achieve etching of the TiO2 film, forming a nanopillar array with a height of about 200 nm and occupying 25% of the projected area of the titanium dioxide film layer, which is confirmed by SEM measurement. Finally, the uniformity of the nanopillar diameter (standard deviation <5 nm) is detected by scanning electron microscopy (SEM, Hitachi SU8010), and the surface roughness (RMS <4 nm) is measured by atomic force microscopy (AFM) to ensure quality control.

[0031] 2. Metal nanoparticle deposition After obtaining the titanium dioxide nanopillar array silicon wafer, a layer of patterned photoresist mask is first formed on the surface of the titanium dioxide nanopillar array silicon wafer. Then, the chip with the photoresist mask is used as the working electrode and placed in a three-electrode system containing HAuCl4 electrolyte for electrochemical deposition. Due to the blocking effect of the photoresist, gold nanoparticles can only be deposited in the "open area" of the photoresist opening, while the "shielded area" covered by the photoresist will not form gold nanoparticles, thereby achieving precise control of the spacing of the gold nanoparticle array.

[0032] The steps for forming the photoresist mask include: 1) Photoresist selection and preparation: A positive photoresist AZ5214E is selected because it can quickly dissolve in the developing solution in the exposed area under 365 nm ultraviolet light, which is suitable for building high-precision patterns. Before use, the photoresist is spin-coated in a clean centrifuge tube at a speed of 4000 rpm to ensure that no bubbles or impurities are mixed in, maintaining the uniformity of the photoresist.

[0033] 2) Silicon wafer surface pretreatment: The titanium dioxide nanorod array silicon wafer is again ultrasonically rinsed with deionized water for 5 minutes to remove any residual nanoparticles or impurities. After nitrogen blowing to dry, it is placed in a 120°C oven for 10 minutes to completely remove surface moisture and enhance the adhesion of the photoresist. Subsequently, the silicon wafer is treated in a hexamethyldisilazane (HMDS) gas phase environment for 30 seconds at a temperature of 200-250°C to change the surface from hydrophilic to hydrophobic, ensuring that the photoresist can be closely attached.

[0034] 3) Photoresist spin coating: Dynamic spin coating is used. First, the silicon wafer is fixed on the spin coater turntable, and a suitable amount of photoresist is slowly added while rotating at a low speed of 500 rpm to ensure uniform spreading of the photoresist on the silicon wafer surface. Subsequently, the speed is quickly increased to 4000 rpm for 30 seconds to uniformly distribute the photoresist and form a 2μm thick film using centrifugal force. After spin coating, the silicon wafer is left to stand at room temperature for 5 minutes to allow the solvent in the photoresist to evaporate initially.

[0035] 4) Soft baking: The photoresist-coated silicon wafer is placed on a vacuum hot plate and baked at 85-120°C for 30-60 seconds. The purpose of soft baking is to remove about 4-7% of the solvent in the photoresist, enhance the adhesion of the photoresist to the silicon wafer surface, release the internal stress of the photoresist film, and prevent the photoresist from warping or falling off during subsequent operations.

[0036] 5) Photomask preparation: A chromium plate photomask is selected. Before use, the mask needs to be purged with high-purity nitrogen for 10 minutes to remove surface dust particles and avoid affecting the exposure accuracy.

[0037] 6) Alignment and exposure: The pre-alignment system of the photoetching machine is used to perform automatic laser coarse alignment by recognizing the notch or flat features on the silicon wafer. Subsequently, based on the alignment marks (AlignMark) on the scribe line, sub-micron level fine alignment is performed to ensure accurate positioning of the mask pattern and the titanium dioxide nanorod array silicon wafer. Exposure is performed using a wavelength of 365nm ultraviolet light with an energy setting of 200mJ / cm². The exposure time is optimized according to the photoresist photosensitivity and the complexity of the mask pattern, generally 10-30 seconds, to ensure that the exposed area of the photoresist undergoes sufficient photochemical reaction.

[0038] 7) Post-baking: The exposed silicon wafer is immediately post-baked on a 95-110°C vacuum hot plate for 2-3 minutes. Post-baking can promote the further cross-linking or decomposition of the photoresist molecules in the exposed area, enhance the solubility difference of the photoresist in the developing solution between the exposed and unexposed areas, and improve the pattern resolution.

[0039] 8) Developing operation: The post-baked silicon wafer was immersed in the developer AZ300MIF, and the developing time was precisely controlled for 60 seconds. During the developing process, the silicon wafer was rotated at a constant speed of 50 rpm to ensure that the developer was uniformly applied to the surface of the silicon wafer. Due to the positive photoresist characteristics, the photoresist in the exposed area dissolved and separated from the silicon wafer in the developer, while the photoresist in the unexposed area remained, thereby forming a periodic array of hole patterned photoresist mask corresponding to the mask on the surface of the titanium dioxide nanorod array silicon wafer.

[0040] 9) Post-development inspection and cleaning: After the developing was completed, the surface of the silicon wafer was immediately rinsed with deionized water to remove the residual developer. After being dried by nitrogen, the integrity of the photoresist pattern was preliminarily inspected by an optical microscope to ensure that there was no pattern missing, fracture or adhesion. Subsequently, the critical dimensions (such as hole diameter and hole pitch) of the pattern were measured by a scanning electron microscope (SEM), and the error should be controlled within ± 5 nm to ensure that the pattern accuracy met the requirements.

[0041] The step of electrochemically depositing gold nanoparticles includes: preparing an electrolyte containing 0.5 mM HAuCl4 and 0.1 M KCl at pH 4.0; using the titanium dioxide nanorod array silicon wafer obtained in the above steps as a working electrode, a platinum sheet as a counter electrode, and a saturated calomel electrode as a reference electrode to build a three-electrode system; providing a deposition potential of -0.35 V for electrochemical deposition, and the deposition time is 10 min to control the gold particle size to be 10 nm, and the electrolyte can be slightly stirred during the deposition process.

[0042] 3. Surface modification The modifier is prepared in a Teflon container, and the modifier is an ethanol solution containing 0.5 mM mercaptoacetic acid; the initial chip obtained in the above steps is immersed in the modifier, and adsorption is carried out at room temperature until the concentration of mercaptoacetic acid in the solution no longer changes. Thus, the mass spectrometry chip is obtained.

[0043] In this process, the mercapto group of mercaptoacetic acid forms an Au-S covalent bond (bond length 2.35 Å, S2p binding energy 163.5 ± 0.2 eV by XPS detection) with the gold nanoparticles, and the carboxyl group (-COOH) is oriented to be exposed to the solution phase. According to the XPS quantitative analysis, the atomic percentage of surface sulfur element is increased from 0% to 1.2-1.5% (corresponding to a modification layer thickness of 2-3 nm), and the water contact angle after modification is reduced from 85° (unmodified) to 35° (mercapto), which proves that the hydrophilicity is significantly improved, which is beneficial to the spreading of polar samples.

[0044] 4. Chip solidification (enhancement of interlayer adhesion) The chip obtained by the above steps is heated to 120°C at 5°C / min in a nitrogen atmosphere, and kept for 30 min. The modifier molecules form stable chemical bonds with the substrate, and at the same time, the interface hydroxyl groups of the semiconductor layer and the substrate are promoted to condense to form Si-O-Ti bonds (for TiO2 / silicon substrate system), and the physical adsorption (van der Waals force is increased by 30%) of the metal nanoparticles and the semiconductor surface is enhanced (AFM force curve measurement).

[0045] SEM characterization shows that the standard deviation of the particle size distribution of the metal nanoparticles is less than 2 nm, and the standard deviation of the interval distribution is less than 10 nm, ensuring that the performance of each region of the chip is consistent. Figure 1 SEM image of the three-dimensional structure.

[0046] Comparative Example 1 Single titanium dioxide film chip A titanium dioxide film chip with a titanium dioxide film layer is prepared using the same semiconductor layer preparation process as in Example 1.

[0047] Comparative Example 2 Primary chip without surface modification An initial chip without surface modification is prepared using the same semiconductor layer preparation process and metal nanoparticle deposition as in Example 1.

[0048] Comparative Example 3 A commercial traditional planar structure MALDI chip is selected as Comparative Example 3, which mainly consists of a stainless steel substrate coated with a layer of α-cyano-4-hydroxycinnamic acid on the surface.

[0049] Comparative Example 4 A non-step mixed preparation process is used to mix TiO2 nanoparticles, Au nanoparticles and surface modifier, and then directly coat them on the surface of a silicon wafer, followed by heat treatment and other subsequent steps to prepare the chip.

[0050] Chip performance test 1. Sensitivity test The detection sensitivity reflects the response ability of the chip to low concentration target substances, and is usually measured by "limit of detection (LOD)". Prepare 10 -12 mol / L, 10 -11 mol / L, 10 -10 mol / L, 10 -8 mol / L and 10⁻ 6 mol / L of aconitine solution as a standard solution.

[0051] 1-2 μL of the standard solution is added to the surface of the chip, dried at room temperature (5 minutes), and a uniform sample film is formed.

[0052] Laser desorption ionization: laser intensity 20-40% (355 nm wavelength), pulse frequency 10 Hz, positive / negative ion mode switching (selected according to the polarity of the drug). Mass spectrometric detection: scan range m / z 50-2000, resolution 70,000 (FWHM), scan number 100 times / point.

[0053] Record the signal intensity (peak height or peak area) of the characteristic peak of the target in the mass spectrum. Draw a standard curve with signal intensity as the vertical coordinate and concentration as the horizontal coordinate, and calculate the linear correlation coefficient (R²). Define the "lowest detection limit (LOD)" as the concentration of the target corresponding to the signal intensity reaching 3 times the background noise (S / N=3), and the lower the LOD, the higher the sensitivity.

[0054] 2. Resolution evaluation Resolution (R) reflects the ability of the chip to distinguish ions with similar mass-to-charge ratios (m / z). The calculation formula is: R=m / Δm. Where m is the mass-to-charge ratio of the target ion, and Δm is the mass-to-charge ratio difference between the adjacent two distinguishable peaks.

[0055] Select a mixed standard containing ions with similar m / z (such as peptide segment mixture: Glu-fibrinopeptide B (m / z 1570.677) and Angiotensin II (m / z 1046.542), or custom mixed system). Spot the standard on the chip, and scan on the mass spectrometer with the same parameters (high resolution mode is recommended, such as TOF-MS). Repeat the detection 3 times for each chip.

[0056] Select two adjacent and distinguishable characteristic peaks in the mass spectrum, and measure their m / z values and peak full width at half maximum (FWHM). Calculate the resolution according to the above formula, and take the average value. The larger the resolution value, the stronger the chip's ability to distinguish similar ions.

[0057] 3. Signal stability evaluation method Signal stability reflects the reproducibility of the signal in multiple detections, which is usually measured by "relative standard deviation (RSD)".

[0058] Select 10 -10 mol / L aconitine solution, and spot it on different areas (at least 5 evenly distributed points) on the same chip surface. Keep the mass spectrometric parameters consistent, and record the signal intensity (peak height or peak area) of the characteristic peak of the target. Calculate the average value and standard deviation (SD) of the signal intensity of multiple detections.

[0059] Relative standard deviation (RSD)=100%×standard deviation / average value, the smaller the RSD, the better the signal stability.

[0060] 4. Specific surface area evaluation Based on the physical adsorption of nitrogen at low temperature (77K), by measuring the nitrogen adsorption amount at different relative pressures (P / P0, 0.05~0.3 range), the monolayer adsorption amount of the chip surface is calculated, and the specific surface area is derived based on the BET equation.

[0061] 5、Results Table 1

[0062] Compared with Comparative Example 1 containing only a TiO2 film, the laser energy conversion efficiency of Example 1 is significantly improved to 12.3% due to the introduction of the plasmonic resonance effect of metal nanoparticles, and the detection sensitivity is also three orders of magnitude higher. In addition, although Comparative Example 2 uses a TiO2-Au heterostructure, the amount of polar molecule adsorption is only 7.2 μg / cm2, which is 42% lower than the 12.5 μg / cm2 of Example 1, further proving the key role of the surface modification layer in specific adsorption.

[0063] In terms of three-dimensional structure and step-by-step process, the traditional MALDI chip of Comparative Example 3 relies on an organic matrix, resulting in a matrix peak covering the m / z<500 region, which makes the detection of small molecules severely interfered by the background. In contrast, the nano-pillar array used in Example 1 increases the laser absorption sites and has no matrix pollution, and the signal-to-noise ratio is improved by more than 10 times. The non-step process of Comparative Example 4 leads to the agglomeration of metal nanoparticles, with standard deviations of particle size and spacing of 5 nm and 25 nm, respectively, and the signal stability RSD is as high as 25%, which is much higher than the 8.2% of Example 1. This fully proves the decisive role of step-by-step assembly in ensuring the uniformity of the structure.

[0064] Application of drug mass spectrometry analysis Angelica has complex chemical components, including ligustilide, butylphthalide, senkyunolide A, senkyunolide H, senkyunolide I, 6,7-epoxy ligustilide, ferulic acid, chlorogenic acid, caffeic acid, vanillic acid, adenosine, guanosine, L-tryptophan, proline, linoleic acid, linolenic acid, and fagarine phthalide, which have high requirements for the enrichment capacity, sensitivity, resolution, and anti-matrix interference ability of the mass spectrometry detection chip. The mass spectrometry detection chip of Example 1 and Comparative Examples 1-4 was used to analyze the components of Angelica, and the specific process was as follows: 1、Specific analysis process (1) Sample pretreatment The angelica medicinal material was first crushed and passed through a 60-mesh sieve, and then 0.5 g of the powder was weighed. Next, 10 mL of a solution containing 80% methanol and 0.1% formic acid was added to the powder, and ultrasonic extraction was performed for 30 min (power 300 W, frequency 40 kHz), and this process was repeated twice. Subsequently, the extraction liquid was combined and centrifuged at 4000 rpm for 10 min, and the supernatant was taken and filtered through a 0.22 μm organic filter membrane, and was ready for use.

[0065] (2) Chip processing and mass spectrometry detection 10 μL of the above-mentioned test solution was loaded onto the surface of the mass spectrometry detection chip of Example 1 and Comparative Examples 1-4, respectively (the chips were all pretreated: ultrasonic cleaning with ultrapure water for 10 min, and nitrogen blowing dry).

[0066] Based on the differences in the properties of the chip surfaces (for example, the specific surface area of Example 1 is larger, and the surface modification is better), after loading, enrichment was performed for 10 min (target components were enriched by adsorption / covalent action on the chip surface), and then the chip surface was washed twice with 0.1% formic acid water (to remove matrix impurities), and was dried with nitrogen.

[0067] Instrument: ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS, model: Waters ACQUITY UPLC-Xevo TQ-S); Chromatographic column: ACQUITY UPLC BEH C18 (2.1 x 100 mm, 1.7 μm); Mobile phase: A phase (0.1% formic acid water), B phase (acetonitrile), gradient elution (0-5 min: 5% B→20% B; 5-15 min: 20% B→50% B; 15-20 min: 50% B→90% B); Flow rate: 0.3 mL / min; column temperature: 30°C; injection volume: 5 μL; Mass spectrometry conditions: electrospray ion source (ESI+), positive and negative ion modes were collected simultaneously (switching time <50 ms). Positive ion mode: Spray voltage: 3.5 kV Capillary temperature: 320°C Sheath gas (N2): 40 arb Auxiliary gas (N2): 10 arb Scan range: m / z 100-1000 Acquisition mode: FullMS / dd-MS² (data-dependent secondary scan) Collision energy: 15, 30, 45 eV (ladder collision energy, to improve fragment information) Negative ion mode: Spray voltage: -3.0 kV Other parameters are the same as positive ion mode. High resolution mass spectrometry (such as Q-TOF, Orbitrap) is used, and the resolution is ≥ 35,000 FWHM.

[0068] Dynamic exclusion time: 30 s (to avoid repeated acquisition of high abundance ions).

[0069] Data acquisition and analysis: MassLynx V4.2 software is used to collect data, and the components are identified by comparing the retention time, secondary fragment ions (such as paeoniflorin m / z 481→225, ferulic acid m / z 194→134) and database (such as TCMSP) matching. Calculate the key indicators: the total number of detected components, the signal-to-noise ratio (S / N) of the target component, the relative standard deviation (RSD, n=6) of the peak area, and the separation degree (resolution) of adjacent peaks.

[0070] 3. Data and results Figures 3-7 The mass spectra of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4, respectively.

[0071] Table 2 Number of detected components

[0072] As shown in Table 2, Example 1 uses a multi-level structure design of titanium dioxide nanocolumn and gold particle surface modification to obtain a chip, which realizes full component coverage, especially has a significant synergistic enrichment ability for components with large polarity differences (such as phthalides and organic acids). In contrast, Comparative Example 1 only has a single TiO2 film, lacks gold particle deposition and surface modification, and cannot detect non-polar fatty acid components. The chip provided by Comparative Example 2 is not surface modified, which leads to the inability to detect polar components (such as nucleosides and amino acids).

[0073] Table 3 Comparison of signal-to-noise ratio (S / N) of key components

[0074] As shown in Table 3, the multi-level structure of Example 1 significantly improves the ionization efficiency, and the signal-to-noise ratio is improved by 3-5 times, which has obvious advantages for low content components, such as L-tryptophan. In contrast, due to the interference of matrix peaks, the signal-to-noise ratio of ligustilide and other components provided by the commercial MALDI chip of Comparative Example 3 decreased by 65%. The chip provided by Comparative Example 4 has large signal fluctuations due to the non-uniformity of the nanostructure, and the average signal-to-noise ratio is only 30% of Example 1.

[0075] As can be seen from Table 4, Example 1 enhances the interlayer bonding force inside the chip through the curing step of forming Si-O-Ti bonds, and the RSD is less than 3%, meeting the requirements of quantitative analysis. The chip preparation process provided by Comparative Example 2 has no curing step, and the internal structure of the chip provided by Comparative Example 4 is disordered, and the RSD of both is more than 10%, which cannot be used for precise quantification.

[0076] As can be seen from Table 5, the chip provided by Example 1 has a periodic nanostructure, which can improve the separation degree through the steric hindrance effect, and the resolution is 50%-100% higher than that of the comparative example. The commercial MALDI chip provided by Comparative Example 3 cannot separate (resolution <1.0) isomers (such as ligustilide A / H) due to uneven matrix crystallization.

[0077] Table 4 reproducibility (peak area RSD, n=6) comparison

[0078] Table 5 adjacent peak separation (resolution) comparison

[0079] Table 6 anti-matrix interference ability (background noise level)

[0080] As can be seen from Table 6, Example 1 significantly reduces the background noise by 80% through the hydrophilic surface of the titanium dioxide nanocolumn and the selective adsorption design, and eliminates the interference peaks in the low molecular weight region. In contrast, the matrix peaks (e.g. m / z 147, 190) in Comparative Example 3 seriously interfere with the detection of ligustilide (m / z 191) and other components.

[0081] In summary, the titanium dioxide nanocolumn and gold particle cooperative adsorption system developed in this study can cover 17 components with large differences in polarity, showing ultra-high sensitivity. The specific surface area of the nanocolumn array is increased by 50 times, and the surface plasmon resonance (SPR) effect of the gold particles enhances the ionization efficiency, and the signal-to-noise ratio (S / N) is more than 1200. In addition, the system has high reproducibility, and the curing process enhances the interlayer bonding force, with a relative standard deviation (RSD) of less than 3%, meeting the requirements of quantitative analysis. In terms of precise separation, the periodic nanostructure improves the resolution through the steric hindrance effect, achieving baseline separation of isomers. In terms of anti-interference ability, the selective adsorption design reduces the background noise by 80% and has no matrix interference in the low molecular weight region. In contrast, Comparative Examples 1-4 have significant shortcomings in component coverage, sensitivity, reproducibility, etc. due to single structure, lack of modification or process defects.

[0082] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A metallic heterostructure, characterized in that, The material includes a substrate formed of a semiconductor material and metal nanoparticles loaded on the substrate. The semiconductor material is TiO2 with a band gap of 3.2 eV, and the metal nanoparticles are gold nanoparticles with a particle size of 1 to 20 nm. The metal heterostructure achieves a tight bond between TiO2 and gold through lattice matching design, thereby constructing a plasmonic resonance-enhanced heterostructure interface.

2. A mass spectrometry detection chip based on metallic heteromaterials, characterized in that, include: The substrate comprises a semiconductor layer, a metal nanoarray, and a surface modification layer. The semiconductor layer is an array of titanium dioxide nanopillars formed on the substrate. The metal nanoarray is a gold array formed by gold nanoparticles deposited on the surface of the semiconductor layer. The surface modification layer is formed on the chip surface by valence grafting of thiol functional groups and covers the heterogeneous interface between the semiconductor layer and the gold nanoparticles.

3. The chip according to claim 2, characterized in that, The substrate is a hydroxylated silicon wafer; the semiconductor layer is a titanium dioxide nanopillar array with a thickness of 200 nm; and the gold nanoparticles have a particle size of 10 nm.

4. A method for fabricating a mass spectrometry detection chip, characterized in that, include: Provide a base; A titanium dioxide nanopillar array is formed on the substrate to serve as a semiconductor layer; A gold array formed of gold nanoparticles is formed on the surface of the semiconductor layer; The initial chip, comprising the substrate, the semiconductor layer, and the gold array, is immersed in an ethanol solution containing mercaptoacetic acid to perform thiol functional group grafting to obtain the mass spectrometry detection chip.

5. The preparation method according to claim 4, characterized in that the step of forming a titanium dioxide nanopillar array on the substrate as a semiconductor layer includes: Titanium dioxide nanoparticles are magnetron sputtered onto the surface of the substrate to form a titanium dioxide thin film. The titanium dioxide thin film was prepared into a titanium dioxide nanopillar array using a nanoimprint lithography process.

6. The preparation method according to claim 4, characterized in that the step of forming a gold array of gold nanoparticles on the surface of the semiconductor layer includes: A patterned photoresist mask is formed on the surface of a titanium dioxide nanopillar array silicon wafer having the semiconductor layer; Using a chip with a photoresist mask as the working electrode, the gold array is formed by electrochemical deposition in a three-electrode system containing HAuCl4 electrolyte.

7. The preparation method according to claim 6, characterized in that the step of forming the photoresist mask includes: Positive photoresist AZ5214E ​​is available; After ultrasonic cleaning and drying, the titanium dioxide nanopillar array silicon wafer is treated in a hexamethyldisilazane gas phase environment for 30 seconds, with the temperature controlled at 200-250℃. The positive photoresist AZ5214E ​​was dynamically spin-coated onto the treated titanium dioxide nanopillar array silicon wafer; Place the photoresist-coated silicon wafer on a vacuum hot plate and bake it at 85-120℃ for 30-60 seconds; Provide chrome plate photomasks; The chromium plate photomask is aligned with the treated silicon wafer coated with photoresist, and exposed with ultraviolet light of wavelength 365nm at an energy of 200mJ / cm². The exposed silicon wafers are immediately post-baked by baking on a vacuum hot plate at 95-110℃ for 2-3 minutes. After post-baking, the silicon wafer is immersed in developer AZ300MIF, and the development time is precisely controlled to be 60 seconds. After development, immediately rinse the silicon wafer surface with deionized water to remove residual developer. After drying with nitrogen, conduct a preliminary inspection of the photoresist pattern integrity using an optical microscope to ensure that there are no missing, broken, or adhered patterns.

8. The metallic heteromaterial of claim 1, or the chip of claim 2 or 3, and its application in drug mass spectrometry analysis.

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