CN117607464B - A method for real-time detection of progesterone based on a portable blood glucose meter using an aptamer sensor - Google Patents

Abstract

The invention provides an immediate detection method of progesterone based on a nucleic acid aptamer sensor of a portable blood glucose meter, which takes the nucleic acid aptamer of progesterone as a specific recognition element, and realizes the conversion of a non-sugar target progesterone signal into a glucose signal which can be detected by the blood glucose meter through strand displacement and magnetic separation; the nucleic acid aptamer of progesterone is used for binding progesterone. According to the detection method disclosed by the invention, the concentration of the non-sugar target is converted into the concentration of the measurable amylose of the glucometer, so that the instant quantitative detection of the glucometer on the non-sugar target-progesterone is realized, the used instrument only needs to be free of the glucometer, other electrochemical/optical detection instruments and the like are not needed, the detection cost is low, the operation is simple, and a foundation is laid for realizing on-site and accurate progesterone detection and promoting the development of a cow early pregnancy diagnosis kit.

Description

A method for instant detection of progesterone using a nucleic acid aptamer sensor based on a portable blood glucose meter

Technical Field

The invention belongs to the technical field, and in particular relates to a method for real-time detection of progesterone by a nucleic acid aptamer sensor based on a portable blood glucose meter.

Background technique

Progesterone (P4) is a steroid hormone secreted by the corpus luteum of the ovary. It is an important marker for early determination of whether a cow is pregnant after fertilization. The immediate and sensitive detection of progesterone is an important means to diagnose early pregnancy in dairy cows. Simple and accurate on-site diagnosis of early pregnancy can further reduce the detection time and reduce the dependence on professional technicians. Therefore, it is of great significance to establish a portable and instant detection method for progesterone. The antigen-antibody based immunoassay method can realize the instant detection of progesterone, but the limitations of high antibody cost, difficult preparation and harsh use conditions affect its application in early pregnancy diagnosis. As a "chemical antibody" and a new recognition molecule, nucleic acid aptamers have high affinity and specificity comparable to antibodies, as well as unique advantages such as low cost, easy synthesis and stable properties.

Prior art 1: The aggregation and dispersion of gold nanoparticles (AuNPs) are affected by P4 binding aptamers and cationic surfactant hexadecyltrimethylammonium bromide (CTAB). When the target P4 is present, the aptamer binds to P4 to form a complex, and the CTAB in the solution induces the aggregation of AuNPs, causing the AuNPs solution to change from red to blue. When P4 is not present, CTAB binds to the aptamer, and the AuNPs remain dispersed, that is, red. Therefore, P4 can be detected by color change with the naked eye. By monitoring the absorbance and color change, a CTAB-induced colorimetric method for detecting P4 was established. However, only qualitative detection of P4 can be achieved through naked eye observation, and quantitative detection still relies on spectroscopic instruments such as microplate readers.

Prior art 2: Photoelectrochemical biosensor: A composite material of carbon dots and graphene oxide (CDs-GO) with good cathode photocurrent response is used as a photoactive material, and the P4 antibody (Ab) is immobilized on it. At the same time, a high-affinity P4 aptamer is immobilized on Au-CuO-Cu ₂ O as a bioconjugate. When P4 is present, the aptamer-Au-CuO-Cu ₂ O bioconjugate can amplify the cathode photocurrent of the CDs-GO modified electrode through the Ab-P4-aptamer interaction, thereby realizing sensitive detection of P4.

Prior Art 3: A label-free electrochemical progesterone aptamer sensor was successfully developed by covalently fixing the amino-functionalized P4-specific aptamer on the electrode surface. Ni-Au hybrid nanofibers were synthesized by electrospinning. Electrospun NiO-AuNFs were dispersed in a synthesized graphene quantum dot (GQDs) solution to prepare a GQDs-NiO-AuNFs nanocomposite. Screen-printed carbon electrodes (SPCEs) were modified with novel GQDs-NiO-AuNFs nanostructures combined with functionalized multi-walled carbon nanotubes (f-MWCNTs) to construct an effective immobilization matrix with a large number of carboxyl functional groups, and the amino-functionalized P4-specific aptamer was covalently fixed on the electrode surface. The formation of the aptamer-P4 complex resulted in the obstruction of the electron transfer reaction at the sensing interface, thereby reducing the peak current of the redox probe. On this basis, quantitative detection of progesterone can be achieved by monitoring the differential pulse voltammetry (DPV) response of the [Fe(CN)6]3-/4-peak current, which decreases with the increase of progesterone concentration.

Although the above-mentioned sensing system has good sensitivity and linear range, it requires the use of electrochemical/optical detection instruments and professional technicians, which limits its application in on-site real-time detection. The colorimetric sensor based on progesterone aptamers and gold nanoparticles can achieve qualitative detection of progesterone by color change, but accurate quantification cannot be achieved by visual detection alone. That is, the existing literature reports that the on-site instant detection methods of progesterone are mostly qualitative colorimetric methods, which cannot achieve accurate content analysis. Existing instant detection equipment still has problems such as poor practicality and complicated operating steps. How to use portable, easy-to-operate and low-cost instant detection equipment to achieve rapid detection of progesterone content is a technical problem that needs to be solved urgently.

Summary of the invention

The purpose of the present invention is to provide a method for instant detection of progesterone by a nucleic acid aptamer sensor based on a portable blood glucose meter, so as to solve the problems that existing instant detection equipment still has poor practicality and complicated operation steps.

To achieve the above object, the present invention adopts the following technical solutions:

A method for instant detection of progesterone based on a portable blood glucose meter using a nucleic acid aptamer sensor, which uses a progesterone nucleic acid aptamer as a specific recognition element, and converts a non-sugar target progesterone signal into a glucose signal measurable by a blood glucose meter through strand displacement and magnetic separation; the progesterone nucleic acid aptamer is used to bind progesterone (P4); specifically, the method comprises the following steps:

S1. Preparation of nucleic acid aptamer sensor based on portable blood glucose meter;

S2, mixing the nucleic acid aptamer sensor with the solution to be tested, and after the reaction, taking the supernatant after magnetic separation, mixing the supernatant with the amylose solution and incubating them, and then testing with a blood glucose meter to obtain the blood glucose reading of the solution to be tested;

S3, respectively mixing the progesterone standard solution with the nucleic acid aptamer sensor to perform step S2, and performing the above experiment on the blank solution to obtain a blank value; using the progesterone concentration as the horizontal axis, the blood glucose meter reading of the progesterone standard solution and the blank value as the vertical axis to perform linear fitting, and obtain its linear equation, i.e., the standard curve;

S4. Substituting the blood glucose reading of the test solution obtained in step S2 into the standard curve can obtain the concentration of progesterone in the test solution.

As described above, preferably, in step S1, the preparation method of the nucleic acid aptamer sensor is to mix and incubate the conjugate CS-GA of the aptamer complementary chain (CS) and glucoamylase (GA) with the biotin-modified nucleic acid aptamer (Apt) for the first time, add streptavidin magnetic beads for the first oscillation coupling, and then wash with buffer to obtain the nucleic acid aptamer sensor.

Further, preferably, the aptamer complementary chain has a sequence as shown in SEQ ID NO.1, and the 5′ end of the aptamer complementary chain has a —SH-SH- modification; the sequence of the nucleic acid aptamer is shown in SEQ ID NO.2, and the 5′ end of the nucleic acid aptamer has a Biotin modification; the particle size of the streptavidin magnetic beads is 1 μm, and the formula of the buffer solution contains a final concentration of 10 mM Tris-HCl, 10 mM NaCl, 5mM MgCl ₂ and 0.01% Tween-20, and the pH value is 7.3.

As described above, preferably, the preparation method of the conjugate CS-GA is to mix and incubate the aptamer complementary chain with sodium phosphate buffer and tris(2-carboxyethyl)phosphine, transfer to an Amicon-3 K ultrafiltration tube, centrifuge, and then purify with buffer A to obtain activated CS;

The glucoamylase solution was mixed with the sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate solution, incubated, transferred to an Amicon-30 K ultrafiltration tube, centrifuged, and then purified with buffer A to obtain activated glucoamylase;

The activated CS was mixed with the activated glucoamylase, incubated at room temperature, and transferred to an Amicon-100 K ultrafiltration tube. After purification, the conjugate CS-GA was obtained and dissolved in buffer B.

The formula of buffer A is as follows: containing sodium phosphate with a final concentration of 0.1 M and 100 mM NaCl, with a pH value of 7.3; the formula of buffer B is as follows: containing Tris-HCl with a final concentration of 10 mM, 10 mM NaCl and 5 mM MgCl ₂ , with a pH value of 7.3.

According to the method described above, preferably, the conjugate CS-GA is mixed with a biotin-modified nucleic acid aptamer to obtain a mixture; the mixture is shaken-coupled with streptavidin magnetic beads; the first mixing and incubation conditions are: incubate at 95°C for 10 min and then slowly cool to room temperature, and the first shaking coupling time is 0.5-1.5h.

Furthermore, the concentration of the conjugate CS-GA is 6 μM and the amount is 30 μL; the concentration of the biotin-modified nucleic acid aptamer is 5 μM and the amount is 30 μL; the concentration of the streptavidin magnetic beads is 10 mg/mL and the amount is 30 μL.

In the method described above, preferably, in steps S2 and S3, the solvent in the test solution and the progesterone standard solution is buffer C, and the formula of buffer C is: containing Tris-HCl with a final concentration of 10 mM, 10 mM NaCl, 5 mM _MgCl2 and 0.01% Tween-20, and the pH value is 7.3.

In the method as described above, preferably, in step S2, the reaction time is preferably 20-120 min, and most preferably 60 min; the reaction is carried out under shaking conditions, and the shaking rate is 500-800 rpm; the mixing and incubation time is 20-40 min, and most preferably the mixing and incubation time is 30 min.

In the method as described above, preferably, in step S2, the amount of the aptamer sensor is 30 μL, and the volume ratio of the aptamer sensor to the solution to be tested is 1: 1. The concentration of the amylose solution is 2 M, and the amount ratio of the supernatant to the amylose solution is 1: 1.

A kit for detecting progesterone using a nucleic acid aptamer sensor based on a blood glucose meter, comprising the nucleic acid aptamer sensor as described above, wherein the preparation method of the nucleic acid aptamer sensor is to mix and incubate for the first time an aptamer complementary chain with a sequence as shown in SEQ ID NO.1 and a conjugate of glucoamylase CS-GA with a nucleic acid aptamer with a sequence modified with biotin as shown in SEQ ID NO.2, add streptavidin magnetic beads for the first oscillation coupling, and wash with a buffer solution to obtain the nucleic acid aptamer sensor.

Further, preferably, the kit also includes amylose solution and progesterone standard solution.

The beneficial effects of the present invention are:

The present invention provides a blood glucose meter-based nucleic acid aptamer sensor for the quantitative detection of progesterone, which converts the non-sugar target concentration into the amylose concentration measurable by the blood glucose meter, thereby realizing the instant quantitative detection of the non-sugar target-progesterone by the blood glucose meter. The instrument used only needs the blood glucose meter and does not need other electrochemical/optical detection instruments. The detection cost is low and the operation is simple, which can realize on-site and accurate progesterone content detection and lay a foundation for the diagnosis of early pregnancy in dairy cows or humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the aptamer sensing system optimized by fluorescence spectroscopy in Example 1.

FIG. 2 shows the effect of the binding buffer on the detection sensitivity of P4 in Example 1.

FIG. 3 shows the effect of incubation time on P4 detection sensitivity in Example 1.

FIG. 4 shows the effect of magnetic bead particle size on P4 detection sensitivity in Example 1.

FIG. 5 shows the effects of different nucleic acid aptamer sequences and complementary chain sequences on P4 detection sensitivity in Example 1.

FIG. 6 is a schematic diagram of the preparation process of CS-GA in Example 2.

FIG. 7 is the UV spectra of CS, GA and the product CS-GA in Example 2.

FIG. 8 is the hysteresis regression curves of MBs and MBs-Apt-CS-GA in Example 3.

FIG. 9 is the thermogravimetric curves of MBs and MBs-Apt-CS-GA in Example 3.

FIG. 10 is a schematic diagram of the application of the aptamer sensor based on the blood glucose meter in Example 4 to portable progesterone detection.

FIG. 11 is the optimization of catalytic time of amylose by CS-GA in Example 4.

FIG. 12 shows the stability results of MBs-Apt-CS-GA in Example 4.

FIG. 13 is the linear equation of P4 in Example 5.

Detailed ways

The present invention aims to develop a P4 instant detection method based on a portable blood glucose meter and a nucleic acid aptamer sensor, and realizes the conversion of non-sugar target signals to measurable signals of a blood glucose meter through the affinity of the aptamer and P4 and magnetic separation. It effectively solves the problem that conventional sensing systems require large detection terminals and existing POCT devices have poor practicality and complicated operation steps. Among them, the signal conversion chain, i.e., the conjugate of the nucleic acid aptamer complementary chain and glucoamylase (CS-GA), is prepared by reacting the disulfide bond of CS with the carboxyl group of GA and purifying by ultracentrifugation, and the signal conversion chain CS-GA is hybridized with the biotin-modified aptamer chain to form Apt-CS-GA, and Apt-CS-GA is coupled to streptavidin magnetic beads through biotin-streptavidin action to prepare a nucleic acid aptamer sensor. In the constructed sensor, in the presence of P4, the specific affinity of the nucleic acid aptamer and P4 forms a streptavidin magnetic bead-biotin-Apt-P4 complex, and releases CS-GA. After magnetic separation, the supernatant contains CS-GA. The supernatant is used to catalyze the amylose solution to produce a signal that can be measured by a blood glucose meter, thereby converting the non-sugar target P4 signal into a glucose signal.

The key points of the present invention are: design and preparation of the signal conversion chain CS-GA. ① How to select a complementary chain of aptamers with suitable sequences; ② How to prepare a CS-GA signal conversion chain without excess CS.

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

If not otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are analytical grade or above. Among them, progesterone can be purchased from Dr. Ehrenstorfer, Germany. Tris(2-carboxyethyl)phosphine (TCEP) and glucoamylase (GA) were purchased from Sigma-Aldrich (St. Louis, MO, USA); sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and DynabeadsTM MyOneTm streptavidin C1 magnetic beads were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The buffer A used was: containing sodium phosphate with a final concentration of 0.1 M and 100 mM NaCl, with a pH value of 7.3; the buffer B was: containing Tris-HCl with a final concentration of 10 mM, 10 mM NaCl and 5 mM MgCl ₂ , with a pH value of 7.3; the buffer C was: containing Tris-HCl with a final concentration of 10 mM, 10 mM NaCl, 5 mM MgCl ₂ and 0.01% Tween-20, with a pH value of 7.3. The P4 solution was dissolved in buffer C.

Example 1

First, the experimental conditions of the aptamer sensing system were optimized by fluorescence spectroscopy, and the schematic diagram is shown in Figure 1. The specific operations are:

(1) Take 50 μL of 5.0 μM Biotin-modified nucleic acid aptamer (Apt) (dissolved in buffer C), mix it with 50 μL of 6.0 μM FAM-modified aptamer complementary chain (CS) (dissolved in buffer C), incubate it in a 95 ℃ water bath for 10 min, and slowly cool it to room temperature to form a double-stranded chain with complementary base pairing between Apt and CS.

(2) Take 30 μL of 1 μm streptavidin C1 magnetic beads, discard the supernatant after magnetic separation, and add 200 μL buffer C. Use a pipette to repeatedly blow for 1 min. Repeat the above steps three times. After magnetic separation, discard the supernatant and add the double-stranded solution of Apt and CS base complementary pairing in step (1).

(3) The mixture in step (2) was shaken at room temperature for 2 h to obtain a double-stranded structure coupled to magnetic beads. After magnetic separation, the supernatant was discarded, and 200 μL of buffer C was added. The mixture was repeatedly blown with a pipette for 1 min, and the step (3) was repeated for more than 5 times. After discarding the supernatant, 100 μL of 200 ng/mL P4 solution (dissolved in buffer C) was added and shaken (600 rpm) at 37 °C for 1 h. After magnetic separation, 70 μL of the supernatant was placed in a 96-well ELISA plate, and the fluorescence intensity of the supernatant was measured using an ELISA instrument. The excitation wavelength was 488 nm and the emission wavelength was 520 nm. Secondly, the type of buffer that affects the detection sensitivity was optimized, that is, the buffer for dissolving P4. According to the above steps (1)-(3), the P4 buffer was adjusted to Tris-HCl (final concentration of 20 mM Tris-HCl, pH 7.4), Tris-HCl+tween (final concentration of 20 mM Tris-HCl, pH 7.4, volume percentage of 0.01% Tween-20), DPBS (final concentration of 0.9 mmol/L CaCl ₂ , 2.685 mmol/L KCl , 1.47 mmol/L KH ₂ PO ₄ , 0.49 mmol/L MgCl ₂ , 137 mmol/L NaCl, 8.1 mmol/L Na ₂ HPO ₄ , pH 7.5) and buffer C, respectively.

The experimental results are shown in Figure 2, which shows the effect of the binding buffer on the sensitivity of P4 detection. The experimental results show that when the binding buffer is Tris-HCl and DPBS, the fluorescence intensity is low; when the binding buffer is Tris-HCl+tween, its fluorescence intensity is lower than that when the binding buffer is buffer C, which may be related to the type and strength of salt ions in the buffer. Therefore, buffer C was selected as the binding buffer for the next study.

Again, the reaction time of the double-stranded structure coupled with P4 and magnetic beads was optimized. According to the above steps (1)-(3), after adding 100 μL of 200 ng/mL P4 solution, the reaction was shaken at 37°C for 10 min, 20 min, 30 min, 1 h and 2 h respectively. After magnetic separation, the supernatant was taken to measure the fluorescence intensity. The experimental results are shown in Figure 3, which shows the effect of incubation time on the sensitivity of P4 detection. The experimental results show that as the reaction time increases, the fluorescence intensity gradually increases and reaches the highest value at 1 h. Therefore, the reaction time is preferably 20~70 min, and the most preferred is 60 min. The reaction time of 1 h is selected for the next step of research.

The particle size of the magnetic beads was optimized. According to the above steps, 30 μL of streptavidin magnetic beads with particle sizes of 1 μm, 25 μm and 100 μm were taken for the subsequent coupling step. The fluorescence intensity of P4 of the fluorescent sensor prepared with magnetic beads of different particle sizes was compared. The experimental results are shown in Figure 4, which is recorded as the effect of the magnetic bead particle size on the detection sensitivity of P4. The experimental results show that when magnetic beads with larger particle sizes are used, the fluorescence intensity is lower than that of magnetic beads with a particle size of 1 μm. This indicates that large-size magnetic beads are not conducive to the binding of P4 to the aptamer, which may be related to the steric hindrance of large-size magnetic beads. Therefore, 1 μm particle size magnetic beads were selected for the next study.

The selection of aptamer sequence and complementary chain sequence is a key factor affecting the sensitivity of P4 detection. Therefore, we designed different complementary sequences based on two P4 aptamer nucleic acid sequences reported in the literature (one is denoted as P4APT(60), which is the original length P4 aptamer screened in the literature; the other is denoted as P4APT(25), which is the truncated P4 aptamer in the literature), respectively. The complementary sequences to P4APT(60) are CS1-6, and the complementary sequences to P4APT(25) are CST1-3. The specific sequences are shown in Table 1.

Table 1 Nucleic acid aptamers and complementary chain sequences

The above sequences were synthesized by Sangon Biotechnology Co., Ltd.

Take 50 μL of 5.0 μM Biotin-labeled P4APT(60), mix with 50 μL of 6.0 μM FAM-labeled CS1-CS6, incubate in a 95 ℃ water bath for 10 min, and slowly cool to room temperature to form a double-stranded base pairing of P4APT(60) and CS. Take 50 μL of 5.0 μM Biotin-P4APT(25), mix with 50 μL of 6.0 μM FAM-CST1-3, incubate in a 95 ℃ water bath for 10 min, and slowly cool to room temperature to form a double-stranded base pairing of P4APT(25) and CS. Add P4 solution according to the above steps (1)-(3), and shake at 37 ℃ for 1 hour. After magnetic separation, take 70 μL of the supernatant and place it in a 96-well ELISA plate, and measure the fluorescence intensity of the supernatant using an ELISA reader. The experimental results are shown in Figure 5, which shows the effect of different aptamer sequences and complementary chain sequences on the detection sensitivity of P4. The experimental results show that when the aptamer is P4APT(60), the fluorescence intensity of CS4-CS6 is relatively high among the six complementary sequences; but when P4APT(25) is used as the aptamer, among the three complementary sequences CST1-CST3, CST1 shows the highest fluorescence intensity, which is significantly higher than CS4-CS6. Therefore, CST1 (3'-CTAATTGTAATC-5') was selected as the final CS, and P4APT(25) (5'-GATTAACATTAGGGGACCGCCCACC-3') was selected as the final Apt for the subsequent preparation and application of aptamer sensors based on blood glucose meters.

Example 2

CS-GA was prepared using Sulfo-SMCC as a coupling agent, and the preparation process is shown in Figure 6. The specific operation is as follows: CS (SEQ ID NO. 1, with its 5-terminal thiol (-SH-SH) modified) was first dissolved in water as CS dry powder, and TCEP was dissolved in 1.1 M sodium phosphate buffer with a pH of 5.5;

Take 30 μL of 1mM CS, 2 μL of 1M sodium phosphate buffer (pH 5.5) and 2 μL of 30 mM tris(2-carboxyethyl)phosphine (TCEP) and mix them. After incubation at room temperature for 1 h, transfer the mixture to an Amicon-3 K ultrafiltration tube and centrifuge it at 14000 g and 4°C for 15 min in a refrigerated centrifuge. Repeat the centrifugation step and purify CS 8 times using buffer A. Invert the ultracentrifuge tube and centrifuge it at 1000 g for 5 min to recover the product and make it up to 200 μL with buffer A.

1 mg of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfoSMCC) was dissolved in 200 μL ultrapure water, and 400 μL of GA solution dissolved in buffer A at a concentration of 20 mg/mL was added. After vortexing for 5 minutes, the solution was incubated at room temperature for 1 hour, and the mixture was transferred to an Amicon-30 K ultrafiltration tube and centrifuged at 14000g and 4°C for 15 min using a refrigerated centrifuge. After repeating the centrifugation step and purifying CS 8 times using buffer A, the ultracentrifuge tube was inverted and centrifuged at 1000 g for 5 minutes to recover the product and make up to 400 μL with buffer A.

The activated CS and GA solutions were mixed, and the resulting solution was incubated at room temperature for 48 h. In order to remove excess cDNA, the mixture was transferred to an Amicon-100 K ultrafiltration tube and centrifuged at 14,000 g and 4°C for 15 min using a refrigerated centrifuge. After repeating the centrifugation step and purifying CS 8 times using buffer B, the ultracentrifuge tube was inverted and centrifuged at 1,000 g for 5 min to recover the product CS-GA conjugate. The CS-GA conjugate was finally dispersed in buffer B at a concentration of 20 mg/mL.

In order to verify whether the preparation of CS-GA was successful, CS, GA and the product CS-GA were characterized by UV spectroscopy, and the results are shown in Figure 7. CS peaks at 260 nm, GA has a maximum UV absorption at 278 nm, and the coupling product CS-GA is between the two (261 nm). The UV absorption spectrum changes, indicating that the coupling of CS-GA is successful.

Example 3

30 μL of 6 μM CS-GA prepared in Example 2 and 30 μL of 5 μM Apt (SEQ ID NO.2, 5' of which is labeled with biotin) were mixed and incubated at room temperature for 2 h, and 30 μL of streptavidin magnetic beads were added for shaking coupling for 1 h. After magnetic separation, the supernatant was discarded, 200 μL of buffer C was added and repeatedly blown for 1 min, the supernatant was discarded after magnetic separation, and the above washing steps were repeated 5 times to obtain the aptamer sensor (MBs-Apt-CS-GA).

In order to verify whether MBs-Apt-CS-GA was successfully prepared, magnetic analysis and thermogravimetric analysis were performed.

The magnetic changes during the synthesis of MBs-Apt-CS-GA were detected by a vibrating sample magnetometer (VSM), and the results are shown in Figure 8. The magnetic response of MBs-Apt-CS-GA is related to the content of MBs. The hysteresis regression curves of MBs and MBs-Apt-CS-GA in Figure 8 are both "S" shaped and symmetrical at the origin, and have paramagnetism. The saturation magnetization of MBs and MBs-Apt-CS-GA are 16.2 and 16.0 emu/g, respectively. Although the saturation magnetization is slightly reduced due to the coupling of the double chain to the magnetic beads, the retained magnetism is sufficient to achieve rapid separation under the action of an external magnetic field.

MBs and MBs-Apt-CS-GA were further verified by thermogravimetric analyzer (TGA). 10 mg MBs and MBs-Apt-CS-GA were subjected to programmed temperature increase (30-900 °C) at a rate of 10 °C/min under nitrogen atmosphere. The results are shown in Figure 9. Since the prepared materials all have a certain amount of residual moisture, their weights decrease slightly at around 100 °C. The weight loss of MBs-Apt-CS-GA is about more than that of MBs, which is caused by the coupling of Apt-CS-GA.

Example 4

The constructed aptamer sensor was applied to the portable detection of progesterone. 30 μL of the aptamer sensor (MBs-Apt-CS-GA) prepared in device example 3 was mixed with 30 μL of 10 μg/mL P4 in a centrifuge tube and shaken at room temperature for 1 h. After magnetic separation, 10 μL of the supernatant was mixed with 10 μL of 2M amylose solution and incubated at 40 °C for 30 min, and 2 μL was taken for blood glucose meter detection. The detection experiment diagram is shown in Figure 10.

In order to achieve sensitive detection of P4, the catalytic time of GA on amylose was optimized. That is, the prepared aptamer sensor was mixed with 30 μL 10 μg/mL P4 in a centrifuge tube and shaken at room temperature for 1 h. After magnetic separation, 10 μL of the supernatant was mixed with 10 μL 2 M amylose solution at 40 °C for 1, 5, 10, 15, 20, 25, and 30 min, and 2 μL was taken for blood glucose meter detection. The experimental results are shown in Figure 11. When the incubation time was 1 min and 5 min, the detection reading of the blood glucose meter was Low. As the incubation time increased from 5 min to 30 min, the reading of the blood glucose meter gradually increased. Since the highest readable reading of the blood glucose meter is 27, it will not increase on the basis of the incubation time of 30 min. In order to achieve high-sensitivity detection of P4, 30 min was selected as the final incubation time of CS-GA and amylose for subsequent research.

In order to investigate the stability of the constructed sensor, MBs-Apt-CS-GA was placed in 30 μL buffer C and stored at 4°C for different days (1, 2, 3, 4 and 5 days). After magnetic separation, the supernatant was discarded, 30 μL 10 μg/mL P4 was added to the centrifuge tube and mixed, and the mixture was shaken at room temperature for 1 h. After magnetic separation, 10 μL of the supernatant was mixed with 10 μL 2M amylose solution at 40°C for 30 min, and 2 μL was taken for blood glucose meter detection. The experimental results are shown in Figure 12. When stored at 4°C for 1-3 days, the reading of the blood glucose meter did not decrease significantly and remained at 15 mmol/L, which was in a higher reading range. When stored at 4°C for 4-5 days, the reading of the blood glucose meter decreased significantly. In order to ensure the sensitivity of P4 detection, the constructed MBs-Apt-CS-GA can be stored in buffer C at 4°C for three days.

Example 5

According to the operation steps described in Example 4, a linear model of P4 concentration and blood glucose meter signal was established. 30 μL of the prepared aptamer sensor and 30 μL of different concentrations of P4 (3.1, 6.2, 12.5, 20, 50 μg/mL) were mixed in centrifuge tubes and shaken at room temperature for 1 h. After magnetic separation, 10 μL of the supernatant was mixed with 10 μL of 2 M amylose solution at 40 ° C and incubated for 30 min, and then 2 μL was taken for blood glucose meter detection. The detection experiment diagram is shown in Figure 13. With P4 concentration as the horizontal axis and the difference between the blood glucose meter reading and the blank value (blood glucose meter reading when P4 concentration is 0) as the vertical axis, a linear fit was performed, and the linear equation was obtained as Y=0.1035X+0.2724 ( ^R2 =0.992), and each group of experiments was measured three times in parallel. The LOD value was calculated according to the formula LOD = 3.3 δ/S, where δ is the standard deviation of the blank value and S is the slope of the standard curve. The LOD value of the established method was calculated to be 2.25 μg/mL, that is, the detection limit was 7.15 μM.

Example 6

The portable blood glucose meter can be used to detect progesterone in real time without the guidance of professional technicians in combination with a nucleic acid aptamer sensing kit, wherein the kit contains a nucleic acid aptamer sensor based on a portable blood glucose meter prepared according to Example 3, a progesterone standard solution, and an amylose solution; the progesterone content is detected according to the following method.

A method for instant detection of progesterone by a nucleic acid aptamer sensor based on a portable blood glucose meter, specifically comprising the following steps:

1. Mix 30 μL of nucleic acid aptamer sensor (MBs-Apt-CS-GA) and 30 μL of blood to be tested in a centrifuge tube and shake at room temperature for 1 h. After magnetic separation, take 10 μL of supernatant and mix with 10 μL of 2M amylose solution at 40 ℃ for 30 min, then take 2 μL for blood glucose meter detection to obtain the blood glucose reading of the solution to be tested;

2. The progesterone standard solution (such as 3.1, 6.2, 12.5, 20, 50 μg/mL) is mixed with the nucleic acid aptamer sensor to perform step S2, and the blank solution is subjected to the above experiment to obtain a blank value; the progesterone concentration is used as the horizontal axis, and the blood glucose meter reading of the progesterone standard solution and the blank value are linearly fitted with the difference obtained later as the vertical axis to obtain its linear equation, i.e., the standard curve;

3. Substitute the blood glucose reading of the test solution obtained in step 1 into the standard curve to obtain the concentration of progesterone in the test solution.

Claims (9)

1. A method for real-time detection of progesterone by a nucleic acid aptamer sensor based on a portable blood glucose meter, characterized in that it comprises the following steps: S1. Preparation of nucleic acid aptamer sensor based on portable blood glucose meter; S2, mixing the nucleic acid aptamer sensor with the solution to be tested, and after the reaction, taking the supernatant after magnetic separation, mixing the supernatant with the amylose solution and incubating them, and then testing with a blood glucose meter to obtain the blood glucose reading of the solution to be tested; S3, respectively mixing the progesterone standard solution with the nucleic acid aptamer sensor to perform step S2, and at the same time performing the experiment of step S2 on the blank solution to obtain a blank value; using the progesterone concentration as the abscissa and the difference between the blood glucose meter reading of the progesterone standard solution and the blank value as the ordinate to perform linear fitting to obtain its linear equation, i.e., the standard curve; S4, substituting the blood glucose reading of the test solution obtained in step S2 into the standard curve to obtain the concentration of progesterone in the test solution; In step S1, the preparation method of the nucleic acid aptamer sensor is to mix and incubate the aptamer complementary chain with the conjugate of glucoamylase CS-GA and the biotin-modified nucleic acid aptamer for the first time, add streptavidin magnetic beads for the first oscillation coupling, and wash with buffer to obtain the nucleic acid aptamer sensor; the sequence of the aptamer complementary chain is shown in SEQ ID NO.1, and the 5′ end of the aptamer complementary chain is modified with -SH-SH-; the sequence of the nucleic acid aptamer is shown in SEQ ID NO.2, and the 5′ end of the nucleic acid aptamer is modified with Biotin. 2. The instant detection method according to claim 1 is characterized in that the particle size of the streptavidin magnetic beads is 1 μm, the formula of the buffer solution contains Tris-HCl with a final concentration of 10 mM, 10 mM NaCl, 5 mM MgCl ₂ and 0.01% Tween-20, and the pH value is 7.3. 3. The instant detection method according to claim 1, characterized in that the preparation method of the conjugate CS-GA is to mix and incubate the aptamer complementary chain with sodium phosphate buffer and tris(2-carboxyethyl)phosphine, transfer it to an Amicon-3 K ultrafiltration tube, centrifuge it, and then purify it with buffer A to obtain activated CS; The glucoamylase solution was mixed with the sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate solution, incubated, transferred to an Amicon-30 K ultrafiltration tube, centrifuged, and then purified with buffer A to obtain activated glucoamylase; The activated CS was mixed with the activated glucoamylase, incubated at room temperature, transferred to an Amicon-100 K ultrafiltration tube, and the conjugate CS-GA was obtained after purification. 4. The instant detection method according to claim 1 is characterized in that the conjugate CS-GA is mixed with a biotin-modified nucleic acid aptamer to obtain a mixture, and the mixture is shake-coupled with streptavidin magnetic beads; the first mixing and incubation conditions are: incubate at 95°C for 10 min and then slowly cool to room temperature, and the first shaking coupling time is 0.5~1.5h. 5. The instant detection method according to claim 1, characterized in that, in steps S2 and S3, the solvent in the test solution and the progesterone standard solution is buffer C, and the formula of buffer C is: containing Tris-HCl with a final concentration of 10 mM, 10 mM NaCl, 5 mM MgCl2 and 0.01% Tween-20, and the pH value is 7.3. 6. The instant detection method according to claim 1 is characterized in that, in step S2, the reaction time is 20 to 120 min; the reaction is carried out under shaking conditions, and the shaking rate is 500 to 800 rpm; the mixing incubation time is 20 to 40 min. 7. The instant detection method according to claim 1 is characterized in that, in step S2, the amount of the nucleic acid aptamer sensor is 30 μL, and the volume ratio to the solution to be tested is 1:1; the concentration of the amylose solution is 2 M, and the amount ratio of the supernatant to the amylose solution is 1:1. 8. A kit for detecting progesterone based on a nucleic acid aptamer sensor of a blood glucose meter, characterized in that it includes a nucleic acid aptamer sensor, and the preparation method of the nucleic acid aptamer sensor is to mix and incubate the aptamer complementary chain of the sequence shown in SEQ ID NO.1 and the conjugate of glucoamylase CS-GA with the nucleic acid aptamer modified with the sequence shown in SEQ ID NO.2 for the first time, add streptavidin magnetic beads for the first shaking coupling, and wash with a buffer solution to obtain the nucleic acid aptamer sensor. 9. The kit according to claim 8, further comprising an amylose solution and a progesterone standard solution.