A SERS probe biosensor, its preparation method and usage method

Abstract

The present invention discloses a SERS probe biosensor, a preparation method and a usage method thereof, relating to the field of biotechnology. The SERS probe biosensor includes DNA probe 1, DNA probe 2 and an aptamer. At least 5 bases on DNA probe 1 and DNA probe 2 are complementary paired. The remaining bases on DNA probe 1 and DNA probe 2 are complementary paired with the bases on the aptamer. And DNA probe 1, DNA probe 2 and the aptamer are pairwise paired to form a Y shape. DNA probe 1 is also covalently connected to gold nanoparticles modified with Raman reporter molecules, and DNA probe 2 is covalently connected to magnetic beads with streptavidin modified on the surface. The SERS probe biosensor prepared by the present invention has the advantages of high sensitivity, high specificity, less sample consumption and high precision, and can effectively realize the accurate diagnosis of acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a SERS probe biosensor, a preparation method thereof, and a usage method thereof. Background Art

[0002] Specific detection of biomarkers can indicate the presence, severity, or progression of diseases. Generally speaking, a biomarker is a protein representing changes in structural, physiological, genetic, or biochemical parameters, and its abnormal expression is often detected earlier than clinical imaging findings, showing great potential in the field of precision diagnosis. In the past few decades, biomarker detection technologies have attracted great attention due to the unique properties of biomarkers and have been widely used in the diagnosis of different diseases. For example, various detection methods such as fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), western blot, surface plasmon resonance, etc. are used to achieve direct detection of specific biomarkers. However, these traditional technologies still have some problems in terms of sensitivity, stability, and operation complexity. In addition, the biomarkers of diseases are usually present in very small amounts in blood or tissues, especially in the early stage of diseases, which increases the difficulty of accurate detection. In view of this, there is an urgent need to develop a specific, rapid, and ultrasensitive biomarker detection technology for early diagnosis of diseases.

[0003] Surface-enhanced Raman scattering (SERS) technology, as an ultrasensitive analytical detection method, can provide fingerprint information of biological substances at the molecular level, not only can quantify protein concentration, but also can analyze protein structure, thus achieving accurate detection of proteins. Due to the unique plasmon coupling between adjacent nanoparticles in biomarkers, the electromagnetic field is very strong, and the SERS signal can highly respond to the fractal distribution of "hot spots". In addition, due to the SERS-based immunoassay technology of functional metal nanoparticles, which has excellent sensitivity and multiplex detection ability, it has attracted great interest in the field of early disease diagnosis. In the applicant's early work, SERS probes based on antibody recognition have been successfully fabricated and can sensitively detect several representative protein biomarkers, thus accurately diagnosing prostate cancer and acute myocardial infarction. These findings indicate that the SERS detection technology for rapid and sensitive disease diagnosis often becomes the research focus by performing rapid, in-situ, and non-destructive detection of biomarkers. Compared with antibodies, aptamers have the advantages of low immunogenicity, repeatable synthesis, low cost, easy modification, strong structure conversion ability, and good long-term stability. Therefore, the applicant's recent research is dedicated to constructing SERS-based aptamer biosensors through the strong binding of DNA aptamers to target analytes to achieve selective detection of biomarkers in complex mixed samples.

[0004] Regarding acute kidney injury (AKI), it first triggers biological and molecular changes and then progresses to cellular injury, which can be detected by specific biomarkers before the increase in serum creatinine used for diagnosing AKI, especially in the elderly and malnourished patients.

[0005] Currently, a series of biomarkers have been discovered, such as neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18, cystatin C (Cys C), N-acetyl-β-D-glucosaminidase, and netrin-1, which are present in disease proteins related to the functional kidney. For example, as a tubular protein biomarker that is immediately upregulated after injury, NGAL has been confirmed by a large number of studies to exhibit significant early diagnostic performance in different clinical situations. In addition, Cys C is an essential cysteine protease inhibitor in the human body and has been proposed as an attractive biomarker for glomerular filtration rate for AKI detection. However, due to the presence of many other biological interfering components in blood or tissues, a single biomarker always fails to achieve the required clinical specificity.

[0006] Therefore, considering the key roles of NGAL and Cys C in the occurrence and progression of AKI, the development of SERS-based detection techniques for dual detection of NGAL and Cys C is of great significance for the accurate diagnosis of AKI. Summary of the Invention

[0007] A SERS probe biosensor provided by the present invention, its preparation method and usage method aim to solve the problems existing in the above background technology.

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

[0009] A SERS probe biosensor includes DNA probe 1, DNA probe 2, and an aptamer. At least 5 bases on DNA probe 1 and DNA probe 2 are complementary paired. The remaining bases on DNA probe 1 and DNA probe 2 are complementary paired with the bases on the aptamer, and DNA probe 1, DNA probe 2, and the aptamer are paired pairwise to form a Y shape. DNA probe 1 is also covalently connected to gold nanoparticles modified with a Raman reporter molecule, and DNA probe 2 is covalently connected to magnetic beads with streptavidin modified on the surface.

[0010] Preferably, the Raman reporter molecule is isothiocyanato-malachite green (MGITC). MGITC is selected as the Raman reporter molecule because its absorption in the π-conjugated form through the Au-S covalent bond of gold nanoparticles is stable under toxic matrix conditions. In addition, using the characteristic Raman peak (aromatic ring stretching) of MGITC at 1618 cm -1 can achieve SERS quantification.

[0011] Further, the DNA probe 1 is covalently linked to the Raman reporter molecule-modified gold nanoparticles through Au-S bonds, and the DNA probe 2 is covalently linked to the streptavidin-modified magnetic beads on the surface through SA-Biotin reaction.

[0012] The present invention also provides a preparation method of the above SERS probe biosensor, which mainly includes the following steps:

[0013] S1 Preparation of gold nanoparticles: Add sodium citrate solution and chloroauric acid solution into a reactor for reaction to obtain gold nanoparticles;

[0014] S2 Preparation of Raman reporter molecule-labeled gold nanoparticles: Add Raman reporter molecules to the gold nanoparticles prepared in step S1, stir and react at room temperature until the Raman reporter molecules are fixed on the surface of the gold nanoparticles through Au-S bonds, and then centrifuge to remove the unbound Raman reporter molecules;

[0015] S3 Preparation of DNA-functionalized SERS probes: Mix and incubate DNA probe 1 with tris(2-carboxyethyl)phosphine to obtain mixture 1, then add the Raman reporter molecule-labeled gold nanoparticles prepared in step S2 into mixture 1 and incubate overnight to obtain mixture 2, and then gradually add PBS to mixture 2 within 24 hours, and wash with double-distilled water to remove the unbound DNA to prepare DNA-functionalized SERS probes;

[0016] S4 Preparation of DNA-functionalized streptavidin-modified magnetic beads: Mix and incubate DNA probe 2 with streptavidin-modified magnetic beads on the surface, oscillate at room temperature, wash with pure water, and disperse in PBS to obtain DNA-functionalized streptavidin-modified magnetic beads on the surface;

[0017] S5 Preparation of SERS probe biosensor: Disperse the DNA-functionalized SERS probes prepared in step S3 into the DNA-functionalized streptavidin-modified magnetic beads prepared in step S4, add aptamers, and incubate together overnight at room temperature, and wash with pure water to obtain Y-shaped SERS probe biosensors.

[0018] Among them, the DNA probe 1, DNA probe 2, and aptamers also include a pretreatment step before use: Disperse the DNA probe 1, DNA probe 2, and aptamers in TE buffer, heat to 95°C in a water bath for 5 minutes, then cool to room temperature and store at 4°C for further use.

[0019] As one embodiment of the present invention, in step S3, the volume ratio of DNA probe 1 to tris(2-carboxyethyl)phosphine is 10:1, and the molar ratio is 1:2000.

[0020] As one embodiment of the present invention, in step S4, the volume ratio of DNA probe 2 to streptavidin-coated magnetic beads is 1:2, and the molar ratio is 100:3.

[0021] As one embodiment of the present invention, the concentrations and molar amounts of DNA probe 1, DNA probe 2, and the aptamer are all equal.

[0022] The present invention also provides a method for detecting protein biomarkers in acute kidney injury using the above SERS probe biosensor, including: mixing and incubating a sample with the SERS probe biosensor, then separating the supernatant, and detecting whether the supernatant contains SERS signals.

[0023] Preferably, the detection of the protein biomarker is a dual detection of neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (Cys C).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention develops a sensitive and accurate AKI diagnosis technology. Through a unique Y-shaped aptamer-assisted SERS sensing platform, based on magnetic separation, a recognition and release mechanism is used to detect dual biomarkers of AKI, which has the advantage of high sensitivity.

[0026] 2. The present invention functionalizes gold nanoparticles with DNA-functionalized Raman reporters, and then mixes the SERS probe with DNA-functionalized conjugated magnetic beads and selective aptamers of biomarkers to form a Y-shaped DNA structure. When detecting AKI-related biomarkers NGAL and Cys C, it is detected by the SERS probe released from the pre-constructed Y-shaped structure on the magnetic beads into the supernatant, and then the supernatant is scanned by a Raman spectrometer for SERS to perform sensitive quantitative detection of the dual biomarkers, which has clinical specificity and is of great significance for the accurate diagnosis of AKI.

[0027] 3. The SERS probe biosensor uses the hot spot effect of metal nanoparticles to enhance Raman signals, effectively achieving highly sensitive detection;

[0028] 4. The SERS probe biosensor is not affected by other non-target biomarker organisms for the recognition of specific biomarkers, and has the advantage of high specificity;

[0029] 5. The SERS probe biosensor requires a small amount of sample for single detection, solving the problem of sample consumption by current large-scale clinical detection instruments.

[0030] 6. The SERS probe biosensor can analyze dual biomarkers, achieving precise diagnosis of acute kidney injury with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the detection process of the SERS probe biosensor in the present invention;

[0032] Figure 2 is the synthesis schematic diagram of the Raman-active SERS probe in the present invention;

[0033] Figure 3 is the DLS intensity map of gold nanoparticles in the construction process of the SERS probe biosensor in the present invention;

[0034] Figure 4 is the electrophoresis property characterization data of the Y-shaped aptamer in the SERS probe biosensor in the present invention;

[0035] Figure 5 is the feasibility verification diagram of biomarker sensing of the SERS probe biosensor in the present invention;

[0036] Figure 6 is the selectivity schematic diagram of the SERS probe biosensor in the present invention;

[0037] Figure 7 is the working curve diagram of the SERS probe biosensor for dual AKI biomarkers in the present invention;

[0038] Figure 8 is the detection diagram of the SERS probe for biomarkers in the blood of rats after drug induction in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and do not limit the present invention. Any other embodiments obtained by non-creative substitution or transformation on the main design concept of the present invention are within the protection scope of the present invention.

[0040] Example 1: Preparation of the SERS Probe Biosensor

[0041] Synthesis and preparation of gold nanoparticles:

[0042] Add 75 mL of 2.2 mM sodium citrate to a three-necked round bottom and heat to boiling, while using a condenser to prevent solvent evaporation. After this step, add 0.5 mL of 25 mM HAuCl4 And it was boiled with stirring. It was found that the color of the solution first changed from light yellow to grayish blue within 15 minutes and finally to pink. The boiling liquid was immediately cooled in the same container until the temperature reached 90 °C. Next, 0.5 mL of 60 mM sodium citrate and 0.5 mL of 25 mM HAuCl 4 solution were added successively for about 2 minutes. By repeating this process, gold particles with gradually increasing sizes in 12 generations could be grown. After that, the solution was stirred at 90 °C for another 30 minutes and cooled to room temperature.

[0043] Preparation of Raman reporter molecule-labeled gold nanoparticles:

[0044] The Raman reporter molecule MGITC (1 μL, 10 -4 M) was added to 5 mL of gold nanoparticles and stirred vigorously at room temperature for 1 hour until MGITC was immobilized on the gold nanoparticle surface through Au-S bonds. A centrifugation process (8000 rpm, 25 minutes) was carried out to remove the unbound MGITC. Finally, the prepared MGITC-labeled gold nanoparticles were stored at 4 °C for further use.

[0045] Pretreatment of DNA probe 1, DNA probe 2 and aptamer:

[0046] DNA probe 1, DNA probe 2 and aptamer were dispersed in TE buffer (5 mL of 1 M Tris-HCl, pH = 8.00, 1 mL of 5 mM EDTA) and heated in a water bath at 95 °C for 5 minutes. It was cooled to room temperature and stored at 4 °C for further use.

[0047] Among them, the Y-shaped aptamer SERS probe sequences for detecting NGAL and the Y-shaped aptamer SERS probe sequences for detecting Cys C are shown in Table 1 and Table 2 below.

[0048]

[0049] Table 2: Y-shaped aptamer SERS probe sequences for detecting Cys C

[0050]

[0051]

[0052] Preparation of DNA-functionalized SERS probes:

[0053] First, 100 μL of 10 μM thiolated DNA probe 1 was incubated with 10 μL of 20 mM tris(2-carboxyethyl)phosphine (TCEP) for 60 minutes to activate it at room temperature, obtaining mixture 1. Then, 0.5 mL of Raman reporter-labeled gold nanoparticles was incubated in mixture 1 overnight at room temperature, obtaining mixture 2. 120 μL of PBS (saline) was gradually added to mixture 2 within 24 hours. Subsequently, the MGITC-labeled gold nanoparticles were washed with double-distilled water to remove unbound DNA. Finally, DNA-functionalized SERS probes were obtained and stored in PBS at 4 °C for further use.

[0054] Gold nanoparticles are easy to synthesize, and the overlap between the plasmon absorption of gold nanoparticles and the incident laser (633 nm) significantly increases the signal intensity. The synthesis of Raman-active SERS probes is as Figure 2 shown. Briefly, gold nanoparticles were prepared by reducing HAuCl with sodium citrate under boiling conditions, and through 12 rounds of seed growth, determined by the corresponding hydrodynamic particle size ( 4 ). Figure 2 b) The final average diameter of the gold nanoparticles was 50 nm, and it had a maximum UV-vis absorption at 520 nm ( Figure 2 a). Figure 2 c shows the TEM image of the gold nanoparticles, showing good dispersion and morphology at a diameter of about 40 nm ( Figure 2 d).

[0055] To attach the negatively charged DNA probe 1 to the negatively charged MGITC-labeled gold nanoparticles, a salt aging method was used. Salt was gradually added to the mixture 2 of thiol (SH)-DNA probe 1 and MGITC-labeled gold nanoparticles to reduce charge repulsion. After adding MGITC and DNA probe 1 in the washing step, it was found that the maximum DLS intensity ( Figure 3 ) of the gold nanoparticles increased by about 10 nm. These results indicate that the Raman reporter and probe DNA were successfully bound to the gold nanoparticles.

[0056] Preparation of DNA-functionalized magnetic beads modified with streptavidin:

[0057] Biotinylated DNA probe 2 was bound to the magnetic beads modified with surface-modified streptavidin at the 3'-end of DNA probe 2 through the SA-Biotin reaction. First, 100 μL of 10 μM DNA probe 2 was incubated with 200 μL of magnetic beads modified with surface-modified streptavidin (300 nm) and shaken at room temperature for 1 hour to obtain DNA-functionalized magnetic beads modified with surface-modified streptavidin.

[0058] Preparation of SERS probe biosensor:

[0059] The prepared DNA-functionalized SERS probe was dispersed into the prepared DNA-functionalized magnetic beads modified with streptavidin, and then 100 μL of 10 μM aptamer was added and incubated together overnight at room temperature. Finally, the solution was washed three times with pure water to obtain a Y-shaped SERS probe biosensor, which was stored at 4 °C before use.

[0060] Example 2: Electrophoretic Characterization of Y-shaped Aptamer

[0061] To determine the formation of the Y-shaped aptamer probe, the Y-shaped DNA probe was first studied by gel electrophoresis. When DNA probe 1, DNA probe 2, and the aptamer were present simultaneously, new bands were observed, indicating successful hybridization of the Y-shaped structure. As Figure 4 shown in a. The corresponding DNA bands appeared at 1-6. Lane 1 was DNA probe 1 and the NGAL aptamer, lane 2 was DNA probe 2 and the NGAL aptamer, lane 3 was DNA probe 1, DNA probe 2, and the NGAL aptamer. When the aptamer was incubated with DNA probe 1 and DNA probe 2 separately, new bands of 89 bp and 89 bp appeared in lanes 1 and 2, respectively. When the aptamer was present together with DNA probe 1 and DNA probe 2, a series of new bands with a length of 103 bp were observed in lane 3. When the Cys C aptamer was incubated with DNA probe 1' and DNA probe 2' separately, new bands were observed in lanes 5 and 6, located at 96 bp and 96 bp, respectively. When the aptamer was incubated with DNA probe 1' and DNA probe 2' separately, a series of new bands were observed in lane 6 at 110 bp. These results indicate that when the aptamer was co-incubated with DNA probe 1 and DNA probe 2 according to the experimental design, the Y-shaped DNA structure was successfully hybridized.

[0062] Example 3: Feasibility of SERS Probe Biosensor

[0063] Three groups of hybrid DNA patterns were designed, as Figure 5 shown in a. After activating the thiol-modified DNA probe 1 with TCEP, DNA probe 1 and DNA probe 2 were respectively coupled to the surfaces of MGITC-modified gold nanoparticles and magnetic beads modified with streptavidin on the surface. Then, a specific aptamer was added and co-incubated with the MGITC-modified gold nanoparticles and the magnetic beads modified with streptavidin on the surface to form a model Y-shaped SERS probe biosensor.

[0064] For comparison, the applicant introduced DNA strand S2, which has no complementary pairing ability with either DNA probe 1 or the aptamer bases, to replace DNA probe 2 as sample 1. In addition, the applicant also attempted the DNA hybridization pattern of DNA probe 1 and DNA probe 2 as sample 2 in the absence of the aptamer. To evaluate their binding ability, the samples were placed on a magnetic centrifuge for a few seconds, and then the surface-modified streptavidin magnetic beads would aggregate under the magnetic force. The red color in sample 1 indicates that the free MGITC-modified gold nanoparticles are dispersed in the supernatant, indicating that DNA probe 2 on the surface of the streptavidin magnetic beads is one of the necessary conditions for forming a stable nanostructure ( Figure 5 b). The same result was also observed in sample 2, which is mainly attributed to the weak binding of DNA probe 1 and DNA probe 2 in the absence of the aptamer. However, it can be clearly found that the supernatant in sample 3 becomes colorless, indicating that all the MGITC-modified gold nanoparticles are tightly bound to the magnetic beads ( Figure 5 b). This can be explained by the high affinity between the aptamer and its complementary sequences (DNA probe 1 and DNA probe 2), which are respectively immobilized on the surfaces of the gold nanoparticles and the magnetic beads, inducing the assembly to form a Y-shaped structure, thus keeping the MGITC-modified gold nanoparticles and the magnetic beads close to each other.

[0065] Example 4: Detection process of the SERS probe biosensor

[0066] To improve the detection sensitivity, MGITC-labeled gold nanoparticles were used as the common SERS probes for the aptasensor assay of protein biomarkers. As Figure 1 shown, the surfaces of the gold nanoparticles and the magnetic beads were functionalized with probe ssDNA respectively. Each ssDNA sequence consists of two parts, one part of the ssDNA sequence (DNA probe 1) is complementary to the other part of the DNA sequence (DNA probe 2). Conversely, the other part of the probe ssDNA sequence (DNA probe 1 and DNA probe 2) is complementary to the two parts of the aptamer sequence. Then, the thiolated DNA-modified gold nanoparticles (DNA probe 1), biotinylated DNA-functionalized magnetic beads (DNA probe 2), and the aptamer were mixed in a centrifuge tube to form a Y-shaped aptasensor. In the presence of the target protein, due to the specific recognition of the aptamer and the unstable 7bp hybridized strand of DNA probe 1 and DNA probe 2 at room temperature, the Y-shaped structure will disintegrate. As the concentration of NGAL or Cys C increases, the number of gold nanoparticles on the magnetic beads decreases, and more dispersed gold nanoparticles appear in the supernatant, resulting in a significant increase in the SERS signal of the supernatant. Then, the quantitative analysis of the AKI biomarker is achieved by measuring the calibration curve of different concentrations of the biomarker and the corresponding SERS signal.

[0067] Example 5: Selectivity of the SERS probe biosensor

[0068] In this biosensor, the determination of AKI biomarkers is achieved based on the specific recognition of corresponding aptamers. In actual detection, other common proteins may coexist in serum. Therefore, by studying the cross-reactivity of potential interfering proteins, the detection specificity of the constructed biosensor is further evaluated. For the NGAL aptamer sensor, almost no Raman signal was observed at the characteristic peaks of interfering components such as HSA and Cys C at 1618 cm -1 . On the contrary, NGAL caused a significant change in the Raman signal intensity, showing good detection sensitivity for NGAL in the developed assay. This can be attributed to the favorable structure of the Y-shaped design, which endows the aptamer sensor with selective binding ability and high affinity for the NGAL biomarker. Similarly, for the Cys C aptamer sensor, it also showed excellent selectivity for Cys C detection and almost no response to HSA and NGAL( Figure 6 ).

[0069] Example 6: Quantitative SERS Analysis of AKI Biomarkers

[0070] To evaluate the detection performance of the aptamer sensor, a quantitative analysis of AKI biomarkers was carried out. The characteristic Raman peak of MGITC (1618 cm -1 ) was selected to monitor the levels of NGAL and Cys C. Figure 7 a shows the SERS signals of testing various concentrations of NGAL using the proposed assay. It can be found that as the concentration of NGAL in the system increases, the Raman signal intensity gradually increases, representing a sensitive and activatable signal response to NGAL. In the range of 1 ng / mL to 10 ng / mL, the standard curve plotted based on the increasing Raman signal intensity and the NGAL concentration shows a good linear relationship between the changing Raman intensity and the NGAL concentration, with a correlation coefficient (R 2 ) of 0.9791( Figure 7 b). In addition, a similar increasing trend was also obtained from the SERS spectra for detecting Cys C( Figure 7 c). The SERS intensity as a function of Cys C, with concentrations ranging from 100 to 1000 ng / mL -1 is plotted in Figure 7 d and shows a good linear response (R 2 = 0.9836). In addition, the limits of detection (LOD) of the developed aptasensing platform for NGAL and Cys C were calculated to be 0.054 ng / mL and 0.32 ng / mL, respectively, which can well meet the requirements of clinical applications.

[0071] Example 7: Dual Detection and Application in Rat Plasma Matrix

[0072] To validate the performance of the dual-strand assay and its application in rat plasma matrix, we first established a rat AKI model and investigated it by observing H&E staining of renal tissue sections and SERS measurement at different time points after drug treatment. Most importantly, Figure 8 H&E staining of the kidney section shown in a showed that the brush border and transparent cast disappeared 24 hours after cisplatin treatment. In contrast, histological staining of kidney sections from the control group and the blank group showed normal renal tissue structure, which proved that AKI rat modeling was successful. The standard equation of SERS spectrum is derived from the characteristic peak 1618cm -1 Intensity obtained (NGAL: y = 7224.4x + 309.05, R 2 =0.994; Cys C: y=5772.93x+337.12, R 2 =0.983). After incubation with the probe, the rat plasma supernatant was taken for SERS detection. 1618 Substitute the values ​​into the equation to obtain the concentrations of NGAL and Cys C, as Figure 8 As shown in Figure 8c, the target protein concentration level in the rat blood increased significantly between 4 and 6 hours of AKI, which enabled SERS to detect changes in the target protein concentration level earlier than fluorescence imaging in rats. This also suggests that the Y-type aptamer probe can be used for in vitro SERS detection of AKI rats, with obvious responses in the early stages of AKI.

[0073] Example 8: Quasi-serum sample detection

[0074] To evaluate the performance of dual detection of NGAL and Cys C in clinical scenarios. The concentrations selected within the linear dynamic range of the standard curve were 0.1, 1, 10 ng / μL and 10, 100, 1000 ng / μL. The biomarkers at different concentrations were mixed and added to the plasma. After dual detection of the spiked samples using the Y-type aptamer sensor, the SERS signal intensity increased as expected with the increase in NGAL and Cys C concentrations. The corresponding test line was monitored at 1618 cm -1 The SERS signal intensity at 400 nm was used to quantify the spiked NGAL and Cys C samples. The linear regression equations derived from the NGAL and Cys C calibration curves were used to calculate the recovered concentrations of the biomarkers in the spiked samples based on the SERS signal intensity.

[0075] Then, the approximate concentrations of NGAL and Cys C for each sample can be determined by the proposed biosensor based on the established calibration curve. These results are summarized in Table 3. It can be found that the concentration ranges of NGAL and Cys C in AKI patients are 28.93 to 44.01 ng / mL and 904 to 1201 ng / mL. By analyzing the consistency between the concentrations measured by two different assays, we found that the NGAL and Cys concentrations measured by the SERS assay were highly correlated with those measured by the ELISA assay. In addition, the detection deviation calculated by (C SERS -C ELISA ) / C ELISA × 100% is shown in Table 3. These results indicate that SERS detection can achieve the detection accuracy of ELISA for NGAL and Cys C. In addition, compared with the commonly used ELISA method (100 μL), SERS detection has a larger dynamic range (10 -2 -10 5 ng / mL) and requires less blood sample (only 5 μL). These exciting performances of the SERS probe-based biosensor suggest that this method has the potential to become an alternative tool for the detection of NGAL and Cys C in clinical practice.

[0076] Table 3 SERS-based determination of NGAL and Cys C (n = 5).

[0077]

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be considered within the protection scope of the present invention. Sequence Listing <110> Hainan Medical University <120> A SERS Probe Biosensor and Its Preparation Method and Usage Method <130> 2022.04.12 <160> 24 <170> SIPOSequenceListing 1.0 <210> 1 <211> 75 <212> DNA <213> Artificial sequence <400> 1 agcagcacag aggtcagatg gcgctggata gcaagatcac gttatcatcg taaaccctat 60 gcgtgctacc gtgaa 75 <210> 2 <211> 14 <212> DNA <213> Artificial sequence <400> 2 ctgtgactgc tgct 14 <210> 3 <211> 14 <212> DNA <213> Artificial sequence <400> 3 acctcgtgtc acag 14 <210> 4 <211> 14 <212> DNA <213> Artificial sequence <400> 4 ctgtgaccca tctg 14 <210> 5 <211> 14 <212> DNA <213> Artificial sequence <400> 5 tccagcggtc acag 14 <210> 6 <211> 14 <212> DNA <213> Artificial sequence <400> 6 ctgtgacctt gcta 14 <210> 7 <211> 14 <212> DNA <213> Artificial sequence <400> 7 acgtgatgtc acag 14 <210> 8 <211> 14 <212> DNA <213> Artificial sequence <400> 8 ctgtgacgat gata 14 <210> 9 <211> 14 <212> DNA <213> Artificial sequence <400> 9 ggtttacgtc acag 14 <210> 10 <211> 14 <212> DNA <213> Artificial sequence <400> 10 ctgtgaccgc atag 14 <210> 11 <211> 14 <212> DNA <213> Artificial sequence <400> 11 ggtagcagtc acag 14 <210> 12 <211> 82 <212> DNA <213> Artificial sequence <400> 12 cctaaccgat atcacactca cgaactgtcg gaactcgggc caaatggacg agcgaccatt 60 ggttgttcgt cattggagta tc 82 <210> 13 <211> 14 <212> DNA <213> Artificial sequence <400> 13 ctgtgacggt tagg 14 <210> 14 <211> 14 <212> DNA <213> Artificial sequence <400> 14 tgatatcgtc acag 14 <210> 15 <211> 14 <212> DNA <213> Artificial sequence <400> 15 ctgtgacgtg agtg 14 <210> 16 <211> 14 <212> DNA <213> Artificial sequence <400> 16 acagttcgtc acag 14 <210> 17 <211> 14 <212> DNA <213> Artificial sequence <400> 17 ctgtgacagt tccg 14 <210> 18 <211> 14 <212> DNA <213> Artificial sequence <400> 18 tggcccggtc acag 14 <210> 19 <211> 14 <212> DNA <213> Artificial sequence <400> 19 ctgtgacgtc catt 14 <210> 20 <211> 14 <212> DNA <213> Artificial sequence <400> 20 gtcgctcgtc acag 14 <210> 21 <211> 14 <212> DNA <213> Artificial sequence <400> 21 ctgtgacacc aatg 14 <210> 22 <211> 14 <212> DNA <213> Artificial sequence <400> 22 acgaacagtc acag 14 <210> 23 <211> 14 <212> DNA <213> Artificial sequence <400> 23 ctgtgaccaa tgac 14 <210> 24 <211> 14 <212> DNA <213> Artificial sequence <400> 24 gatactcgtc acag 14

Claims

1. A method for detecting protein biomarkers in acute kidney injury using a SERS probe biosensor for non-diagnostic purposes, characterized in that, it includes the steps of: mixing and incubating a sample with a SERS probe biosensor, then separating the supernatant, and detecting whether SERS signals are contained in the supernatant; the protein biomarker is neutrophil gelatinase lipocalin (NGAL) or cystatin C (Cys C); the SERS probe biosensor includes DNA probe 1, DNA probe 2 and an aptamer. At least 5 bases on DNA probe 1 and DNA probe 2 are complementary paired. The remaining bases on DNA probe 1 and DNA probe 2 are complementary paired with the bases on the aptamer. And DNA probe 1, DNA probe 2 and the aptamer are paired pairwise to form a Y shape. DNA probe 1 is also covalently connected to gold nanoparticles modified with a Raman reporter molecule, and DNA probe 2 is covalently connected to magnetic beads with streptavidin modified on the surface; A method for preparing a SERS probe biosensor includes the following steps: S1 Preparation of gold nanoparticles: Adding a sodium citrate solution and a chloroauric acid solution into a reactor for reaction to obtain gold nanoparticles; S2 Preparation of Raman reporter molecule-labeled gold nanoparticles: Adding a Raman reporter molecule to the gold nanoparticles prepared in step S1, stirring and reacting at room temperature until the Raman reporter molecule is fixed on the surface of the gold nanoparticles through Au-S bonds, and then centrifuging to remove the unbound Raman reporter molecule; S3 Preparation of DNA-functionalized SERS probes: Mixing and incubating DNA probe 1 with tris(2-carboxyethyl)phosphine to obtain mixture 1, then adding the Raman reporter molecule-labeled gold nanoparticles prepared in step S2 into mixture 1 and incubating overnight to obtain mixture 2, and then gradually adding PBS to mixture 2 within 24 hours, and then washing with double-distilled water to remove the unbound DNA to prepare DNA-functionalized SERS probes; S4 Preparation of DNA-functionalized magnetic beads with streptavidin modified on the surface: Mixing and incubating DNA probe 2 with magnetic beads with streptavidin modified on the surface, oscillating at room temperature, washing with pure water, and dispersing into PBS to obtain DNA-functionalized magnetic beads with streptavidin modified on the surface; S5 Preparation of SERS probe biosensors: Dispersing the DNA-functionalized SERS probes prepared in step S3 into the DNA-functionalized magnetic beads with streptavidin modified on the surface prepared in step S4, adding an aptamer, and incubating overnight together at room temperature, and washing with pure water to obtain a Y-shaped SERS probe biosensor.

2. The method according to claim 1, characterized in that: the Raman reporter molecule is isothiocyanato-malachite green (MGITC).

3. The method according to claim 1, characterized in that: DNA probe 1 is covalently connected to the gold nanoparticles modified with the Raman reporter molecule through Au-S bonds, and DNA probe 2 is covalently connected to the magnetic beads with streptavidin modified on the surface through SA-Biotin reaction.

4. The method according to claim 1, It is characterized in that Before use, the DNA probe 1, DNA probe 2 and aptamer further include a pretreatment step: dispersing the DNA probe 1, DNA probe 2 and aptamer in TE buffer, heating them in a water bath to 95 °C for 5 minutes, then cooling them to room temperature and storing them at 4 °C for further use.

5. The method according to claim 1, It is characterized in that: In the step S3, the volume ratio of the DNA probe 1 to tris(2-carboxyethyl)phosphine is 10:1, and the molar ratio is 1:2000.

6. The method according to claim 1, It is characterized in that: In the step S4, the volume ratio of the DNA probe 2 to the magnetic beads surface-modified with streptavidin is 1:2, and the molar ratio is 100:

3.

7. The method according to claim 1, It is characterized in that: The concentrations and molar amounts of the DNA probe 1, DNA probe 2 and aptamer are all equal.

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