Method for multi-target detection based on magnetic-assisted cell counting technology

Abstract

The present invention discloses a method for multi-target detection based on magnetic-assisted cell counting technology, comprising the following steps: (1) preparing an aptamer nucleic acid hybridization chain AS-TDO5; (2) incubating AS-TDO5 with Fe304 nanoparticles modified with streptavidin, and obtaining Fe304@AS-TDO5 by magnetic separation and washing; (3) incubating Fe304@AS-TDO5 with suspended cells, and obtaining a conjugate sensor of Fe304 and cells by magnetic separation and washing; (4) incubating the conjugate sensor of Fe304 and cells with exonuclease III, and then magnetic separation and washing, and distinguishing between exonuclease III and no exonuclease III groups by monitoring the number of cells captured by Fe304 nanoparticles, and at the same time distinguishing the number of suspended cells captured by Fe304 nanoparticles after incubation with different concentrations of exonuclease III. This method is simple to operate, has low detection cost, high sensitivity, and low signal-to-noise ratio.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically is a method for multi-target detection based on magnetic-assisted cell counting technology. Background Art

[0002] The main goal of biological detection is to develop simple, sensitive, highly selective, and cost-effective biosensing platforms that can detect biomolecules (such as DNA, RNA, or proteins) or whole cells. At present, the detection of biomolecules mainly includes polymerase chain reaction and enzyme-linked immunosorbent assay, which are quite advanced in nucleic acid and protein detection. However, they have several disadvantages: 1) The complex detection system takes a lot of time and increases the demand for equipment; 2) Most detection methods can only be applied to limited targets; 3) Due to insufficient sensitivity, the measurement of extremely low levels of biomolecules requires the signal to be amplified many times. Therefore, current biomolecule detection methods are still a key challenge in many clinical diagnostic applications. Summary of the invention

[0003] The purpose of the present invention is to provide a method for multi-target detection based on magnetic-assisted cell counting technology in view of the shortcomings of the prior art. The method is simple to operate, low in detection cost, high in sensitivity, and low in signal-to-noise ratio.

[0004] The technical solution for achieving the purpose of the present invention is:

[0005] A method for multi-target detection based on magnetic-assisted cell counting technology comprises the following steps:

[0006] (1) The biotin-modified anchor chain (AS) and the aptamer (TDO5) chain with the same concentration were shaken and annealed to obtain AS-TDO5;

[0007] (2) incubating the AS-TDO5 obtained in step (1) with streptavidin-modified Fe304 nanoparticles, and obtaining Fe304@AS-TDO5 by magnetic separation and washing;

[0008] (3) incubating the Fe304@AS-TDO5 obtained in step (2) with suspended cells, and preparing a Fe304-cell conjugate sensor by magnetic separation and washing;

[0009] (4) The Fe304-cell conjugate sensor obtained in step (3) is incubated with exonuclease III, followed by magnetic separation and washing. By monitoring the number of cells captured by Fe304, the exonuclease III and no exonuclease III groups can be distinguished. At the same time, the number of suspended cells captured by Fe304 after incubation with different concentrations of exonuclease III can be distinguished.

[0010] The Fe304-cell conjugate sensor in step (3) is obtained by the following method:

[0011] (1) The AS nucleic acid chain and the TDO5 aptamer chain were diluted to the same concentration with PBS. The AS nucleic acid chain and the TDO5 aptamer chain formed an AS-TDO5 double chain through base complementary pairing;

[0012] (2) AS-TDO5 was modified onto the surface of magnetic nanoparticles through the affinity of streptavidin and biotin, and then Ramos cells were captured through the TDO5 aptamer to form a conjugate sensor of Fe304 and cells to detect exonuclease III.

[0013] The preparation process of the Fe304 and cell conjugate sensor described in step (3) is as follows:

[0014] (1) Mix 45-55 µL AS nucleic acid chain and 45-55 µL TDO5 aptamer chain respectively, shake well, place in a 90-100°C water bath, turn off the water bath after 4-6 minutes, wait for the water bath temperature to drop to room temperature, and take out to obtain AS-TDO5 hybrid nucleic acid chain;

[0015] (2) Add 10-50 µL of AS-TDO5 hybrid nucleic acid chain to 20-100 µL of Fe304 nanoparticles, add 10-50 µL of incubation buffer, shake well and incubate for 1-2 h, then separate by magnetic separation, and rinse three times with washing buffer to obtain Fe304@AS-TDO5;

[0016] (3) Take 100-500 µL Ramos cell suspension and add 10-50 µL Fe304@ AS-TDO5, blow evenly, place in a refrigerator at 1-5°C for incubation for 30-60 min, then perform magnetic separation and discard the supernatant, wash three times with PBS to obtain the Fe304 and cell conjugate sensor.

[0017] The concentrations of the AS nucleic acid chain and the TDO5 aptamer chain were both 20 µM.

[0018] The concentration of AS-TDO5 was 10 µM.

[0019] The concentration of the Fe304 nanoparticles is 10 mg / mL.

[0020] The diameter of the Fe304 nanoparticles is 180-200 nm.

[0021] The concentration of the Ramos cells was 1×10 6 / mL.

[0022] The step (4) described in which the change in the number of cells captured by the Fe304 nanoparticles is specifically as follows:

[0023] If the number of cells captured by Fe304 nanoparticles is small, exonuclease III is present, and the number of captured cells is inversely proportional to the concentration of exonuclease III;

[0024] If the number of cells captured by Fe304 nanoparticles does not change much, exonuclease III does not exist.

[0025] Cells have a complex membrane-nuclear structure. The cell membrane is composed of a phospholipid bilayer and is embedded with a large number of receptor proteins and other biological molecules that control the entry and exit of external substances. Different cells have characteristic proteins that regulate information conduction between cells. Due to the characteristics of tumor cells, circulating tumor cells are often used as detection targets, but it is the first time to use them as signal probes. This technical solution captures circulating tumor cells by modifying the Fe304 nanoparticles of the aptamer to form a biosensor, and then cuts the aptamer chain through the target to achieve a cell signal output from "on" to "off". This technical solution uses Fe304 nanoparticles with high sensitivity, easy to obtain, and simple to operate to capture circulating tumor cells as a biosensor to detect biological molecules.

[0026] Principle of the present technical scheme: In the present technical scheme, streptavidin-modified Fe304 nanoparticles are co-incubated with biotin-modified nucleic acid chain hybrid aptamer chains to obtain aptamer-modified Fe304 nanoparticles; the functionalized Fe304 nanoparticles are connected to the surface of tumor cells through the specific recognition of the aptamer, and the Fe304 and cell conjugate sensors are obtained after magnetic separation and washing; when exonuclease III is added to the Fe304 and cell conjugate sensor, after incubation at 37°C for a period of time, the cells are dissociated from the Fe304 nanoparticles due to the cleavage of the 3' end of the complementary nucleic acid chain by exonuclease III, and after magnetic separation and washing, the suspended cells are washed away with the supernatant, the number of cells captured by the Fe304 nanoparticles decreases, and the number of cells captured by the Fe304 nanoparticles is inversely proportional to the concentration of exonuclease III.

[0027] Compared with the prior art, the beneficial technical effects of this technical solution are:

[0028] The technical solution and method are simple to operate and have low detection cost. Cells are used as probe signals, and their signals are larger than fluorescent signals, easier to observe, easy to prepare and obtain, and have high sensitivity. When the target is present, Fe304 nanoparticles can be separated from cells, and the number of cells captured by Fe304 nanoparticles can be observed after magnetic separation, thereby distinguishing the groups with and without exonuclease III incubation, and the concentration of exonuclease III can be detected.

[0029] This method is simple to operate, has low detection cost, high sensitivity and low signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the microscopic morphology of Fe304 nanoparticles, Ramos cells and the prepared Fe304 and cell conjugate sensor used in the examples, a is a TEM image of multiple Fe304 nanoparticles; b is a confocal image of Ramos cells; c is a confocal image of multiple Fe304 and cell conjugate sensor;

[0031] Figure 2 Zeta potential diagram of Fe304 nanoparticles (0.2 mg / mL), Fe304@TDO5 (0.2 mg / mL) and Fe304@Sgc-8 (0.2 mg / mL) in the examples;

[0032] Figure 3 These are confocal images of the Hoechst33342 staining channel of the Fe304 nanoparticles and different concentrations of Fe304@TDO5 in the embodiment, respectively, capturing the change in the number of Ramos cells; a is a confocal image of the number of Ramos cells captured by Fe304 nanoparticles; b is a confocal image of the number of Ramos cells captured by 0.1 mg / ml Fe304@TD05; c is a confocal image of the number of Ramos cells captured by 0.5 mg / ml Fe304@TD05; d is a confocal image of the number of Ramos cells captured by 1 mg / ml Fe304@TD05; e is a confocal image of the number of Ramos cells captured by 3 mg / ml Fe304@TD05;

[0033] Figure 4 The figure is a bar graph of the change in the number of Ramos cells (Cell Number) captured by Fe304@TDO5 at different concentrations in the embodiment. a is a bar graph made based on the confocal image of the Hoechst33342 staining channel; b is a bar graph made based on the cell count by the hemocytometer method;

[0034] Figure 5 The confocal images of the Hoechst33342 staining channel in which Fe304 nanoparticles and Fe304@AS-TDO5 respectively capture the change in the number of Ramos cells in the embodiment, a is a confocal image of Fe304 nanoparticles before incubation and washing with Ramos cells; b is a confocal image of Fe304 nanoparticles after incubation and washing with Ramos cells; c is a confocal image of Fe304@AS-TDO5 before incubation and washing with Ramos cells; d is a confocal image of Fe304@AS-TDO5 after incubation and washing with Ramos cells; e is a confocal image of Fe304@AS-TDO5 after incubation and washing with Ramos cells and the addition of exonuclease III;

[0035] Figure 6It is the agarose electrophoresis diagram of AS, TDO5, AS-TDO5 hybrid chain and AS-TDO5 hybrid chain after enzyme digestion in the embodiment, band 1 is AS nucleic acid chain; band 2 is TDO5 aptamer chain; band 3 is AS-TDO5 hybrid chain; band 4 is AS-TDO5 hybrid chain after enzyme digestion;

[0036] Figure 7 The bar graph of the change in the number of cells captured by Fe304 nanoparticles (Cell Number) after incubation with different proteins or enzymes according to the confocal images in the embodiment;

[0037] Figure 8 The graphs and linear graphs of the number of cells captured by Fe304 nanoparticles and the change of three exonuclease concentrations according to the confocal images in the embodiment, a is the graph of the number of cells captured by Fe304 nanoparticles and the change of three exonuclease concentrations; b is the linear graph of the number of cells captured by Fe304 nanoparticles and the change of three exonuclease concentrations;

[0038] Fig. 9 The conjugate sensor of Fe304 and cells is counted according to the confocal image in the embodiment. After the sensor and exonuclease III are incubated in fetal bovine serum and PBS, magnetic separation and washing are performed, the bar graph of the change in the number of cells captured by Fe304 nanoparticles (Cell Number). DETAILED DESCRIPTION

[0039] The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0040] Embodiment 1:

[0041] A method for multi-target detection based on magnetic-assisted cell counting technology comprises the following steps:

[0042] (1) Preparation of Fe304-cell conjugate sensor:

[0043] AS nucleic acid chain (50 μL, 20 μM) and TDO5 (50 μL, 20 μM) aptamer chain were mixed and shaken, placed in a 95℃ water bath, and the water bath was turned off after 5 minutes. The water bath was taken out after the temperature dropped to room temperature to obtain AS-TDO5; in a 1.5 mL centrifuge tube, 250μL AS-TDO5 (10 μM) was added to 500μL Fe304 (10 mg / mL) nanoparticles, and 100μL incubation buffer was added. After shaking and incubating for 1 hour, magnetic separation was performed, and after rinsing with washing buffer three times, Fe304@AS-TDO5 (10 mg / mL) was resuspended in 500μL PBS; in a 15mL centrifuge tube, 1mL Ramos cell suspension (1×10 6 / mL) and 500µL Fe304@ AS-TDO5 were pipetted evenly, placed in a 4℃ refrigerator for incubation for 1h, and then magnetically separated and the supernatant was discarded. The cells were washed three times with PBS, and finally resuspended with 500µL PBS and pipetted evenly to obtain the Fe304-cell conjugate sensor.

[0044] Figure 1 Schematic diagram of the microscopic morphology of Fe304 nanoparticles, Ramos cells and the prepared Fe304 and cell conjugate sensor used in the embodiment (a is a TEM image of Fe304 nanoparticles; b is a confocal image of Ramos cells; c is a confocal image of the Fe304 and cell conjugate sensor). It can be seen from Figure a that the diameter of Fe304 nanoparticles is about 200 nm; it can be seen from Figure b that the surface of Ramos cells is smooth and free of impurities; it can be seen from Figure c that the cell surface is coated with Fe304 nanoparticles;

[0045] (2) Preparation of Fe304@TDO5: Place the TDO5 aptamer chain (50 μL, 20 μM) dissolved in PBS into a 95℃ water bath. After 5 minutes, turn off the water bath and wait for the water bath temperature to drop to room temperature before taking out the annealed TDO5 aptamer chain (50 μL, 20 μM). Take 10 μL of the annealed TDO5 aptamer chain (20 μM) and add it to 20 μL of Fe304 (10 mg / mL) nanoparticles. Add 10 μL of incubation buffer, shake well and incubate for 1 hour, then perform magnetic separation. After rinsing with washing buffer three times, resuspend it in 20 μL PBS to obtain Fe304@TDO5 (10 mg / mL). Add 980 μL PBS, shake well and sonicate for 15 minutes to obtain Fe304@TDO5 (0.2 mg / mL).

[0046] (3) Preparation of Fe304@Sgc-8: Place the Sgc-8 aptamer chain (50 μL, 20 μM) dissolved in PBS into a 95℃ water bath. After 5 minutes, turn off the water bath and wait for the water bath temperature to drop to room temperature before taking out the annealed Sgc-8 aptamer chain (50 μL, 20 μM). Take 10 μL of the annealed Sgc-8 aptamer chain (20 μM) and add it to 20 μL of Fe304 (10 mg / mL) nanoparticles. Add 10 μL of incubation buffer, shake well and incubate for 1 hour, then separate by magnetic separation. Rinse three times with washing buffer, resuspend in 20 μL PBS to obtain Fe304@Sgc-8 (10 mg / mL). Add 980 μL PBS, shake well and sonicate for min to obtain Fe304@Sgc-8 (0.2 mg / mL).

[0047] Figure 2Zeta potential diagram of Fe304 nanoparticles (0.2 mg / mL), Fe304@TDO5 (0.2 mg / mL) and Fe304@Sgc-8 (0.2 mg / mL). Figure 2 It can be seen that Fe304@TDO5 and Fe304@Sgc-8 have more negative potential values ​​due to the modification of negatively charged aptamer chains;

[0048] (4) Preparation of Fe304@TDO5: Place the TDO5 aptamer chain (50 μL, 20 μM) dissolved in PBS into a 95℃ water bath. After 5 minutes, turn off the water bath and wait for the water bath temperature to drop to room temperature before taking out the annealed TDO5 aptamer chain (50 μL, 20 μM). Take 25 μL of the annealed TDO5 aptamer chain (20 μM) and add it to 50 μL of Fe304 (10 mg / mL) nanoparticles. Add 10 μL of incubation buffer, shake well, incubate for 1 hour, and then separate by magnetic separation. After rinsing with washing buffer three times, resuspend in 50 μL PBS to obtain Fe304@TDO5 (10 mg / mL). Then dilute Fe304@TDO5 with PBS to concentrations of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 3 mg / mL, respectively.

[0049] (5) Preparation of stained Ramos cells: Ramos cells were transferred from the culture flask to a 15 mL centrifuge tube, and then centrifuged at 1000 r / min in a centrifuge. The supernatant was discarded, 900 µL PBS and 100 µL Hoechst33342 were added, and the tube was evenly mixed and placed in a 37°C CO2 incubator for incubation for 30 min. The tube was then centrifuged at 1000 r / min in a centrifuge, and the supernatant was discarded. 1 mL PBS was added and the tube was evenly mixed to obtain stained Ramos cells.

[0050] (6) Hemocytometer: Take a 600µL centrifuge tube and add 180µL PBS and 20µL The Ramos cells stained in step (5) are blown and evenly distributed; take a blood cell counting plate, cover the counting area with a cover glass, use a pipette to absorb a small amount of stained Ramos cells in the centrifuge tube, and pick a small drop from the grooves on both sides of the middle platform of the counting plate along the lower edge of the cover glass, so that the Ramos cell liquid can fill the counting area by the surface tension of the liquid, and do not allow bubbles to be generated, and use absorbent paper to absorb the excess cell suspension flowing out of the groove; let it stand for a while to allow the cells to settle on the counting plate and no longer drift with the liquid, place the blood cell counting plate on the stage of the microscope and clamp it firmly, first find the counting area under the low-power microscope, and then switch to the high-power microscope for observation and counting; when counting, count the number of cells in the four large squares (each large square consists of 16 small squares) in the upper left, lower left, upper right, and lower right diagonally. The number of cells in 1 mL of stained Ramos cells in step (5) = n÷4×10 5 (n = total number of cells in 4 large squares), and then the stained Ramos cells were diluted to 2 × 10 6 ;

[0051] (7) Different concentrations of Fe304@TDO5 in step (4) and Ramos cells (2×10 6 ) were placed in 5 wells of a 96-well plate in the following manner, pipetted evenly, then incubated for 30 minutes, magnetically separated and the supernatant discarded, and washed three times with PBS, and finally resuspended with 50 µL PBS, pipetted evenly, and photographed on a confocal microscope:

[0052] Control: 100 µL Ramos (concentration 2×10 6 )+50µL Fe304(1mg / ml)

[0053] 1. 100µL Ramos (concentration 2×10 6 )+50µLFe304@TDO5(0.1mg / ml)

[0054] 2. 100µL Ramos (concentration 2×10 6 )+50µLFe304@TDO5(0.5mg / ml)

[0055] 3. 100µL Ramos (concentration 2×10 6 )+50µLFe304@TDO5(1mg / ml)

[0056] 4. 100µL Ramos (concentration 2×10 6 )+50µLFe304@TDO5(3mg / ml)

[0057] Figure 3 Confocal images of the Hoechst33342 staining channel showing changes in the number of Ramos cells captured by Fe304 nanoparticles and different concentrations of Fe304@TDO5 (a is a confocal image of the number of Ramos cells captured by Fe304 nanoparticles; b is a confocal image of the number of Ramos cells captured by 0.1 mg / ml Fe304@TDO5; c is a confocal image of the number of Ramos cells captured by 0.5 mg / ml Fe304@TDO5; d is a confocal image of the number of Ramos cells captured by 1 mg / ml Fe304@TDO5; e is a confocal image of the number of Ramos cells captured by 3 mg / ml Fe304@TDO5). Figure 3 It can be seen that the number of Ramos cells captured by Fe304 nanoparticles is basically zero, and the number of Ramos cells captured by Fe304@TDO5 gradually increases with the increase of concentration;

[0058] (8) Using the confocal images taken in step (7), capture the confocal images of the Hoechst33342 staining channel of the number of Ramos cells with different concentrations of Fe304@TDO5. Randomly take three confocal images for each group, count the number of cells in each confocal image, and take the average of the three groups of data. Repeat this three times to make a bar graph;

[0059] (9) Using the blood cell counting method used in step (6), the number of Ramos cells captured by Fe304@TDO5 at different concentrations was counted. Each group was repeated three times, and the average value of the three groups of data was taken. Repeat the process three times to make a bar graph.

[0060] Figure 4 To capture the Ramos cell number changes (Cell Number) bar graph according to different concentrations of Fe304@TDO5 (a is a bar graph made based on the confocal image of the Hoechst33342 staining channel; b is a bar graph made based on the cell count by hemocytometer), from Figure 4 It can be seen that the number of Ramos cells captured by Fe304@ TD05 gradually increases with the increase of concentration;

[0061] (10) Preparation of Fe304@AS-TDO5: AS nucleic acid chain (50 μL, 20 μM) and TDO5 (50 μL, 20 μM) aptamer chain were mixed and shaken, and placed in a 95°C water bath. After 5 min, the water bath was turned off and taken out after the water bath temperature dropped to room temperature to obtain AS-TDO5 (10 μM). In a 0.6 mL centrifuge tube, 10 μL of AS-TDO5 (10 μM) was added to 20 μL of Fe304 (10 mg / mL) nanoparticles, and 10 μL of incubation buffer was added. The mixture was shaken and incubated for 1 h, followed by magnetic separation. After rinsing with washing buffer three times, the mixture was resuspended in 20 μL of PBS to obtain Fe304@AS-TDO5 (10 mg / mL).

[0062] (11) Use the Ramos cells stained in step (5) and dilute them to 1×10 6 / mL; add 100 μL of stained Ramos cells (1×10 6 ) and 20μL Fe304 (10 mg / mL) were pipetted evenly, then incubated for 30 min, magnetic separation was performed, supernatant was discarded, and the cells were washed three times with PBS, and finally resuspended with 50μL PBS and pipetted evenly for confocal microscopy; 100μL stained Ramos cells (1×10 6 ) and 20µL of Fe304@ AS-TDO5 (10 mg / mL) were pipetted evenly, then incubated for 30 minutes, magnetic separation was performed, the supernatant was discarded, and the cells were washed three times with PBS. Finally, the cells were resuspended with 50µL PBS, pipetted evenly, and confocal microscopy was performed. Then, the supernatant was aspirated by magnetic separation, 100µL exonuclease III (4U / µL) was added, and the cells were incubated at 37℃ for 30 minutes. Then, the supernatant was discarded by magnetic separation, and the cells were washed three times with PBS. Finally, the cells were resuspended with 50µL PBS, pipetted evenly, and confocal microscopy was performed.

[0063] Figure 5 Confocal images of the Hoechst33342 staining channel for Fe304 nanoparticles and Fe304@AS-TDO5 capturing the changes in the number of Ramos cells, respectively (a is the confocal image before Fe304 nanoparticles were incubated and rinsed with Ramos cells; b is the confocal image after Fe304 nanoparticles were incubated and rinsed with Ramos cells; c is the confocal image before Fe304@AS-TDO5 were incubated and rinsed with Ramos cells; d is the confocal image after Fe304@AS-TDO5 were incubated and rinsed with Ramos cells; e is the confocal image after Fe304@AS-TDO5 was incubated and rinsed with Ramos cells and exonuclease III was added). Figure 5It can be seen that after washing, the number of Ramos cells captured by Fe304 nanoparticles is almost zero, and Fe304@AS-TDO5 can capture a large number of Ramos cells. After incubation with exonuclease III, since exonuclease III cuts the AS-TDO5 hybrid chain to separate Fe304 nanoparticles from Ramos cells, the number of Ramos cells captured by Fe304 nanoparticles after incubation and washing with exonuclease III is almost zero.

[0064] (12) Preparation of TAE buffer solution: Weigh 48 g of Tris-acetic acid, 18 g of magnesium acetate and 3 g of EDTA into a 1 L beaker, dilute to 1 L, stir evenly and adjust the pH to 8.0 to prepare a TAE buffer solution;

[0065] (13) Preparation of agarose gel: Weigh 4 g of agar powder and place it in a 100 mL beaker. Add 100 mL of Tris-boric acid and heat it in a microwave oven for 3 min. Take it out and shake it continuously to eliminate bubbles. Pour it into a mold to prepare agarose gel.

[0066] (14) Take four 600 µL centrifuge tubes and add the samples as follows. Shake well and place in a 95°C water bath. After 5 minutes, turn off the water bath and wait until the water bath temperature drops to room temperature before taking out:

[0067] 1. AS (20µM) 10µL + TAE buffer solution 2µL + ultrapure water 8µL;

[0068] 2. TDO5 (20µM) 10µL + TAE buffer solution 2µL + ultrapure water 8µL;

[0069] 3. AS (20µM) 2µL + TDO5 (20µM) 2µL + TAE buffer solution 2µL + ultrapure water 14µL;

[0070] 4. AS (20µM) 2µL + TDO5 (20µM) 2µL + TAE buffer solution 2µL + ultrapure water 14µL;

[0071] (15) Take 10µL of sample No. 4, add 2µL of exonuclease III and 2µL of exonuclease III incubation buffer, and incubate at 37℃ water bath for 30min to obtain the AS-TDO5 hybrid chain after enzyme digestion; take 10µL of sample No. 4 incubated with exonuclease III, respectively, and place them in four new 600µL centrifuge tubes, labeled as 1', 2', 3' and 4', add 2µL of SG-I to each of the four new centrifuge tubes, incubate at 37℃ water bath for 20min, and then add 6µL of Loading Buffer; use a pipette to take 10µL of the prepared 1', 2', 3' and 4' samples, respectively, and place them in the four wells of the agarose gel, and then place the agarose gel with samples in a bath at 100V for 45min;

[0072] Figure 6 Agarose electrophoresis diagram of AS, TDO5, AS-TDO5 hybrid chain and AS-TDO5 hybrid chain after enzyme digestion (band 1 is AS nucleic acid chain; band 2 is TDO5 aptamer chain; band 3 is AS-TDO5 hybrid chain; band 4 is AS-TDO5 hybrid chain after enzyme digestion). Figure 6 It can be seen that AS has fewer bases, smaller molecular weight and moves farther, while AS-TDO5 hybrid chain has larger molecular weight and moves closer. After enzyme digestion, the AS-TDO5 hybrid chain is decomposed by exonuclease tri-decomposition, so the band color is lighter, the molecular weight is smaller than the AS-TDO5 hybrid chain and moves farther.

[0073] Example 2

[0074] A method for multi-target detection based on magnetic-assisted cell counting technology comprises the following steps:

[0075] The Fe304 and cell conjugate sensor prepared in Example 1 was used and diluted ten times by adding PBS; Preparation of each group of enzyme or protein samples:

[0076] 1.EXO-III: Add 70µL PBS + EXO-III mixed solution: 5µL EXO-III (20U / µL) + 10µL EXO-III Buffer 10X + 15µL PBS (final concentration 1U / µL)

[0077] 2.EXO-I: Add 70µL PBS + EXO-I mixed solution: 5µL EXO-I (20000U / mL, i.e. 20U / µL) + 10µL EXO-I Buffer 10X + 15µL PBS (final concentration 1U / µL)

[0078] 3. BSA: Add 70µL PBS + BSA mixed solution: 10µL BSA (10mg / ml) + 20µL PBS (final 1mg / ml)

[0079] 4.T4-PNK: Add 70µL PBS + T4-PNK mixed solution: 10µL T4-PNK (10000U / mL, i.e. 10U / µL) + 10µL Buffer 10X + 10µL PBS (final concentration 1U / µL)

[0080] 5. Klenow: Add 70µL PBS + Klenow mixed solution: 20µL DNA polymerase 1 (Klenow) (5000U / mL, i.e. 5U / µL) + 10µL NE Buffer (final concentration 1U / µL)

[0081] 6. Add 100 µL of PBS

[0082] Add the prepared 6 groups of enzyme or protein samples to each of the 6 wells of a 96-well plate, and then add 50µL of the conjugate sensor of Fe304 and cells, and blow evenly; then perform magnetic separation and discard the supernatant, place the six groups of samples into 6 wells of a 96-well plate, blow evenly, incubate at 37°C for 30 minutes, and then perform magnetic separation, wash three times with PBS, and finally resuspend with 50µL PBS, blow evenly, and take confocal images. For each group of data, take three random confocal images, count the number of cells in each confocal image, take the average, and repeat three times to make a bar graph.

[0083] Figure 7 The cell number bar graph is a bar graph of the changes in the number of cells captured by Fe304 nanoparticles after incubation with different proteins or enzymes according to the confocal images. Figure 7 It can be seen that when the Fe304 and cell conjugate sensor is incubated with exonuclease III, exonuclease III will cut the nucleic acid aptamer chain, causing the Fe304 nanoparticles to separate from the cells; when incubated with other enzymes or proteins, it has no effect on the nucleic acid aptamer chain, and the binding force between the Fe304 nanoparticles and the cells is still very strong.

[0084] Example 3

[0085] A method for multi-target detection based on magnetic-assisted cell counting technology comprises the following steps:

[0086] The Fe304 and cell conjugate sensor prepared in Example 1 was used and diluted ten times by adding PBS. 100 μL of the Fe304 and cell conjugate sensor was added to each of the 10 wells of a 96-well plate. After magnetic separation and supernatant aspiration of the obtained ten groups of Fe304 and cell conjugate sensors, different concentrations of exonuclease 3 were added respectively:

[0087] 1. Add 80µL PBS + 20µL exonuclease mixed solution (20U / µL) (final concentration 4U / µL)

[0088] 2. Add 80µL PBS + 20µL exonuclease mixed solution (15U / µL) (final concentration 3U / µL)

[0089] 3. Add 80µL PBS + 20µL exonuclease mixed solution (10U / µL) (final concentration 2U / µL)

[0090] 4. Add PBS 80µL + exonuclease mixed solution 20µL (5U / µL) (final concentration 1U / µL)

[0091] 5. Add 80µL PBS + 20µL exonuclease mixed solution (2.5U / µL) (final concentration 0.5U / µL)

[0092] 6. Add PBS 80µL + exonuclease mixed solution 20µL (1.25U / µL) (final concentration 0.25U / µL)

[0093] 7. Add PBS 80µL + exonuclease mixed solution 20µL (0.5U / µL) (final concentration 0.1U / µL)

[0094] 8. Add PBS 80µL + exonuclease mixed solution 20µL (0.3U / µL) (final concentration 0.06U / µL)

[0095] 9. Add 80µL PBS + 20µL exonuclease mixed solution (0.1U / µL) (final concentration 0.02U / µL)

[0096] 10. Add PBS 80µL + exonuclease mixed solution 20µL (0U / µL) (final concentration 0U / µL)

[0097] After being evenly blown and incubated at 37℃ for 30min, the cells were magnetically separated and washed three times with PBS. Finally, they were resuspended in 50µL PBS and blown evenly and confocal images were taken. Three confocal images were randomly taken for each group of data, and the number of cells in each confocal image was counted and the average value was taken. This was repeated three times to make a bar graph.

[0098] Figure 8 The number of cells captured by Fe304 nanoparticles and the change curve and linear graph of the three exonuclease concentrations according to the confocal image count (a is the change curve of the number of cells captured by Fe304 nanoparticles and the change curve of the three exonuclease concentrations; b is the linear graph of the number of cells captured by Fe304 nanoparticles and the change curve of the three exonuclease concentrations), by Figure 8 It can be seen that the number of cells captured by Fe304 nanoparticles decreases as the concentration of exonuclease III increases, and the number of cells captured by Fe304 nanoparticles is linearly related to the concentration of exonuclease III.

[0099] Example 4

[0100] A method for multi-target detection based on magnetic-assisted cell counting technology comprises the following steps:

[0101] The Fe304 and cell conjugate sensor prepared in Example 1 was used and diluted ten times by adding PBS; 100 µL of Fe304 and cell conjugate sensor was added to each of two wells of a 96-well plate for magnetic separation. After aspirating the supernatant, exonucleases of different substrates were added respectively, and the mixture was incubated at 37°C for 30 min, followed by magnetic separation, and washed three times with PBS. Finally, the mixture was resuspended with 50 µL PBS, and the mixture was evenly blown and photographed by confocal microscopy:

[0102] 1. Add 80µL PBS + Exonuclease III mixed solution: 10µL Exonuclease III (20U / µL) + 10µL Exonuclease III Buffer (final concentration 2U / µL);

[0103] 2. Add 80µL of fetal bovine serum (40µL ultrapure water + 40µL serum) + Exonuclease III mixed solution: 10µL Exonuclease III (20U / µL) + 10µL Exonuclease III Buffer (final concentration 2U / µL).

[0104] Fig. 9 The conjugate sensor of Fe304 and cells is counted according to the confocal image in the embodiment. After the sensor and exonuclease 3 are incubated in fetal bovine serum and PBS, magnetic separation and washing are performed, and the cell number (Cell Number) captured by Fe304 nanoparticles is bar graphed. Fig. 9 It can be seen that the number of cells captured by Fe304 nanoparticles after exonuclease III incubation when fetal bovine serum was used as a substrate was less than that when PBS was used as a substrate, indicating that the exonuclease III digestion efficiency was higher in fetal bovine serum.

[0105] The Fe304 and cell conjugate sensor of the present invention can also be used for the detection of various biological molecules. Through the specific recognition effect of the aptamer, the functionalized Fe304 nanoparticles are connected to the surface of tumor cells, and the Fe304 and cell conjugate sensor is obtained after magnetic separation and washing. In the presence of a target, the Fe304 nanoparticles can be separated from the cells, and the number of cells captured by the Fe304 nanoparticles can be observed after magnetic separation, thereby distinguishing whether the biological molecules to be detected exist in the sample.

Claims

1. A method for multi-target detection based on magnetic-assisted cell counting technology, characterized in that: The following steps are involved: 1) Preparation of aptamer nucleic acid hybrid chain AS-TDO5; 2) incubating the AS-TDO5 obtained in step 1) with Fe304 nanoparticles modified with streptavidin, and preparing Fe304@AS-TDO5 by magnetic separation and washing; 3) Incubate the Fe304@AS-TDO5 obtained in step 2) with suspended cells, and prepare a Fe304-cell conjugate sensor by magnetic separation and washing; 4) incubating the Fe304-cell conjugate sensor obtained in step 3) with exonuclease III, followed by magnetic separation and washing, and distinguishing the exonuclease III and no exonuclease III groups by monitoring the number of cells captured by Fe304 nanoparticles. At the same time, the number of suspended cells captured by Fe304 nanoparticles after incubation with different concentrations of exonuclease III can be distinguished; The preparation process of the Fe304-cell conjugate sensor in step 3) is as follows: (1) Mix 45-55 µL AS nucleic acid chain and 45-55 µL TDO5 aptamer chain respectively, shake well, place in a 90-100°C water bath, turn off the water bath after 4-6 minutes, wait for the water bath temperature to drop to room temperature, and take out to obtain AS-TDO5 hybrid nucleic acid chain; (2) Add 10-50 µL of AS-TDO5 hybrid nucleic acid chain to 20-100 µL of Fe304 nanoparticles, add 10-50 µL of incubation buffer, shake well and incubate for 1-2 h, then separate by magnetic separation, and rinse three times with washing buffer to obtain Fe304@AS-TDO5; (3) Take 100-500 µL Ramos cell suspension and add 10-50 µL Fe304@ AS-TDO5, blow evenly, place in a refrigerator at 1-5°C for incubation for 30-60 min, then perform magnetic separation and discard the supernatant. Wash three times with PBS to obtain the Fe304-cell conjugate sensor.

2. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The Fe304-cell conjugate sensor described in step 3) is obtained by the following method: (1) The AS nucleic acid chain and the TDO5 aptamer chain were diluted to the same concentration with PBS. The AS nucleic acid chain and the TDO5 aptamer chain formed an AS-TDO5 hybrid nucleic acid chain through base complementary pairing; (2) AS-TDO5 was modified onto the surface of magnetic nanoparticles through the affinity of streptavidin and biotin, and then Ramos cells were captured through the TDO5 aptamer to form a conjugate sensor of Fe304 and cells to detect exonuclease III.

3. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The concentrations of the AS nucleic acid chain and the TDO5 aptamer chain were both 20 µM.

4. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The concentration of AS-TDO5 was 10 µM.

5. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The concentration of the Fe304 nanoparticles is 10 mg / mL.

6. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The diameter of the Fe304 nanoparticles is 180-200 nm.

7. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The concentration of the Ramos cells was 1×10 6 / mL.

8. The method for multi-target detection based on magnetic-assisted cell counting technology according to claim 1, characterized in that: The step 4) described in the method of monitoring the change in the number of cells captured by Fe304 nanoparticles is as follows: If the number of cells captured by Fe304 nanoparticles is small, exonuclease III is present, and the number of captured cells is inversely proportional to the concentration of exonuclease III; If the number of cells captured by Fe304 nanoparticles does not change much, exonuclease III does not exist.

Images