A detection device and detection method for prostate-specific antigen

Abstract

The present invention discloses a detection device and a detection method for prostate-specific antigen based on surface-enhanced Raman scattering technology. It relates to the highly selective and highly sensitive SERS detection of prostate-specific antigen (PSA), a specific marker produced by organs during the diagnosis of prostate cancer (PCa). First, an Au nano-finger array structure is obtained through nanoimprinting; then, the aptamer DNA of prostate-specific antigen PSA is modified on the Au nano-finger structure through thiol hydrogen bonds. During the evaporation process of the DNA solution, a finger array structure with tetramers as structural units is formed; finally, serum containing PSA is dropped onto the Au nano-finger / DNA / Au nano-finger coupling structure substrate pre-placed on a biological carrier slide to achieve highly selective and highly sensitive detection of PSA in serum. The detection limit is 0.05 ng / mL, and it has high specificity and stability. Compared with the existing methods for determining prostate-specific antigen, the method of the present invention does not require labeling during preparation and determination, and is easy to operate.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a surface-enhanced Raman detection device and a detection method for highly selective and highly sensitive detection requirements of prostate-specific antigen (PSA), a specific biomarker produced by organs during the diagnosis of prostate cancer. Background Art

[0002] Cancer is a disease with a high mortality rate worldwide and is a major public health problem faced by the world. Therefore, accurately detecting tumor biomarkers is of great significance for the early diagnosis, monitoring, and treatment of diseases. Prostate-specific antigen (PSA) is a glycoprotein with a molecular weight of 33-34 kDa, mainly produced by the prostate. This antigen is a specific biomarker produced by organs during the diagnosis of prostate cancer (PCa). The normal critical value of prostate-specific antigen in serum should be less than 4 ng / mL, and the concentration of prostate-specific antigen usually increases in the case of prostate cancer or other prostate diseases. The successful treatment of these diseases mainly depends on the highly sensitive and selective detection of prostate-specific antigen at an early stage.

[0003] Raman spectroscopy technology is a promising non-invasive detection technology because it provides chemical fingerprint information of analytes and has specific detection characteristics. However, according to the selection law of quantum mechanics, the scattering cross-section of the Raman effect is only one in ten billion of the fluorescence signal, and the signal is very weak. With the development of micro-nano optics, it has been found that the free electrons in the conduction band near the Fermi level of metal micro-nano structures couple with incident light, and the collective coherent fluctuations of surface free electrons will form a local electromagnetic mode, concentrating the incident light field energy within a sub-wavelength spatial range, forming a local enhanced electromagnetic field near the metal surface. Using this enhanced electromagnetic field can significantly increase the intensity of Raman scattering of molecules located near the metal surface, realizing surface-enhanced Raman scattering (SERS) detection.

[0004] In the SERS application of the plasmonic optical effect on the surface of metal micro-nano structures, how to regulate the excitation of the local optical field and obtain a strong local optical field state density is crucial for enhancing the SERS efficiency. Compared with the resonance excitation of a single nanostructure, metal coupled structures can generate a stronger local optical field due to the gap plasmon coupling effect between the structures, and thus have received extensive attention. In addition, from the perspective of the application of SERS in biomedical detection, how to achieve highly selective detection is crucial. In the actual detection environment, in addition to the substance to be detected, there are also a large number of other substances. Since Raman characteristic peaks mainly reveal the vibration information of different molecular parts, other substances in the mixture will cause great interference to the molecule to be detected, thus affecting the true detection results. For the specific biomarker PSA produced by organs during the diagnosis of prostate cancer (PCa), in addition to PSA in serum, there are also a large number of other glycoproteins. How to achieve high selectivity for PSA and high sensitivity for SERS detection is the key point of the application.

[0005] In our previous work, we combined technologies such as EBL, lithography, and nanoimprinting to achieve 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 titrated with a solvent, and the surface tension during its evaporation process will cause the flexible nano-fingers that are very close to each other to collapse and lean against each other. Through this technology, we proposed that the corresponding aptamer DNA modification technology for PSA can be combined to achieve highly selective and sensitive SERS detection of PSA, especially the direct highly sensitive detection of PSA in the serum of actual patients.

[0006] First, the aptamer DNA of prostate-specific antigen is combined with Au nanofingers through a covalent sulfur-gold bond to form an extended conformation on the Au surface. Secondly, during the subsequent collapse process, the Au nanofingers with aptamer DNA attached to the Au surface come close to each other, thus forming an Au nanofinger / aptamer DNA / Au nanofinger coupling structure. The coupling gap is the sub-nanometer aptamer DNA. Such an ultra-small gap will trigger the optimal coupled electromagnetic field regulated by quantum plasmonics, which is crucial for the enhancement of SERS. Finally, the serum containing DNA is dropped into the coupling structure. In the liquid environment, the DNA will capture PSA in the serum and localize PSA near the Au nanofingers, while having no effect on other substances such as proteins in the serum. Thus, as long as the incident laser spot is focused on the Au nanofinger surface, the collected Raman signals are only from the aptamer DNA and PSA. By distinguishing the Raman characteristic peaks of the two, the high-sensitivity (determined by sub-nanometer DNA) and high-selectivity (aptamer DNA captures PSA) detection of PSA in serum can be achieved. Our detection limit is 0.05 ng / mL, with high specificity and stability. This method is used for the determination of prostate-specific antigen in human serum samples, and its advantages are that it does not require prior purification of the sample, can use a lower sample volume (100 μL), and the process is easy to operate. Compared with the existing methods for determining prostate-specific markers, the preparation and determination do not require labeling, the operation is simple, and it can be completed in only 30 minutes. Summary of the Invention

[0007] Provided is a surface-enhanced Raman detection method for highly selective and highly sensitive detection requirements of the specific biomarker PSA produced by organs during prostate cancer diagnosis, belonging to the technical field of analytical detection. This method is simply and reasonably prepared and can be prepared in large quantities at low cost. By using the ordered structure of a flexible Au nanofinger array and binding with PSA aptamer DNA molecules, an Au nanofinger / DNA antibody molecule / Au nanofinger coupling structure is first constructed. Since the spatial longitudinal dimension of the antibody is at the sub-nanometer level, the formed coupling structure can achieve an enhanced electromagnetic field regulated by quantum plasmonics. Secondly, by using the specific interaction between the aptamer DNA in this coupling structure and PSA molecules, PSA molecules are directly captured and localized from the patient's serum to the SERS-enhanced substrate surface, and the high-selectivity and high-sensitivity detection of PSA are realized by using the SERS characteristic peaks different from those of the aptamer DNA in PSA molecules.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A detection device for prostate-specific antigen, characterized in that it is prepared by the following steps:

[0010] 1) Prepare an ordered array structure of Au nanogap arrays by nanoimprinting, Au thin film deposition, and reactive ion beam etching;

[0011] 2) Perform thiol activation and reduction treatment on the aptamer DNA solution for prostate-specific antigen PSA. Then heat all DNA samples in hot water at 90 °C for 20 minutes, and then rapidly cool in an ice-water bath to achieve uniform DNA amplification;

[0012] 3) Drop the aptamer single-stranded DNA solution processed in step 2 onto the ordered array structure of Au nanogap arrays prepared in step 1 for incubation, and place it in a refrigerator at 4 °C for 12 hours, allowing the aptamer single-stranded DNA to bind to the Au nanogap arrays through Au-S bonds. The single-stranded DNA extends in a conformation through Au-S covalent bonds on the gold surface;

[0013] 4) Take out the ordered array structure of Au nanogap arrays modified with single-stranded DNA processed in step 3, rinse with ultrapure water to remove the unconnected and excess single-stranded DNA solution, then soak in ultrapure water for two hours, and take it out to dry under natural conditions;

[0014] 5) During the evaporation of the solution, the micro surface tension causes four adjacent nanogap arrays to come together to form a collapsed Au nanogap array structure with tetramers as the structural unit. A sub-nanogap with a size twice that of DNA is formed between the nanogap structures, which can generate an enhanced coupled electromagnetic field regulated by quantum plasmonics. Moreover, the enhanced electromagnetic field in the tetramer coupling structure can detect different concentrations of PSA solutions without relying on the polarization of the incident light.

[0015] Titrate different known concentrations of PSA solutions on the coupled structure of the Au / DNA / Au nanogap arrays obtained in step 5, which is used as a SERS-enhanced substrate, and let it stand for 20 minutes for SERS measurement. The excitation light source uses a 785-nm laser. In this way, the detection limit of the device can be obtained.

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

[0017] A method for detecting prostate-specific antigen, characterized in that the aforementioned detection device is adopted, and the following steps are added:

[0018] 6) Titrate the serum of a patient on the coupled structure of the Au / DNA / Au nanogap arrays obtained in step 5, which is used as a SERS-enhanced substrate. Place the substrate in the groove of a biological glass slide. At this time, the prostate-specific antigen molecule PSA will be captured by the DNA aptamer, and is identified by the PSA Raman characteristic peak at 1650 cm -1 to identify.

[0019] The height of a single nano-finger in the Au nano-finger ordered array structure in step 1 is 300 nanometers, including a 250-nanometer polymer pillar and 50 nanometers of Au on top, with a diameter of 80 nanometers and a distance of 110 nanometers between adjacent fingers, in order to form a tetramer coupling structure in the subsequent collapse process.

[0020] The thiolated single-stranded DNA sequence in the above step 2 is 5'-HS-(CH2)6-TTT TTA ATT AAA GCT CGCCAT CAA ATA GCT TT-3', wherein T, G, A, and C are base molecules with different structures, and is reduced with tri-(2-formylethyl)phosphine hydrochloride (TCEP) reducing agent for use.

[0021] The DNA incubation in step 3 above is carried out in a refrigerator at 4°C with high humidity overnight for 12 hours, so that one end of the single-stranded DNA is tightly connected to the Au surface through a sulfur-hydrogen bond, and the single-stranded DNA is in a conformation on the gold surface extended by the sulfur-gold covalent bond;

[0022] The substrate in step 6 above is placed in a biological carrier sheet to specifically detect PSA in a serum liquid environment.

[0023] The present invention is based on a flexible Au nano-finger array structure combined with PSA-specific aptamer DNA modification technology to construct a coupling structure with an ultra-small gap of sub-nanometer DNA size. The interaction between PSA and the corresponding aptamer DNA is utilized to achieve the capture and localization of PSA on the surface of Au nano-finger. By distinguishing the Raman characteristic peaks of the aptamer DNA and PSA, high-sensitivity (determined by sub-nanometer DNA) and high-selectivity (aptamer DNA captures PSA) detection of PSA in serum can be achieved. Our detection limit is 0.05 ng / mL.

[0024] Beneficial effects of the present invention:

[0025] 1. By combining Au flexible nano-fingers with aptamer DNA incubation technology, a sub-nanometer gap with uniform gap size is obtained under the action of microcapillary force. The gap size is determined by twice the longitudinal dimension of DNA, rather than the resolution of nanoimprinting, and has the characteristic of gap size protection.

[0026] 2. Due to the sub-nanometer longitudinal size characteristics of the antibody, the optimal coupling electromagnetic field under quantum plasmon regulation can be achieved, achieving a huge SERS enhancement factor.

[0027] 3. It can realize the rapid and specific detection of prostate antigen molecules. In addition, it realizes the specific capture and detection of prostate-specific antigens in complex biological environments in serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 (a) The left is a schematic diagram of SERS detection of prostate-specific antigen PSA in serum based on the Au flexible nano-finger array structure, and the right is the substrate placed in a biological carrier slide for specific PSA detection in a serum liquid environment. (b) Detection results. First, from the Raman spectra of pure DNA and PSA, it can be seen that PSA has a characteristic peak at 1650 cm-1 that is different from DNA. Second, the PSA in serum is identified through the PSA detection results on DNA-modified Au nano-fingers.

[0029] Figure 2 This is a scanning electron microscope image of the closed array structure of gold nano-fingers belonging to the present invention.

[0030] Figure 3 It is a concentration-gradient Raman spectrogram measured by the binding of DNA-bound gold nano-fingers and antigens at different concentrations.

[0031] Figure 4 SERS detection of prostate-specific antigen in a liquid environment was carried out, and measurements in the range of 0.05 - 100 ng / ml were achieved.

[0032] Figure 5 For the sample detection of patient serum, in a liquid environment, we measured the Raman signal peak of prostate-specific antigen. In the measurement of serum, we observed other Raman peaks in addition to DNA and prostate-specific antigen, and these Raman peaks originated from other complex substances in the serum. Detailed implementation method

[0033] Example 1

[0034] A preparation method for the Au nano-finger / aptamer DNA / Au nano-finger coupling structure includes the following steps:

[0035] Step 1: Use nanoimprinting, Au thin film deposition, and reactive ion beam etching to prepare an ordered array structure of Au nano-fingers. The height of a single nano-finger is 300 nanometers, including a 250-nanometer polymer column and 50 nanometers of Au on it, the diameter is 80 nanometers, and the distance between adjacent fingers is 110 nanometers;

[0036] Step 2: Centrifuge the dry film DNA and configure it into a 10 μM solution with ultrapure water, and reduce all thiolated DNA oligomers with tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) reducing agent;

[0037] Step 3: Heat the DNA solution in hot water at 90 °C for 20 minutes, and then quickly cool it in an ice-water bath to achieve uniform DNA amplification for better obtaining SERS spectra;

[0038] Step 4: Drop DNA and prostate-specific antigen on two pieces of gold film respectively, conduct Raman detection, and calibrate the characteristic peaks;

[0039] Step 5: Place two pieces of gold nanofinger array structures obtained in Step 2 into 30 μL of the treated DNA solution, and place them in a refrigerator at 4 °C for 12 hours to allow the DNA to bind to the gold nanofingers via gold-sulfur bonds;

[0040] Step 6: Then take out the gold nanofinger array structure, rinse it with ultrapure water to remove the excess DNA, soak it in ultrapure water for two hours, and after taking it out and drying, the fingers collapse due to the liquid surface tension, and the DNA molecules are captured between the finger structures, finally forming a tetramer structure, as specifically shown in Figure 2 shown.

[0041] Example 2

[0042] Highly selective and sensitive detection of PSA based on the Au nanofinger / aptamer DNA / Au nanofinger coupling structure, including the following steps:

[0043] Step 1: After obtaining the coupling structure of Au / DNA / Au nanofinger array, the detection of prostate-specific antigen solutions with different concentrations can be carried out. Drop the solutions onto the chip in sequence from low concentration to high concentration, and measure the SERS spectra of prostate-specific antigen with different concentrations, as specifically shown in Figure 3 shown;

[0044] Step 2: Detection of prostate-specific antigen in a liquid environment was carried out. Take 100 μL of the prostate-specific antigen solution diluted with ultrapure water and soak it in the gold nanofinger array structure modified with DNA aptamer placed in a biological glass slide. Finally, the measurement in a liquid environment was realized, as specifically shown in Figure 4 shown;

[0045] Step 3: Drop the treated patient serum on another piece of gold nanofinger array structure obtained in Step 6 for Raman detection, and wash it with deionized water and then conduct Raman detection again, as specifically shown in Figure 5 shown.

Claims

1. A detection device for prostate-specific antigen, characterized in that, It is prepared through the following steps: 1) Prepare an ordered array structure of Au nano-fingers by using nanoimprinting, Au thin film deposition, and reactive ion beam etching; 2) Perform thiol activation and reduction treatment on the aptamer DNA solution of prostate-specific antigen PSA, then heat all DNA samples in hot water at 90 °C for 20 minutes, and then rapidly cool in an ice-water bath to achieve uniform DNA amplification; 3) Drop the aptamer single-stranded DNA solution treated in step 2 onto the ordered array structure of Au nano-fingers prepared in step 1 for incubation, place it in a refrigerator at 4 °C for 12 hours, and allow the aptamer single-stranded DNA to bind to the Au nano-fingers by thiol-gold bonds. The single-stranded DNA extends through thiol-gold covalent bonds and conforms on the gold surface; 4) Take out the ordered array structure of Au nano-fingers modified with single-stranded DNA treated in step 3, rinse it with ultrapure water to remove the unconnected and excess single-stranded DNA solution, soak it in ultrapure water for two hours, take it out and dry it under natural conditions; 5) During the evaporation of the solution, the micro-surface tension causes four adjacent nano-fingers to lean against each other, forming a collapsed Au finger array structure with tetramers as the structural unit. A sub-nano gap of 2 times the DNA size is formed between the finger structures, generating an enhanced coupled electromagnetic field regulated by quantum plasmonics. Moreover, the enhanced electromagnetic field in the tetramer coupling structure does not depend on the polarization of the incident light, and thus the detection of PSA solutions with different concentrations can be carried out; 6) In the coupling structure of the Au / DNA / Au nano-finger array obtained in step 5, the serum of the patient is titrated on the SERS-enhanced substrate. The substrate is placed in the groove of the biological glass slide. At this time, the prostate-specific antigen molecule PSA will be captured by the DNA aptamer, and is identified by the PSA Raman characteristic peak at 1650 cm -1 -1.

2. The detection device according to claim 1, characterized in that: In step 1), the height of a single nano-finger in the ordered array structure of Au nano-fingers is 300 nm, including a 250-nm polymer column and 50 nm of Au on it, the diameter is 80 nm, and the distance between adjacent fingers is 110 nm.

3. The detection device according to claim 1, wherein, In step 2), the thiolated single-stranded DNA sequence is 5’ -HS-(CH2)6-TTT TTA ATT AAA GCT CGC CAT CAA ATAGCT TT–3′, where T, G, A, and C are base molecules with different structures, and it is treated with a reducing agent tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) for standby.

Images