Brain natriuretic peptide detection device and detection method based on surface enhanced Raman scattering technology

Abstract

The present invention discloses a device and method for detecting brain natriuretic peptide, a serological marker secreted by acute myocardial infarction, based on surface enhanced Raman scattering technology. First, the antibody molecules corresponding to BNP are modified by nano-imprinting and chemical bonds to complete the preparation of SERS substrate chip; during the volatilization process of the BNP antibody solution, a collapsed finger array structure with hexamers or polymers as structural units is formed. The present invention can simultaneously achieve high selectivity and high sensitivity SERS detection of BNP. By dropping serum into a coupling structure substrate pre-placed in a biological carrier chip and directly optically focusing the Au finger surface, high selectivity and high sensitivity measurement of specific BNP in a liquid serum environment within 5 seconds can be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a device and method for detecting brain natriuretic peptide, a secretion marker of acute myocardial infarction, based on surface enhanced Raman scattering technology. Background Art

[0002] The incidence of cardiovascular diseases represented by acute myocardial infarction (AMI) is increasing year by year. AMI has a rapid onset, rapid progression of the disease, and arrhythmia at any time, leading to sudden cardiac death. It has a high mortality rate and disability rate and is a cardiovascular emergency. Currently, AMI has surpassed lung cancer to become the main disease with the highest mortality rate.

[0003] At present, the fastest clinical biochemical test for AMI in the world is the test of myoglobin and troponin, which usually takes 20 minutes. The "Troponin I Rapid Test Card" developed and produced by Shanghai Chuanzhi Biotechnology Co., Ltd. can achieve 15 minutes of visual observation of the test results. The test time of 15 to 20 minutes is still not fast enough compared to the extreme speed of life passing away. The acute myocardial infarction detection scheme adopted by the hospital still has some defects. For example, several immunoassay techniques, including fluorescence detection, chemiluminescence, electrochemical detection, etc., have been used to simultaneously quantify myoglobin (Mb), troponin (TnT), creatine kinase isoenzyme (CK-MB) and pro-brain natriuretic peptide (pro-BNP) in patient serum, among which pro-BNP is generally referred to as brain natriuretic peptide (BNP). Since the rescue time of patients with acute myocardial infarction is very critical, the National Health Commission hopes to reduce the detection time of myocardial infarction markers. Therefore, how to quickly realize the fingerprint recognition of AMI marker molecules plays a very critical role in medical treatment.

[0004] Brain Natriuretic Peptide (BNP), also known as B-type Natriuretic Peptide and brain natriuretic peptide, is another member of the natriuretic peptide system after atrial natriuretic peptide (ANP). It was first isolated from pig brain by Japanese scholar Sudoh et al. in 1988, hence the name, and is mainly derived from the ventricle. BNP has important pathophysiological significance, can promote sodium excretion and urination, has a strong vasodilator effect, and can counteract the vasoconstriction effect of the renin-angiotensin-aldosterone system (RAAS). Like ANP, it is a major endocrine system for the human body to resist excessive volume load and hypertension. Cardiac dysfunction can greatly activate the natriuretic peptide system, and increased ventricular load leads to BNP release. At present, although the content of brain natriuretic peptide has a high value in the diagnosis of AMI, the determination of brain natriuretic peptide levels has not been well popularized, mainly due to the limitations of the detection method. The methodological research of brain natriuretic peptide detection has always been a hot topic in brain natriuretic peptide research. The main methods for detecting brain natriuretic peptide include radioimmunoassay (RIMA) and immunofluorescence. RIMA is not suitable for acute diagnosis of AMI and small sample testing due to its short half-life and long detection time. Biosite's immunofluorescence method for determining brain natriuretic peptide levels has achieved the purpose of rapid detection. Bayer Diagnostics and Abbott have also launched brain natriuretic peptide assay reagents, but the costs are relatively high.

[0005] The Raman scattering effect was discovered by Indian scientist CV Raman in 1928. It is mainly based on the inelastic scattering of incident light and matter, which causes the incident light to lose some energy (depending on the structure of the molecule itself), thereby identifying the fingerprint information of the molecule. Compared with other detection methods (such as fluorescence detection), it does not require sample processing, is water-insensitive (especially useful for medical biological detection), and has the characteristics of micro-area trace detection and completion within a few seconds. It has important application prospects in the rapid fingerprint detection of disease marker molecules. However, according to the selection law of quantum mechanics, the scattering cross section of the Raman effect is only one billionth of the fluorescence signal, and the signal is very weak. With the development of micro-nano optics, people have found that the free electrons on the conduction band near the Fermi level of metal micro-nano structures are coupled with the incident light, and the collective coherent fluctuations of the surface free electrons will form a local electromagnetic mode, which will gather the energy of the incident light field in a sub-wavelength spatial range, and form a local enhanced electromagnetic field near the metal surface. The use of this enhanced electromagnetic field will significantly increase the intensity of Raman scattering of molecules near the metal surface, and realize surface-enhanced Raman scattering (SERS) detection. The emergence of SERS technology provides an effective way for rapid detection, trace detection and even single-molecule detection of BNP in AMI serum (needed for early warning and early diagnosis).

[0006] In the SERS application of surface plasmon optical effects on metal micro-nanostructures, how to regulate the excitation of the local light field and obtain a strong local light field state density is crucial to its enhanced SERS efficiency. Compared with the resonant excitation of a single nanostructure, metal coupled structures will produce a stronger local light field due to the gap plasmon coupling effect between the structures, and therefore have attracted widespread attention. In 1999, Academician Xu Hongxing of Wuhan University discovered for the first time that the experimental phenomenon of SERS single-molecule detection (Phys. Rev. Lett., 1999, 83 (21): 4357-4360) was caused by the coupled electromagnetic field in the ultra-small gap between Ag nanoparticles, and the field enhancement can reach 10 3 times, making the SERS enhancement factor reach 10 12 , thus explaining the single-molecule detection phenomenon of SERS. In recent years, Zhang Shuang et al. from the University of Birmingham in the UK used a curled nanobowl combined with a medium and gold nanoparticles to deposit a gold coupling structure, achieving single-molecule SERS detection in a wide frequency band (Nat. Commun., 2018, 9(1): 5248-5255).

[0007] Based on the above-mentioned physical mechanism of SERS enhancement, at present, the application of SERS in the detection of BNP molecules, a marker in AMI, needs to solve two problems at the same time: first, how to achieve high sensitivity and specificity detection of BNP: in addition to BNP molecules, there are more than thousands of other biological proteins in the patient's serum, how to detect only the marker molecules to be tested in the serum; second, how to achieve rapid detection of BNP, a marker of AMI: this is very important for the early diagnosis and early warning of AMI. Highly selective detection requires that the metal structure can capture molecules, and high-sensitivity detection requires that the captured molecules are in the enhanced electromagnetic field area of ​​the metal structure. The simultaneous realization of both is the key to the application of SERS-based cardiovascular disease marker BNP molecule detection.

[0008] In the early stage, we combined EBL, lithography and nanoimprinting techniques to realize an ordered structure of flexible metal nano-finger arrays with precisely adjustable structural units (ACS Nano, 2017, 11(6): 5836-5843; Small, 2018, 14: 1801146-1801155; ZL201710825180.0; ZL201910666854.6). This metal nano-finger array can be used to collapse the flexible nano-fingers that are close to each other by titrating the solvent and utilizing the surface tension during its evaporation process. Through this technology, the BNP antibody modification technology can be combined with the Au surface in advance, and then collapsed and brought together to form an Au nano-finger / BNP antibody / Au nano-finger coupling structure. Since the spatial longitudinal size of the BNP antibody is at the sub-nanometer level, the coupling structure formed can achieve an enhanced electromagnetic field under the control of quantum plasmons; in this coupling structure, the antibody molecule acts as a coupling structure gap layer, which can not only capture the cardiovascular disease marker BNP molecules, but also localize the marker BNP molecules in the gap layer. Since the coupling enhanced electromagnetic field is mainly distributed in the gap layer, it can simultaneously achieve high selectivity and high sensitivity SERS detection of BNP molecules. In specific applications, by dripping serum into the nano-finger coupling structure substrate pre-placed in the biological carrier, high selectivity and high sensitivity measurement of specific cardiovascular disease marker molecules in a liquid serum environment can be achieved, and the detection time is about 5 seconds, which is much shorter than the current hospital standard BNP detection time (10-15 minutes). Summary of the invention

[0009] In order to solve the shortcomings of the prior art, a method for preparing a surface enhanced Raman substrate for high selectivity and high sensitivity detection of AMI marker molecule BNP is provided, which belongs to the field of analytical detection technology. This method is simple and reasonable to prepare, can be prepared in large quantities and at low cost, and can achieve high selectivity and high sensitivity detection of BNP in AMI patient serum for BNP monitoring, and can be applied to portable devices such as handheld Raman loaded in ambulances. By using the ordered structure of the flexible Au nano-finger array and combining with BNP antibody molecules, firstly, the Au nano-finger / BNP antibody molecule / Au nano-finger coupling structure is constructed. Since the spatial longitudinal size of the antibody is at the sub-nanometer level, the coupling structure formed can realize the enhanced electromagnetic field under the control of quantum plasmons; secondly, by using the specific interaction between the antibody and BNP molecules in this coupling structure, the BNP molecules are directly captured and localized from the patient serum to the surface of the SERS enhanced substrate, and the SERS characteristic peaks in the BNP molecules that are different from those corresponding to the antibody molecules are used to achieve high selectivity and high sensitivity quantitative detection of the BNP molecules.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The brain natriuretic peptide detection device based on surface enhanced Raman scattering technology is characterized in that it is prepared by the following steps:

[0012] 1) Prepare the ordered array structure of Au nano-finger by nanoimprinting, Au thin film deposition and reactive ion beam etching;

[0013] 2) dropping the antibody molecule solution corresponding to BNP onto the Au nano-finger ordered array structure prepared in step 1 and incubating for 8 hours to allow the antibody molecule to bind to Au;

[0014] 3) Take out the Au nano-finger ordered array structure incubated in step 2, dry it naturally under room temperature and air environment, rinse it with ultrapure water to remove the unconnected and excess BNP antibody molecules, soak it in ultrapure water for 30 minutes, take it out and dry it under natural conditions;

[0015] 4) During the evaporation of the solution, the micro-surface tension causes six or more adjacent nano-fingers to approach each other, forming a collapsed Au finger array structure with hexamers or polymers as structural units. A sub-nanometer gap twice the size of the BNP antibody is formed between the finger structures, which can generate an enhanced coupled electromagnetic field regulated by quantum plasmons. In addition, the antibody molecules are distributed on the surface of the Au nano-fingers and can capture the BNP molecules into the coupled electromagnetic field.

[0016] The present invention also adopts the following technical solution:

[0017] The method for detecting brain natriuretic peptide based on surface enhanced Raman scattering technology is characterized by using the aforementioned detection device and adding the following steps:

[0018] 5) Place the SERS enhanced substrate based on the Au nano-finger closed array structure obtained in step 4 into a biological carrier, then drop serum into it, cover the carrier, and let it stand for 5 minutes. At this time, the BNP molecules will be captured and localized on the finger surface;

[0019] 6) The Raman spectrometer is used to directly focus on the surface of the nano-finger array in step 5, and only BNP and antibody molecules are detected by Raman, so that the characteristic peaks of the antibody and BNP can be directly obtained. -1 、1453cm -1 、1657cm -1 To realize the fingerprint recognition detection of BNP.

[0020] The height of a single nano-finger in the Au nano-finger ordered array structure in step 1 is 350 nanometers, including a 300-nanometer polymer pillar and 50 nanometers of Au on top, with a diameter of 75 nanometers and a distance of 110 nanometers between adjacent fingers, in order to form a hexameric coupling structure in the subsequent collapse process.

[0021] The incubation time in step 2 above is 8 hours.

[0022] The standing time in step 5 above is 5 minutes.

[0023] The present invention is based on a flexible Au nano-finger array structure combined with antibody modification technology, and constructs a coupling structure with an ultra-small gap of sub-nanometer antibody size. The BNP is used to interact with the corresponding antibody to capture and localize the BNP molecules on the Au nano-finger surface, directly optically focus the Au finger surface, and achieve high sensitivity and high specificity detection of BNP molecules in serum through the Raman characteristic peak of the BNP molecule. The detection time is 5 seconds. In our design, since the longitudinal size of the antibody is at the sub-nanometer level, when the nano-fingers are close together, the localized surface plasmon dipole modes of a single nano-fingers will couple with each other to form two coupling modes of bonding and anti-bonding. The bonding coupling mode will induce an ultra-strong local enhanced electromagnetic field. In our design, the enhancement factor of this local enhanced electromagnetic field is 10 9 In addition, in the preparation of the flexible Au nanofinger array structure, by designing the appropriate size and period, we can realize the coupling structure with hexamer and polymer as the structural unit. This polymer structure has more coupling hot spots.

[0024] During the volatilization of the BNP antibody solution, due to the specific size design of the nano-finger, the micro-surface tension makes six or more adjacent nano-fingers close to each other, forming a collapsed finger array structure with hexamer or polymer as the structural unit. The coupling structure of the hexamer can obtain more coupling hotspots; in the collapsed finger array structure formed, the gap between the fingers is determined by the size of 2 times the BNP antibody, which is at the sub-nanometer level. Therefore, between the Au fingers, an enhanced electromagnetic field regulated by quantum plasmons is formed, making the SERS enhancement factor reach 10 9 In this coupling structure, the antibody molecule acts as the gap layer of the coupling structure, which can capture BNP and localize BNP in the gap layer. Since the coupling enhanced electromagnetic field is mainly distributed in the gap layer, it can simultaneously achieve high selectivity and high sensitivity SERS detection of BNP. By dropping serum into the Au nano-finger / BNP antibody / Au nano-finger coupling structure substrate pre-placed in the biological carrier, and directly optically focusing the Au finger surface, it can achieve high selectivity and high sensitivity measurement of specific BNP in a liquid serum environment within 5 seconds.

[0025] Beneficial effects of the present invention:

[0026] 1. By combining Au flexible nano-fingers with antibody incubation technology, a sub-nanometer antibody gap with uniform gap size is obtained under the action of micro-capillary force. The gap size is determined by twice the longitudinal size of the antibody, rather than the resolution of nano-imprinting, and has the characteristics of gap size protection.

[0027] 2. Due to the sub-nanometer longitudinal size characteristics of the antibody, the coupled electromagnetic field can achieve electromagnetic field enhancement with the participation of quantum plasmons, and the enhancement multiple can achieve SERS detection at the single-molecule level.

[0028] 3. In addition to being a gap layer, the antibody also plays a role in capturing and localizing BNP molecules in the serum. In the subsequent Raman detection, the Raman signal of BNP can be directly measured in a liquid environment, avoiding the influence of other proteins in the serum. At the same time, considering the enhanced local electric field, high sensitivity and high selectivity detection of BNP can be achieved. Since the Raman detection time is very short, the detection time of BNP can be greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the detection method of brain natriuretic peptide, a secretion marker of mercury acute myocardial infarction, based on the Au flexible nano-finger array structure. First, the BNP antibody solution is titrated onto the Au flexible nano-finger, and then through the collapse effect, an Au nano-finger / BNP antibody / Au nano-finger coupling structure with BNP antibody as the interstitial layer is formed. Finally, by dripping serum containing BNP molecules into the coupling structure, the antibody will specifically capture BNP and localize it in the coupled electromagnetic field, achieving high sensitivity and high selectivity measurement of SERS.

[0030] Figure 2 : The coupled structure array formed after the ordered array of Au flexible nano-finger prepared by nanoimprinting of the present invention is modified by BNP antibody solution and dried, including scanning electron microscope images (SEM) of hexamers and polymers.

[0031] Figure 3 After BNP antibody and antigen are dropped on the gold film respectively, the Raman scanning results of antibody and antigen can be used to calibrate the characteristic peaks of antibody and antigen.

[0032] Figure 4 From bottom to top, the spectrum of gold nano-fingers, the spectrum of BNP antigen, and the spectrum of BNP antigen dropped on gold nano-fingers. The gold nano-fingers array structure can measure BNP signals.

[0033] Figure 5From bottom to top, the Raman spectra of BNP dropped on the system of antibody and gold nano-fingers combined, and the Raman spectra after cleaning; the Raman spectra of patient serum dropped on the system of antibody and gold nano-fingers combined, and the Raman spectra after cleaning. The signal of BNP can be detected in the serum. DETAILED DESCRIPTION

[0034] Example 1

[0035] The preparation method of Au nano-finger / BNP antibody / Au nano-finger coupling structure comprises the following steps:

[0036] Step 1: Prepare an ordered array structure of Au nano-finger by nano-imprinting, Au thin film deposition and reactive ion beam etching. The height of a single nano-finger is 350 nanometers, including a 300-nanometer polymer pillar and 50 nanometers of Au on top, with a diameter of 75 nanometers and a distance of 110 nanometers between adjacent fingers;

[0037] Step 2: Take the brain natriuretic peptide (BNP) antibody out of the -20°C freezer, thaw it at room temperature, and use a 10ul pipette to measure 1ul of the antibody;

[0038] Step 3: Drop the antibody in step 2 on the metal nano-finger array structure obtained in step 1 and incubate for 8 hours. During the incubation process, the antibody is stored in a refrigerator at 4°C and evaporates under natural conditions to form a closed array structure of Au nano-finger, specifically Au nano-finger / BNP antibody / Au nano-finger, where the BNP antibody serves as the gap layer.

[0039] Step 4: Wash the substrate obtained in step 3 with deionized water to remove excess antibodies, and then dry it naturally at room temperature. Figure 2 As shown, hexameric and multimeric structures are finally formed;

[0040] Example 2

[0041] The highly selective and highly sensitive quantitative detection of BNP molecules based on the Au nanofinger / BNP antibody / Au nanofinger coupling structure includes the following steps:

[0042] Step 1: Drop the antibody and the 10% 4 The brain natriuretic peptide was detected by Raman and the characteristic peaks were calibrated. Figure 3 The peak of brain natriuretic peptide is at 2100 cm -1 、2200cm -1 Place.

[0043] Step 2: Drop a 10% 4pg / ml of brain natriuretic peptide was incubated for 5 minutes, and then Raman detection was performed on it. The Raman spectrum was compared with the Raman spectrum of the clean gold nanofinger array and the Raman spectrum of brain natriuretic peptide dropped on the gold film. The Raman spectrum obtained is shown in Figure 4 As shown, it can be seen that our gold nanofinger array can detect brain natriuretic peptide.

[0044] Step 3: Drop 10 pg / ml brain natriuretic peptide on the gold nanofinger array structure obtained in step 2, let it bind at room temperature for one hour, perform Raman detection, then wash the excess brain natriuretic peptide on the surface with deionized water, dry it at room temperature, and then perform Raman detection.

[0045] Step 4: Drop treated human serum (brain natriuretic peptide content 1.451 pg / ml) on another metal nanofinger array structure obtained in step 2, bind at room temperature for one hour, perform Raman detection, and wash with deionized water before performing Raman detection again.

[0046] Step 5: Put the four Raman spectra obtained in step 3 and step 4 into one graph for comparison. Figure 5 shown.

Claims

1. A brain natriuretic peptide detection device based on surface enhanced Raman scattering technology, characterized in that It is prepared by the following steps: (1) Prepare Au nano-finger ordered array structure by nanoimprinting, Au thin film deposition and reactive ion beam etching; (2) dropping the antibody molecule solution corresponding to BNP onto the Au nano-finger ordered array structure prepared in step 1) and incubating for 8 hours to allow the antibody molecules to bind to Au; (3) Take out the Au nano-finger ordered array structure incubated in step 2), dry it naturally under room temperature and air environment, rinse it with ultrapure water to remove the unconnected and excess BNP antibody molecules, soak it in ultrapure water for 30 minutes, take it out and dry it under natural conditions; (4) During the evaporation of the solution, the micro-surface tension causes six or more adjacent nano-fingers to come close to each other, forming a collapsed Au finger array structure with hexamers or polymers as structural units. A sub-nanometer gap twice the size of the BNP antibody is formed between the finger structures, which can generate an enhanced coupled electromagnetic field regulated by quantum plasmons. In addition, the antibody molecules are distributed on the surface of the Au nano-fingers and can capture the BNP molecules into the coupled electromagnetic field. (5) Place the SERS enhanced substrate based on the Au nano-finger closed array structure obtained in step 4) into a biological carrier, then drop serum into it, cover the carrier, and let it stand for 5 minutes. At this time, the BNP molecules will be captured and localized on the finger surface; (6) By directly focusing on the surface of the nano-finger array in step 5) through the Raman spectrometer, only BNP and antibody molecules are detected by Raman, and the characteristic peaks of the antibody and BNP can be directly obtained. The fingerprint characteristic peak of BNP 1001cm -1 、1453cm -1 、1657cm -1 To realize the fingerprint recognition detection of BNP.

2. The detection device according to claim 1, characterized in that: In step 1), the height of a single nano-finger in the Au nano-finger ordered array structure is 350 nanometers, including a 300-nanometer polymer pillar and 50 nanometers of Au on top, with a diameter of 75 nanometers, and the distance between adjacent fingers is 110 nanometers.

3. The detection device according to claim 1, characterized in that: The incubation time in step 2) is 8 hours.

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