Label-free aptamer sensor and method for detecting lactoferrin using the same

Abstract

The application provides a label-free nucleic acid aptamer sensor and a lactoferrin detection method thereof, and belongs to the technical field of analysis and detection. The label-free nucleic acid aptamer sensor is composed of lactoferrin and a nucleic acid aptamer, the nucleic acid aptamer can specifically bind with the lactoferrin, and the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3'. The nucleic acid aptamer does not need to be labeled and has low cost. The method for detecting lactoferrin based on the label-free nucleic acid aptamer sensor has the advantages of simple operation, low cost, high sensitivity, strong specificity and the like, and avoids the defects of the prior art, such as the need for expensive instruments and professional operators, due to the characteristics that the DNA structure selectively embedded dye can be combined with the small groove region of the nucleic acid aptamer to produce strong fluorescence.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and in particular relates to a label-free nucleic acid aptamer sensor and a method for detecting lactoferrin. Background Technology

[0002] Nucleic acid aptamers, as an ideal emerging biosensor recognition element, are DNA or RNA sequences that specifically bind to target molecules, screened from random oligonucleotide libraries using exponential enrichment methods. They possess advantages such as small size, ease of selection, good stability, ease of modification, and broad target range, and have been widely applied in various technical fields including clinical diagnostics, food safety, and biotherapy. Compared with other antibody- or enzyme-based biosensors, nucleic acid aptamer biosensors offer advantages such as flexible design, rapid and simple operation, cost-effectiveness, and high accuracy and sensitivity. Traditional biosensors mainly consist of two parts: biorecognition and signal conversion. Currently, the signal conversion technologies for aptamer sensors mainly include colorimetry, fluorescence, electrochemistry, and chemiluminescence. Among these, aptamer-based fluorescent sensors are the most common type of optical biosensor, widely studied and applied due to their ease of operation, high efficiency and sensitivity, universal applicability, and low cost. Existing fluorescence methods mostly use fluorescent groups to label aptamers or modify bases, which are both time-consuming and expensive, and the affinity, selectivity, and detection sensitivity of the aptamer are affected after labeling. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a label-free nucleic acid aptamer sensor and a method for detecting lactoferrin. The nucleic acid aptamer of the present invention does not require labeling, and the method for detecting lactoferrin is simple to operate and can detect lactoferrin quickly, accurately and specifically.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a label-free nucleic acid aptamer sensor composed of lactoferrin and a nucleic acid aptamer, wherein the nucleic acid aptamer can specifically bind to lactoferrin, and the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3'.

[0006] The present invention also provides a method for preparing the above-mentioned label-free nucleic acid aptamer sensor, wherein the lactoferrin and the nucleic acid aptamer are incubated to obtain the label-free nucleic acid aptamer sensor.

[0007] Preferably, the incubation temperature is 22–28°C and the incubation time is 15–30 min.

[0008] This invention also provides a method for detecting lactoferrin based on the above-described label-free nucleic acid aptamer sensor or the label-free nucleic acid aptamer sensor obtained by the above preparation method, comprising the following steps:

[0009] (1) Add multiple lactoferrin standard solutions of different known concentrations, then add nucleic acid aptamer solutions respectively, mix and incubate, then add DNA structure selective intercalation dye to react, detect the fluorescence intensity of each, and plot a standard curve;

[0010] (2) Add the test sample solution containing lactoferrin of unknown concentration, then add the nucleic acid aptamer solution, mix and incubate, then add DNA structure selective intercalation dye to react, detect the fluorescence intensity, and obtain the concentration of lactoferrin in the test solution according to the standard curve.

[0011] Preferably, the DNA structure-selective intercalation dye includes SYBR Green I or Pico Green.

[0012] Preferably, the reaction time in steps (1) and (2) is 0 to 10 minutes, and the reaction temperature is 22 to 28°C.

[0013] Preferably, the concentration range of the lactoferrin standard solution is 0–3 μmol / L; and the concentration of the nucleic acid aptamer solution is 100 nmol / L.

[0014] Preferably, the concentration of the DNA structure selectively intercalating dye is 1 to 20×.

[0015] Preferably, the incubation temperature in steps (1) and (2) is 22-28°C, and the incubation time is 15-30 min.

[0016] Preferably, the fluorescence detection conditions are: excitation wavelength 485nm and emission wavelength 528nm.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a label-free nucleic acid aptamer sensor, wherein the nucleic acid aptamer does not require labeling and is low in cost. The method for detecting lactoferrin based on this label-free nucleic acid aptamer sensor utilizes the characteristic that DNA structure-selectively intercalating dyes can bind to the minor groove region of the nucleic acid aptamer of this invention to produce strong fluorescence. This method offers advantages such as simple operation, low cost, high sensitivity, and strong specificity, avoiding the drawbacks of existing detection methods that require expensive instruments and specialized operators. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the principle of detecting lactoferrin using a label-free nucleic acid aptamer fluorescent sensor.

[0020] Figure 2 To determine the detection range and standard curve of lactoferrin standard solution, (A) the detection range and standard curve of lactoferrin standard solution detected by DNA structure selective intercalation dye PG; (B) the detection range and standard curve of lactoferrin standard solution detected by DNA structure selective intercalation dye SG I.

[0021] Figure 3 The matrix standard curves are shown in Figure 1. (A) Matrix standard curve detected by DNA structure-selective intercalation dye PG; (B) Blank matrix standard curve detected by DNA structure-selective intercalation dye SG I.

[0022] Figure 4 The results show the specificity detection results of the aptamer sensor: (A) aptamer sensor with DNA structure selectively intercalating dye PG; (B) aptamer sensor with DNA structure selectively intercalating dye SG I.

[0023] Figure 5 The results show the optimized concentrations of DNA structure-selective intercalation dyes SG I and PG. (A) Optimized concentration of DNA structure-selective intercalation dye PG; (B) Optimized concentration of DNA structure-selective intercalation dye SG I. Detailed Implementation

[0024] This invention provides a label-free nucleic acid aptamer sensor composed of lactoferrin and a nucleic acid aptamer, wherein the nucleic acid aptamer can specifically bind to lactoferrin, and the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3'.

[0025] This invention also provides a method for preparing the above-mentioned label-free nucleic acid aptamer sensor, wherein the lactoferrin and the nucleic acid aptamer are incubated to obtain the label-free nucleic acid aptamer sensor. In this invention, the incubation temperature is preferably 22-28°C, more preferably 24-26°C; the incubation time is preferably 15-30 min, more preferably 16-19 min.

[0026] This invention also provides a method for detecting lactoferrin based on the above-described label-free nucleic acid aptamer sensor or the label-free nucleic acid aptamer sensor obtained by the above preparation method, comprising the following steps:

[0027] (1) Add multiple lactoferrin standard solutions of different known concentrations, then add nucleic acid aptamer solutions respectively, mix and incubate, then add DNA structure selective intercalation dye to react, detect the fluorescence intensity of each, and plot a standard curve;

[0028] (2) Add the test sample solution containing lactoferrin of unknown concentration, then add the nucleic acid aptamer solution, mix and incubate, then add DNA structure selective intercalation dye to react, detect the fluorescence intensity, and obtain the concentration of lactoferrin in the test solution according to the standard curve.

[0029] In this invention, multiple lactoferrin standard solutions of different known concentrations are added. The concentration range of the lactoferrin standard solutions is preferably 0–3 μmol / L, more preferably 0–500 nmol / L. As a preferred embodiment, the concentration gradient of the lactoferrin standard solutions is 0, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 2000, and 3000 nmol / L. In this invention, after adding the lactoferrin standard solutions, nucleic acid aptamer solutions are added separately and then mixed and incubated. The nucleic acid aptamer solution is preferably prepared by dissolving and diluting it in ultrapure water to a certain concentration. The concentration of the nucleic acid aptamer is preferably 100 nmol / L. The concentration of the nucleic acid aptamer in this invention is sufficient to bind lactoferrin in the lactoferrin-containing sample solution and can be used for fluorescence detection. In this invention, the incubation temperature in steps (1) and (2) is preferably 22–28°C, more preferably 24–26°C; the incubation time is preferably 15–30 min, more preferably 16–19 min. In this invention, after mixed incubation, a DNA structure-selective intercalation dye is added for reaction, and the fluorescence intensity of each dye is detected to plot a standard curve. In this invention, the DNA structure-selective intercalation dye preferably includes SYBR Green I or Pico Green. In this invention, when the DNA structure-selective intercalation dye is SYBR Green I, the detection linear range of lactoferrin is preferably 20–500 nmol / L; when the DNA structure-selective intercalation dye is Pico Green, the detection linear range of lactoferrin is preferably 20–500 nmol / L. The DNA structure-selective intercalation dyes SYBR Green I or Pico Green of this invention have low toxicity and emit only weak fluorescence in the free state, but can produce a strong fluorescence signal after binding to single-stranded DNA. In this invention, the concentration of the DNA structure-selective intercalation dye is preferably 1–20×, more preferably 5×. In this invention, the reaction time in steps (1) and (2) is preferably 0–10 min, more preferably 3–7 min; the reaction temperature is preferably 22–28 °C, more preferably 24–26 °C. As a preferred embodiment, when the lactoferrin-containing sample solution is infant formula, the pretreatment of the infant formula includes: dissolving 0.02–0.06 g of infant formula in 0.8–1.2 mL of 45–55 mmol / L acetic acid, vortexing to mix, centrifuging to obtain the intermediate supernatant, centrifuging the intermediate supernatant again, obtaining the supernatant, diluting, and setting aside; the centrifugation method is preferably 7500–8500 rpm for 5–15 min. In this invention, the fluorescence detection conditions are preferably: excitation wavelength 485 nm and emission wavelength 528 nm.

[0030] The principle of the method for detecting lactoferrin in this invention is as follows: Figure 1 As shown: Lactoferrin and nucleic acid aptamers are incubated at room temperature to form a label-free nucleic acid aptamer sensor. Then, SYBR Green I or Pico Green dyes are added to the label-free nucleic acid aptamer sensor, respectively. After the nucleic acid aptamers bind to the target (lactoferrin), the remaining free nucleic acid aptamers will bind to the dyes SYBR Green I or Pico Green, producing a strong fluorescence signal. The higher the concentration of lactoferrin, the more nucleic acid aptamers in the system bind to lactoferrin, and the fewer nucleic acid aptamers can bind to the dyes SYBR Green I and Pico Green, resulting in a weakening of the fluorescence signal. Quantitative analysis is performed based on the above principle.

[0031] The method for detecting lactoferrin of the present invention is used to detect the concentration of lactoferrin in infant formula.

[0032] The method for detecting lactoferrin in this invention has high sensitivity, high specificity, and high accuracy.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] In the following embodiments, the DNA structure-selective intercalation dyes SYBR Green I and Pico Green were both purchased from Thermo Fisher Scientific (China) Co., Ltd., wherein the DNA structure-selective intercalation dye SYBR Green I is hereinafter referred to as SG I, and the DNA structure-selective intercalation dye Pico Green is hereinafter referred to as PG.

[0035] Example 1

[0036] A method for preparing a label-free nucleic acid aptamer sensor, the specific steps of which are as follows:

[0037] Label-free nucleic acid aptamer sensors were prepared by incubating a sample solution containing lactoferrin with 100 nmol / L nucleic acid aptamers at 25°C for 18 min.

[0038] In this embodiment, the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3' (SEQ ID No. 1).

[0039] Example 2

[0040] A method for preparing a label-free nucleic acid aptamer sensor, the specific steps of which are as follows:

[0041] Label-free nucleic acid aptamer sensors were prepared by incubating a sample solution containing lactoferrin with 100 nmol / L nucleic acid aptamers at 22°C for 30 min.

[0042] In this embodiment, the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3' (SEQ ID No. 1).

[0043] Example 3

[0044] A method for preparing a label-free nucleic acid aptamer sensor, the specific steps of which are as follows:

[0045] Label-free nucleic acid aptamer sensors were prepared by incubating a sample solution containing lactoferrin with 100 nmol / L nucleic acid aptamers at 28°C for 15 min.

[0046] In this embodiment, the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3' (SEQ ID No. 1).

[0047] Example 4

[0048] A method for detecting lactoferrin based on a label-free nucleic acid aptamer sensor, comprising the following steps:

[0049] (1) Dissolve lactoferrin standards in ultrapure water to prepare lactoferrin standard solutions of 0, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 2000, and 3000 nmol / L. Take 60 μL of each lactoferrin standard solution into an ELISA plate, add 30 μL of 100 nmol / L nucleic acid aptamer solution, mix and incubate at 25 °C for 18 min, and finally add 10 μL of 5×SG I. Mix and react at 25 °C for 5 min, detect the fluorescence intensity of each, and plot the standard curve.

[0050] (2) Take 60 μL of the sample solution containing lactoferrin into the ELISA plate, then add 30 μL of 100 nmol / L nucleic acid aptamer solution, mix and incubate at 25 °C for 18 min, finally add 10 μL of 5×SG I, mix and react at 25 °C for 5 min, and detect the fluorescence intensity.

[0051] In this embodiment, the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3' (SEQ ID No. 1).

[0052] Depend on Figure 2 It can be seen that lactoferrin exhibits a good linear relationship with fluorescence intensity in the concentration range of 20–500 nmol / L, and the detection limit of SG I is 2 nmol / L.

[0053] Example 5

[0054] The difference between this embodiment and embodiment 4 is that in this embodiment, steps (1) and (2) are "adding 10 μL of 5×PG at the end", while the other steps are the same as in embodiment 4.

[0055] Depend on Figure 2 It can be seen that lactoferrin exhibits a good linear relationship with fluorescence intensity in the concentration range of 20–500 nmol / L, and the detection limit for PG is 2.0 nmol / L.

[0056] Example 6

[0057] A method for detecting lactoferrin based on a label-free nucleic acid aptamer sensor, comprising the following steps:

[0058] (1) Dissolve lactoferrin standards in ultrapure water to prepare lactoferrin standard solutions of 0, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 2000, and 3000 nmol / L. Take 60 μL of each lactoferrin standard solution into an ELISA plate, add 30 μL of 100 nmol / L nucleic acid aptamer solution, mix and incubate at 25 °C for 18 min, and finally add 10 μL of 5×SG I. Mix and react at 22 °C for 7 min, detect the fluorescence intensity of each, and plot the standard curve.

[0059] (2) Take 60 μL of the sample solution containing lactoferrin into the ELISA plate, then add 30 μL of 100 nmol / L nucleic acid aptamer solution, mix and incubate at 25 °C for 18 min, finally add 10 μL of 5×SG I, mix and react at 22 °C for 7 min, and detect the fluorescence intensity.

[0060] In this embodiment, the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3' (SEQ ID No. 1).

[0061] Example 7

[0062] A method for detecting lactoferrin based on a label-free nucleic acid aptamer sensor, comprising the following steps:

[0063] (1) Dissolve lactoferrin standards in ultrapure water to prepare lactoferrin standard solutions of 0, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 2000, and 3000 nmol / L. Take 60 μL of each lactoferrin standard solution into an ELISA plate, add 30 μL of 100 nmol / L nucleic acid aptamer solution, mix and incubate at 25 °C for 18 min, and finally add 10 μL of 5×SG I. Mix and react at 25 °C for 5 min, detect the fluorescence intensity of each, and plot the standard curve.

[0064] (2) Take 60 μL of the sample solution containing lactoferrin into the ELISA plate, then add 30 μL of 100 nmol / L nucleic acid aptamer solution, mix and incubate at 25 °C for 18 min, finally add 10 μL of 5×SG I, mix and react at 28 °C for 3 min, and detect the fluorescence intensity.

[0065] In this embodiment, the nucleotide sequence of the nucleic acid aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3' (SEQ ID No. 1).

[0066] Example 8: Actual Sample Testing

[0067] Weigh 0.04 g of blank milk powder, add 1 mL of 50 mmol / L acetic acid, vortex to mix, centrifuge at 8000 r / min for 10 min, collect the intermediate supernatant, repeat twice, filter the intermediate supernatant through a 0.22 μm filter membrane to obtain the test sample solution, i.e., the test sample solution containing lactoferrin. Determine the lactoferrin in the milk powder sample according to the method for detecting lactoferrin in Example 4 or 5. Results are as follows... Figure 3 As shown, the concentration of lactoferrin in the matrix solution showed a good linear relationship with the fluorescence intensity in the range of 20–500 nmol / L.

[0068] The lactoferrin detection method described in Example 4 or 5 was used for spiked recovery determination. The spike concentrations were 50, 100, and 200 nmol / L. The recovery results are shown in Table 1.

[0069] Table 1 Results of spiked recovery determination

[0070]

[0071]

[0072] The results in Table 1 show that the average recovery rate of lactoferrin is between 90.8% and 95.8%, and the relative standard deviation (RSD) is less than 10%, indicating that the method for detecting lactoferrin in this invention has good accuracy and stability and is suitable for the detection of lactoferrin in infant formula.

[0073] Example 9: Confirmation of Detection Results

[0074] Three types of infant formula milk powder with different lactoferrin content were selected. 0.04 g of each of the three types of infant formula milk powder with different lactoferrin content was weighed out, and 1 mL of 50 mmol / L acetic acid was added to each. After vortexing and mixing, the mixture was centrifuged at 8000 r / min for 10 min. The intermediate supernatant was collected, and this process was repeated twice. The intermediate supernatant was filtered through a 0.22 μm filter membrane to obtain the test sample solutions containing lactoferrin. The three milk powder samples with different lactoferrin contents were analyzed according to the method for detecting lactoferrin in Example 4, and the results were compared with those obtained by high-performance capillary electrophoresis. The results are shown in Table 2.

[0075] Table 2 Comparison of detection results between the method of the present invention and high-performance capillary electrophoresis (HPCE)

[0076]

[0077] The results show that, compared with high-performance capillary electrophoresis, the method of the present invention for detecting lactoferrin yields lactoferrin content similar to that detected by high-performance capillary electrophoresis. Therefore, the method of the present invention for detecting lactoferrin has high accuracy.

[0078] Example 10 Specificity Analysis Experiment

[0079] α-lactalbumin (α-La), β-lactoglobulin (β-Lg), bovine serum albumin (BSA), mixture 1 (α-La, β-Lg, BSA, 1:1:1) and mixture 2 (LF, α-La, β-Lg, BSA, 1:1:1:1) were selected as the detection targets, and the determination was carried out according to the method for detecting lactoferrin in Example 4, and the fluorescence signals were collected.

[0080] The results are as follows Figure 4 As shown, the fluorescence signal change induced by the target compound lactoferrin was very significant, while the fluorescence signal changes of other protein solutions were very small. This result indicates that the method for detecting lactoferrin based on a label-free nucleic acid aptamer fluorescent sensor has high specificity and specificity.

[0081] Example 11: Determination of SGI and PG concentrations

[0082] Lactoferrin aptamer was diluted to a concentration of 100 nmol / L. The concentrations of SG I and PG were 1×, 3×, 5×, 7×, 10×, and 20×, respectively. Ultrapure water was used instead of sample solution to determine the concentrations of SG I and PG. First, 30 μL of the above aptamer solution was added to each well, followed by 60 μL of sample solution. After mixing, the mixture was incubated at 25°C for 18 min. Then, 10 μL of SG I and PG at different concentrations were added, mixed, and reacted at 25°C for 5 min. The fluorescence intensity was then detected. The results are shown below. Figure 5 As shown.

[0083] Depend on Figure 5 The results showed that the fluorescence intensity was highest when the concentrations of SG I and PG were 5×.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting lactoferrin based on a label-free aptamer sensor, characterized in that, The method comprises the following steps: (1) adding a plurality of different known concentrations of lactoferrin standard solution, respectively adding aptamer solution, mixing and incubating, then adding DNA structure selective intercalating dye for reaction, detecting the fluorescence intensity of each, and drawing a standard curve; (2) adding an unknown concentration of lactoferrin sample solution, respectively adding aptamer solution, mixing and incubating, then adding DNA structure selective intercalating dye for reaction, detecting the fluorescence intensity, and obtaining the concentration of lactoferrin in the sample solution according to the standard curve; The concentration of the aptamer solution is 100 nmol / L; The concentration of the DNA structure selective intercalating dye is 5x; The DNA structure selective intercalating dye is SYBR Green I or Pico Green; The label-free aptamer sensor is composed of lactoferrin and aptamer, the aptamer can specifically bind to lactoferrin, and the nucleotide sequence of the aptamer is 5'-TGGTGCTGCCCCTAGTCTCCGGCTGATAGCTGCTTCTTGG-3'.

2. The method for detecting lactoferrin according to claim 1, characterized by, The label-free aptamer sensor is prepared by incubating the lactoferrin and the aptamer.

3. The method of claim 1, wherein the lactoferrin is detected by, The reaction time in steps (1) and (2) is 3-7 min, and the reaction temperature is 22-28℃.

4. The method of claim 1, wherein the lactoferrin is detected by, The concentration of the lactoferrin standard solution ranges from 0 to 3 μmol / L.

5. The method of claim 1, wherein the lactoferrin is detected by, The incubation temperature in steps (1) and (2) is 22-28℃, and the incubation time is 15-30 min.

6. The method of claim 1, wherein the lactoferrin is detected by, The fluorescence detection conditions are: excitation wavelength 485 nm, and emission wavelength 528 nm.

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