A detection kit and method for a cow milk allergenic protein

Abstract

The application discloses a detection kit and a detection method for cow milk allergen protein, wherein the detection kit comprises 4-mercapto benzoic acid (4-MBA) labeled gold nanoparticles, beta-lactoglobulin aptamer, complementary aptamer, carboxyl functionalized magnetic nanoparticles, beta-lactoglobulin standard and buffer. The application combines SERS signal amplification technology, aptamer specific recognition and magnetic separation technology to construct a gold nanoparticle-magnetic nanosphere composite SERS aptamer sensor. The method has good linearity in the range of 0.1-20 mu g / mL (R²=0.9953), the detection limit is 0.038 mu g / mL, the quantification limit is 0.114 mu g / mL, and the method has the advantages of high specificity, high sensitivity, rapid response, simple operation and low cost. The detection kit prepared by the method can be used for quantitative detection of cow milk allergen beta-lactoglobulin.

Description

Technical Field

[0001] This invention relates to the field of allergen detection, specifically to a method for detecting β-lactoglobulin (β-Lg) based on a surface-enhanced Raman scattering (SERS) aptamer sensor. This invention also relates to a detection kit using the above method. Background Technology

[0002] Food allergies have become a major global public health problem, affecting approximately 3-10% of the population, with a higher incidence in children. Milk is one of the most common food allergens, and β-lactoglobulin (β-Lg) is the main whey protein in milk that triggers allergic reactions. It is a small globular protein (18.3 kDa) belonging to the lipid transporter superfamily and possesses resistance to pepsin digestion, which enhances its sensitizing potential. Even trace amounts of β-Lg residue in processed foods can trigger adverse reactions ranging from urticaria to life-threatening anaphylactic shock in sensitive individuals. Therefore, developing sensitive, reliable, and rapid methods for detecting β-Lg is crucial for food safety and allergen labeling compliance.

[0003] Traditional methods for β-Lg detection include enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While these methods offer high sensitivity and specificity, they typically suffer from limitations such as long operation times, the need for specialized equipment and personnel, and high operating costs. These shortcomings highlight the necessity of developing simpler, faster, and more cost-effective alternative detection strategies.

[0004] Surface-enhanced Raman scattering (SERS) has attracted widespread attention as a powerful analytical technique, possessing unique advantages such as ultra-high sensitivity, rapid response, molecular fingerprint specificity, and compatibility with portable Raman instruments. SERS relies on the significant amplification of molecular Raman signals adsorbed on plasmonic nanostructures (typically noble metal gold or silver nanoparticles). In recent years, SERS-based biosensors have been extensively explored for food safety detection, including the detection of pathogens, contaminants, and allergens.

[0005] Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Similar to antibodies, they offer advantages such as simple chemical synthesis, low batch-to-batch variability, and excellent stability. Integrating aptamers with SERS nanoprobes has led to the development of performance-enhanced aptamer-based SERS sensors. Magnetic nanoparticles (MNPs), particularly Fe3O4 nanospheres, offer additional advantages such as ease of separation, strong enrichment capabilities, and compatibility with SERS platforms. Summary of the Invention

[0006] The purpose of this invention is to provide a detection kit for milk allergenic proteins, aiming to at least partially solve one of the technical problems in related technologies. To this end, one objective of this invention is to provide a detection method for a SERS aptamer sensor based on a gold nanoparticle (Au NP)-magnetic nanosphere (Fe3O4) composite structure. This method has advantages such as high specificity, high sensitivity, rapid response, simple operation, and low cost, and can be used for the quantitative detection of the milk allergen β-lactoglobulin.

[0007] A detection kit for bovine milk allergenic proteins includes: 4-mercaptobenzoic acid (4-MBA) labeled gold nanoparticles (Au@4-MBA NPs), β-lactoglobulin aptamers (Apt), a sequence complementary to the aptamer (cApt), carboxyl-functionalized magnetic nanoparticles (MNPs, Fe3O4), β-lactoglobulin standards, and buffer. The Au@4-MBA NPs are surface-modified with 4-MBA as a Raman reporter molecule and further coupled with a thiol-modified β-lactoglobulin aptamer (Apt-SH) to form a signal probe (Au@4-MBA@Apt). The carboxyl-functionalized magnetic nanoparticles are coupled with the complementary aptamer (cApt) via amide bonds to form a capture probe (MNPs@cApt). The signal probe and the capture probe are assembled via DNA hybridization to form an Au@4-MBA / MNPs nanocomplex.

[0008] The 4-MBA, acting as a Raman reporter molecule, generates a characteristic SERS signal at 1078 cm⁻¹. The β-lactoglobulin aptamer specifically recognizes β-lactoglobulin. When β-lactoglobulin is present, the competitive binding between the aptamer and the target causes Au@4-MBA NPs to be released from the magnetic substrate, resulting in a decrease in the SERS signal. Quantitative detection of β-lactoglobulin is achieved by differential SERS signal (ΔI = I0 − I).

[0009] A method for detecting bovine milk allergenic proteins, comprising: a. Preparation of signal probe: Thiol-modified β-lactoglobulin aptamer (Apt-SH) was incubated with 4-MBA-labeled gold nanoparticles (Au@4-MBA NPs) to form Au@4-MBA@Apt signal probe through Au-S bonds; b. Preparation of capture probe: Carboxyl-functionalized magnetic nanoparticles (MNPs) were activated by EDC / NHS and then incubated with complementary aptamers (cApt) to form MNPs@cApt capture probes through amide bonds; c. Assembling the nanocomposite: The Au@4-MBA@Apt signal probe and the MNPs@cApt capture probe were mixed and incubated in a certain ratio, and Au@4-MBA / MNPs nanocomposite was formed by DNA hybridization. d. β-lactoglobulin detection: The sample to be tested was incubated with Au@4-MBA / MNPs nanocomposite. β-lactoglobulin specifically binds to the aptamer, causing some Au@4-MBA NPs to dissociate from the magnetic nanoparticles. After magnetic separation, the SERS signal of the remaining nanocomposite was measured. Quantitative detection was achieved based on the linear relationship between the differential SERS signal (ΔI = I0 − I) and the concentration of β-lactoglobulin.

[0010] In step a, the average particle size of Au NPs is 40 nm, the concentration of 4-MBA is 1 mM, and the incubation time is 1 h.

[0011] In step a, the concentration of Apt-SH is 100 μM, the incubation time is 2 h, and the temperature is 37 ℃.

[0012] The beneficial effects of this invention are as follows: According to the kit and detection method of this invention, by combining SERS signal amplification technology, aptamer-specific recognition, and magnetic separation technology, it has advantages over traditional ELISA and PCR methods, including faster detection speed, simpler operation, higher sensitivity, stronger specificity, and lower cost. This kit uses a portable Raman spectrometer for detection, enabling rapid on-site detection. It has a wide linear detection range (0.1~20 μg / mL), a low limit of detection (0.038 μg / mL), a limit of quantitation of 0.114 μg / mL, strong specificity, good selectivity for common interfering proteins, and satisfactory recovery rates in actual food samples. It is widely applicable to the detection of the milk allergen β-lactoglobulin. Additional aspects and advantages of this invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the principle of SERS aptamer sensor detecting β-lactoglobulin in an embodiment of the present invention.

[0014] Figure 2 This is a diagram showing the results of optimized detection conditions in an embodiment of the present invention.

[0015] Figure 3 This is a standard curve for β-lactoglobulin detection in an embodiment of the present invention. Detailed Implementation

[0016] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0017] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0018] Example 1 Fabrication of SERS aptamer sensors: (1) Synthesis of gold nanoparticles (Au NPs): Au NPs were synthesized by sodium citrate reduction. 4.24 mL of HAuCl4 solution (4 g / L) was added to 100 mL of ultrapure water, heated to boiling and stirred vigorously. 2.3 mL of 1% (w / v) sodium citrate solution was quickly added, and the solution color changed from light yellow to dark red, indicating the formation of Au NPs. After the color stabilized, heating and stirring were stopped, and the solution was cooled to room temperature. The resulting Au NPs solution was stored at 4 °C.

[0019] (2) Raman reporter molecular marker: 20 μL of 1 mM 4-MBA solution was added to 10 mL of Au NPs solution, and the reaction was carried out with gentle stirring at room temperature for 1 h. After the reaction, the excess 4-MBA was removed by centrifugation at 6500 rpm for 10 min. The precipitate was washed twice with ultrapure water and resuspended in 2 mL of ultrapure water to obtain Au@4-MBA NPs, which were stored at 4 ℃.

[0020] (3) Preparation of signal probe (Au@4-MBA@Apt): 15 μL of 100 μM thiol-modified β-lactoglobulin aptamer (Apt-SH) was added to 1 mL of Au@4-MBA NPs suspension and incubated at 37 °C for 2 h to allow the aptamer to attach to the surface of Au NPs via Au-S bonds. Excess aptamer was removed by centrifugation at 8000 rpm for 10 min, and the precipitate was resuspended in 1 mL of PBS buffer (10 mM, pH 7.4) to obtain Au@4-MBA@Apt, which was stored at 4 °C.

[0021] (4) Preparation of capture probe (MNPs@cApt): 12 μL of 0.1 M NHS and 12 μL of 0.1 M EDC were added to 1.0 mL of 1.0 mg / mL carboxyl-functionalized MNPs, and the carboxyl groups were activated by reacting at room temperature for 15 min. Excess EDC and NHS were removed by magnetic separation, and the activated MNPs were resuspended in 1 mL of PBS buffer (10 mM, pH 7.4). 20 μL of 100 μM complementary aptamer (cApt) was added, and the mixture was incubated with gentle shaking at 37 °C for 2 h to allow cApt to bind to the surface of MNPs via amide bonds. Unbound cApt was removed by magnetic separation and washing twice with ultrapure water to obtain MNPs@cApt, which was then resuspended in 1 mL of PBS buffer and stored at 4 °C.

[0022] (5) Assembly of the nanocomposite (Au@4-MBA / MNPs): Au@4-MBA@Apt and MNPs@cApt were mixed at a molar ratio of 50:1 and incubated at 37 °C for 1 h for DNA hybridization. After magnetic separation, the mixture was washed with PBS buffer and resuspended in PBS to obtain the SERS aptamer sensor.

[0023] When using this assay kit, the β-lactoglobulin sample is incubated with 100 μL of aptasensor solution at 37 °C for 1 h. Due to the specific binding of the aptamer to β-lactoglobulin, some Au@4-MBA NPs dissociate from the MNPs. After magnetic separation to remove the dissociated Au@4-MBA NPs, the remaining nanocomplex is resuspended in 10 μL of PBS buffer, and the SERS signal is measured using a portable Raman spectrometer (785 nm excitation laser, 100 mW power, integration time 10 s). Within a certain concentration range, the SERS signal (1078 cm⁻¹) gradually decreases with increasing β-lactoglobulin concentration. Quantitative detection of β-lactoglobulin is achieved by demonstrating the linear relationship between the differential SERS signal and concentration. For details, please refer to [link to relevant documentation]. Figure 1In the figure, without β-Lg, the Au@4-MBA / MNPs nanocomposite generates a strong SERS signal at 1078 cm⁻¹ (I0); after the addition of β-Lg, the aptamer binds specifically to the target, causing Au@4-MBA NPs to be released from MNPs, and the SERS signal decreases (I). The differential signal ΔI is proportional to the concentration of β-Lg.

[0024] Example 2 The key experimental parameters were systematically optimized to obtain the best detection performance. Figure 2 ).

[0025] (1) Optimization of aptamer (Apt) and complementary aptamer (cApt) concentrations: The concentrations of Apt and cApt were optimized to ensure sufficient recognition element loading while reducing reagent consumption. The optimal concentrations were determined to be 10 μM and 30 μM, respectively. Figure 2 Optimization of Apt concentration in A Figure 2 (Optimization of cApt concentration in B).

[0026] (2) Optimization of the molar ratio of capture probe to signal probe: The optimal ratio of MNPs@cApt to Au@4-MBA NPs@Apt was determined to be 1:50, at which point the differential SERS signal (ΔI) reaches its maximum. Figure 2 (Molar ratio optimization in C).

[0027] (3) Optimization of β-lactoglobulin incubation time: The effect of incubation time on detection efficiency was investigated. When the incubation time exceeded 60 min, the SERS signal did not change significantly, indicating that β-lactoglobulin had been basically recognized and bound by the aptamer within 60 min. Therefore, 60 min was selected as the optimal incubation time. Figure 2 (Optimization of incubation time).

[0028] Optimization of detection conditions: The key experimental parameters were systematically optimized to obtain the best detection performance.

[0029] (1) Optimization of aptamer (Apt) and complementary aptamer (cApt) concentrations: The concentrations of Apt and cApt were optimized to ensure sufficient recognition element loading while reducing reagent consumption. The optimal concentrations were determined to be 10 μM and 30 μM, respectively.

[0030] (2) Optimization of the molar ratio of capture probe to signal probe: The molar ratio of MNPs@cApt to Au@4-MBA NPs@Apt was examined, and the optimal ratio was determined to be 1:50, at which point the differential SERS signal (ΔI) reaches its maximum.

[0031] (3) Optimization of β-lactoglobulin incubation time: The effect of incubation time on detection efficiency was investigated. When the incubation time exceeded 60 min, the SERS signal did not change significantly, indicating that β-lactoglobulin was basically recognized and bound by the aptamer within 60 min. Therefore, 60 min was selected as the optimal incubation time.

[0032] Example 3 Under optimal conditions, the SERS aptamer sensor described in this invention was used to detect β-lactoglobulin standard solutions of different concentrations. Figure 3 A standard curve was plotted with β-lactoglobulin concentration on the x-axis (x, μg / mL) and differential SERS signal (ΔI = I0 − I) on the y-axis.

[0033] The results showed that ΔI had a good linear relationship with β-lactoglobulin concentration in the range of 0.1 to 20 μg / mL, with a linear regression equation of y = −1828.60x + 4790.52 and a correlation coefficient R² = 0.9953.

[0034] Three parallel measurements were performed on a blank sample (0 μg / mL), and the signal values ​​were 7352.88, 7314.78, and 7348.40, respectively. The standard deviation σ was calculated to be 20.82.

[0035] The limit of detection (LOD) is calculated using the formula: LOD = 3.3 × σ / S = 3.3 × 20.82 / 1828.60 = 0.038 μg / mL, and the limit of quantitation (LOQ) is calculated using the formula: LOD = 10 × σ / S = 10 × 20.82 / 1828.60 = 0.114 μg / mL.

[0036] Standard curve of β-lactoglobulin: Under optimal conditions, the SERS aptamer sensor described in this invention was used to detect β-lactoglobulin standard solutions of different concentrations. A standard curve was plotted with β-lactoglobulin concentration as the abscissa (x, μg / mL) and SERS signal as the ordinate (y).

[0037] The results showed that within the range of 0.1–20 μg / mL, the SERS signal exhibited a good linear relationship with the concentration of β-lactoglobulin, with a linear regression equation of y = −1828.60x + 4790.52 and a correlation coefficient R² = 0.9953. The limit of detection (LOD) was 0.038 μg / mL, and the limit of quantitation (LOQ) was 0.114 μg / mL. These results indicate that the SERS aptamer sensor described in this invention has high sensitivity and can meet the requirements for trace β-lactoglobulin detection.

[0038] Example 4 Specificity evaluation of SERS aptamer sensors: The SERS aptamer sensor described in this invention was used to detect β-lactoglobulin and common interfering proteins, including bovine serum albumin (BSA), ovalbumin, casein, and lysozyme. The results showed that only β-lactoglobulin caused significant changes in the SERS signal, while the responses from interfering proteins were negligible, indicating that the SERS aptamer sensor based on aptamer recognition has high specificity.

[0039] This method is highly sensitive and specific, and can be performed using a portable Raman spectrometer without the need for large instruments or equipment. It is simple and quick to operate.

[0040] β-lactoglobulin of known concentration was added to actual food matrix samples and detected using the SERS aptamer sensor described in this invention. The recovery rate was calculated using a standard curve, and the results are shown in Table 1.

[0041] Table 1 Results of β-lactoglobulin spiked recovery experiment (n=3) 0.5 0.48 ± 0.03 96.0 5.2 2.0 1.94 ± 0.08 97.0 4.1 10.0 10.28 ± 0.36 102.8 3.5 20.0 19.12 ± 0.67 95.6 3.5 As shown in Table 1, within the spiked concentration range of 0.5–20 μg / mL, the spiked recoveries of β-lactoglobulin were 95.6%–102.8%, and the relative standard deviations (RSDs) were 3.5%–5.2%. This indicates that the SERS aptamer sensor has good accuracy and precision in food matrices and can meet the requirements for quantitative detection of β-lactoglobulin in actual samples.

[0042] Example 5 Repeatability and stability evaluation of SERS aptamer sensors: (1) Intra-batch repeatability: The SERS aptamer sensor prepared in the same batch was used to perform five parallel measurements on the 10 μg / mL β-lactoglobulin standard solution under the same conditions. The SERS signal intensity at 1078 cm⁻¹ was recorded, and the relative standard deviation (RSD) was calculated to be 4.2%.

[0043] (2) Inter-batch repeatability: Five different batches of SERS aptamer sensors were prepared and tested against 10 μg / mL β-lactoglobulin standard solution. The RSD was 5.8%. The results show that the intra-batch and inter-batch RSDs are both less than 10%, indicating that the SERS aptamer sensor has good precision and reproducibility.

[0044] (3) Storage stability: The prepared SERS aptamer sensor was stored in the dark at 4 ℃, and the SERS signal intensity was measured on days 0, 3, 7, 14, 21 and 30. The results showed that after 30 days of storage in the dark at 4 ℃, the signal retention rate of the SERS aptamer sensor was still higher than 85%, indicating that it has good storage stability and can meet the needs of practical detection applications.

[0045] In summary, this invention achieves highly sensitive and specific quantitative detection of the milk allergen β-lactoglobulin by constructing a SERS aptamer sensor with a gold nanoparticle-magnetic nanosphere composite structure, combined with SERS signal amplification, aptamer-specific recognition, and magnetic separation technology. The reagent kit and detection method obtained by this invention are simple to operate, and can be rapidly detected on-site using a portable Raman spectrometer. It exhibits high sensitivity, strong specificity, and objective result interpretation, and can be used for quantitative and qualitative determination of the content of the milk allergen β-lactoglobulin in food, possessing extremely high value.

[0046] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0047] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A detection kit for bovine milk allergenic proteins, characterized in that: The formulation includes 4-mercaptobenzoic acid (4-MBA) labeled gold nanoparticles (Au@4-MBA NPs), β-lactoglobulin aptamers (Apt), a sequence complementary to the aptamer (cApt), carboxyl-functionalized magnetic nanoparticles (MNPs, Fe3O4), β-lactoglobulin standards, and buffer. The Au@4-MBA NPs are modified with 4-MBA as a Raman reporter molecule and further coupled with thiol-modified β-lactoglobulin aptamers to form a signal probe (Au@4-MBA@Apt). The carboxyl-functionalized MNPs are coupled with complementary aptamers via amide bonds to form a capture probe (MNPs@cApt). The signal probe and the capture probe are assembled through DNA hybridization to form an Au@4-MBA / MNPs nanocomplex.

2. The detection kit for bovine milk allergenic proteins according to claim 1, characterized in that: The 4-MBA generates a characteristic SERS signal at 1078 cm⁻¹; the β-lactoglobulin aptamer can specifically recognize β-lactoglobulin, and through competitive binding, it causes Au@4-MBA NPs to be released from the magnetic substrate, resulting in a decrease in the SERS signal and enabling quantitative detection.

3. The detection method for bovine milk allergenic proteins as described in claim 1, characterized in that... Includes the following steps: a. Preparation of signal probe: Thiol-modified β-lactoglobulin aptamers were incubated with 4-MBA-labeled gold nanoparticles to form Au@4-MBA@Apt signal probes through Au-S bonds; b. Preparation of capture probe: After activation by EDC / NHS, carboxyl-functionalized magnetic nanoparticles are incubated with complementary aptamers to form MNPs@cApt capture probes through amide bonds; c. Assembling the nanocomposite: The Au@4-MBA@Apt signal probe and the MNPs@cApt capture probe are mixed and incubated in a certain ratio. The Au@4-MBA / MNPs nanocomposite is assembled by DNA hybridization. d. β-lactoglobulin detection: The sample to be tested is incubated with the nanocomposite. The specific binding of β-lactoglobulin to the aptamer causes some Au@4-MBA NPs to dissociate. After magnetic separation, the SERS signal of the remaining nanocomposite is measured. Quantitative detection is achieved based on the differential SERS signal.

4. The method for detecting bovine milk allergenic proteins according to claim 3, characterized in that: In step a, the average particle size of Au NPs is 40 nm, the concentration of 4-MBA is 1 mM, and the concentration of Apt-SH is 100 μM.

5. The method for detecting bovine milk allergenic proteins according to claim 3, characterized in that: In step c, the molar ratio of Au@4-MBA@Apt to MNPs@cApt is 50:

1.

6. The method for detecting bovine milk allergenic proteins according to claim 3, characterized in that: The SERS detection conditions in step d are: 785 nm excitation laser, power 100 mW, integration time 10 s.

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