A multi-channel paper chip device and its application in detection of small extracellular vesicle membrane proteins

Abstract

The application belongs to the technical field of biomedical engineering, and particularly relates to a multi-channel paper chip device and application thereof in detection of small extracellular vesicle membrane proteins. The core component of the multi-channel paper chip device disclosed by the application is a paper chip co-modified by a nucleic acid aptamer and a metal organic framework (MOF). The device is to grow the MOF material on the paper chip in situ to provide an amino modification site of the small extracellular vesicle membrane protein aptamer, reduce non-specific adsorption of the paper to impurity proteins, and serve as a reference for ratio type detection. Then, the aptamer is modified on the MOF to capture small extracellular vesicles expressing corresponding proteins through immunological affinity. Meanwhile, a detection probe DP composed of a small extracellular vesicle membrane protein CD63 aptamer and a G-quadruplex is developed to detect the content of the captured small extracellular vesicles by colorimetry. The device can be used to detect the small extracellular vesicle membrane proteins in multiple channels, and realize precise diagnosis and typing of cancers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, and particularly relates to a multi-channel paper chip device and application thereof in detection of small extracellular vesicle membrane proteins. BACKGROUND

[0002] Hepatocellular carcinoma is one of the most common malignant tumors in the world, and its incidence and mortality have been high for many years. In recent years, with the development of science and technology, the understanding of hepatocellular carcinoma has been gradually deepened, but the mortality rate of hepatocellular carcinoma patients remains high, and it is estimated that more than 1 million people will be affected by hepatocellular carcinoma every year by 2025. The reason is that cancer is usually discovered in the middle and late stages, and the treatment effect is usually not ideal. At present, the focus and difficulty of hepatocellular carcinoma research is early diagnosis. Among them, B-ultrasound examination and serum alpha-fetoprotein (AFP) determination are the main methods for diagnosing hepatocellular carcinoma at present. However, these detection methods often cannot timely detect early symptoms of hepatocellular carcinoma, and can only detect symptoms after morphological changes. Therefore, it is urgent to explore and develop new biomarkers to provide new ideas for improving the diagnosis and treatment of hepatocellular carcinoma.

[0003] Biomarkers are indicators that can objectively measure and evaluate the physiological or pathological processes of organs, tissues, cells, and even organelles. They are widely present in body fluids such as blood and tissue fluid, and the abnormal expression level is usually closely related to the occurrence and development of cancer. Among them, small extracellular vesicles (sEVs) have become the most potential biomarkers for early diagnosis and treatment monitoring of cancer due to their high abundance, universal presence and excellent stability in various body fluids. Therefore, developing excellent biomarker detection technology based on small extracellular vesicles has important significance for the diagnosis and treatment of cancer, reducing the mortality rate of patients and carrying out basic research related to cancer.

[0004] Small extracellular vesicles are a new biomarker for cancer diagnosis, which can reveal the phenotype information of organisms in depth, so real-time detection of small extracellular vesicles has important significance for large-scale screening of cancer. However, the current separation method of small extracellular vesicles and the analysis method of small extracellular vesicle surface membrane proteins are still limited by complicated operation and expensive large instruments, making it difficult to popularize the use. Therefore, it is urgent to develop a low-cost, efficient and simple-to-operate small extracellular vesicle detection method. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a multi-channel paper chip device and a small extracellular vesicle membrane protein detection method, which can determine the protein expression profile of small extracellular vesicle membranes in various cancer cell culture liquids and serum of cancer patients at a low cost, and realize accurate diagnosis and typing of cancer.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0007] The first aspect of the present application provides a multi-channel paper chip device (Exopp-PAD), the core component of the device is a plurality of aptamer-modified paper chips C@MOF@Paper, the C@MOF@Paper includes MOF@Paper and nucleic acid aptamer, the MOF@Paper is a MOF@Paper on which UiO-66-NH2 is grown in situ on filter paper, and the nucleic acid aptamer is used for specifically recognizing small extracellular vesicle membrane proteins.

[0008] Preferably, the preparation method of the device is as follows: a plurality of holes are formed on a resistant plate, different C@MOF@Paper are respectively filled into each hole as test points, MOF@Paper without aptamer modification is filled as a reference point, a filter membrane is used to cover the resistant plate, then the resistant plate is loaded into a replaceable membrane filter, and then the filter is connected to a syringe at both ends, thereby obtaining the multi-channel paper chip device.

[0009] Preferably, the nucleic acid aptamer includes an EpCAM aptamer shown in SEQ ID No: 2, a PTK7 aptamer shown in SEQ ID No: 3, a CEA aptamer shown in SEQ ID No: 4, and a PD-L1 aptamer shown in SEQ ID No: 5.

[0010] Preferably, the preparation method of the MOF@Paper includes the following steps:

[0011] S1, dissolving ZrCl4 in a mixed solution of formic acid and ethanol, placing filter paper in the obtained mixed solution, and standing to make the paper sheet surface fully adsorb Zr 4+ ions,

[0012] S2, dissolving BDC-NH2 in a mixed solution of formic acid, ethanol and water, adding the BDC-NH2 solution to the solution of step S1, standing at room temperature to generate MOF nanoparticles on the paper sheet surface, and then washing and drying to obtain the MOF@Paper.

[0013] More preferably, the filter paper is Whatman No. 4 filter paper.

[0014] More preferably, in the mixed solution of formic acid and ethanol, the volume ratio of formic acid to ethanol is 6-9:20; and in the mixed solution of formic acid, ethanol and water, the volume ratio of formic acid, ethanol and water is 6-9:20:7-10.

[0015] The second aspect of the present application provides a kit for detecting small extracellular vesicle membrane proteins, the kit including the multi-channel paper chip device of the first aspect and a detection probe DP, the detection probe DP having a nucleotide sequence as shown in SEQ ID No: 1.

[0016] The application grows a MOF material with an amino group in situ on filter paper, and further modifies a series of aptamers capable of specifically recognizing small extracellular vesicle membrane proteins on the surface of the MOF to obtain a C@MOF@Paper paper chip, which can specifically capture small extracellular vesicles, improve the anti-protein non-specific adsorption performance of the filter paper through the special surface properties of the MOF material, and can use the color of the MOF itself as a reference for subsequent ratio-type detection. Meanwhile, a DP nucleic acid probe is prepared by using a G-quadruplex sequence and a CD63 aptamer sequence, based on the affinity reaction of the aptamer and the specific binding of the small extracellular vesicle membrane protein CD63, the structure of the DP is changed, thereby releasing the G-quadruplex catalytic color development activity. Then it is combined with Hemin and exhibits peroxidase catalytic activity to catalyze the color development of TMB, obtaining a colorimetric detection signal of small extracellular vesicles, thereby realizing the specific recognition and detection of small extracellular vesicle membrane proteins. In addition, by integrating paper chips targeting multiple membrane proteins into a multi-channel chip, and by using a needle cylinder for capture, washing and detection, etc., the determination of the expression profile of small extracellular vesicle membrane proteins can be realized simply and inexpensively.

[0017] Preferably, the method for using the kit is as follows: using a syringe at one end of the multi-channel paper chip device to suck the body fluid sample to be tested, slowly pushing while pulling the syringe at the other end, so that the liquid flows completely into the syringe at the other end, and so on to make the sample fully bind to the device, then replacing the body fluid sample to be tested with a washing liquid, a DP detection liquid and a Hemin solution in turn, and operating in the same way, finally taking out the multi-channel paper chip device, adding TMB single-component color developing liquid at each test point and reference point, observing the color development result and taking a photo for analyzing the Hue value for quantitative analysis.

[0018] More preferably, the body fluid sample to be tested is a serum sample.

[0019] More preferably, the method for quantitative analysis is as follows: using the self-calibration characteristic that the color of the reference point remains unchanged to construct a ratio-type Hue value detection signal: R-ΔHue = ΔHue sample / ΔHue reference, ΔHue = Hue after reaction - blank Hue, and then making R-ΔHue and the logarithm of the concentration of small extracellular vesicles into a standard curve.

[0020] More preferably, the endurance plate is a PC endurance plate.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application discloses a multi-channel small extracellular vesicle membrane protein detection method and a matching paper chip device (Exopp-PAD), which realizes cancer cell typing through detection of the expression profile of small extracellular vesicle membrane proteins in a body fluid sample. The core component of the device is a paper chip co-modified by aptamers and metal organic frameworks (MOF). The preparation method of the device is as follows: first, the MOF material is grown in situ on the paper chip to provide an amino modification site for the small extracellular vesicle membrane protein aptamer, thereby reducing the non-specific adsorption of the paper to impurity proteins and serving as a reference for ratio type detection. Then, the aptamer is modified on the MOF to capture small extracellular vesicles expressing corresponding proteins through immunological affinity. Meanwhile, a detection probe (DP) composed of a small extracellular vesicle membrane protein CD63 aptamer and a G-quadruplex is developed for colorimetric detection of the content of the captured small extracellular vesicles. In addition, the paper chips for different membrane protein targets are assembled into a multi-channel capture chip, and the capture, washing and detection processes of the small extracellular vesicles are completed by the action of the paper chip with the sample, the washing liquid and the DP detection liquid controlled by a needle cylinder. As can be seen, the device can be used to determine the protein expression profile of the small extracellular vesicle membrane in a plurality of cancer cell culture liquids and the serum of cancer patients at a low cost, and the device is expected to realize precise diagnosis and typing of cancer.

[0023] Specifically, the application has the following advantages: (1) the aptamer-based paper chip and detection probe are low in cost and stable in performance, and can improve the performance-price ratio and stability of detection and diagnosis; (2) the metal organic framework modification can improve the capture efficiency and purity of the paper chip and improve the detection performance; (3) the developed paper chip device does not require expensive and complex equipment and professional skills, and can detect small extracellular vesicle membrane proteins in multiple channels, thereby realizing precise typing diagnosis of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Fig. 1 is a schematic diagram of the preparation of the paper chip, wherein (a) is a small extracellular vesicle separation and analysis device; (b) is a schematic diagram of the preparation of the paper chip; (c) is a schematic diagram of DP detection; (d) is a schematic diagram of the principle of the Exopp-PAD used for small extracellular vesicle capture and membrane protein detection.

[0025] Figure 2 Fig. 2 is a SEM image of (a, b) an unmodified paper chip (Paper) and (c, d) a MOF-modified paper chip (MOF@Paper);

[0026] Figure 3 Fig. 3 is a PXRD spectrum of (a) MOF@Paper, Paper, MOF powder and standard UiO-66-NH2; (b) ATR-FTIR spectrum of MOF@Paper, Paper and MOF powder;

[0027] Figure 4(a) SEM images of MOF@Paper and (b) EDS elemental mapping of MOF@Paper;

[0028] Figure 5 (a) Photos of unmodified paper chip and MOF@Paper before and after adsorbing BSA and reacting with CBB; (b) Hue values and (c) AHue values of each sample in (a);

[0029] Figure 6 (a) PXRD spectra of aptamer-modified MOF@Paper (C0@MOF@Paper) and MOF@Paper without aptamer modification; (b) ATR-FTIR spectra of C0@MOF@Paper and MOF@Paper;

[0030] Figure 7 (a) Photos of MOF@Paper without EDC / NHS activation and MOF@Paper with EDC / NHS activation before and after fixing G-quadruplex sequence and developing with TMB; (b) Columnar chart of Hue values of each sample in (a);

[0031] Figure 8 SEM images of C1@MOF@Paper capturing small extracellular vesicles;

[0032] Figure 9 Circular dichroism spectra of DP reacting with CD63 protein, small extracellular vesicles and blank control;

[0033] Figure 10 Fluorescence spectra of DP reacting with CD63 protein, small extracellular vesicles and blank control;

[0034] Figure 11 UV-Vis absorption spectra of DP reacting with CD63 protein, small extracellular vesicles and blank control;

[0035] Figure 12 (a) Optimization of hemin concentration; (b) Optimization of incubation time of hemin and G4;

[0036] Figure 13 Relationship between small extracellular vesicle concentration and absorbance;

[0037] Figure 14 (a) Color development results of DP binding with small extracellular vesicles of different concentrations on C@MOF@Paper; (b) Relationship chart of AHue values and small extracellular vesicle concentrations;

[0038] Figure 15(a) Schematic diagram of the assembly of Exopp-PAD; (b) Color development results of different kinds of small extracellular vesicles after binding with DP in Exopp-PAD;

[0039] Figure 16 (a) Color development results of different concentrations of small extracellular vesicles after binding with DP in Exopp-PAD and R-ΔHue, and (b) relationship diagram of different concentrations of HepG2 (i), HuH-7 (ii), and LO2 (iii) small extracellular vesicles;

[0040] Figure 17 R-ΔHue values of each target protein on HepG2 / HuH-7 / LO2 small extracellular vesicles;

[0041] Figure 18 (a) Photos of actual samples after analysis using Exopp-PAD; (b) Heat map of protein expression in actual samples. DETAILED DESCRIPTION

[0042] The specific embodiments of the present application are further described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0043] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0044] The present application provides a multi-channel paper chip device and its application in small extracellular vesicle membrane protein detection, including the following contents:

[0045] 1. Construction of small extracellular vesicle separation and enrichment and analysis device

[0046] Amino-modified MOF material is grown in situ on filter paper, and a series of aptamers that can specifically recognize small extracellular vesicle membrane proteins are further modified on the surface of the MOF to obtain C@MOF@Paper (C represents nucleic acid aptamer), which has the function of specifically capturing small extracellular vesicles. Then the obtained series of composite paper chips are assembled into Exopp-PAD, and then two pieces of polycarbonate (PC) filter membrane with a pore size of 200 nm are assembled into a replaceable membrane filter, and then the filter is connected with a syringe, thereby constructing a small extracellular vesicle separation and enrichment and analysis device, as shown in Figure 1 a, b.

[0047] 2. Design of detection probe DP and verification analysis of its combination with small extracellular vesicles

[0048] (1) As shown in Figure 1 c, a hairpin type detection probe DP is designed, which is connected by a G-quadruplex sequence and a CD63 aptamer sequence, which is used for specific binding with CD63 protein highly expressed on the surface of small extracellular vesicles. After binding, the structure of the probe changes and releases the G-quadruplex sequence, and then the content of each protein in the Exopp-PAD captured small extracellular vesicles can be reflected by detecting the content of G-quadruplex.

[0049] (2) By using the characteristics that G-quadruplex emits fluorescence under specific wavelength excitation after binding with ThT and exhibits peroxidase catalytic activity after forming a complex with Hemin, the existence of G-quadruplex is verified by circular dichroism spectrum, fluorescence spectrum and ultraviolet-visible spectrum, and the combination of DP with CD63 and small extracellular vesicles is tested.

[0050] (3) By using the peroxidase catalytic activity of G-quadruplex sequence after binding with Hemin, different concentration gradients of Hemin and different reaction temperature gradients are set to optimize the combination conditions of G-quadruplex sequence and Hemin.

[0051] (4) Linear analysis of color development results of detection probe DP after binding with small extracellular vesicles is carried out on 96-well plates and C@MOF@Paper respectively, different concentrations of small extracellular vesicle suspensions are added, and then detection probe DP, Hemin and TMB are added in turn. For the 96-well plate, the standard curve is drawn after detecting the absorbance at 595 nm by the enzyme-labeled instrument. For C@MOF@Paper, the Hue value is recorded by taking a picture and reading, and the standard curve of Hue value and small extracellular vesicle concentration is drawn.

[0052] 3. Multi-channel detection of small extracellular vesicle proteins based on Exopp-PAD

[0053] The color development results of HepG2, HuH-7 and L02 small extracellular vesicles with known concentrations are tested by using Exopp-PAD and detection probe DP, and the Hue values are analyzed by taking pictures and using ImageJ for quantitative analysis. The specific process is shown in Figure 1 d.

[0054] In order to further clearly present the process of constructing a multi-channel paper chip device and detecting small extracellular vesicle membrane proteins, the following embodiments are described in detail.

[0055] 1. Preparation and characterization of MOF@Paper

[0056] (1) In-situ growth of MOF on paper chip

[0057] Whatman 4 filter paper was cut into 6mm diameter round paper pieces by puncher for later use. 0.5mmol of ZrCl4 was dissolved in 0.7mL formic acid and 2.0mL ethanol, and after ultrasonic treatment for 1h, the paper pieces were added to the ZrCl4 solution and left to stand for 2h to allow the paper pieces to fully adsorb Zr 4+ ions on the surface. On the other hand, 0.2mmol of BDC-NH2 was dissolved in 0.7mL formic acid, 2.0mL ethanol and 0.8mL pure water, and ultrasonic treatment was carried out for 2h. After ultrasonic treatment, the BDC-NH2 solution was added to the ZrCl4 solution containing the paper pieces, and after mixing gently, the reaction was left to stand at room temperature for 8h to generate MOF nanoparticles on the surface of the paper pieces. After that, the MOF@Paper was washed with anhydrous ethanol and water for 3 times respectively, and dried at 37℃ for 10min to obtain the UiO-66-NH2 functionalized paper pieces, which were finally stored in a sealed bag for later use.

[0058] (2) SEM and energy spectrum characterization of paper pieces

[0059] The MOF modified paper pieces (Paper) and MOF modified paper pieces (MOF@Paper) were adhered to the sample table with conductive adhesive, vacuum dried in a vacuum drying oven for 12h, and then gold was sprayed on the sample for 60s using a particle sputtering instrument. SEM was used to characterize the samples at an acceleration voltage of 3kV. As shown in Figure 2 , the SEM results show that the paper pieces before and after growing MOF both show an interlaced porous fiber structure, as shown in Figure 2 a and 2c, that is, the growth of MOF does not change the porous three-dimensional structure of the paper itself. Compared with the smooth fibers of the paper itself ( Figure 2 b), the MOF@paper forms uniform nanoparticles on the surface of the fibers ( Figure 2 d). Through the characterization of SEM, it can be known that after the modification of UiO-66-NH2, the MOF@Paper still retains the original porous structure and filtering function of the paper, so that the sample liquid can pass through the paper pieces, ensuring the ability of the aptamer to fully contact and act with the subsequent modification of the paper pieces.

[0060] (3) PXRD characterization and ATR-FTIR characterization of paper pieces

[0061] In order to verify that the nanoparticles generated on the surface of the paper are the target MOF material UiO-66-NH2, Paper, MOF@Paper and pure MOF powder were characterized by X-ray powder diffraction, respectively. The experimental parameters were as follows: scanning speed 6° per minute, scanning range 5° to 40°, step size 0.02°. The results are shown in Figure 3As shown in Figure a, MOF@Paper exhibits characteristic PXRD peaks of the Paper crystal plane and the UiO-66-NH2(111) and (002) at 2θ = 7.4° and 8.5°, indicating that the structure generated in the synthesized MOF@Paper is UiO-66-NH2.

[0062] Meanwhile, ATR-FTIR was used to perform infrared measurements on Paper, MOF@Paper, and the synthesized MOF powder, with the following experimental parameters: scan range 500 cm⁻¹. -1 Up to 2500cm -1 Step length 1cm -1 Scanned 16 times. (For example...) Figure 3 As shown in b, MOF@Paper exhibits the characteristic absorption peak of UiO-66-NH2 at 768 cm⁻¹. -1 The peak at 1374 cm⁻¹ belongs to the Zr-O stretching vibration. -1 and 1567cm -1 The peak at this point is attributed to the stretching vibration of the carbonyl group. These results further demonstrate that UiO-66-NH2 nanoparticles were successfully grown in situ on paper fibers.

[0063] In addition, such as Figure 4 As shown, the SEM image of MOF@Paper ( Figure 4 a) and EDS element distribution map ( Figure 4 (b) The widespread distribution of Zr on its surface indicates that UiO-66-NH2 nanoparticles are uniformly and extensively grown on paper fibers. These results confirm that UiO-66-NH2 nanoparticles grow and are widely and uniformly distributed on paper fibers, providing easily modifiable amino-active sites for the paper.

[0064] (4) Investigation into the ability of paper chips to resist non-specific protein adsorption

[0065] Using BSA as a protein model molecule, Paper and MOF@Paper (6 mm diameter discs) were first incubated with BSA solution (20 μL, 10 mg / mL) for 20 minutes at room temperature and thoroughly washed. Then, Coomassie Brilliant Blue solution (10 μL, 1 μM) was added and the reaction was carried out at room temperature for 30 minutes. The BSA adsorbed on the paper was then visualized. The results are as follows: Figure 5 As shown in Figure a, Paper exhibits a significant color change, while MOF@Paper shows almost no color change. Further quantification using ImageJ software to determine the Hue value of the color change reveals that Paper's ΔHue value is 10.6 times that of MOF@Paper. Figure 5 b, Figure 5c). The above results indicate that a large amount of BSA is nonspecifically adsorbed on the surface of unmodified paper fibers, while UiO-66-NH2 modification can greatly reduce the nonspecific adsorption of proteins on paper.

[0066] 2. Paper chip modified aptamers and their characterization

[0067] The amino groups on the paper chip were coupled to aptamers with 5'-terminal carboxyl groups using EDC-NHS. Specifically, 1.0 mmol NHS and 4.0 mmol EDC were dissolved in 10 mL of water to prepare a mixed solution of 0.1 M NHS and 0.4 M EDC. The MOF@Paper was then immersed in the freshly prepared EDC-NHS solution for 10 min, followed by washing three times with anhydrous ethanol and water, and then immersed in 5 mL of 1.0 μM capture aptamers (C1-C4) and a reference (Ctrl) solution for 3 h. After washing three times with PBST, the paper chip was dried at 37 °C for 15 min to obtain the aptamer-modified paper chip (C@MOF@Paper, C1-C4 being nucleic acid aptamers of EpCAM, PTK7, CEA, and PD-L1, respectively, as shown in Table 1). Finally, it was placed in a sealed bag and stored at -20 °C for later use. The MOF@Paper and C@MOF@Paper (C1@MOF@Paper was used as the paper chip for feasibility verification) were characterized by PXRD and ATR-FTIR.

[0068] To verify the successful immobilization of the carboxyl-modified aptamer onto MOF@Paper, the 5' carboxyl-modified G-quadruplex sequence C0, possessing both enzymatic activity and covalent linkage function, was bound to MOF@Paper via EDC / NHS to obtain C0@MOF@Paper for further characterization. First, C0@MOF@Paper was characterized by PXRD and ATR-FTIR, with results as follows: Figure 6 As shown. Compared with the MOF@Paper of the unincubated aptamer, the PXRD patterns of the two are basically consistent. Figure 6 a) This demonstrates that the structure of the MOF remained unchanged after the aptamer was modified. The ATR-FTIR spectra of both ( Figure 6 b) It can be seen that C0@MOF@Paper is at 1640cm -1 A new infrared absorption peak was observed, which should be attributed to the characteristic peak of the carbonyl group in the amide bond. This is because the carboxyl group at C0 undergoes an amidation reaction with EDC / NHS, forming an amide bond. These results preliminarily demonstrate that the aptamer can be successfully immobilized on MOF@Paper under the bridging of EDC / NHS.

[0069] G-quadruplex sequence can exhibit peroxidase catalytic activity after forming complex with Hemin, and then catalyze TMB / H2O2 to develop color reaction. According to this, a probe G4 (1.0 μM, 5 mL) composed of G-quadruplex sequence and modified with carboxyl at 5' end was designed, and then incubated with MOF@Paper activated and unactivated by EDC / NHS at room temperature for 3 h. After washing, Hemin solution (4 μM, 20 μL) was added for incubation (37 °C, 30 min), and after sufficient washing, TMB developing solution (1 mM, 20 μL) was added for reaction (37 °C, 30 min), and the color change was observed and photographed Figure 7 a). The Hue value was quantified using ImageJ software Figure 7 b), and it was found that after EDC / NHS activation treatment, the final discoloration of the paper chip was obvious, while the paper chip without EDC / NHS activation treatment basically did not develop color reaction. The above results further showed that nucleic acid sequence can be covalently modified on MOF@Paper by EDC / NHS reaction, and G-quadruplex sequence modified on MOF@Paper can develop color reaction on paper.

[0070] Table 1 nucleic acid sequences used in the experiment

[0071]

[0072] 3, verification of paper chip loading and enrichment of small extracellular vesicles

[0073] Exosomes were collected from HepG2 cell culture medium by ultracentrifugation. After the cells grew to about 70% of the area of the cell culture bottle, the culture medium was replaced with DMEM medium containing 1.5% exosome-free FBS and cultured for 48 h. Then, the cell culture medium suspension was transferred to a centrifuge tube, and the cell pellet was avoided. The relative centrifugal force was 300 g at 4 °C for 10 min to remove large particles and cells. Then the supernatant was transferred to a new centrifuge tube, and the relative centrifugal force was 2000 g at 4 °C for 20 min to remove cell debris and larger particles. Subsequently, the supernatant was transferred to a new centrifuge tube, and the relative centrifugal force was 10000 g at 4 °C for 30 min to remove large vesicles and protein aggregates. The obtained supernatant was centrifuged at 120000 g at 4 °C for 90 min, and the supernatant was carefully removed with a pipette. The exosome pellet was washed with PBS and resuspended, and then centrifuged at 120000 g at 4 °C for another 90 min. Then the supernatant was carefully removed, 200 μL of PBS was added to resuspend the exosome pellet, and then it was aliquoted at 10 μL per portion and stored at -80 °C for use.

[0074] Take 10 μL of HepG2 small extracellular vesicles, drop onto C@MOF@Paper (take C1 modified paper chip as a feasibility verification paper chip), incubate at room temperature for 1 h, wash away the unbound small extracellular vesicles with PBST, dry the paper chip and then paste it on the SEM sample stage with conductive glue, vacuum dry for 12 h, and characterize by SEM. Whether C@MOF@Paper captures small extracellular vesicles is verified by SEM, taking SEM characterization of C1@MOF@Paper as an example, the results are shown in Figure 8 From which it can be seen that after binding with small extracellular vesicles, MOF on the paper fiber captures circular nanovesicles smaller than 200 nm. This result shows that the device can load and enrich small extracellular vesicles.

[0075] 4. Feasibility verification of detection probe binding to CD63 and small extracellular vesicles

[0076] (1) Circular dichroism spectrum verification

[0077] The DP solution (1 μM, 20 μL) was incubated (37°C, 30 min) with 20 μL of recombinant CD63 protein (1 μg / mL), small extracellular vesicles (Exos, 10 8 particles / mL) and PBS buffer solution (0.1 mM) respectively, and the circular dichroism spectrum was detected, and the results are shown in Figure 9 Compared with the blank control, when CD63 and small extracellular vesicles are present, there are negative peaks at 245 nm and positive peaks at 270 nm in the circular dichroism spectrum of the detection probe DP, which is because the parallel G-quadruplex spatial structure is formed, proving the feasibility of the binding of DP to CD63 and small extracellular vesicles.

[0078] (2) Fluorescence spectrum verification

[0079] After the DP was incubated with recombinant CD63 protein, small extracellular vesicles and blank control respectively, an equal amount of thioflavin T (ThT, 10 μM, 20 μL) solution was added, incubated (37°C, 30 min) and diluted with PBS to 100 μL, and then the fluorescence emission spectrum was measured under 425 nm excitation light, as shown in Figure 10 Compared with the blank control, after the binding of DP with CD63 protein and small extracellular vesicles, there is a fluorescence emission peak at 495 nm in the presence of ThT, which further proves the feasibility of the binding of DP to CD63 and small extracellular vesicles.

[0080] (3) UV-Vis absorption spectrum verification

[0081] The same amount of Hemin (Hemin solution) solution was added to the solution after DP was incubated with recombinant CD63 protein, small extracellular vesicles and blank control respectively, incubated (37°C, 30 min) and added with TMB single-component color developing solution for full reaction, and then added with HC1 solution to terminate color development, and the ultraviolet-visible absorption spectrum was measured as shown in Figure 11 Compared with the blank control, after DP was combined with CD63 protein and small extracellular vesicles, incubated with Hemin and added with TMB for color development reaction, there was a strong absorption peak at 450 nm. This further verified the feasibility of the combination of DP with CD63 and small extracellular vesicles.

[0082] 5. Optimization of experimental conditions for the combination of G-quadruplex with Hemin

[0083] G-quadruplex nucleic acid (G4, see Table 1) was dissolved and diluted to 10.0 μM with 1xTE Buffer, and Hemin powder was dissolved and diluted to 50.0 μM with PBS buffer for standby use. First, 80 μL of 10 mixtures with G4 concentration of 1.0 μM and Hemin concentration of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 μM respectively were prepared in a 96-well plate, incubated at room temperature for 30 min, and then 20 μL of TMB single-component color developing solution was added, reacted at room temperature for 20 min, and then 20 μL of 1.0 M HC1 was added to terminate the reaction. After that, the absorbance at 450 nm was measured by an enzyme marker, and the results are shown in Figure 12 a. The absorbance first increased with the increase of Hemin concentration, and when the Hemin concentration exceeded 4.0 μM, the absorbance did not change significantly, so the optimal concentration of Hemin was selected as 4.0 μM.

[0084] The Hemin concentration optimized in the previous step was used, and the incubation time of G4 with Hemin was set to 10, 20, 30, 60 and 90 min respectively. After incubation, 20 μL of TMB single-component color developing solution was added, reacted at room temperature for 20 min, and then 20 μL of 1.0 M HC1 was added to terminate the reaction. After that, the absorbance at 450 nm was measured by an enzyme marker and plotted as shown in Figure 12 b. The absorbance increased with the increase of incubation time, and after 30 min, the absorbance did not change significantly, so the optimal time for incubation of Hemin with G-quadruplex was 30 min.

[0085] 6. Linear analysis of color development results after the combination of detection probe DP with small extracellular vesicles

[0086] HepG2 small extracellular vesicles were diluted with PBS to concentrations of 0, 1, 5, 10, 50 and 100 x 10 6 -1 ​Diluent was diluted 20 μL in each well of a 96-well plate, and then incubated with 20 μL of 1.0 μM DP solution for 45 min. Next, 20 μL of 4.0 μM Hemin solution was added and incubated for 30 min. The plates were washed three times with PBST, and then 20 μL of TMB single-component chromogenic reagent was added and reacted for 20 min. The absorbance was then measured at 595 nm using a microplate reader, and a standard curve was plotted based on the concentration of small extracellular vesicles and the absorbance: Abs. = 0.0673 × lg[Exos] – 0.0383 (R 2 =0.98), such as Figure 13 As shown in the figure, it can be seen that when the DP concentration remains constant and other conditions are kept consistent, the absorbance of the solution has a good linear correlation with the logarithm of the concentration of small extracellular vesicles.

[0087] Subsequently, the linear relationship between DP and small extracellular vesicles on C@MOF@Paper was examined. Different concentrations (0, 1, 5, 10, 50, 100 × 10⁻⁶) were used. 6 pcs·mL -1 20 μL of each of the HepG2 small extracellular vesicle dilution buffers was added to C@MOF@Paper (6 mm diameter discs). After incubation for 1 h, the plates were washed three times with PBST, and then 20 μL of 1.0 μM DP solution was added and incubated for 45 min. Next, 20 μL of 4.0 μM Hemin solution was added and incubated for 30 min. After washing three times with PBST, 20 μL of LMB single-component chromogenic reagent was added and reacted for 20 min. The color change after the reaction was recorded by photography (14a). A standard curve was plotted based on Hue value and small extracellular vesicle concentration: ΔHue=8.03×lg[Exos]–7.11(R 2 =0.96), such as Figure 14 As shown in b, it can be seen that when the DP concentration remains constant and other conditions are kept consistent, the ΔHue value of the paper chip color change has a good linear correlation with the logarithm of the small extracellular vesicle concentration.

[0088] 7. Multichannel detection of small extracellular vesicle proteins based on Exopp-PAD

[0089] First, a device for enriching and analyzing small extracellular vesicles and their proteins was constructed. This device consists of three main components: a syringe, a 25mm diameter replaceable membrane filter, and a vertical small extracellular vesicle enrichment and membrane protein detection chip (Exopp-PAD). The specific preparation method is as follows: Five 6mm diameter holes were cut into a 25mm diameter, 0.2mm thick PC endurance plate. Four pre-prepared paper chips modified with four aptamers (Table 1) were filled in as four test points. Unmodified MOF@Paper chips were filled in as reference points, forming the Exopp-PAD. This was then covered with two 25mm diameter, 200nm pore size filter membranes, and finally placed into a 25mm diameter replaceable membrane filter. The two ends of the filter were then connected to the syringe. During operation, draw 2 mL of sample solution with one end of the syringe and slowly push it while simultaneously pulling with the other end. Repeat this operation until the liquid has completely flowed into the other end, ensuring thorough binding of the Exopp-PAD with the sample. Afterward, replace the sample solution with washing buffer, DP detection buffer, and Hemin solution in the same manner.

[0090] The colorimetric results of HepG2, HuH-7, and LO2 small extracellular vesicles at known concentrations were tested using Exopp-PAD and DP, respectively, to establish standard curves for EpCAM, PTK7, CEA, and PD-L1 on each type of small extracellular vesicle. The specific procedure is as follows: Different concentrations of liver / hepatocellular carcinoma-derived small extracellular vesicles (0, 5.0 × 10⁻⁶) were used. 6 –5.0×10 8 pcs·mL -1 Small extracellular vesicle serum samples were prepared by adding FBS containing 50% PBS. Figure 15 The Exopp-PAD was assembled sequentially, and then serum samples from small extracellular vesicles were introduced into the Exopp-PAD using a syringe. When the sample passed through the filter, the membrane proteins on the surface of the small extracellular vesicles specifically bound to C@MOF@Paper, causing the small extracellular vesicles to be captured at the test sites. After incubation and washing, the detection probe DP was added to the Exopp-PAD using a syringe. After sufficient reaction, Hemin was added to the Exopp-PAD using a syringe. After washing, the Exopp-PAD was removed, and TMB single-component chromogenic solution was added to each test site and reference point. The chromogenic results were observed, photographed, and the Hue values ​​were analyzed for quantitative analysis. Figure 15The color of the reference point was basically the same in different types and concentrations of small extracellular vesicles. For small extracellular vesicles of hepatoma cells HepG2 and HuH-7, the color change degree of the test point increased with the increase of the concentration of small extracellular vesicles, and hepatocyte L02 did not show a large color change. Subsequently, using the self-calibration characteristics of the unchanged color of the reference point, a ratio type Hue value detection signal (R-ΔHue = ΔHue sample / ΔHue reference, ΔHue = Hue after reaction - blank Hue) was constructed, and a standard curve of R-ΔHue and the logarithm of the concentration of small extracellular vesicles was drawn and the detection limit was calculated (Table 2). Figure 16 b, Table 2).

[0091] Different small extracellular vesicles of different cells showed different membrane protein expressions. The images of the multi-channel chip with a concentration of 5.0×10 8 individual·mL -1 were taken and columnar charts were drawn as shown in Figure 17 . The expression of membrane proteins of each cell small extracellular vesicle was as follows: HepG2: PTK7>PD-L1>EpCAM>CEA; HuH-7: CEA>PD-L1>PTK7>EpCAM; L02: low expression of the four proteins. The results of the high expression proteins in HepG2 and HuH-7 were consistent with the recent reports. The above results showed that Exopp-PAD could accurately detect the membrane protein expression of cell small extracellular vesicles by a simpler method.

[0092] Table 2 Linear relationship of R-ΔHue and small extracellular vesicle concentration

[0093]

[0094]

[0095] 8. Analysis of actual samples

[0096] According to the established detection probe DP and Exopp-PAD combined device, 5 serum samples were analyzed, including 2 healthy persons (#1, #2) and 3 liver cancer patients (#3, #4, #5, from the biological sample library of the Seventh Affiliated Hospital of Sun Yat-sen University). The results are shown in Figure 18 a. In #1, #2 samples, the color of the test point was almost unchanged, and the expression of the marker protein corresponding to the test point was low; #3 sample had a higher response to PTK7 and a lower response to PD-L1; #4 sample had a lower response to EpCAM and #5 had a lower response to PD-L1.

[0097] The R-ΔHue of the 5 samples was quantitatively analyzed and a heat map was drawn Figure 18b), further supporting the above observations. The comprehensive comparison shows that the expression levels of all four small extracellular vesicle membrane proteins of healthy individuals (#1, #2) are relatively low, consistent with the expression levels of small extracellular vesicle membrane proteins obtained from LO2 cell culture medium, indicating that samples #1 and #2 are very likely to be LO2 cell subtypes. For sample #4, its small extracellular vesicle membrane protein expression is CEA > PD-L1 > PDK7 > EpCAM, which is consistent with the expression of small extracellular vesicle membrane proteins obtained from HuH-7 cell culture medium, indicating that sample #4 is very likely to be a cell subtype of HuH-7. For sample #3, its small extracellular vesicle membrane protein expression is PTK7 » EpCAM ≈ CEA > PD-L1, and sample #5 has a small extracellular vesicle membrane protein expression of EpCAM ≈ CEA > PTK7 > PD-L1. This feature is not similar to those cell subtypes we measured, so samples #3 and #5 may belong to other cell subtypes, not HepG2 or HuH-7. In summary, Exopp-PAD has certain application prospects in the non-invasive diagnosis and subtype classification of hepatocellular carcinoma.

[0098] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A kit for small extracellular vesicle membrane protein detection, characterized by, The kit comprises a multi-channel paper chip device and a detection probe DP having a nucleotide sequence as shown in SEQ ID No: 1, and the core component of the multi-channel paper chip device is a plurality of aptamer-modified paper chips C@MOF@Paper, which comprises MOF@Paper and nucleic acid aptamers for specifically recognizing small extracellular vesicle membrane proteins, wherein the MOF@Paper comprises MOF@Paper and nucleic acid aptamers for specifically recognizing small extracellular vesicle membrane proteins, wherein the MOF@Paper is grown in situ on filter paper. The method for using the kit is as follows: a syringe at one end of the multi-channel paper chip device is used to suck the body fluid sample to be tested, and the other end of the syringe is slowly pushed while the other end of the syringe is pulled, so that the liquid is completely flowed into the other end of the syringe, and the sample is fully combined on the device by such reciprocating operation, then the body fluid sample to be tested is replaced by a washing liquid, a DP detection liquid and a Hemin solution in sequence, and the operation is performed in the same way, finally the multi-channel paper chip device is taken out, TMB single-component color developing liquid is added at each test point and reference point, the color developing result is observed and photographed, and the Hue value is analyzed for quantitative analysis.

2. The kit for detecting small extracellular vesicle membrane proteins according to claim 1, wherein, The preparation method of the device is as follows: a plurality of holes are formed on a resistant plate, different C@MOF@Paper are filled into the holes as test points, MOF@Paper without aptamer modification is filled as a reference point, the resistant plate is covered with a filter membrane, and then the multi-channel paper chip device is prepared by connecting the filter membrane to the syringes at both ends.

3. The kit for detecting small extracellular vesicle membrane proteins according to claim 1, wherein, The nucleic acid aptamer comprises an EpCAM aptamer as shown in SEQ ID No: 2, a PTK7 aptamer as shown in SEQ ID No: 3, a CEA aptamer as shown in SEQ ID No: 4 and a PD-L1 aptamer as shown in SEQ ID No:

5.

4. The kit for detecting small extracellular vesicle membrane proteins according to claim 1, wherein, The preparation method of the MOF@Paper comprises the following steps: S1, dissolve ZrCl4 in a mixture of formic acid and ethanol, then place filter paper in the resulting mixture, and let stand to allow the paper sheet surface to sufficiently adsorb Zr 4+ ions, S2, BDC-NH2 is dissolved in a mixed solution of formic acid, ethanol and water, the BDC-NH2 solution is added to the solution of step S1, MOF nanoparticles are generated on the surface of the paper sheet at room temperature, and then the MOF@Paper is prepared by washing and drying.

5. The kit for detecting small extracellular vesicle membrane proteins according to claim 4, wherein, The filter paper is Whatman No. 4 filter paper.

6. The kit for detecting small extracellular vesicle membrane proteins according to claim 4, wherein The volume ratio of formic acid to ethanol in the mixed solution of formic acid and ethanol is 6-9:20, and the volume ratio of formic acid, ethanol and water in the mixed solution of formic acid, ethanol and water is 6-9:20:7-10.

7. The kit for detecting small extracellular vesicle membrane proteins according to claim 1, wherein, The body fluid sample to be tested is a serum sample.

8. The kit for detecting small extracellular vesicle membrane proteins according to claim 1, wherein, The method for quantitative analysis is as follows: the self-calibration characteristics of the reference point with constant color are used to construct a ratio-type Hue value detection signal: R-ΔHue=ΔHue sample / ΔHue reference, ΔHue=Hue after reaction – blank Hue, and then a standard curve is drawn by taking R-ΔHue and the logarithm of the concentration of small extracellular vesicles.

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