Chromatographic assay for detection of various biogenic amines based on aptamer displacement binding

Abstract

This invention discloses a chromatographic analysis method for detecting multiple biogenic amines based on nucleic acid aptamer replacement. The method utilizes the DSN shearing process to cyclically amplify the signals from biogenic amines and combines the sheared long and short nucleic acid probes to achieve signal separation of multiple biogenic amines on HPLC, effectively performing the replacement of target signal sources and realizing rapid identification and detection of multiple biogenic amines in a single run. Compared with the prior art, this invention has the following advantages: (1) The method reduces the detection limits of the four target biogenic amines to 0.25 pM, 0.21 pM, 0.27 pM and 0.19 pM respectively by negligible background signal and cumulative signal amplification, with a linear range of 1 pM-1 μM. (2) The established method is applied to food and drug detection, and the biogenic amine content in actual samples such as gentamicin sulfate, fish and pork samples is measured to prove its applicability. (3) This invention has the advantages of high throughput, high sensitivity, high selectivity and high detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of food and drug testing technology, and relates to a method for achieving high-throughput simultaneous detection of multiple biogenic amines, specifically a chromatographic analysis method based on nucleic acid aptamer replacement combination for detecting multiple biogenic amines. Background Technology

[0002] Biogenic amines are a collective term for a class of biologically active, nitrogen-containing, low-molecular-weight organic substances. Low levels of biogenic amines are crucial components of biological proteins, playing a vital role in regulating the chemical composition of nucleic acids and proteins, as well as in the stability of biological membranes. However, some high levels of biogenic amines are classified as harmful compounds, potentially causing disease or adverse symptoms in sensitive individuals. High intake of biogenic amines (exceeding 100 mg / kg in food) may pose a threat to human health. The main external sources of biogenic amines are common foods and pharmaceuticals, such as antibiotics and meat products. Therefore, accurate detection of excessive levels of biogenic amines (especially histamine, tyramine, spermine, and tryptamine) in food and pharmaceuticals is of great significance.

[0003] Traditional strategies for detecting biogenic amines mainly include chromatography, spectrometry, electrophoresis, colorimetry, chemiluminescence, and electrochemical methods. Commonly used chromatographic methods include thin-layer chromatography, ion-exchange chromatography, gas chromatography, and high-performance liquid chromatography (HPLC), combined with different detectors. However, traditional chromatographic methods for detecting multiple biogenic amines require complex derivatization steps and cumbersome sample pretreatment. Furthermore, existing chromatographic methods sometimes cannot detect multiple different types of biogenic amines in a single run, necessitating multiple analyses of the same sample using different chromatographic methods. Simultaneously, conventional chromatographic detection methods cannot effectively address the problem of matrix interference in antibiotic mixtures; when the target biogenic amine concentration is low, the target peak is often submerged in the impurity spectrum, making effective identification impossible. In addition, narrow linear ranges and high limits of detection (LODs) are also major factors limiting the accurate identification of multiple biogenic amines using traditional chromatographic methods.

[0004] Nucleic acid aptamers, as a novel type of detection probe, are widely used in various detection and analytical techniques. A nucleic acid aptamer is an oligonucleotide sequence that can bind to specific target substances with high specificity and selectivity. Currently, aptamers targeting biogenic amines have been extensively screened and applied in detection methods. The selective binding and separation of biogenic amines by nucleic acid aptamers can effectively eliminate the influence of matrix effects, thereby improving the selectivity and sensitivity of detection.

[0005] Signal amplification methods can further enhance detection sensitivity and have become an effective means of detecting trace targets. Summary of the Invention

[0006] Technical problem solved: To overcome the shortcomings of existing technologies, this invention utilizes a DSN shearing process to cyclically amplify signals from biogenic amines. Combined with sheared long and short nucleic acid probes, it achieves signal separation of multiple biogenic amines on HPLC, effectively replacing the target signal source and enabling rapid identification and detection of multiple biogenic amines in a single run. This has been applied to the identification and detection of multiple biogenic amines in antibiotics and meat.

[0007] Technical solution: A chromatographic analysis method for detecting multiple biogenic amines based on nucleic acid aptamer substitution, including the following steps:

[0008] S1. Biotin-labeled aptamers Aptamer 1, Aptamer 2, Aptamer 3, and Aptamer 4 are coupled to the surface of streptavidin-coated magnetic beads to obtain four aptamer-magnetic bead conjugates. The aptamer sequences are SEQ ID NO:1-4, as shown in Table 1. Biotin-labeled probes Probe 1, Probe 2, Probe 3, and Probe 4 are coupled to the surface of streptavidin-coated magnetic beads to obtain four probe-magnetic bead conjugates. The probe sequences are SEQ ID NO:9-12, as shown in Table 1.

[0009] S2. Add the four aptamer-magnetic bead conjugates to a centrifuge tube, then add Trigger 1, Trigger 2, Trigger 3, and Trigger 4 sequentially (SEQ ID NO: 5-8, as shown in Table 1) and reaction buffer. After reacting at 37°C for 1 hour, add tyramine, histamine, spermine, and tryptamine sequentially, and continue reacting at 37°C for 2 hours. After the reaction is complete, place the tube on a magnetic rack, take the supernatant and add it to a brown centrifuge tube containing the four probe-magnetic bead conjugates, then add DSN and reaction buffer. After the reaction mixture is thoroughly mixed, incubate at 40°C, add 2×DSN to terminate the reaction, place the reaction solution on a magnetic rack, and inject the supernatant into HPLC for detection.

[0010] S3. A 5% acetonitrile and 100mM triethylamine acetate solution was used as mobile phase A, and methanol was used as mobile phase B. The column temperature was set to 35℃, the flow rate was 1.0mL / min, and the proportion of methanol was increased from 10% to 20% within 20min under gradient elution mode. The excitation wavelength and emission wavelength of the fluorescence detector were set to 488nm and 520nm, respectively, to perform fluorescence detection on the target analyte.

[0011] Table 1 Nucleic Acid Sequences

[0012]

[0013]

[0014] Preferably, the specific preparation method of the aptamer-magnetic bead conjugate is as follows: Add magnetic beads to a centrifuge tube, centrifuge, place on a magnetic rack, discard the solvent and keep the magnetic beads in the centrifuge tube, then wash with 1×B&W buffer at low speed and place on a magnetic rack, discard the solvent and keep the magnetic beads; resuspend the magnetic beads in 2×B&W buffer, add 4 kinds of aptamers at the same time, and vortex at room temperature to obtain the aptamer-magnetic bead conjugate.

[0015] Preferably, the specific preparation method of the probe-magnetic bead conjugate is as follows: add magnetic beads to a centrifuge tube, centrifuge, place on a magnetic rack, discard the solvent and keep the magnetic beads in the centrifuge tube, then wash with 1×B&W buffer at low speed and place on a magnetic rack, discard the solvent and keep the magnetic beads; resuspend the magnetic beads in 2×B&W buffer, add 4 kinds of probes at the same time, and vortex at room temperature to obtain the probe-magnetic bead conjugate.

[0016] Preferably, the optimal coupling time and coupling efficiency of the four probes and magnetic beads in S2 are determined by detecting the fluorescence value of the supernatant. The lower the fluorescence value of the supernatant, the higher the coupling efficiency of the probe on the magnetic beads.

[0017] Preferably, the reaction buffer solution is prepared from 50 mM Tris-HCl and 30 mM MgCl2, with a pH of 7.

[0018] Preferably, the DSN can specifically cleave the DNA in the heteroduplex, with the trigger strand being RNA and the probe strand being DNA. After being cleaved by the DSN, the released trigger strand re-participates in the next cycle, achieving signal accumulation and amplification. The released long and short nucleic acid probes carrying fluorescent labels are then detected by HPLC.

[0019] Preferably, the HPLC uses a Phenomenex fully porous hybrid silica column. A 3μm Oligo-RP column with an inner diameter of 50×4.6mm and a particle size of 3μm was used for data processing using LCsolution software.

[0020] Preferably, the detection limits of the method for tyramine, histamine, spermine and tryptamine are reduced to 0.25 pM, 0.21 pM, 0.27 pM and 0.19 pM, respectively, with a linear range of 1 pM-1 μM.

[0021] Application of any of the methods described above in food and drug testing.

[0022] Preferably, the method can simultaneously detect the content of biogenic amines in gentamicin sulfate, fish, and pork samples.

[0023] Beneficial effects: (1) The method can reduce the detection limits of the four target biogenic amines to 0.25 pM, 0.21 pM, 0.27 pM and 0.19 pM respectively by negligible background signal and cumulative signal amplification, with a linear range of 1 pM-1 μM. (2) The established method is applied to food and drug detection, and the biogenic amine content in actual samples such as gentamicin sulfate, fish and pork samples is determined to prove its applicability. (3) The present invention has the advantages of high throughput, high sensitivity, high selectivity and high detection efficiency. Attached Figure Description

[0024] Figure 1 Retention of long and short probes with different base sequences in HPLC.

[0025] Figure 2 Chromatograms of four DNA probes after orthogonal optimization.

[0026] Figure 3 Feasibility analysis. Peak areas: (a) blank, (b) 0.9 U DSN, (c) 1 μM tyramine, histamine, spermine, tryptophan, (d) 1 μM tyramine + 0.9 U DSN, (e) 1 μM histamine + 0.9 U DSN, (f) 1 μM spermine + 0.9 U DSN, (g) 1 μM tryptophan + 0.9 U DSN, (h) 1 μM tyramine, histamine, spermine, tryptophan + 0.9 U DSN. Experimental conditions: 1 μM aptamer and promoter, 100 μM probe, 30 mM Mg 2+ The pH was 7.0, and the mixture was incubated at 40°C for 210 minutes. The error bars represent the standard deviations of three independent experiments.

[0027] Figure 4 (A) Chromatograms of the target analyte at different concentrations; (B) Resolution of the four signal peaks in HPLC.

[0028] Figure 5 This is a schematic diagram of a chromatographic method for detecting various biogenic amines by combining nucleic acid aptamer displacement signals with cyclic amplification. Detailed Implementation

[0029] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0030] Example 1

[0031] A chromatographic analysis method for detecting multiple biogenic amines based on nucleic acid aptamer substitution includes the following steps:

[0032] First, biotin-labeled aptamers (Aptamer 1, Aptamer 2, Aptamer 3, and Aptamer 4) were coupled to the surface of streptavidin-coated magnetic beads, resulting in aptamer-magnetic bead conjugates, which were stored at 4°C for later use. Subsequently, using the same procedure as coupling the aptamers to the magnetic beads, biotin-labeled probes (Probe 1, Probe 2, Probe 3, and Probe 4) were coupled to the surface of streptavidin-coated magnetic beads and stored at 4°C for later use.

[0033] Add 60 μL of the Aptamer 1, Aptamer 2, Aptamer 3, and Aptamer 4 conjugates with magnetic beads to a centrifuge tube. Then, add 15 μL of 1 μM Trigger 1, Trigger 2, Trigger 3, and Trigger 4 and 80 μL of reaction buffer sequentially, and react at 37°C for 1 hour. Next, add 15 μL of 1 μM tyramine, histamine, spermine, and tryptamine sequentially, and react at 37°C for 2 hours. After the reaction is complete, place the tube on a magnetic rack for 90 seconds, and collect the supernatant. Add the supernatant to a brown centrifuge tube containing 60 μL of the Probe 1, Probe 2, Probe 3, and Probe 4 conjugates with magnetic beads. Then, add 0.9 U DSN and 60 μL of reaction buffer. Vortex the reaction mixture for 10 seconds to mix thoroughly. After incubation at 40°C for 210 minutes, the DNA strand cleavage by DSN was stopped with 5 μL of 2×DSN stop solution. The sample was then placed on a magnetic rack for 90 seconds, and the supernatant was injected into an HPLC system for detection.

[0034] A 5% acetonitrile and 100 mM triethylamine acetate (TEAA, pH = 7.0) solution was used as the mobile phase (A), and methanol was used as the mobile phase (B). The column temperature was set to 35 °C, and the flow rate was 1.0 mL / min. In gradient elution mode, the proportion of methanol was increased from 10% to 20% over 20 min. The excitation and emission wavelengths of the fluorescence detector were set to 488 nm and 520 nm, respectively, for fluorescence detection of the target analyte.

[0035] The specific method for modifying magnetic beads with biogenic amine aptamers is as follows: First, add 30 μL of magnetic beads to a 2 mL centrifuge tube, centrifuge for 90 seconds, and then place it on a magnetic rack for 90 seconds. Discard the solvent and retain the magnetic beads in the centrifuge tube. Then, wash the tube three times with 1×B&W buffer on low speed, and again place it on a magnetic rack for 90 seconds, discarding the solvent and retaining the magnetic beads. Resuspend the magnetic beads in 2×B&W buffer, and simultaneously add four different aptamers. Gently vortex at room temperature for a period of time. After vortexing, store the resulting aptamer-magnetic bead conjugate at 4°C for later use.

[0036] The optimal coupling time and coupling efficiency of the biogenic amine aptamer modified on the magnetic beads are determined by adding the aptamer labeled with fluorescein at the 3' end into the centrifuge tube after the same magnetic bead washing steps as above, gently vortexing at room temperature for different times, placing it on the magnetic rack for 90 seconds, taking the supernatant to measure the fluorescence value. The lower the fluorescence value of the supernatant, the more probes are coupled. After 30 minutes, the fluorescence value of the supernatant does not decrease significantly, indicating that 30 minutes is the optimal coupling time. Subsequently, four fluorescein-labeled aptamers are added to four centrifuge tubes respectively, and then the other three unlabeled aptamers are added. After vortexing for 30 minutes, the supernatant is taken to measure the fluorescence value. The coupling efficiency (IE) and percentage content (IP) of the DNA probe on the magnetic beads can be estimated by the ratio of the fluorescence intensity of the supernatant to that of the control group.

[0037] The buffer solution is prepared from 50 mM Tris-Hcl and 30 mM Mgcl2, with a pH of 7.

[0038] The DSN can specifically cleave the DNA in the heteroduplex. All Trigger strands in this method are RNA, and all probe strands are DNA. After DSN cleavage, the released Trigger strands participate in the next round of cycles again to achieve signal accumulation and amplification. The released fluorescently labeled long and short nucleic acid probes enter HPLC for detection.

[0039] As Figure 1 shown, when the number of bases in the nucleic acid strand is the same, the retention order of C, A, and T is C4 < A4 < T4. For C and A, the electrostatic interaction between them and the ion pair increases with the increase of the nucleic acid length. Therefore, the hydrophobic interaction between the obtained ion pair and the stationary phase also increases with the increase of the number of C and A bases. Therefore, with the increase of the nucleic acid length of C and A, their retention time increases. Considering the separation efficiency and experimental cost, from Figure 1 we selected C4, C 12 , C 30 and T4 as the tails of the DNA probe, and their retention times increase in turn.

[0040] After optimization by orthogonal experiment, it is selected that the proportion of methanol increases from 10% to 20% within 0 - 20 minutes, the column temperature is 35 °C, and the flow rate is 1.0 mL / min as the HPLC analysis method for this experiment. As Figure 2 shown, under the optimized chromatographic conditions, C4, C 12 , C 30 and T4 are injected at the same concentration, and the resolution of the chromatographic peaks are 2.059, 2.102, and 5.072 respectively, achieving complete separation between the four peaks.

[0041] To verify the feasibility of this method, different samples were tested for the presence / absence of biogenic amines or DSN under the same reaction conditions. The results are as follows: Figure 3 As shown. A corresponding signal only appears when both the target biogenic amine and the DSN are present. When all four biogenic amines and the DSN are present, a significant increase in the peak area of ​​the chromatographic peaks corresponding to these four target compounds can be observed. Figure 3 h). The above results demonstrate that this method can achieve highly sensitive detection of tyramine, histamine, spermine, and tryptamine in a single injection.

[0042] To evaluate the performance of this method for detecting biogenic amines, we investigated the signal responses produced by different concentrations of biogenic amines under optimal reaction conditions. Figure 4 A). The retention times of the DNA lysis probes corresponding to tyramine, histamine, spermine, and tryptamine were 5.69 min, 9.26 min, 13.40 min, and 16.10 min, respectively. Figure 4 As shown in Figure B, to evaluate the resolution of these four peaks, the resolution of the chromatographic peaks corresponding to the biogenic amine at 1 μM was calculated. The values ​​were R1 = 3.06, R2 = 3.62, and R3 = 2.19. All resolutions were greater than 1.5, indicating that the four peaks were completely separated. At a concentration of 1 pM, the chromatographic peaks corresponding to the four target compounds could still be clearly separated.

[0043] Example 2

[0044] This example demonstrates the detection of four biogenic amines (tyramine, histamine, spermine, and tryptamine) in gentamicin sulfate, pork, and fish:

[0045] Fresh grass carp and pork purchased from the supermarket were tested, and the results are shown in Table 2. Tyramine (12.321 nM) and histamine (14.676 nM) were detected in the fish; histamine (16.982 nM), spermine (17.756 nM), and tryptamine (13.172 nM) were detected in the pork. Since the content of biogenic amines in meat samples increases sharply during storage, reaching a high level after three days, fish stored for three days was selected for testing. To assess the matrix effect, standard solutions of tyramine (1 nM) and histamine (10 nM), spermine (10 nM), and tryptamine (10 nM), respectively, were added to the pretreated fish and pork solutions, and the relative recoveries ranged from 101.2% to 104.5%. The relative standard deviation (RSD) of this method ranged from 1.5% to 4.3%. The same method was used to detect biogenic amines in gentamicin sulfate, and the results in Table 3 show that spermine was detected in gentamicin sulfate. Table 4 lists some common methods for detecting biogenic amines and compares their detection performance. As can be seen from Table 3, our method has a lower LOD and a wider linear range, fully demonstrating its superior detection performance.

[0046] Table 2. Detection of biogenic amines in fresh pork and fish.

[0047]

[0048]

[0049] a Relative recovery rate = (total concentration - blank concentration) / incorporation concentration

[0050] Table 3. Detection of biogenic amines in gentamicin sulfate

[0051]

[0052] a Relative recovery rate = (total concentration - blank concentration) / incorporation concentration

[0053] Table 4 Comparison of various biogenic amine detection strategies

[0054]

Claims

1. Chromatographic assay for the detection of a plurality of biogenic amines based on nucleic acid aptamer displacement binding, characterized in that, The method comprises the steps of: S1, coupling biotin-labeled aptamers Aptamer 1, Aptamer 2, Aptamer 3 and Aptamer 4 to the surface of streptavidin-coated magnetic beads to obtain four kinds of aptamer-magnetic bead conjugates, and the aptamer sequences are SEQ ID NO: 1-4; coupling biotin-labeled probes Probe 1, Probe 2, Probe 3 and Probe 4 to the surface of streptavidin-coated magnetic beads to obtain four kinds of probe-magnetic bead conjugates, and the probe sequences are SEQ ID NO: 5-8; S2, adding the four kinds of aptamer-magnetic bead conjugates into a centrifuge tube, then sequentially adding Trigger 1, Trigger 2, Trigger 3, Trigger 4, the sequences are SEQ ID NO: 9-12 and reaction buffer, and then sequentially adding tyramine, histamine, spermine and tryptamine after 37°C reaction for 1 hour; continue to react at 37°C for 2 hours; after the reaction is completed, place the reaction mixture on a magnetic stand, take the supernatant and add it into a brown centrifuge tube containing the four kinds of probe-magnetic bead conjugates, then add DSN and reaction buffer, mix the reaction mixture uniformly, then incubate at 40°C, add 2×DSN to terminate the reaction, place the reaction mixture on a magnetic stand, take the supernatant and inject it into HPLC for detection; S3, using 5% acetonitrile and 100 mM triethylamine acetate solution as mobile phase A, methanol as mobile phase B, setting the column temperature to 35°C, the flow rate to 1.0 mL / min, increasing the proportion of methanol from 10% to 20% within 20 min in gradient elution mode, setting the excitation wavelength and emission wavelength of the fluorescence detector to 488 nm and 520 nm respectively, and performing fluorescence detection on the target substance; The HPLC uses a full-porous hybrid silica gel column Phenomenex Clarity®3 μm Oligo-RP chromatographic column with an inner diameter of 50 × 4.6 mm and a particle size of 3 μm, and the LCsolution software is used for data processing.

2. The chromatographic assay for detecting multiple biogenic amines based on aptamer displacement binding according to claim 1, wherein, The specific preparation method of the aptamer-magnetic bead conjugate is as follows: take magnetic beads and add them into a centrifuge tube, centrifuge and then place them on a magnetic stand, discard the solvent and retain the magnetic beads in the centrifuge tube, then use 1×B&W buffer to low-grade shake and clean the magnetic beads, place them on a magnetic stand, discard the solvent and retain the magnetic beads; resuspend the magnetic beads in 2×B&W buffer, add the four kinds of aptamers, and vortex at room temperature to obtain the aptamer-magnetic bead conjugate.

3. The chromatographic assay for detecting multiple biogenic amines based on aptamer displacement binding according to claim 1, wherein, The specific preparation method of the probe-magnetic bead conjugate is as follows: take magnetic beads and add them into a centrifuge tube, centrifuge and then place them on a magnetic stand, discard the solvent and retain the magnetic beads in the centrifuge tube, then use 1×B&W buffer to low-grade shake and clean the magnetic beads, place them on a magnetic stand, discard the solvent and retain the magnetic beads; resuspend the magnetic beads in 2×B&W buffer, add the four kinds of probes, and vortex at room temperature to obtain the probe-magnetic bead conjugate.

4. The chromatographic assay for detecting multiple biogenic amines based on aptamer displacement binding according to claim 1, wherein, The optimal coupling time and coupling efficiency of the four probes and the magnetic bead conjugate in the judgment S2 are determined by detecting the fluorescence value of the supernatant, and the lower the fluorescence value of the supernatant, the higher the coupling efficiency of the probe on the magnetic bead.

5. The chromatographic assay for detecting multiple biogenic amines based on aptamer displacement binding according to claim 1, wherein, The reaction buffer is prepared by 50 Mm Tris-Hcl, 30mM Mgcl2, pH=7.

6. The chromatographic assay for detecting multiple biogenic amines based on aptamer displacement binding according to claim 1, wherein, The DSN can specifically cleave the DNA in the heterologous double-stranded chain, the trigger chain is RNA, and the probe chain is DNA. After being cleaved by the DSN, the released trigger chain participates in the next cycle again, realizes the cumulative amplification of the signal, and the released long and short nucleic acid probe carrying the fluorescent label enters the HPLC for detection.

7. The chromatographic assay for detecting multiple biogenic amines based on aptamer displacement binding according to claim 1, wherein, The detection limit of tyramine, histamine, spermine and tryptamine is reduced to 0.25 pM, 0.21 pM, 0.27 pM and 0.19 pM, and the linear range is 1 pM-1 μM.

8. The application of the chromatographic analysis method based on nucleic acid aptamer displacement binding for detecting various biological amines according to any one of claims 1-7 in food and drug detection.

9. Use according to claim 8, characterized in that, The method can simultaneously detect the content of biological amines in kanamycin sulfate, fish and pork samples.

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