CN119936379A - A multi-channel paper chip device and its application in the detection of small extracellular vesicle membrane proteins - Google Patents
A multi-channel paper chip device and its application in the detection of small extracellular vesicle membrane proteins Download PDFInfo
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Abstract
本发明属于生物医学工程技术领域，具体涉及一种多通道纸芯片装置及其在小细胞外囊泡膜蛋白检测中的应用。本发明公开的多通道纸芯片装置的核心组件是核酸适配体与金属有机框架(MOF)共同修饰的纸芯片。该装置为先在纸芯片上原位生长MOF材料，以提供小细胞外囊泡膜蛋白适配体的氨基修饰位点，同时减少纸对杂质蛋白的非特异性吸附，并作为比率型检测的参比。然后在MOF上修饰适配体，以通过免疫亲和力捕获表达相应蛋白的小细胞外囊泡。同时，研发了一种由小细胞外囊泡膜蛋白CD63适配体和G‑四链体组成的检测探针DP，用于比色检测捕获的小细胞外囊泡含量。利用该装置可多通道检测小细胞外囊泡膜蛋白，实现癌症的精准诊断与分型。
The present invention belongs to the field of biomedical engineering technology, and specifically relates to a multi-channel paper chip device and its application in the detection of small extracellular vesicle membrane proteins. The core component of the multi-channel paper chip device disclosed in the present invention is a paper chip modified with a nucleic acid aptamer and a metal organic framework (MOF). The device first grows MOF material in situ on a paper chip to provide amino modification sites for small extracellular vesicle membrane protein aptamers, while reducing the nonspecific adsorption of paper to impurity proteins, and serving as a reference for ratio-type detection. The aptamer is then modified on the MOF to capture small extracellular vesicles expressing the corresponding protein by immunoaffinity. At the same time, a detection probe DP composed of a small extracellular vesicle membrane protein CD63 aptamer and a G-quadruplex has been developed for colorimetric detection of the captured small extracellular vesicle content. The device can be used to detect small extracellular vesicle membrane proteins in multiple channels to achieve accurate diagnosis and typing of cancer.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering, and particularly relates to a multichannel paper chip device and application thereof in detecting small extracellular vesicle membrane proteins.
Background
Liver cancer is one of the most common malignant tumors worldwide, and the morbidity and mortality of the liver cancer are high and difficult throughout the year. In recent years, the understanding of liver cancer is gradually deepened along with the development of science and technology, but the death rate of liver cancer patients is still high, and it is expected that over 100 tens of thousands of people are affected by the liver cancer every year in 2025, and the reason is that the discovery of cancer is usually in middle and late stages, and the treatment effect is usually not ideal. At present, the important point and difficulty in liver cancer research is early diagnosis. Among them, the B-ultrasonic examination and the measurement of serum Alpha Fetoprotein (AFP) are the main methods of liver cancer diagnosis at present. However, these detection means often cannot timely detect early symptoms of liver cancer, and only can detect symptoms after morphological changes. Therefore, a new biomarker needs to be explored and developed, and a new idea is provided for improving diagnosis and treatment of liver cancer.
Biomarkers refer to indicators that can objectively measure and evaluate physiological or pathological processes of organs, tissues, cells, and even organelles. They are widely found in body fluids such as blood and interstitial fluid of the human body, and the abnormal expression level thereof is often closely related to the occurrence and development of cancer. Among them, small extracellular vesicles (sEV) are the most potential biomarkers for early diagnosis and therapeutic monitoring of cancer due to high abundance, ubiquitous and excellent stability in various body fluids. Therefore, the development of excellent biomarker detection technology based on small extracellular vesicles has important significance for diagnosis and treatment of cancer, reduction of mortality of patients and development of basic research related to cancer.
Small extracellular vesicles are a new biomarker for cancer diagnosis and can deeply reveal biological phenotype information, so that the instant detection of small extracellular vesicles is of great significance for large-scale screening of cancers. However, the existing methods for separating small extracellular vesicles and analyzing surface membrane proteins of small extracellular vesicles are still limited by complicated operations and expensive large-scale instruments, so that the methods are difficult to popularize and use. Based on this, development of a small extracellular vesicle detection method that is low in cost, number-effective and easy to handle is urgently needed.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a multi-channel paper chip device and a small extracellular vesicle membrane protein detection method, which can be used for measuring protein expression profiles of small extracellular vesicle membranes in various cancer cell culture solutions and serum of cancer patients with lower cost and realizing accurate diagnosis and typing of cancers.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
In a first aspect the invention provides a multi-channel paper chip device (Exopp-PAD) whose core is a plurality of paper chips C@MOF@paper modified with an aptamer, said C@MOF@paper comprising MOF@paper grown in situ on filter paper with UIO-66-NH 2 and a nucleic acid aptamer for specific recognition of small extracellular vesicle membrane proteins.
Preferably, the device is prepared by forming a plurality of holes in a endurance plate, filling different C@MOF@paper into each hole as a test point, filling MOF@paper modified by an aptamer-free reference point, coating the endurance plate with a filter membrane, loading the endurance plate into a replaceable membrane filter, and connecting two ends of the filter into a syringe to prepare the multichannel paper chip device.
Preferably, the nucleic acid aptamer comprises an EpCAM aptamer shown as SEQ ID No. 2, a PTK7 aptamer shown as SEQ ID No.3, a CEA aptamer shown as SEQ ID No.4 and a PD-L1 aptamer shown as SEQ ID No. 5.
Preferably, the preparation method of the MOF@paper comprises the following steps:
S1, dissolving ZrCl 4 in a mixed solution of formic acid and ethanol, placing filter paper in the obtained mixed solution, standing to enable the surface of a paper sheet to fully adsorb Zr 4+ ions,
S2, dissolving BDC-NH 2 in a mixed solution of formic acid, ethanol and water, adding the BDC-NH 2 solution into the solution in the step S1, standing at room temperature to generate MOF nano particles on the surface of the paper sheet, and washing and drying to obtain the MOF@paper.
More preferably, the filter paper is Whatman No. 4 filter paper.
More preferably, 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.
In a second aspect, the invention provides a kit for detecting small extracellular vesicle membrane proteins, which comprises the multi-channel paper chip device of the first aspect and a detection probe DP, wherein the detection probe DP has a nucleotide sequence shown as SEQ ID No. 1.
According to the invention, through in-situ growth of an MOF material with amino on filter paper and further modification of a series of aptamers capable of specifically recognizing small extracellular vesicle membrane proteins on the MOF surface, a C@MOF@paper chip is obtained, and the paper chip can specifically capture small extracellular vesicles, and simultaneously can also promote the protein nonspecific adsorption performance of the filter paper through the special surface property of the MOF material, and can be used as a reference by taking the color of the MOF as a follow-up ratio detection. Meanwhile, a DP nucleic acid probe is prepared by using the G-quadruplex sequence and the CD63 aptamer sequence, and the structure of the DP is changed based on the affinity reaction of the aptamer and the specific combination with the small extracellular vesicle membrane protein CD63, so that the G-quadruplex catalytic chromogenic activity is released. And then combining the small extracellular vesicles with Hemin to show the catalytic activity of peroxidase to catalyze TMB to develop color, so as to obtain colorimetric detection signals of the small extracellular vesicles, and further realize specific identification and detection of the small extracellular vesicle membrane proteins. In addition, by integrating paper chips targeting various membrane proteins into one multi-channel chip and performing operations such as capturing, washing, detecting and the like through a syringe, the measurement of the expression profile of the small extracellular vesicle membrane protein can be simply and cheaply realized.
Preferably, the kit is used by sucking the body fluid sample to be tested by using an injector at one end of the multi-channel paper chip device, pushing slowly, pulling the injector at the other end at the same time, enabling the liquid to flow into the injector at the other end completely, circulating in such a way that the sample is fully combined on the device, then sequentially changing the body fluid sample to be tested into a washing liquid, a DP detection liquid and a Hemin solution, operating in the same way, finally taking out the multi-channel paper chip device, adding TMB single-component color development liquid into each test point and a reference drop, observing the color development result, photographing, and analyzing the Hue value of the color development result for quantitative analysis.
More preferably, the body fluid sample to be tested is a serum sample.
More preferably, the quantitative analysis method is to construct a ratio type Hue value detection signal by utilizing the self-calibration characteristic that the color of a reference point is kept unchanged, wherein R-Deltahue=Deltahue sample/Deltahue reference, deltahue=hue-blank Hue after reaction, and then taking the logarithm of R-Deltahue and the concentration of small extracellular vesicles as a standard curve.
More preferably, the endurance plate is a PC endurance plate.
Compared with the prior art, the invention has the beneficial effects that:
The invention discloses a multi-channel small extracellular vesicle membrane protein detection method and a matched paper chip device (Exopp-PAD), and cancer cell typing is realized by detecting a small extracellular vesicle membrane protein expression profile in a body fluid sample. The core component of the device is a paper chip co-decorated with an aptamer and a Metal Organic Framework (MOF). The preparation method of the device comprises the steps of firstly growing MOF materials on a paper chip in situ to provide amino modification sites of small extracellular vesicle membrane protein aptamer, reducing the non-specific adsorption of paper on impurity proteins, and taking the paper as a reference for ratio detection. The aptamer was then modified on the MOF to capture small extracellular vesicles expressing the corresponding protein by immunoaffinity. Meanwhile, a Detection Probe (DP) consisting 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, paper chips aiming at different membrane protein targets are assembled into a multi-channel capturing chip, and the processes of capturing, washing, detecting and the like of small extracellular vesicles are completed by controlling the actions of a sample, a washing liquid, a DP detection liquid and the paper chips through a needle cylinder. Therefore, the device can be used for measuring protein expression profiles of small extracellular vesicle membranes in various cancer cell culture solutions and serum of cancer patients at low cost, and is expected to realize accurate diagnosis and typing of cancers.
The invention has the advantages that (1) the cost of the paper chip and the detection probe based on the aptamer is low, the performance is stable, the cost performance and the stability detection diagnosis of the detection can be improved, (2) the capture efficiency and the purity of the paper chip can be improved by modifying the metal organic framework, the detection performance is improved, and (3) the developed paper chip device does not need expensive and complex equipment and professional skills, and can detect the small extracellular vesicle membrane protein in a multi-channel manner, thereby realizing the accurate diagnosis of cancer.
Drawings
FIG. 1 is a schematic diagram of (a) a small extracellular vesicle separation and analysis device, (b) a paper chip preparation schematic diagram, (c) a DP detection schematic diagram, and (d) Exopp-PAD for capturing small extracellular vesicles and detecting membrane proteins;
FIG. 2 is an SEM image of (a, b) unmodified Paper chips (Paper) and (c, d) MOF modified Paper chips (MOF@paper);
FIG. 3 shows the PXRD spectra of (a) MOF@paper, paper, MOF powder and standard UiO-66-NH 2, (b) ATR-FTIR spectra of MOF@paper, paper and MOF powder;
FIG. 4 is an SEM image of (a) MOF@paper and an EDS element distribution diagram of (b) MOF@paper;
FIG. 5 is a photograph of (a) an unmodified paper chip and a MOF@paper reacted with CBB before and after adsorption of BSA;
FIG. 6 is a graph showing the PXRD spectra of (a) aptamer modified MOF@Paoper (C0@MOF@paper) and MOF@paper without aptamer modification, (b) ATR-FTIR spectra of C0@MOF@paper and MOF@paper;
FIG. 7 is a photograph of (a) MOF@paper not activated by EDC/NHS and MOF@paper activated by EDC/NHS before and after the reaction with TMB after the immobilization of the G quadruplex sequence;
FIG. 8 is an SEM image of C1@MOF@paper capturing small extracellular vesicles;
FIG. 9 is a circular dichroism spectrum of DP reacted with CD63 protein, small extracellular vesicles and a blank;
FIG. 10 is a fluorescence spectrum of DP reacted with CD63 protein, small extracellular vesicles and a blank;
FIG. 11 is an ultraviolet-visible absorbance spectrum of DP reacted with CD63 protein, small extracellular vesicles, and a blank;
FIG. 12 shows (a) optimization of Hemin concentration, (b) optimization of Hemin incubation time with G4;
FIG. 13 is a plot of extracellular vesicle concentration versus absorbance;
FIG. 14 is a graph of (a) the results of color development on a C@MOF@paper after binding of small extracellular vesicles of different concentrations to DP, (b) DeltaHue values versus small extracellular vesicle concentration;
FIG. 15 is a schematic diagram of the assembly of (a) Exopp-PAD, (b) the color development results in Exopp-PAD after binding of different types of small extracellular vesicles to DP;
FIG. 16 is a graph showing the results of color development in Exopp-PAD after binding of (a) small extracellular vesicles of different concentrations to DP, as a function of the log of the different concentrations of R-. DELTA.hue to (b) HepG2 (i), huH-7 (ii), LO2 (iii) small extracellular vesicles;
FIG. 17 shows the R-. DELTA.Hue values for each target protein on HepG2/HuH-7/LO2 small extracellular vesicles;
FIG. 18 is a photograph showing (a) the actual sample after analysis using Exopp-PAD, and (b) a heat map showing the protein expression in the actual sample.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The invention provides a multichannel paper chip device and application thereof in detecting small extracellular vesicle membrane proteins, comprising the following contents:
1. construction of small extracellular vesicles separation, enrichment and analysis device
Amino-modified MOF materials are grown on filter paper in situ, and a series of aptamers capable of specifically recognizing small extracellular vesicle membrane proteins are further modified on the surface of the MOF to obtain a C@MOF@paper (c represents a nucleic acid aptamer) which has the function of specifically capturing small extracellular vesicles. And then assembling the obtained serial composite paper chips on an acrylic plate to form Exopp-PAD, then assembling the acrylic plate and two Polycarbonate (PC) filter membranes with the pore diameters of 200nm in a replaceable membrane filter, and connecting the filter with a syringe to construct the device for separating, enriching and analyzing the small extracellular vesicles, as shown in figures 1a and b.
2. Design of detection probes DP and validated analysis of binding to small extracellular vesicles
(1) As shown in FIG. 1c, a hairpin detection probe DP is designed, and the probe is formed by connecting a G-quadruplex sequence and a CD63 aptamer sequence, wherein the sequence is used for specifically binding with CD63 protein which is highly expressed on the surface of a small extracellular vesicle, and the structure of the probe is converted after the binding and the G-quadruplex sequence is released, so that the content of each protein in the small extracellular vesicle captured by Exopp-PAD can be reflected by detecting the content of the G-quadruplex.
(2) The presence of the G-quadruplex is verified and detected by circular dichroism spectrum, fluorescence spectrum and ultraviolet-visible spectrum by utilizing the fluorescence emitted by the combination of the G-quadruplex and the THT under the excitation of specific wavelength and the catalytic activity characteristic of peroxidase which is shown after the combination of the G-quadruplex and the THT forms a compound with the Hemin, and the combination of DP, CD63 and small extracellular vesicles is verified.
(3) And (3) performing optimization on the combination condition of the G-quadruplex sequence and the Hemin by utilizing the combination of the G-quadruplex sequence and the Hemin to show the catalytic activity of peroxidase and setting the Hemin with different concentration gradients and different reaction temperature gradients.
(4) Linear analysis of the color development results after binding of the detection probes DP and the small extracellular vesicles was performed on 96-well plates and c@mof@paper, respectively, small extracellular vesicle suspensions of different concentrations were added, followed by sequential reaction with the detection probes DP, hemin, TMB, and for 96-well plates, standard curves were drawn after absorbance was detected at 595nm with an enzyme-labeled instrument, and for c@mof@paper, hue values were recorded and read by photographing, and standard curves of Hue values and small extracellular vesicle concentrations were drawn.
3. Multi-channel detection of small extracellular vesicle proteins based on Exopp-PAD
The chromogenic results of HepG2, huH-7 and LO2 small extracellular vesicles with known concentrations were tested using Exopp-PAD and detection probe DP, respectively, photographed and analyzed for Hue values for quantitative analysis using imageJ, the detailed flow scheme is shown in FIG. 1 d.
In order to further clearly demonstrate the process of constructing the multi-channel paper chip device and detecting small extracellular vesicle membrane proteins of the present invention, a detailed description is provided below in connection with the examples.
1. Preparation and characterization of MOF@paper
(1) In situ paper chip growth MOF
Whatman No. 4 filter paper was cut by a perforator into round pieces of paper 6mm in diameter for use. 0.5mmol of ZrCl 4 is dissolved in a mixed solution of 0.7mL of formic acid and 2.0mL of ethanol, after 1h of ultrasonic treatment, a paper chip is added into the ZrCl 4 solution and is kept stand for 2h, so that Zr 4+ ions are fully adsorbed on the surface of the paper chip. On the other hand, 0.2mmol of BDC-NH 2 was dissolved in a mixture of 0.7mL of formic acid, 2.0mL of ethanol and 0.8mL of pure water, and sonicated for 2h. Adding BDC-NH 2 solution into ZrCl 4 solution containing paper chips after ultrasonic treatment, slightly shaking and uniformly mixing, standing at room temperature for reaction for 8 hours to generate MOF nano particles on the surfaces of the paper chips, respectively washing 3 times with absolute ethyl alcohol and water, drying at 37 ℃ for 10 minutes to obtain UIO-66-NH 2 functionalized paper chips (MOF@paper), and finally placing in a sealed bag for standby.
(2) SEM and Spectrum characterization of paper chips
The filter Paper (Paper) before MOF modification and the filter Paper (MOF@paper) after MOF modification are adhered to a sample table by conductive adhesive, dried in vacuum for 12h in a vacuum drying oven, and after the sample is sprayed with gold for 60s by a particle sputtering instrument, the sample is characterized by using SEM under an accelerating voltage of 3 kV. As shown in fig. 2, SEM results indicate that the paper core showed a staggered porous fibrous structure before and after MOF growth, as shown in fig. 2a and 2c, i.e. the MOF growth did not alter the porous three-dimensional structure of the paper itself. The mof@paper forms uniform nanoparticles on the surface of the fiber (fig. 2 d) compared to the smooth fiber of the paper itself (fig. 2 b). According to the SEM characterization, after UiO-66-NH 2 is modified, the MOF@paper still maintains the original porous structure and filtering function of the paper, so that sample liquid can pass through the paper chip, and the capability of full contact and action with an aptamer modified later on of the paper chip is ensured.
(3) PXRD characterization and ATR-FTIR characterization of paper chips
In order to verify that the nano particles generated on the surface of the Paper are the target MOF material UiO-66-NH 2, X-ray powder diffraction characterization is respectively carried out on Paper, MOF@paper and pure MOF powder, and experimental parameters are that the scanning speed is 6 degrees per minute, the scanning range is 5 degrees to 40 degrees, and the step size is 0.02 degrees. As a result, as shown in FIG. 3a, the MOF@paper exhibited peaks of crystal plane characteristics PXRD of Paper and UiO-66-NH 2 (111) and (002) at 2θ=7.4° and 8.5 °, indicating that the structure generated in the synthesized MOF@paper was UiO-66-NH 2.
Meanwhile, the infrared test is carried out on Paper, MOF@paper and synthesized MOF powder by using ATR-FTIR respectively, wherein the experimental parameters are that the scanning range is 500cm -1 to 2500cm -1, the step length is 1cm -1, and the scanning is 16 times. As shown in FIG. 3b, the MOF@paper shows a characteristic absorption peak of UiO-66-NH 2, the peak at 768cm -1 being assigned to the Zr-O stretching vibration, the peaks at 1374cm -1 and 1567cm -1 being assigned to the carbonyl stretching vibration. The above results further demonstrate the successful in situ growth of UiO-66-NH 2 nanoparticles on paper fibers.
In addition, as shown in FIG. 4, the SEM image (FIG. 4 a) and EDS element profile (FIG. 4 b) of the MOF@paper show a broad distribution of Zr on its surface, indicating uniform and broad growth of UiO-66-NH 2 nanoparticles on the paper fibers. The above results demonstrate that UiO-66-NH 2 nanoparticles grow and are widely and uniformly distributed on paper fibers, providing an amino active site for the paper sheet that is easy to modify.
(4) Investigation of the ability of paper chips to resist protein non-specific adsorption
Using BSA as a protein model molecule, the Paper and MOF@paper (6 mm diameter disc) were first incubated (20. Mu.L, 10 mg/mL) with BSA solution (20 min at RT) and washed thoroughly, and then coomassie brilliant blue solution (10. Mu.L, 1. Mu.M) was added and reacted at RT for 30min to develop the adsorbed BSA on the Paper, as shown in FIG. 5 a. The Paper was visibly discolored, while the MOF@paper was hardly discolored. Further quantification of the color change Hue value using ImageJ software, it was found that the Δhue value of Paper was 10.6 times that of mof@paper (fig. 5b, fig. 5 c). The results show that a large amount of BSA is nonspecifically adsorbed on the surface of unmodified paper fibers, and UiO-66-NH 2 modification can greatly reduce the nonspecific adsorption of proteins by the paper.
2. Paper chip modified aptamer and characterization thereof
The amino groups on the paper chip were coupled with 5' -end modified carboxyl group aptamer using EDC-NHS. Specifically, a mixed solution of 0.1M NHS and 0.4M EDC was prepared by dissolving 1.0mmol NHS and 4.0mmol EDC in 10mL of water, and then MOF@paper was immersed in a freshly prepared EDC-NHS solution for 10min, and then washed 3 times with absolute ethanol and water, and then immersed in 45 mL of 1.0 μm capture aptamer (C1-C4) and reference (Ctrl) solutions for 3h in batches, followed by washing 3 times with PBST, and drying at 37℃for 15min to obtain aptamer-modified paper chips (C@MOF@paper, and C1-C4 are in this order of EpCAM, PTK7, CEA and PD-L1 nucleic acid aptamers, see Table 1 for details). Finally, the mixture is placed in a sealed bag and stored at-20 ℃ for standby. And PXRD and ATR-FTIR characterization were performed on mof@paper and c@mof@paper (take c1@mof@paper as paper chips for feasibility verification).
To verify that the carboxyl modified aptamer was successfully immobilized on the MOF@paper, the 5' -terminal carboxyl modified G-quadruplex sequence C0 with the pseudoenzymatic activity and the covalent linkage function was combined with the MOF@paper by EDC/NHS to obtain C0@MOF@paper for relevant characterization. First, PXRD characterization and ATR-FTIR characterization were performed on c0@mof@paper, and the results are shown in fig. 6. In contrast to mof@paper without the aptamer incubated, the PXRD patterns of the two were substantially identical (fig. 6 a), demonstrating that the structure of the MOF was unchanged after the aptamer was modified. As can be seen from the ATR-FTIR spectra of both (FIG. 6 b), C0@MOF@paper has a new infrared absorption peak at 1640cm -1 due to the characteristic peak attributed to the carbonyl group in the amide bond, which is due to the amidation reaction of the carboxyl group on C0 with EDC/NHS, forming an amide bond. The above results can initially demonstrate that the aptamer can be successfully immobilized to the mof@paper under EDC/NHS bridging.
The G-quadruplex sequence can show peroxidase catalytic activity after forming a complex with Hemin, and further catalyze TMB/H 2O2 to carry out chromogenic reaction. The 5' -carboxyl modified probe G4 (1.0. Mu.M, 5 mL) consisting directly of the G-quadruplex sequence was designed and then incubated with MOF@paper with and without EDC/NHS activation for 3h at room temperature. After washing, a further incubation (37 ℃ C., 30 min) with Hemin solution (4. Mu.M, 20. Mu.L) was added, and after extensive washing, the reaction (37 ℃ C., 30 min) was performed with TMB chromogenic solution (1 mM, 20. Mu.L) and the change in color was recorded by observation and photographing (FIG. 7 a). Further quantification of the Hue value using ImageJ software (fig. 7 b) revealed that the final color change of the paper chips was evident after EDC/NHS activation treatment, whereas the paper chips without EDC/NHS activation treatment did not substantially undergo a color reaction. The above results further indicate that the nucleic acid sequence can be covalently modified on MOF@paper by EDC/NHS reaction, and that the G-quadruplex sequence modified on MOF@paper can be subjected to catalytic chromogenic reaction on paper.
TABLE 1 nucleic acid sequences used in experiments
3. Verification of paper chip load enrichment small extracellular vesicles
Exosomes were collected from HepG2 cell culture broth using ultracentrifugation. After the cells were grown to about 70% of the area of the cell culture flask, the medium was changed to DMEM medium containing 1.5% of apocrine FBS for culturing for 48 hours. Then, the cell culture suspension was collected while avoiding cell sedimentation and transferred to a centrifuge tube, and centrifuged at 4℃for 10min with a relative centrifugal force of 300g to remove large particles and cells, and then the supernatant was transferred to a new centrifuge tube, and centrifuged at 4℃for 20min with a relative centrifugal force of 2000g to remove cell debris and larger particles. Subsequently, the supernatant was transferred to a new centrifuge tube and centrifuged at 10000g of relative centrifugal force at 4 ℃ for 30min to remove large vesicles and protein aggregates. The resulting supernatant was centrifuged at 120000g for 90min at 4℃and the supernatant carefully removed with a pipette, the exosome pellet washed with PBS and resuspended, and centrifuged again at 120000g for 90min at 4 ℃. The supernatant was then carefully removed, 200. Mu. LPBS was added to resuspend the exosome pellet and 10. Mu.L each was dispensed and stored at-80 ℃.
10 Mu L of HepG2 small extracellular vesicles are taken and dripped on a C@MOF@paper (a C1 modified paper chip is taken as a paper chip for feasibility verification), incubated for 1h at room temperature, unbound small extracellular vesicles are washed off by using PBST, the paper chip is attached to an SEM sample table by using a conductive adhesive after drying, and then vacuum drying is carried out for 12h and is characterized by SEM. It was verified by SEM whether the c@mof@paper captured small extracellular vesicles, as exemplified by SEM characterization in c1@mof@paper, and the results are shown in fig. 8. It can be seen that MOFs on paper fibers captured circular nanovesicles less than 200nm after binding to small extracellular vesicles. The results indicate that the device can load and enrich small extracellular vesicles.
4. Feasibility verification of detection probe binding to CD63 and small extracellular vesicles
(1) Circular dichroism spectrum verification
The DP solution (1. Mu.M, 20. Mu.L) was incubated (37℃for 30 minutes) with 20. Mu.L of recombinant CD63 protein (1. Mu.g/mL), small extracellular vesicles (Exos, 10 8 parts/mL) and PBS buffer, respectively, and the results were shown in FIG. 9. In comparison with the blank, there was a negative peak at 245nm and a positive peak at 270nm in the circular dichroism spectrum of the detection probe DP when CD63 and small extracellular vesicles were present, because parallel G-quadruplex steric structures were formed, demonstrating the feasibility of DP binding to CD63 and small extracellular vesicles.
(2) Fluorescence spectrum verification
Equivalent amounts of thioflavin T (ThT, 10 μm,20 μl) solution were added to the solutions after DP incubation with recombinant CD63 protein, small extracellular vesicles and blank control, respectively, and after incubation (37 ℃ for 30 minutes) and dilution with PBS to 100 μl, their fluorescence emission spectra were measured at 425nm excitation light, as shown in fig. 10. In comparison with the blank, after DP bound to CD63 protein and small extracellular vesicles, there was a fluorescence emission peak at 495nm in the presence of ThT, further demonstrating the feasibility of DP binding to CD63 and small extracellular vesicles.
(3) Ultraviolet-visible absorption spectrum verification
Equal amounts of a Hemin (Hemin solution) solution was added to the solutions after DP incubation with recombinant CD63 protein, small extracellular vesicles and a blank, respectively, incubation (37 ℃ for 30 minutes) and addition of TMB single-component chromogenic solution for sufficient reaction, followed by addition of HCl solution to terminate the chromogenic reaction, and the uv-vis absorption spectra were measured as shown in fig. 11. Compared with blank control, DP is combined with CD63 protein and small extracellular vesicles, and after the incubation of Hemin and the addition of TMB for color reaction, the DP has a stronger absorption peak at 450 nm. This further verifies the feasibility of DP binding to CD63 and small extracellular vesicles.
5. Experimental condition optimization of G-quadruplex and Hemin combination
G-quadruplex nucleic acid (G4, see Table 1) was dissolved and diluted to 10.0. Mu.M with 1 XTE Buffer, and Hemin powder was dissolved and diluted to 50.0. Mu.M with PBS Buffer for use. Firstly, 10 parts of mixed solution with the volume of 80 mu L, the G4 concentration of 1.0 mu M and the Hemin concentration of 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 mu M are sequentially prepared in a 96-well plate, after incubation for 30min at room temperature, 20 mu L of TMB single-component color development solution is added, after reaction for 20min at room temperature, 20 mu L of 1.0M HCl is added to terminate the reaction, and then an enzyme-labeled instrument is used for measuring the absorbance at 450nm, and the result is shown in FIG. 12 a. The absorbance increases with the concentration of the Hemin, and when the concentration of the Hemin exceeds 4.0 mu M, the absorbance does not change obviously, so that the optimal concentration of the Hemin is 4.0 mu M.
With the concentration of Hemin optimized in the previous step, the incubation time of G4 and Hemin is set to be 10,20,30,60 and 90min respectively, 20 mu L of TMB single-component color development liquid is added after the incubation is completed, the reaction is stopped by adding 20 mu L of 1.0M HCl after the reaction is performed for 20min at room temperature, and then the absorbance at 450nm is measured by an enzyme-labeling instrument and plotted as shown in FIG. 12 b. Absorbance increased with increasing incubation time and there was no significant change in absorbance after 30min, so the optimal time for Hemin incubation with G-quadruplex was 30min.
6. Linear analysis of the color development results after binding of detection probes DP to small extracellular vesicles
HepG2 small extracellular vesicles were diluted with PBS to a concentration of 0,1,5,10,50,100 X10: 10 6.mL -1, 20. Mu.L of each was sequentially added to a 96-well plate, and then incubated with 20. Mu.L of 1.0. Mu.M DP solution for 45min, and then 20. Mu.L of 4.0. Mu.M Hemin solution was added for 30min, and PBST was washed 3 times, and then 20. Mu.L of LTMB single-component color-developing agent was added for 20min. Absorbance was then measured at 595nm with a microplate reader and a standard curve was drawn from small extracellular vesicle concentration and absorbance: abs.=0.0673×lg [ Exos ] -0.0383 (R 2 =0.98) as shown in fig. 13. It can be seen that the absorbance of the solution is linearly related to the logarithm of the extracellular vesicle concentration, with the DP concentration unchanged and other conditions kept the same.
Subsequently, the linear relationship of DP to small extracellular vesicles on c@mof@paper was examined. 20 mu L of HepG2 small extracellular vesicle diluents with different concentrations (0,1,5,10,50,100 multiplied by 10 6. Mu.mL -1) are respectively dripped on a C@MOF@paper (disc with the diameter of 6 mm), after incubation for 1h, PBST is washed 3 times, 20 mu L of 1.0 mu M DP solution is dripped for 45min, 20 mu L of 4.0 mu M Hemin solution is dripped for 30min, PBST is washed 3 times, and then 20 mu LTMB single-component color reagent is added for reaction for 20min. The color change after the reaction was recorded by photographing (14 a), and a standard curve was drawn based on Hue values and small extracellular vesicle concentrations, Δhue=8.03×lg [ Exos ] -7.11 (R 2 =0.96), as shown in fig. 14 b. From this, it was found that the Δhue value of the paper chip discoloration correlated well with the logarithm of the small extracellular vesicle concentration, with the DP concentration unchanged and other conditions being consistent.
7. Multi-channel detection of small extracellular vesicle proteins based on Exopp-PAD
First, a device for enriching small extracellular vesicles and analyzing small extracellular vesicle proteins is constructed, which comprises three main components, namely a syringe, a replaceable membrane filter with the diameter of 25mm, a vertical small extracellular vesicle enrichment and a membrane protein detection chip (Exopp-PAD) thereof. The preparation method comprises the steps of digging 5 holes with the diameter of 6mm on a PC (polycarbonate) endurance plate with the diameter of 25mm and the thickness of 0.2mm, filling 4 paper chips C@MOF@paper modified by the prepared aptamer (table 1) as 4 test points, filling MOF@paper modified by the aptamer-free type test point as a reference point to form Exopp-PAD, coating the PC endurance plate with two filter membranes with the diameter of 25mm and the aperture of 200nm, filling the PC endurance plate with the filter membranes with the diameter of 25mm, and connecting two ends of the filter into a syringe. In operation, 2mL of sample solution is withdrawn with one end syringe, pushed slowly while the other end is pulled, and this operation is repeated as the solution flows completely into the other end, and so on and so forth in a cycle to allow Exopp-PAD to bind well with the sample. The sample solution was then replaced with a wash solution, a DP detection solution, a Hemin solution in the same manner.
The chromogenic results for HepG2, huH-7 and LO2 small extracellular vesicles with 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 procedure was as follows, by adding different concentrations of liver/liver cancer cell-derived small extracellular vesicles (0,5.0 ×10 6–5.0×108 ·ml -1) to FBS containing 50% pbs, small extracellular vesicle serum samples were prepared. Assembling Exopp-PAD in the sequence shown in FIG. 15a, introducing a serum sample of the small extracellular vesicles into Exopp-PAD through a syringe, specifically combining membrane proteins on the surface of the small extracellular vesicles with C@MOF@paper when the sample passes through a filter, capturing the small extracellular vesicles on a test point, incubating and washing, adding a detection probe DP into Exopp-PAD through the syringe, fully reacting, adding Hemin into Exopp-PAD through the syringe, washing, taking out Exopp-PAD, adding TMB single-component color development liquid into each test point and reference spot, observing color development results, photographing, and analyzing Hue values for quantitative analysis. As shown in fig. 15b and 16a, the color of the reference spot is substantially the same in the small extracellular vesicles of different types and different concentrations, and the color change degree of the test spot is increased with the increase of the small extracellular vesicle concentration for liver cancer cells HepG2 and HuH-7, and the liver cell LO2 does not show a large color change. Subsequently, using the self-calibration property that the color of the reference spot remains unchanged, a ratiometric Hue value detection signal (R- Δhue=Δhue sample/Δhue reference, Δhue=post-reaction Hue-blank Hue) was constructed, a standard curve was made with the logarithm of R- Δhue and the small extracellular vesicle concentration and the detection limit was calculated (fig. 16b, table 2).
Small extracellular vesicles from different cells show different membrane protein expression. Images of the multichannel chip at a concentration of 5.0X10 8. Multidot.mL -1 were taken as a bar graph, as shown in FIG. 17. The expression condition of each small extracellular vesicle membrane protein is as follows, hepG2, PTK7> PD-L1> EpCAM > CEA, huH-7, CEA > PD-L1> PTK7> EpCAM, and LO2, 4 proteins are all expressed in low. The results for high expression of proteins in HepG2 and HuH-7 are consistent with recent reports. These results indicate that Exopp-PAD can accurately detect the membrane protein expression of small extracellular vesicles by a simpler method.
TABLE 2 Linear relationship of R-DeltaHue to small extracellular vesicle concentration
8. Analysis of actual samples
According to the established combination of detection probes DP and Exopp-PAD, 5 serum samples, including 2 healthy subjects (# 1, # 2) and 3 liver cancer patient samples (# 3, #4, # 5), were analyzed from the university of Zhongshan affiliated seventh hospital biological sample library. As a result, as shown in FIG. 18a, the color of the test spot was almost unchanged in samples #1, #2, and the expression level of the marker protein corresponding to the test spot was low, sample #3 had a higher response to PTK7, a lower response to PD-L1, and sample #4 had a lower response to EpCAM and a lower response to PD-L1 in sample # 5.
Quantitative analysis of R-. DELTA.hue was performed on these 5 samples and a heat map was drawn (FIG. 18 b), further supporting the above observation results. The overall comparison showed that the expression levels of all four small extracellular vesicle membrane proteins of healthy individuals (# 1, # 2) were 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 highly likely to be LO2 cell subtypes. For sample #4, the small extracellular vesicle membrane protein expression profile was CEA > PD-L1> PDK7> EpCAM, which is consistent with the small extracellular vesicle membrane protein expression profile obtained from HuH-7 cell culture medium, indicating that sample #4 is most likely a cell subtype of HuH-7. For sample #3, the small extracellular vesicle membrane protein expression profile was PTK7> EpCAM+.CEA > PD-L1, while the small extracellular vesicle membrane protein expression profile of sample #5 was 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, rather than HepG2 or HuH-7. Overall, exopp-PAD has a certain application prospect in non-invasive diagnosis and subtype classification of hepatocellular carcinoma.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (10)
1. A multi-channel paper chip device, characterized in that the core component of the device is a plurality of paper chips c@mof@paper modified by an aptamer, wherein the c@mof@paper comprises a mof@paper obtained by growing UiO-66-NH 2 in situ on filter paper and a nucleic acid aptamer, and the nucleic acid aptamer is used for specifically identifying small extracellular vesicle membrane proteins.
2. The multi-channel paper chip device according to claim 1, wherein the device is prepared by forming a plurality of holes in a endurance plate, filling different C@MOF@paper into each hole as test points, filling MOF@paper without aptamer modification as reference points, coating the endurance plate with a filter membrane, loading the endurance plate into a replaceable membrane filter, and connecting two ends of the filter into a syringe.
3. The multi-channel paper chip device of claim 1, wherein the nucleic acid aptamer comprises an EpCAM aptamer shown as SEQ ID No. 2, a PTK7 aptamer shown as SEQ ID No. 3, a CEA aptamer shown as SEQ ID No. 4, and a PD-L1 aptamer shown as SEQ ID No. 5.
4. The multi-channel paper chip device according to claim 1, wherein the preparation method of the mof@paper comprises the following steps:
S1, dissolving ZrCl 4 in a mixed solution of formic acid and ethanol, placing filter paper in the obtained mixed solution, standing to enable the surface of a paper sheet to fully adsorb Zr 4+ ions,
S2, dissolving BDC-NH 2 in a mixed solution of formic acid, ethanol and water, adding the BDC-NH 2 solution into the solution in the step S1, standing at room temperature to generate MOF nano particles on the surface of the paper sheet, and washing and drying to obtain the MOF@paper.
5. The multi-channel paper chip device of claim 4, wherein the filter paper is Whatman No.4 filter paper.
6. The multi-channel paper chip device 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. A kit for detecting small extracellular vesicle membrane proteins, characterized in that it comprises the multi-channel paper chip device of any one of claims 1-6 and a detection probe DP having a nucleotide sequence as shown in SEQ ID No. 1.
8. A kit for detecting small extracellular vesicle membrane protein according to claim 7, wherein the kit is used by sucking the sample of body fluid to be detected by a syringe at one end of a multi-channel paper chip device, pushing slowly while pulling the syringe at the other end to make the liquid flow into the syringe at the other end completely, repeating the cycle so as to make the sample be detected fully combined on the device, then changing the sample of body fluid to be detected into a washing liquid, a DP detection liquid and a Hemin solution in sequence, operating in the same way, finally taking out the multi-channel paper chip device, adding TMB single-component color development liquid at each test point and reference drop, observing the color development result and analyzing the Hue value for quantitative analysis after photographing.
9. The kit for detecting small extracellular vesicle membrane protein according to claim 8, wherein the body fluid sample to be detected is a serum sample.
10. The kit for detecting small extracellular vesicle membrane protein according to claim 8, wherein the quantitative analysis method is to construct a ratio type Hue value detection signal by utilizing the self-calibration characteristic that the color of the reference point is kept unchanged, wherein R-Deltahue=Deltahue sample/Deltahue reference, deltahue=hue-blank Hue after reaction, and taking the logarithm of R-Deltahue and the concentration of small extracellular vesicle as a standard curve.
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