CN104178568A - Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe - Google Patents

Abstract

The invention discloses a method for detecting target substances in samples to be tested based on nucleic acid aptamer probe fluorescence sensing analysis. The method provided by the present invention comprises the following steps: (1) co-incubating the functional magnetic beads with the sample to be tested; the functional magnetic beads are obtained by hybridizing (a) and (b); (a) the surface is modified with a specific recognition target substance Magnetic beads of nucleic acid aptamers; (b) probes connected with streptavidin and markers; (2) after completing step (1), magnetic separation is carried out to collect the supernatant; (3) Under the excitation of the corresponding excitation wavelength of the substance, the supernatant obtained in the step (2) is detected by using a solid-phase chip modified with desthiobiotin or biotin on the surface, and it is judged according to the signal value whether the target substance is contained in the sample to be tested. The method of the present invention has the advantages of low cost, simplicity and speed, maintains the advantages of extensive target molecules of the aptamer, good specificity and high sensitivity, and can be used stably for more than 300 times.

Description

A method for detecting target substances in samples to be tested based on nucleic acid aptamer probe fluorescence sensing analysis

technical field

The invention relates to a method for detecting target substances in samples to be tested based on nucleic acid aptamer probe fluorescence sensing analysis.

Background technique

Sensing analysis methods using nucleic acid aptamers as biorecognition materials have developed rapidly in the past two decades. Aptamers are RNA or single-stranded DNA fragments that specifically recognize target contaminants. As early as the 1990s, Nature and Science journals reported nucleic acid aptamer materials and their screening techniques for the first time almost simultaneously. After more than 20 years of development, the reported nucleic acid aptamer materials have been able to detect hundreds of substances in the environment.

Sensing analysis methods using nucleic acid aptamers as biological recognition elements include two methods, homogeneous and heterogeneous (or heterogeneous) aptamer sensing and analysis. At present, the homogeneous nucleic acid aptamer sensing technology is still the mainstream. For example, Lu Yi's research group at the University of Illinois at Urbana-Champaign used the immobilization of fluorescent groups and quenching groups on nucleic acid aptamer probes to enhance or weaken the fluorescent signal based on the DNA conformational changes after target substance recognition. , so as to establish the quantitative relationship between the target concentration and the fluorescence signal. The homogeneous reaction is fast and the detection process is simple, but the reagent consumption is large and the detection cost is high. The solid-phase method is usually based on the immobilization of single-stranded DNA probes, and the detection is based on the difference in the affinity of the DNA probes to the hybridized strand and the target. For multiple uses, hybridized DNA strands or targets need to be eluted. The reported elution methods include: acid wash/alkali wash, heating, high salt elution, EDTA chelation, surfactant elution, etc. The disadvantage is that the number of times it can be reused is very limited, usually between 5-15 times. The reason is that the tertiary conformation of DNA is very fragile, and it is difficult to return to the initial state under various elution conditions. Such a low number of stable repeated uses also greatly limits the development and scope of application of solid-phase DNA technology.

Contents of the invention

The purpose of the present invention is to provide a method for detecting target substances in samples to be tested based on nucleic acid aptamer probe fluorescence sensing analysis.

The invention provides a method for detecting whether a sample to be tested contains a target substance, comprising the following steps:

(1) co-incubating the functional magnetic beads with the sample to be tested; the functional magnetic beads are obtained by hybridizing (a) and (b); the (a) is a nucleic acid suitable for surface modification with a specific recognition target substance The magnetic beads of the ligand; the (b) is a probe connected with streptavidin and a label; the probe is a single-stranded nucleic acid molecule partially complementary to the nucleic acid aptamer;

(2) After completing step (1), perform magnetic separation and collect the supernatant;

(3) Under excitation at the excitation wavelength corresponding to the marker, use a solid-phase chip modified with desthiobiotin or biotin on the surface to detect the supernatant obtained in step (2), and judge whether the sample to be tested contains target substance.

The preparation method of the solid-phase chip whose surface is modified with desthiobiotin comprises the following steps:

(1) Take the solid-phase chip and make its surface hydroxylated;

(II) After completing step (I), take the solid-phase chip to modify the surface of the chip with APTS;

(IV) After completing step (II), take the solid-phase chip and modify its surface with desthiobiotin.

The method of "taking the solid-phase chip and hydroxylating its surface" is as follows: ①Immerse the solid-phase chip in piraha solution (98% concentrated H ₂ SO ₄ :H ₂ O ₂ =3:1, volume ratio) and react at 120°C 1h; ② After completing step ①, take out the solid-phase chip, wash it with ultrapure water until the pH value is neutral, dry it with nitrogen at room temperature, and then store it in a vacuum oven at 70°C for 1 hour.

The method of "taking a solid-phase chip to modify its surface with APTS" is as follows: ①Take a solid-phase chip and place it in APTS solution for 1 hour of reaction; ②After completing step ①, take out the solid-phase chip, rinse it with anhydrous toluene, Blow dry with nitrogen, and then bake at 200 °C for 1 h. APTS solution: the solute is APTS, the solvent is anhydrous toluene, and the concentration of the solute is 2% (volume ratio).

The method of "taking a solid-phase chip and modifying its surface with dethiobiotin" is as follows: ①Take a solid-phase chip and place it in a glutaraldehyde solution at room temperature for 1 hour, then wash it with ethanol, and then place it in an ethylenediamine solution at room temperature. React for 1.5h, then place in NaBH ₄ solution for 15min at room temperature; ②Take 25mg desthiobiotin, 50mg EDC and 50mg NHS, dissolve in 1000μl DMF, react at room temperature for 3h, then add 200μl pH8.7, 1M NaHCO ₃ -Na ₂ CO ₃ buffer solution and mix well to obtain a mixed solution; ③ After completing step ①, take out the solid-phase chip, add it to the mixed solution obtained in step ②, and react at room temperature for 12 hours; ④ After completing step ③, take out the solid-phase chip and use Rinse with ethanol, then place in PBS buffer containing 2mg/mL BSA, and react at room temperature for 1h. Glutaraldehyde solution: the solute is glutaraldehyde, the solvent is ethanol, and the volume percentage concentration of the solute is 2.5%. Ethylenediamine solution: the solute is ethylenediamine, the solvent is ethanol, and the volume percentage concentration of the solute is 10%. NaBH ₄ solution: the solute is NaBH ₄ , the solvent is ethanol, and the solute concentration is 10 mg/ml.

The preparation method of the "probe linked with streptavidin and a label" is as follows: ① Synthesize the probe, modify the 5' end with a sulfhydryl group, and carry out a label modification at the 3' end, named SH-DNA -marker; ②Add 0.67μL TCEP solution to 10μl 1mM SH-DNA-label, and react at room temperature in the dark for 1h to activate the thiol; ③Take Amicon-3K, add the whole reaction system obtained after completing step ②, and then 200μL Buffer A-1, then centrifuge at 12000rpm for 10min, wash Amicon-3K with buffer A-2, and collect components with a molecular weight greater than 3K (solution form); ④Take 133μL 20mg/mL streptavidin aqueous solution, add 0.33mg Sulfo -SMCC, vortexed for 5min, then reacted at room temperature for 1h, centrifuged at 12000rpm for 5min, and took the supernatant; -2 Wash Amicon-10K, collect components with a molecular weight greater than 10K (solution form); ⑥Add the solution obtained in step ⑤ to the solution obtained in step ③, and react at room temperature in the dark for 48h; ⑦Take Amicon-10K, add to complete step ⑥ Then add 200μL Buffer A-1 to the whole reaction system, centrifuge at 12000rpm for 10min, wash Amicon-10K with bufferA-2, and collect components with a molecular weight greater than 10K (in the form of solution), which are those containing streptavidin and labeled probe solution. TCEP solution: the solute is TCEP, the solvent is ultrapure water, and the concentration of the solute is 30 mM. Buffer A-1: Aqueous solution containing 0.1M NaCl and 0.1M sodium phosphate, pH7.3. Buffer A-2: Aqueous solution containing 0.1M NaCl, 0.1M sodium phosphate and 0.05% (volume ratio) Tween 20, pH7.3.

The preparation method of the "surface-modified magnetic beads with nucleic acid aptamers that specifically recognize target substances" includes the following steps: ① Dissolve 38.4 mg of EDC in 2 mL of pH 7.0, 0.1 M methylimidazole buffer, and then add 0.65 nmol The nucleic acid aptamers were mixed thoroughly to obtain a mixed solution; ② Add the mixed solution obtained in step ① to about 20 mg of magnetic beads, shake well at room temperature and react for 24 hours, then magnetically separate and collect the magnetic beads, and use 2 ml of pre-hybridization buffer The unreacted carboxyl group on the surface of the magnetic bead is blocked by the liquid to obtain the magnetic bead whose surface is modified with a nucleic acid aptamer that specifically recognizes the target substance. Pre-hybridization buffer: the solvent is Tris buffer at pH 7.4, 0.1M, containing 0.005M EDTA, 0.5% (volume ratio) N-lauroyl sarcosine and 1g/100mL BSA.

The preparation method of the functional magnetic beads specifically includes the following steps: ① Take about 20 mg of magnetic beads whose surface is modified with nucleic acid aptamers that specifically recognize target substances, suspend them in 2 mL of hybridization buffer, and warm them in a water bath at 60 ° C for 1 hour; ② Take 10 μL of probe solution containing streptavidin and markers, dissolved in 2mL of hybridization buffer, warmed in a 60°C water bath for 1h; ③Mix the magnetic bead suspension obtained in step ① with the solution obtained in step ② , shake well at room temperature and react for 2.5 hours to obtain a suspension of functional magnetic beads. Hybridization buffer: aqueous solution containing 10mM Tris, 120mM NaCl, 5mM KCl, 20mM CaCl ₂ , pH 8.5.

The marker can specifically be a fluorescent marker, more specifically Cy5.5. The excitation wavelength corresponding to the marker may specifically be 650nm.

The step (3) is specifically carried out in an all-fiber evanescent wave biosensor. The operating procedure of the all-fiber evanescent wave biosensor is as follows: turn on the laser, and the excitation wavelength is 650nm; wash the solid-phase chip with desthiobiotin or biotin on the surface with PBS buffer until the baseline is stable; after the baseline is stable, take the solution to be tested , turn on the peristaltic pump for sample injection for 25s, then stop the peristaltic pump to allow the solution to be tested to react in the reaction tank for 120s; turn on the peristaltic pump again, and feed the eluent (0.5g/100mL SDS aqueous solution, adjust the pH to 1.9) for 90 seconds; finally Inject PBS buffer until the baseline is stable.

The magnetic beads can specifically be carboxyl magnetic beads.

Specifically, the solid-phase chip can be an optical fiber.

The target substance is ochratoxin A.

The nucleic acid aptamer is specifically shown in sequence 1 of the sequence listing. The probe is specifically shown in sequence 2 of the sequence listing.

The present invention also protects a kit for detecting whether the sample to be tested contains a target substance, including functional magnetic beads and a solid-phase chip whose surface is modified with desthiobiotin or biotin; the functional magnetic beads are (a) and ( b) obtained by hybridization; said (a) is a magnetic bead whose surface is modified with a nucleic acid aptamer that specifically recognizes a target substance; said (b) is a probe connected with streptavidin and a marker; said The probe is a single-stranded nucleic acid molecule partially complementary to the nucleic acid aptamer.

The invention also protects a kit for detecting whether a sample to be tested contains a target substance, including "a solid-phase chip whose surface is modified with desthiobiotin or biotin", "a nucleic acid aptamer whose surface is modified with a specific recognition target substance" magnetic beads" and "probes connected with streptavidin and labels"; the probes are single-stranded nucleic acid molecules partially complementary to the nucleic acid aptamers.

The preparation method of the solid-phase chip whose surface is modified with desthiobiotin comprises the following steps:

(1) Take the solid-phase chip and make its surface hydroxylated;

(II) After completing step (I), take the solid-phase chip to modify the surface of the chip with APTS;

(IV) After completing step (II), take the solid-phase chip and modify its surface with desthiobiotin.

The method of "taking the solid-phase chip and hydroxylating its surface" is as follows: ①Immerse the solid-phase chip in piraha solution (98% concentrated H ₂ SO ₄ :H ₂ O ₂ =3:1, volume ratio) and react at 120°C 1h; ② After completing step ①, take out the solid-phase chip, wash it with ultrapure water until the pH value is neutral, dry it with nitrogen at room temperature, and then store it in a vacuum oven at 70°C for 1 hour.

The method of "taking a solid-phase chip to modify its surface with APTS" is as follows: ①Take a solid-phase chip and place it in APTS solution for 1 hour of reaction; ②After completing step ①, take out the solid-phase chip, rinse it with anhydrous toluene, Blow dry with nitrogen, and then bake at 200 °C for 1 h. APTS solution: the solute is APTS, the solvent is anhydrous toluene, and the concentration of the solute is 2% (volume ratio).

The method of "taking a solid-phase chip and modifying its surface with dethiobiotin" is as follows: ①Take a solid-phase chip and place it in a glutaraldehyde solution at room temperature for 1 hour, then wash it with ethanol, and then place it in an ethylenediamine solution at room temperature. React for 1.5h, then place in NaBH ₄ solution for 15min at room temperature; ②Take 25mg desthiobiotin, 50mg EDC and 50mg NHS, dissolve in 1000μl DMF, react at room temperature for 3h, then add 200μl pH8.7, 1M NaHCO ₃ -Na ₂ CO ₃ buffer solution and mix well to obtain a mixed solution; ③ After completing step ①, take out the solid-phase chip, add it to the mixed solution obtained in step ②, and react at room temperature for 12 hours; ④ After completing step ③, take out the solid-phase chip and use Rinse with ethanol, then place in PBS buffer containing 2mg/mL BSA, and react at room temperature for 1h. Glutaraldehyde solution: the solute is glutaraldehyde, the solvent is ethanol, and the volume percentage concentration of the solute is 2.5%. Ethylenediamine solution: the solute is ethylenediamine, the solvent is ethanol, and the volume percentage concentration of the solute is 10%. NaBH ₄ solution: the solute is NaBH ₄ , the solvent is ethanol, and the concentration of the solute is 10 mg/ml.

The preparation method of the "probe linked with streptavidin and a label" is as follows: ① Synthesize the probe, modify the 5' end with a sulfhydryl group, and carry out a label modification at the 3' end, named SH-DNA -marker; ②Add 0.67μL TCEP solution to 10μl 1mM SH-DNA-label, and react at room temperature in the dark for 1h to activate the thiol; ③Take Amicon-3K, add the whole reaction system obtained after completing step ②, and then 200μL Buffer A-1, then centrifuge at 12000rpm for 10min, wash Amicon-3K with buffer A-2, and collect components with a molecular weight greater than 3K (solution form); ④Take 133μL 20mg/mL streptavidin aqueous solution, add 0.33mg Sulfo -SMCC, vortexed for 5min, then reacted at room temperature for 1h, centrifuged at 12000rpm for 5min, and took the supernatant; -2 Wash Amicon-10K, collect components with a molecular weight greater than 10K (solution form); ⑥Add the solution obtained in step ⑤ to the solution obtained in step ③, and react at room temperature in the dark for 48h; ⑦Take Amicon-10K, add to complete step ⑥ Then add 200μL Buffer A-1 to the whole reaction system, centrifuge at 12000rpm for 10min, wash Amicon-10K with bufferA-2, and collect components with a molecular weight greater than 10K (in the form of solution), which are those containing streptavidin and labeled probe solution. TCEP solution: the solute is TCEP, the solvent is ultrapure water, and the concentration of the solute is 30 mM. Buffer A-1: Aqueous solution containing 0.1M NaCl and 0.1M sodium phosphate, pH7.3. Buffer A-2: Aqueous solution containing 0.1M NaCl, 0.1M sodium phosphate and 0.05% (volume ratio) Tween 20, pH7.3.

The preparation method of the "surface-modified magnetic beads with nucleic acid aptamers that specifically recognize target substances" includes the following steps: ① Dissolve 38.4 mg of EDC in 2 mL of pH 7.0, 0.1 M methylimidazole buffer, and then add 0.65 nmol The nucleic acid aptamers were mixed thoroughly to obtain a mixed solution; ② Add the mixed solution obtained in step ① to about 20 mg of magnetic beads, shake well at room temperature and react for 24 hours, then magnetically separate and collect the magnetic beads, and use 2 ml of pre-hybridization buffer The unreacted carboxyl group on the surface of the magnetic bead is blocked by the liquid to obtain the magnetic bead whose surface is modified with a nucleic acid aptamer that specifically recognizes the target substance. Pre-hybridization buffer: the solvent is Tris buffer at pH 7.4, 0.1M, containing 0.005M EDTA, 0.5% (volume ratio) N-lauroyl sarcosine and 1g/100mL BSA.

The preparation method of the functional magnetic beads specifically includes the following steps: ① Take about 20 mg of magnetic beads whose surface is modified with nucleic acid aptamers that specifically recognize target substances, suspend them in 2 mL of hybridization buffer, and warm them in a water bath at 60 ° C for 1 hour; ② Take 10 μL of the probe solution containing streptavidin and markers, dissolved in 2 mL of hybridization buffer, warmed in a 60°C water bath for 1 hour; ③Mix the magnetic bead suspension obtained in step ① with the solution obtained in step ② , shake well at room temperature and react for 2.5 hours to obtain a suspension of functional magnetic beads. Hybridization buffer: aqueous solution containing 10mM Tris, 120mM NaCl, 5mM KCl, 20mM CaCl ₂ , pH 8.5.

The marker can specifically be a fluorescent marker, more specifically Cy5.5. The excitation wavelength corresponding to the marker may specifically be 650nm.

The magnetic beads can specifically be carboxyl magnetic beads.

Specifically, the solid-phase chip can be an optical fiber.

The target substance is ochratoxin A.

The nucleic acid aptamer is specifically shown in sequence 1 of the sequence listing. The probe is specifically shown in sequence 2 of the sequence listing.

The purpose of the present invention is to propose a highly stable and highly reusable nucleic acid aptamer probe fluorescence sensing analysis method. The main features of the invention are: the nucleic acid aptamer is used as a biorecognition probe, solid-phase sensing, laser-induced fluorescence, and highly stable repeated use times. Specifically (see FIG. 1 ): the nucleic acid aptamer is immobilized on the surface of the magnetic bead, and a complementary strand is hybridized, and this complementary DNA strand is conjugated with streptavidin and a fluorescent marker. At the same time, desthiobiotin was modified at the interface of the solid-phase chip. When there is a target pollutant in the system, the complementary chain is competed and combined with desthiobiotin modified at the solid phase interface, the fluorescent substance is excited, and the obtained signal has a quantitative relationship with the target pollutant in the system, so as to achieve detection the goal of. The method of the invention has the advantages of low cost, simplicity and speed for measuring the concentration of target pollutants, and maintains the advantages of wide range of target molecules of the aptamer, good specificity and high sensitivity, and can be used stably for more than 300 times.

Description of drawings

Fig. 1 is a schematic diagram of the principle of the method provided by the present invention.

FIG. 2 is a schematic flow chart of Step 1 of Embodiment 1.

FIG. 3 is a schematic flow chart of Step 2 of Example 1.

FIG. 4 is a schematic flow chart of Step 3 of Embodiment 1.

Fig. 5 is the result of embodiment 2.

Fig. 6 is the result of embodiment 3.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. The PBS buffer used in the examples, unless otherwise specified, is a PBS buffer of pH 7.4 and 10 mM. Desthiobiotin: sigma, Cat. No. D1411. Streptavidin: Thermo, Cat. No. prod#21135. Ochratoxin A: pribolab, Cat. No. IAC-040-3. The full name of APTS is "(3-aminopropyl) triethoxysilane". The full name of TCEP in Chinese is "(tris(2-carboxyethyl)phosphine), and the full name in English is "Tris(2-carboxyethyl)phosphine). The Chinese full name of Sulfo-SMCC is "4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt".

DNA molecule A is a nucleic acid aptamer specifically binding to ochratoxin A, and its nucleotide sequence (sequence 1 in the sequence listing) is 5-NH ₂ -A ₆ GATCGGGTGTGGGTGGCGTAAAGG GAGCATCGGACA -3'. DNA molecule B is a single-stranded DNA molecule partially complementary to DNA molecule A, and its nucleotide sequence (sequence 2 in the sequence listing) is 5'-AAAAAAAAAAAA TGTCCGATGCTC -3'.

For the evanescent wave detection instrument (all-fiber evanescent wave biosensor) used in this embodiment, see patent ZL200610089497.4 (CN1873450A).

Embodiment 1, the preparation of kit

1. Preparation of optical fiber surface modified with desthiobiotin

The flow diagram is shown in Figure 2.

1. Take the optical fiber and make its surface hydroxylated.

Specific method: (1) Immerse the optical fiber (570/600/900, purchased from Nanjing Chunhui Technology Industrial Co., Ltd.) in piraha solution (98% concentrated H ₂ SO ₄ :H ₂ O ₂ =3:1, volume ratio) and React at 120°C for 1 hour; (2) After completing step (1), take out the optical fiber, wash it with ultrapure water until the pH value is neutral, dry it with nitrogen at room temperature, and store it in a vacuum oven at 70°C for 1 hour.

2. After completing step 1, take the optical fiber and modify its surface with APTS.

Specific methods: (1) After completing step 1, take the optical fiber and place it in the APTS solution for 1 hour of reaction; (2) After completing step (1), take out the optical fiber, rinse it with anhydrous toluene, dry it with nitrogen at room temperature, and then Bake at 200°C for 1h. APTS solution: the solute is APTS, the solvent is anhydrous toluene, and the concentration of the solute is 2% (volume ratio).

3. After completing step 2, take the optical fiber and modify the surface with desthiobiotin.

Specific method: (1) After completing step 2, take the optical fiber, place it in a glutaraldehyde solution for 1 hour at room temperature, then wash it with ethanol, then place it in ethylenediamine solution for 1.5 hours at room temperature, and then place it in a NaBH ₄ solution React at room temperature for 15 minutes; (2) Take 25mg desthiobiotin, 50mg EDC and 50mg NHS, dissolve them in 1000μl DMF, react at room temperature for 3h (mix and rotate), then add 200μl pH8.7, 1M NaHCO ₃ -Na ₂ CO ₃ buffer and mix well to obtain a mixed solution; (3) after completing step (1), take out the optical fiber, add it to the mixed solution obtained in step (2), and react at room temperature for 12 hours; (4) after completing step (3), take out the optical fiber, Rinse with ethanol, then place in PBS buffer containing 2mg/mL BSA, react at room temperature for 1h.

Glutaraldehyde solution: the solute is glutaraldehyde, the solvent is ethanol, and the volume percentage concentration of the solute is 2.5%.

Ethylenediamine solution: the solute is ethylenediamine, the solvent is ethanol, and the volume percentage concentration of the solute is 10%.

NaBH ₄ solution: the solute is NaBH ₄ , the solvent is ethanol, and the concentration of the solute is 10 mg/ml.

2. Preparation of probes linked with streptavidin and fluorescent markers

The flow diagram is shown in Figure 3.

1. Synthesize DNA molecule B, carry out sulfhydryl modification at the 5' end, and carry out Cy5.5 modification at the 3' end, named SH-DNA-Cy5.5.

2. Add 0.67 μL TCEP solution to 10 μl 1mM SH-DNA-Cy5.5, and react for 1 hour at room temperature in the dark to activate sulfhydryl groups. TCEP solution: the solute is TCEP, the solvent is ultrapure water, and the concentration of the solute is 30 mM.

3. Take Amicon-3K (Amicon, article number is UFC500396-1), add the whole reaction system obtained after completing step 2, then add 200μL Buffer A-1, then centrifuge at 12000rpm for 10min, wash Amicon-3K with buffer A-2, Fractions with a molecular weight greater than 3K were collected (in solution).

Buffer A-1: Aqueous solution containing 0.1M NaCl and 0.1M sodium phosphate, pH7.3.

Buffer A-2: Aqueous solution containing 0.1M NaCl, 0.1M sodium phosphate and 0.05% (volume ratio) Tween 20, pH7.3.

4. Take 133μL 20mg/mL streptavidin aqueous solution, add 0.33mg Sulfo-SMCC, vortex for 5min, react at room temperature for 1h, centrifuge at 12000rpm for 5min, and take the supernatant.

5. Take Amicon-10K (Amicon, article number UFC501096-1), add the supernatant obtained in step 4, then add 200 μL Buffer A-1, centrifuge at 12000 rpm for 10 minutes, wash Amicon-10K with buffer A-2, and collect Components of 10K (in solution).

6. Add the solution obtained in step 5 to the solution obtained in step 3, and react at room temperature for 48 hours in the dark.

7. Take Amicon-10K, add the whole reaction system obtained after completing step 6, then add 200μL BufferA-1, centrifuge at 12000rpm for 10min, wash Amicon-10K with buffer A-2, and collect components with a molecular weight greater than 10K (solution form) , named as STV-probe-Cy5.5 solution. In terms of streptavidin, the concentration of STV-probe-Cy5.5 solution is 200mg/mL.

3. Preparation of magnetic beads with surface-modified nucleic acid aptamers

The flow diagram is shown in Figure 4.

1. Take 1mL carboxyl magnetic beads (Bangs Laboratories Inc, BioMag BP618; in the form of suspension, the concentration of magnetic beads is 20.3mg/ml), magnetically separate and discard the supernatant, and wash the magnetic beads 2-3 times with DEPC water (each time After washing, the supernatant was discarded by magnetic separation).

2. Dissolve 38.4mg EDC in 2mL pH 7.0, 0.1M methylimidazole buffer, then add 0.65nmol DNA molecule A, mix thoroughly to obtain a mixture.

3. Add the mixture obtained in step 2 to the magnetic beads obtained in step 1, shake well at room temperature and react for 24 hours, then magnetically separate and collect the magnetic beads, and use 2 ml of pre-hybridization buffer to block the unreacted carboxyl groups on the surface of the magnetic beads (incubating at room temperature for 4 h) to obtain a magnetic bead-nucleic acid aptamer conjugate mixture.

Pre-hybridization buffer: the solvent is Tris buffer at pH 7.4, 0.1M, containing 0.005M EDTA, 0.5% (volume ratio) N-lauroyl sarcosine and 1g/100mL BSA.

4. Hybridization of magnetic bead-nucleic acid aptamer conjugate with STV-probe-Cy5.5

The flow diagram is shown in Figure 4.

1. Take 2 mL of the magnetic bead-aptamer conjugate mixture (containing about 20 mg magnetic beads) prepared in step 3, discard the supernatant after magnetic separation, wash with hybridization buffer three times, and then suspend with 2 mL of hybridization buffer , warmed in a 60°C water bath for 1h.

2. Take 10 μL of the STV-probe-Cy5.5 solution obtained in step 2, dissolve it in 2 mL of hybridization buffer, and warm it in a 60°C water bath for 1 hour.

3. Mix the magnetic bead suspension obtained in step 1 and the solution obtained in step 2, shake well at room temperature and react for 2.5 hours to obtain a functional magnetic bead suspension.

Hybridization buffer: aqueous solution containing 10mM Tris, 120mM NaCl, 5mM KCl, 20mM CaCl ₂ , pH 8.5.

Embodiment 2, detection ochratoxin A

Using DMF as solvent, prepare 1 mg/mL stock solution of ochratoxin A.

1. Get the functional magnetic bead suspension (containing about 20 mg magnetic beads) of Example 1, wash it twice with PBS buffer containing 2 mg/mL BSA, and discard the supernatant.

2. After completing step 1, take the magnetic beads, add ochratoxin A stock solution, and add PBS buffer solution containing 2 mg/mL BSA to make the total volume of the reaction system 350 μL, and mix for 15 minutes. The ochratoxin A stock solution is set with different addition amounts, so that the concentration of ochratoxin A at the initial moment of the reaction system is 6.4nM, 38.2nM, 95.4nM, 158.7nM, 222.6nM, 445.3nM, 635.0nM, 1269.6nM , 3174.9nM or 3710.0nM, each concentration set three replicates.

3. After completing step 2, perform magnetic separation and collect the supernatant (solution to be tested).

4. The operation procedure of the all-fiber evanescent wave biosensor: turn on the exciter, and the excitation wavelength is 650nm; wash the optical fiber prepared in step 1 of Example 1 with PBS buffer until the baseline is stable; To measure the solution, turn on the peristaltic pump to inject samples for 25s, then stop the peristaltic pump to allow the solution to be tested to react in the reaction tank for 120s; turn on the peristaltic pump again, and feed the eluent (0.5g/100mL SDS aqueous solution, adjust the pH to 1.9) for 90 seconds ;Finally pass into PBS buffer until the baseline is stable.

After the detection of a solution to be tested is completed, use the eluent and pH7.4, 0.1M PBS buffer solution to alternately wash the optical fiber prepared in step 1 of Example 1, so that STV-probe-Cy5.5 can be obtained from Example 1 The optical fiber prepared in step 1 falls off, and then the next solution to be tested is detected. Through this detection method, the traditional DNA binding and elution process can be converted into a protein elution process, thereby achieving a high number of stable repeated applications .

The results are shown in Figure 5, the detection limit of ochratoxin A is 12nM (4.9ng/mL), and the linear range is 12nM-32μM.

Embodiment 3, the number of reusable times of the optical fiber prepared in step 1 of embodiment 1

Using DMF as solvent, prepare 1 mg/mL stock solution of ochratoxin A.

1. Get the functional magnetic bead suspension (containing about 20 mg magnetic beads) of Example 1, wash it twice with PBS buffer containing 2 mg/mL BSA, and discard the supernatant.

2. After completing step 1, take the magnetic beads, add ochratoxin A stock solution, and add PBS buffer solution containing 2 mg/mL BSA, so that the total volume of the reaction system is 350 μL (the initial moment of the reaction system is ochratoxin A The concentration is 3μM), mixed for 15min.

3. After completing step 2, perform magnetic separation and collect the supernatant (solution to be tested).

4. The operating procedure of the all-fiber evanescent wave biosensor: turn on the laser, and the excitation wavelength is 650nm; wash the optical fiber prepared in step 1 of Example 1 with PBS buffer until the baseline is stable; after the baseline is stable, take the sample obtained in step 3 to be tested Solution, turn on the peristaltic pump to inject samples for 25s, then stop the peristaltic pump to allow the solution to be tested to react in the reaction tank for 120s; turn on the peristaltic pump again, and feed the eluent (0.5g/100mL SDS aqueous solution, adjust the pH to 1.9) for 90 seconds; Finally, PBS buffer was introduced until the baseline was stable.

Repeat the detection of the solution to be tested obtained in step 3. After the detection of a solution to be tested is completed, use the eluent and pH7.4, 0.1M PBS buffer solution to alternately wash the optical fiber prepared in step 1 of Example 1, so that STV-probe-Cy5.5 can be obtained from Example 1 The optical fiber prepared in step 1 falls off, and then the next solution to be tested is detected. Through this detection method, the traditional DNA binding and elution process can be converted into a protein elution process, thereby achieving a high number of stable repeated applications .

The results are shown in Figure 6. The optical fiber involved in the present invention can be reused at least 300 times.

Claims (10)

1. detect a method that whether contains target substance in sample to be tested, comprise the steps:

(1) function magnetic bead and described sample to be tested are hatched altogether; Described function magnetic bead is hybridized and is obtained by (a) with (b); Described (a) has the magnetic bead of the aptamer of specific recognition target substance for finishing; Described (b) is for being connected with the probe of Streptavidin and marker; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part;

(2) after completing steps (1), carry out magnetic separation, collect supernatant liquor;

(3), under exciting with the corresponding excitation wavelength of marker, the supernatant liquor that adopts finishing to have the solid phase chip detecting step (2) of desthiobiotin or vitamin H to obtain, judges in sample to be tested, whether to contain target substance according to signal value.

2. the method for claim 1, is characterized in that: described finishing has the preparation method of the solid phase chip of desthiobiotin to comprise the steps:

(1) get solid phase chip, make its surface hydroxylation;

(2) after completing steps (1), get solid phase chip, make its finishing APTS;

(3) after completing steps (2), get solid phase chip, make its finishing desthiobiotin.

3. method as claimed in claim 1 or 2, is characterized in that: described solid phase chip is optical fiber.

4. as the method as described in arbitrary in claims 1 to 3, it is characterized in that: described target substance is Ochratoxin A.

5. as the method as described in arbitrary in claim 1 to 4, it is characterized in that: described magnetic bead is carboxyl magnetic bead.

6. as the method as described in arbitrary in claim 1 to 5, it is characterized in that: described aptamer is as shown in the sequence 1 of sequence table, and described probe is as shown in the sequence 2 of sequence table.

7. detect a test kit that whether contains target substance in sample to be tested, comprise that function magnetic bead and finishing have the solid phase chip of desthiobiotin or vitamin H; Described function magnetic bead is hybridized and is obtained by (a) with (b); Described (a) has the magnetic bead of the aptamer of specific recognition target substance for finishing; Described (b) is for being connected with the probe of Streptavidin and marker; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part.

8. detect a test kit that whether contains target substance in sample to be tested, comprise " solid phase chip that finishing has desthiobiotin or vitamin H ", " finishing has the magnetic bead of the aptamer of specific recognition target substance " and " being connected with the probe of Streptavidin and marker "; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part.

9. test kit as claimed in claim 7 or 8, is characterized in that: described solid phase chip is optical fiber; Described magnetic bead is carboxyl magnetic bead.

10. as the method as described in arbitrary in claim 7 to 9, it is characterized in that: described target substance is Ochratoxin A; Described aptamer is as shown in the sequence 1 of sequence table, and described probe is as shown in the sequence 2 of sequence table.