CN113943736B

CN113943736B - ssDNA aptamer for specifically recognizing 6' -sialyllactose, and screening method and application thereof

Info

Publication number: CN113943736B
Application number: CN202111235842.1A
Authority: CN
Inventors: 周楠迪; 陈金日; 张雨婷; 王晓丽
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2022-05-03
Anticipated expiration: 2041-10-22
Also published as: CN113943736A

Abstract

The invention utilizes streptavidin modified magnetic bead-SELEX technology to screen by an immobilized DNA library method, obtains 4 ssDNA aptamers with high affinity and specificity with 6' -sialyllactose from 3187 sequencing results, and constructs a fluorescence biosensor for detecting 6' -sialyllactose based on fluorescence resonance energy transfer by taking the ssDNA aptamers as a 6' -sialyllactose biological recognition element, catalyzing self-assembly of hairpins as a signal amplification unit and quantum dots as a signal label. The invention provides a recognition element with high affinity, high specificity and easy modification and marking for the detection of 6' -sialyllactose and a detection method.

Description

ssDNA aptamer for specifically recognizing 6' -sialyllactose, and screening method and application thereof

Technical Field

The invention relates to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry, in particular to a ssDNA aptamer for specifically recognizing 6' -sialyllactose, a screening method and application thereof.

Background

Sialyllactose is a Human Milk Oligosaccharide (HMOs), and plays an important role in promoting the brain development of infants, improving the immunity of infants and the like as a prebiotic. 6 '-sialyllactose and 3' -sialyllactose are the main forms of sialyllactose, both of which have antibacterial activity and immunomodulatory effects. The content of 6' -sialyllactose is an important nutritional index in infant formula milk powder. To date, high performance liquid chromatography, high pH anion exchange chromatography, proton nuclear magnetic resonance spectroscopy, and mass spectrometry have been widely used for the qualitative and quantitative detection of 6' -sialyllactose. However, although these methods are widely used in the food and biomedical fields, they are limited by the disadvantages of long time consumption, high cost, high technical requirements for inspectors, and the like. Therefore, a method for rapidly detecting sialyllactose needs to be developed to meet the market demand, and a high-specificity recognition element is a basis for constructing a detection method.

At present, the biological recognition elements used for constructing sugar biosensors are mainly lectins, antibodies, synthetic molecularly imprinted polymers, borates, and the like. However, due to the complexity of sugars, the above-mentioned biorecognition elements are limited in the construction and application of biosensors. For example, lectins can only recognize specific types of sugars and cannot perform further differential analysis on sugars; the preparation cost of the antibody is high, so that the application of the antibody in sugar detection is limited; the synthesized molecularly imprinted polymer has poor affinity and specificity with borate. Therefore, there is a need to develop a new bio-recognition element to construct a sugar biosensor.

Disclosure of Invention

In order to solve the technical problems, the invention discloses a ssDNA aptamer for specifically recognizing 6 '-sialyllactose, and further constructs a fluorescent biosensor based on the aptamer, wherein the fluorescent biosensor can be used for detecting 6' -sialyllactose and has better sensitivity and accuracy.

The invention discloses a ssDNA aptamer for specifically recognizing 6' -sialyllactose, wherein the nucleotide sequence of the ssDNA aptamer is one of the sequences shown in SEQ ID NO.1-4, and specifically, the sequence of SEQ ID NO.1-4 is as follows:

Apt 3(SEQ ID NO.1)：

5′-TAGGGAATTCGTCGACGGATGCCGTGGCGTCTGCAACGGAAAAGAATTTATCTTGTCCTGCAGGTCGACGCATGCGCCG-3′；

Apt 8(SEQ ID NO.2)：

5′-TAGGGAATTCGTCGACGGATCCATCCCCACGACGGTCAAGGCCGCGTGCCGGTAGGGCTGCAGGTCGACGCATGCGCCG-3′；

Apt 9(SEQ ID NO.3)：

5′-TAGGGAATTCGTCGACGGATCCCGGAGCCACGAGCGAGAGCGCACTACGGCGCCGAACTGCAGGTCGACGCATGCGCCG-3′；

Apt 13(SEQ ID NO.4)：

5′-TAGGGAATTCGTCGACGGATCCGAATACACTATGACTGTCGGAGGTCCGAGTGCGGGCTGCAGGTCGACGCATGCGCCG-3′。

further, the 3 'end or 5' end of the ssDNA aptamer is modified with functional groups or molecules, such as fluorophores, isotopes, electrochemical labels, enzyme labels for providing detection signals, and affinity ligands, thiol groups for forming compositions, which can improve the stability of the ssDNA aptamer.

The ssDNA aptamer is obtained by screening according to the following method:

s1, carrying out PCR amplification on the single-stranded DNA library by using a primer pair with nucleotide sequences shown as SEQ ID NO.8 and SEQ ID NO.9 to construct a double-stranded DNA library;

the sequences in the single-stranded DNA library all structurally conform to the structural characteristics shown in the following general formula: 5 '-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGCCG-3', wherein N represents any one of bases A, T, C, G, N35 represents a random fragment 35 nucleotides in length,

the nucleotide sequence of the upstream primer is as follows:

5′-TAGGGAATTCGTCGACGGAT-3′，

the nucleotide sequence of the downstream primer is as follows:

5′-CGGCGCATGCGTCGACCTG-3′，

one of the upstream primer and the downstream primer modifies a fluorescent group, and the other one modifies biotin;

s2, adding streptavidin modified magnetic beads into the double-stranded DNA library constructed in the S1 step to construct a double-stranded DNA library fixed on the surface of the magnetic beads;

s3, adding target 6' -sialyllactose into the double-stranded DNA library fixed on the surface of the magnetic beads constructed in the step S2 for incubation, obtaining supernatant containing single-stranded DNA through magnetic separation, measuring the fluorescence intensity of the supernatant, and taking the supernatant as a PCR template chain for the next round of PCR amplification to obtain a secondary double-stranded DNA library;

s4, repeating the operations of the steps S2 and S3 for multiple times on the secondary double-stranded DNA library obtained in the step S3, judging the required repetition times by detecting the change of the intensity of the fluorescence signal, and sequencing after the last round of PCR amplification;

s5, screening a sequence with high affinity and high specificity to the 6 '-sialyllactose from the sequencing result obtained in the step S4 to obtain the ssDNA aptamer specifically recognizing the 6' -sialyllactose.

Further, in step S3, 6' -sialyllactose is used as a positive-screen target, and sialic acid and/or lactose is used as a negative-screen target.

Further, the double-stranded DNA library constructed in step S1 is a double-stranded DNA library modified with a fluorophore at one end and biotin at the other end.

The ssDNA aptamer can be used in a composition, a kit, a sensor or a chip for 6 '-sialyllactose detection, and the invention constructs a biosensor for 6' -sialyllactose detection, which comprises one or more ssDNA aptamers, a signal probe completely complementary with the ssDNA aptamer, a hairpin probe Hp1, a hairpin probe Hp2 and quantum dots; the signal probe can trigger the hairpin probe Hp1 and the hairpin probe Hp2 to hybridize to form a DNA double strand, one of the hairpin probe Hp1 and the hairpin probe Hp2 is connected with a fluorescent group, and the other probe can be connected with a quantum dot.

Further, in the hairpin probes, the fluorophore linked to one probe is any measurable fluorescent material such as FAM, Cy5, and the other probe is linked to the quantum dot through a biotin-avidin system. In one embodiment of the invention, biotin is modified on the hairpin probe Hp1, a fluorescent group is modified on the hairpin probe Hp2, and streptavidin is modified on the quantum dots.

Further, the quantum dots are water-soluble quantum dots, such as CdSe/ZnS quantum dots.

Further, hairpin probe Hp1 and hairpin probe Hp2 are both sequences that form a hairpin structure after annealing treatment. The renaturation treatment specifically comprises the steps of carrying out high-temperature denaturation on the hairpin probe sequence at 95 ℃ for 5min, and then placing the hairpin probe sequence in a water bath kettle at 37 ℃ for incubation for 1 h.

Further, the nucleotide sequence of the signal probe is shown as SEQ ID NO.5, and specifically comprises: 5'-CCGTAGTGCGCTCTCGCTCGTGGCT-3' are provided.

Further, the nucleotide sequence of the hairpin probe Hp1 is shown as SEQ ID NO.6, and specifically comprises: 5'-CTCTCGCTCGTGGCTTTTTTTTTTTTTTTTAGCCACGAGCGAGAGCGCACTACGG-3' are provided.

Further, the nucleotide sequence of the hairpin probe Hp2 is shown as SEQ ID NO.7, and specifically comprises: 5'-CCGTAGTGCGCTCTCGCTCGTGGCTAAAAAAAAAAAAAAAAGCCACGAGCGAGAG-3' are provided.

The method for detecting 6' -sialyllactose by adopting the biosensor specifically comprises the following steps:

s1, selecting one or more of the ssDNA aptamers and a signal probe forming a complementary structure with the ssDNA aptamers to construct an aptamer-based nucleic acid molecule hybridization system;

s2, adding a sample to be detected into the nucleic acid molecule hybridization system, releasing the signal probe, separating to obtain a free signal probe, uniformly mixing the free signal probe with a mixture of the hairpin probe Hp1 and the hairpin probe Hp2 which form a hairpin structure, and adding quantum dots;

s3, detecting the change of fluorescence intensity on the hairpin probe connected with the fluorescent group, constructing a standard curve of the fluorescence intensity and the concentration of 6 '-sialyllactose, and calculating to obtain the content of the 6' -sialyllactose in the sample to be detected.

The fluorescence biosensor is used for detecting 6' -sialyllactose, when the 6' -sialyllactose exists, the fluorescence biosensor competitively binds with ssDNA aptamer, changes the secondary structure of the complementary sequence of double-stranded DNA, thereby leading a signal probe to be replaced by the 6' -sialyllactose, when the signal probe is incubated with a hairpin probe which is respectively modified with fluorescent group and biotin, the hairpin probe Hp1 can be triggered to be hybridized and complemented with the hairpin probe Hp2 to form a DNA double chain, the formed DNA double chain is combined with a quantum dot through a biotin-avidin system, a fluorescence resonance energy transfer system based on the quantum dot and the fluorescent group is constructed, because the signal probe can be repeatedly used for many times, a large amount of the hairpin probe Hp1 is triggered to be hybridized and complemented with the hairpin probe Hp2 to form the double chain, thereby leading the fluorescence signal in the constructed fluorescence resonance energy transfer system to be obviously enhanced, the sensitivity of the fluorescence biosensor is improved, and the quantitative detection of the target substance is realized.

By means of the scheme, the invention at least has the following advantages:

(1) according to the invention, by virtue of the bead SELEX technology, ssDNA molecules bound with 6 '-sialyllactose can be enriched when ssDNA in a dsDNA library adsorbed on beads is incubated with target 6' -sialyllactose, and ssDNA aptamers capable of specifically binding with 6 '-sialyllactose can be obtained by screening by taking the ssDNA molecules as a medium for binding and separating without enriching the characteristics of ssDNA not bound with 6' -sialyllactose. Compared with the traditional ssDNA library immobilization method, the dsDNA library immobilized magnetic bead screening method has the advantages of shorter and more efficient operation process, rapidness and convenience, does not need to separate double-stranded DNA generated by PCR into single-stranded DNA, and reduces the influence of reagents on a library system, thereby reducing the generation of false positive.

(2) The ssDNA aptamer specifically bound with 6' -sialyllactose is obtained by screening and is used as a 6' -sialyllactose biological recognition element, the self-assembly of the catalytic hairpin is used as a signal amplification unit, the quantum dot is used as a signal label, and the fluorescence resonance energy transfer-based fluorescence biosensor for detecting 6' -sialyllactose is constructed.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of ssDNA aptamer paramagnetic alternative for 6' -sialyllactose and a detection method based on fluorescence resonance energy transfer and catalytic hairpin self-assembly assisted cycle amplification strategy;

FIG. 2 shows the results of affinity verification of ssDNA aptamers;

FIG. 3 shows the result of ssDNA aptamer specificity verification;

FIG. 4 is a 6' -sialyllactose fluorescence detection standard curve.

Detailed Description

The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

(1) Construction of random ssDNA libraries and their primers:

a. construction of a random ssDNA library of 79 bases in length

5 '-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGC CG-3', wherein N represents any one of bases A, T, C, G.

b. Synthesis of the upstream primer

An upstream primer 1: 5'-TAGGGAATTCGTCGACGGAT-3', respectively;

an upstream primer 2: 5 '-FAM-TAGGGAATTCGTCGACGGAT-3';

c. synthesis of the downstream primer

A downstream primer 1: 5'-CGGCGCATGCGTCGACCTG-3', respectively;

a downstream primer 2: 5 '-biotin-CGGCGCATGCGTCGACCTG-3'.

(2) In vitro screening of aptamers:

s1, carrying out PCR amplification on the single-stranded DNA library with the length of 79nt by using the upstream primer containing the fluorescent group and the downstream primer modified with biotin to construct the double-stranded DNA library for screening the 6' -sialyllactose specific aptamer, wherein a 25 mu L PCR amplification system is shown in Table 1.

TABLE 1PCR amplification System

Raw materials	Concentration of	Volume of
			Upstream primer 2	20μmol·L^-1	1μL
Downstream primer 2	20μmol·L^-1	1μL
			Template DNA	/	1μL
2×PrimeSTARMaxPremix	/	12.5μL
			ddH₂O	/	Make up to 25 μ L system

Amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 15 s; extending for 5min at 72 ℃; 25 cycles.

S2, adding streptavidin modified magnetic beads Str-MMs into the double-stranded DNA library constructed in the step S1, mixing uniformly, constructing a double-stranded DNA library fixed on the surface of the magnetic beads, washing the magnetic beads four times by using buffer solution, and washing away redundant unfixed library, wherein the specific method comprises the following steps:

taking 20 mu L of Str-MMs, washing the Str-MMs for 4 times by using B & W buffer solution, adding 200 mu L of PCR products of the previous round, slightly shaking the mixture, reacting the mixture at room temperature for 2 hours, placing the mixture on a magnetic separation frame, taking out supernatant, washing precipitates for 4 times by using the B & W buffer solution, and adding 200 mu LB & W buffer solution to obtain a double-stranded DNA library fixed on the surface of magnetic beads;

s3, adding 1 mu L of target 6' -sialyllactose (target sialic acid and lactose with the same concentration are used for replacing in the process of reverse screening) into the double-stranded DNA library fixed on the surface of the magnetic beads constructed in the step S2, uniformly mixing, reacting at room temperature for 1h, obtaining supernatant containing single-stranded DNA through magnetic separation, measuring the fluorescence intensity of the supernatant, and simultaneously taking a trace amount of supernatant as a PCR template chain for the next round of PCR amplification to obtain a secondary double-stranded DNA library;

s4, repeating the steps S2 and S3 for multiple times on the secondary double-stranded DNA library obtained in the step S3, judging the required repetition times by detecting the change of the fluorescence signal intensity, and sending the sample to perform high-throughput sequencing after the last round of PCR amplification;

s5, carrying out sequence statistics and comparison analysis on the high-throughput sequencing result obtained in the step S4, and selecting 4 candidate aptamer sequences Apt 3, Apt 8, Apt 9 and Apt 13 from the sequence statistics and comparison analysis to synthesize;

s6, carrying out affinity and specificity tests on the 4 sequences synthesized in the step S5 by adopting a graphene oxide adsorption method, wherein the specific method and results are as follows:

aptamer sequences (50nM,100nM,150nM,200nM,250nM,300nM,500nM) with fluorophore 6-carboxyfluorescein (FAM) modification at different concentrations were added to 200. mu.L of binding buffer, pre-denatured at 90 ℃ for 5min, ice-washed for 10min, and placed at room temperature for 10 min. Then 20. mu.L of graphene oxide was added and incubated for 2h at room temperature. mu.L of sialyllactose (1mM) was added to the mixture, the mixture was gently shaken and reacted at room temperature for 1 hour, and then the mixture was centrifuged at 13000rpm for 5min to obtain a supernatant. Finally, the fluorescence intensity (emission wavelength 520nm, excitation wavelength 494nm) of all the supernatants is measured by a microplate reader. The obtained fluorescence intensity values were plotted on a corresponding curve, and fitted nonlinearly by the equation y ═ Bmax × free ssDNA/(Kd + free ssDNA) to obtain the affinity Kd values of each aptamer, as shown in fig. 2.

Add 200. mu.L of binding buffer to FAM-modified aptamers of the same concentration, followed by pre-denaturation at 90 ℃ for 5min, ice-bath for 10min, and standing at room temperature for 10 min. Adding graphene oxide, and incubating for 2h at room temperature. Adding 1 mu L of sialyllactose, lactose, sucrose, glucose, maltose and a sialic acid solution (the concentration is 1mM) into the solution, slightly shaking, reacting at room temperature for 1h, centrifuging the mixed solution at 13000rpm for 5min, taking supernatant, adding all the supernatant into a 96-well plate, measuring the fluorescence intensity by using an enzyme-linked immunosorbent assay (ELISA) instrument, wherein the emission wavelength is 520nm, and the excitation wavelength is 494 nm. The obtained fluorescence intensity values were plotted as a corresponding curve, as shown in FIG. 3.

Wherein, the sequence of Apt 3 is specifically as follows:

the sequence of Apt 8 is specifically:

the sequence of Apt 9 is specifically:

the sequence of Apt 13 is specifically:

example 2

S1, denaturing the aptamer and the signal probe at a high temperature of 95 ℃ for 5min, carrying out ice bath for 10min, then incubating at 37 ℃, adding streptavidin-modified magnetic beads, carrying out slight oscillation reaction for a period of time, forming a nucleic acid molecule hybridization system based on the aptamer and the signal probe, carrying out magnetic separation on the solution by using a magnet, discarding supernatant, washing the precipitate for 1 time by using PBS (phosphate buffer solution), washing the precipitate for 3 times by using Tris-HCl buffer solution, and then suspending in the Tris-HCl buffer solution;

s2, adding 50 mu L of 6' -sialyllactose with different concentrations into the Tris-HCl buffer solution, mixing uniformly, and incubating for 1h at room temperature; performing magnetic separation by using a magnet to obtain supernatant, wherein the supernatant contains a signal probe, and the sequence of the signal probe is as follows: 5'-CCGTAGTGCGCTCTCGCTCGTGGCT-3', respectively;

s3, respectively taking 200 mu L of 1 mu mol and L^-1Hairpin probe Hp1 with 200. mu.L of 1. mu. mol. L^-1And (3) denaturing the hairpin probe Hp2 at the high temperature of 95 ℃ for 5min, then incubating at the temperature of 37 ℃ for 1h, uniformly mixing the incubated hairpin probes, and simultaneously adding 20 mu L of the solution obtained in the step S2 for uniform mixing, wherein the sequences of the hairpin probe Hp1 and the hairpin probe Hp2 are as follows:

hairpin probe Hp 1:

5′-Biotin-CTCTCGCTCGTGGCTTTTTTTTTTTTTTTTAGCCACGAGCGAGAGCGCACTACGG-3′；

hairpin probe Hp 2:

5′-CCGTAGTGCGCTCTCGCTCGTGGCTAAAAAAAAAAAAAAAAGCCACGAGCGAGAG-Cy5-3′；

s4, mixing 2. mu.L of 1. mu. mol. L^-1Adding the CdSe/ZnS quantum dots into the mixed solution obtained in the step S3, and uniformly mixing, so that fluorescence resonance energy transfer is generated between the quantum dots and the Cy5 fluorescent dye, the fluorescence of Cy5 is enhanced, and the fluorescence of the quantum dots is reduced;

s5, detecting the solution in the step S4 by using a fluorescence spectrophotometer, comparing the solution with a blank sample, reading the change of a fluorescence value, adopting an excitation wavelength of 380nm, measuring a fluorescence signal with an emission wavelength of 670 +/-5 nm, diluting the concentration of 6 '-sialyllactose by times, and drawing a corresponding linear relation curve according to the relation between the measured fluorescence value and the concentration of the added 6' -sialyllactose.

As shown in FIG. 4, the fluorescence intensity of Cy5 increased with the increase in the concentration of 6' -sialyllactose, and the linear regression equation thereof was (F-F)₀)/F₀＝0.9402*lg c-0.775(R²0.985), wherein F and F₀The fluorescence intensity of Cy5 fluorescent material in the presence and absence of 6 '-sialyllactose (670. + -.5 nm), c represents the concentration of 6' -sialyllactose in nmol. L^-1The detection limit of the method is 0.3 nmol.L^-1。

Example 3

In order to further verify the accuracy of the method in the determination of the 6' -sialyllactose content in the actual sample, a milk product treated by centrifuging and discarding the supernatant was selected.

Take 10mL milk samples were centrifuged at room temperature for 5min (5,000 Xg) and then the upper fat layer was removed. The milk preparation was then diluted 20-fold with Tris-HCl buffer. Then adding 6' -sialyllactose with different concentrations and mixing evenly. 5 mu mol/L of^-1The aptamer sequence and the complementary signal probe are subjected to ice bath for 10min after being denatured at high temperature for 5min, then are incubated for 1h at 37 ℃, then supernatant is removed through magnetic separation, the precipitate is washed for 1 time by PBS buffer solution and is resuspended after being washed for 3 times by Tris-HCl buffer solution, and is mixed with 50 mu L of milk products containing 6' -sialyllactose with different concentrations evenly, and the mixture is incubated for 30min under slight shaking at room temperature. And then supernatant is obtained by magnetic separation. Collecting 20 μ L supernatant, adding 200 μ L of 1 μmol/L^-1The hairpin probe Hp1 was reacted with Hp2 at 37 ℃ for 1h, and finally 2. mu.L of 1. mu. mol. L^-1The CdSe/ZnS quantum dots are added and mixed evenly for 3min, then the mixture is placed under a fluorescence spectrophotometer to detect fluorescence, and the fluorescence is substituted into a standard curve to calculate the concentration of 6' -sialyllactose. Specific samples and test results are shown in table 2.

TABLE 26' -Sialyllactose assay accuracy results

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Sequence listing

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Claims

1. The ssDNA aptamer capable of specifically recognizing 6' -sialyllactose is characterized in that the nucleotide sequence of the ssDNA aptamer is one of the sequences shown in SEQ ID NO. 1-4.

2. A kit for detecting 6' -sialyllactose, which is characterized in that: comprising one or more of the ssDNA aptamers of claim 1.

3. A fluorescent biosensor for detecting 6' -sialyllactose, characterized in that: comprising one or more of the ssDNA aptamers of claim 1, further comprising a signaling probe complementary to the ssDNA aptamers, a hairpin probe Hp1, a hairpin probe Hp2, and quantum dots; and the signaling probe can trigger the hybridization of the hairpin probe Hp1 with the hairpin structure and the hairpin probe Hp2 to form a DNA double strand, one of the hairpin probe Hp1 and the hairpin probe Hp2 is connected with a fluorescent group, and the other probe can be connected with the quantum dot.

4. The fluorescence biosensor of claim 3, wherein: the nucleotide sequence of the signal probe is shown as SEQ ID NO. 5.

5. The fluorescence biosensor of claim 3, wherein: the nucleotide sequence of the hairpin probe Hp1 is shown as SEQ ID NO.6, and the nucleotide sequence of the hairpin probe Hp2 is shown as SEQ ID NO. 7.

6. The fluorescence biosensor of claim 3, wherein: the quantum dots are CdSe/ZnS quantum dots.

7. Use of the ssDNA aptamer of claim 1 or the fluorescent biosensor of claims 3-6 in the detection of 6' -sialyllactose.

8. The use of claim 7, wherein the fluorescent biosensor is used for detecting 6' -sialyllactose and comprises the following steps:

s1, selecting one or more ssDNA aptamers as claimed in claim 1 and a signal probe forming a complementary structure with the ssDNA aptamers to construct an aptamer-based nucleic acid molecule hybridization system;

s2, adding a sample to be detected into the nucleic acid molecule hybridization system, separating to obtain a free signal probe, and adding a mixture of a hairpin probe Hp1 forming a hairpin structure, a hairpin probe Hp2 forming the hairpin structure and quantum dots into the free signal probe;