KR20210067916A - Label-free Polydiacetylene Liposome-based Method for Detection of Exosomes - Google Patents

Abstract

According to an embodiment of the present disclosure, disclosed is a method for conveniently detecting exosomes in a sample without labeling using poly-diacetylene liposomes in which a receptor specific for a biomarker present on the surface of the exosomes is immobilized on poly-diacetylene.

Description

Label-free Polydiacetylene Liposome-based Method for Detection of Exosomes

The present disclosure relates to a method for detecting exosomes based on label-free polydiacetylene liposomes. More specifically, the present disclosure provides a method for conveniently detecting exosomes in a sample without labeling using polydiacetylene liposomes in which a receptor specific for a biomarker present on the surface of the exosomes is immobilized (conjugated) to polydiacetylene. it's about how

Cells are known to secrete extracellular vesicles surrounded by lipid bilayers of various sizes. These extracellular vesicles are classified into exosomes, microvesicles, and large oncosomes according to their size. Double exosomes are small vesicles with a membrane structure secreted from various types of cells. It has been reported that the diameter of exosomes is approximately 30 to 100 nm, and studies through electron microscopy show that they originate from specific intracellular compartments called multivesicular bodies (MVBs) rather than directly from the plasma membrane, and are released and released outside the cell. Secretion was observed. That is, when the fusion of the polycystic body and the plasma membrane occurs, these vesicles are released to the environment outside the cell, which is referred to as an exosome.

Although it has not yet been clearly identified by what molecular mechanism the above-described exosomes are formed, various types of immune cells including red blood cells, B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, etc. It has been reported that tumor cells and the like also produce and secrete exosomes in a live state. Exosomes are known to be released separately from many other cell types in both normal and pathological conditions.

Recently, exosomes, which are newly emerging in the field of disease diagnosis, have been spotlighted for their utility as a diagnostic and drug delivery medium for various diseases. That is, exosomes with cell-derived properties are released to the outside of the cell and contained in various types of body fluids such as blood, saliva, urine, etc., by analyzing the exosomes present in the body fluid to identify specific cell-derived biomarkers. method of diagnosing In this regard, exosomes are known to contain about 4,500 kinds of proteins, lipids, and genetic information (eg, microRNA, mRNA, tRNA, rRNA, DNA, etc.) that are indicators of drug response, exosomes A method of diagnosing a disease by detecting the presence or absence and amount of various microRNAs present in the body (eg, Korean Patent Publication No. 2010-0127768, etc.), in samples (blood, saliva, tears, etc.) resulting from cancer By detecting the exosomes and measuring the amount of change in microRNA, a method for predicting and diagnosing association with a specific disease when it increases or decreases compared to the control group (eg, WO2009/015357 A, etc.) has been developed.

However, since exosomes present in various body fluids have low density and small size (from tens of nm to 150 nm), an additional process for separating them is required. In order to simplify the above-described separation procedure, a sensor that selectively detects or detects exosomes in body fluid is being studied. Most of these exosome detection methods use a chip that can flow a liquid containing exosomes into a passageway, and the exosomes coupled with receptors present on the surface of the passageways are subjected to electrical resistance, current, or surface plasmon resonance using electrical resistance, current, or surface plasmon resonance. can be detected (eg, Korean Patent Publication No. 87594, etc.).

However, in the above-described method, a separate chip system must be manufactured, and in the process, complicated procedures and processes must be performed. In addition, expensive equipment is required to obtain a detectable signal, and it is disadvantageous in terms of compatibility and versatility, such as using a labeling material for diagnosis. In particular, the fabrication of a chip system for detecting exosomes is limited in commercial use because separate expensive manufacturing equipment and complicated procedures are involved, and mass production is difficult. In addition, a chip of a fixed standard has a limited application range and is not suitable for application in various fields. In addition, surface plasmon resonance-based devices used to obtain exosome capture signals are expensive equipment and have low versatility.

As such, due to issues related to simplification and cost reduction of complex diagnostic systems, a simple detection principle is essential for commercialization. However, in general, fluorescence and colorimetric principles related to target-specific light sources, filters, prisms and visualization devices are used, and these methods require complex methods and equipment, so there is a need to improve diagnostic convenience. have.

Therefore, there is a need for a method capable of detecting exosomes in a simple way without using a separate label.

In one embodiment according to the present disclosure, unlike the prior art, it is possible to easily and accurately detect or diagnose exosomes in a sample based on an analysis principle detectable through general-purpose means without requiring the use of complicated procedures and expensive equipment. We want to provide a method (or platform).

In one embodiment according to the present disclosure, an object of the present disclosure is to provide a method for detecting exosomes in a sample without labeling without requiring chip fabrication.

According to the first aspect of the present disclosure,

a) providing a poly-diacetylene liposome modified with a receptor having a specific binding ability with respect to a biomarker present on the surface of the exosome; and

b) measuring the change in optical properties induced by the ligand-receptor reaction between the exosome and the modified polydiacetylene liposome in the sample as the modified polydiacetylene liposome is in contact with the exosome-containing sample;

There is provided a method for detecting exosomes comprising a.

According to an exemplary embodiment, the step a) comprises:

a1) self-combining diacetylene monomer and phospholipid to form lipid vesicles having an interior space isolated by a lipid layer membrane;

a2) attaching the receptor to the lipid ER to form a modified lipid ER; and

a3) polymerizing the modified lipid endoplasmic reticulum to form a modified polydiacetylene liposome;

may include.

According to an exemplary embodiment, at least one biomarker is present on the surface of the exosome, and the receptor may be a single-receptor specific for the at least one biomarker.

According to an exemplary embodiment, a plurality of biomarkers are present on the surface of the exosome, and the receptor may be a multi-receptor specific to the plurality of biomarkers.

According to an exemplary embodiment, the receptor is a multiple receptor, wherein step a2) may include attaching any one of the multiple receptors to the lipid endoplasmic reticulum, and then attaching the others sequentially.

According to an exemplary embodiment, the diacetylene monomer is at least one selected from the group consisting of PCDA (pentacosadiyonic acid), TCDA (trocosadiyonic acid), HCDA (heneicosadiynoic acid) and HDDA (heptadecadiynoic acid), and

The phospholipids include dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroyl ethylphosphocholine ( DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleoyloxy)-3 -(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylic acid (DMPA), dipalmitoylphosphatidylic acid (DPPA), distearoylphosphatidylic acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylinositol (DMPS), dipalmitoylphosphatidylserine (DPPS), and dis It may be at least one selected from the group consisting of thearoylphosphatidylserine (DSPS).

According to an exemplary embodiment, the molar ratio of diacetylene monomer to phospholipid in step a1) may range from 2 to 10:1.

According to an exemplary embodiment, the method may further include activating the lipid ER with a chemical functional group prior to step a2).

According to an exemplary embodiment, the receptor may be at least one selected from the group consisting of the whole antibody or a partial antibody containing an antigen binding region and an aptamer.

According to an exemplary embodiment, the biomarker may be a tetraspanin protein.

According to an exemplary embodiment, the tetraspanin protein may be at least one selected from the group consisting of CD-9, CD-63, CD-81 and CD-82.

According to an exemplary embodiment, the optical property change may be measured with the naked eye or a measuring device.

According to an exemplary embodiment, the apparatus for measuring the change in optical properties may be a UV-Vis spectrometer or a fluorescence spectrometer.

The label-free polydiacetylene liposome-based exosome detection method according to an embodiment of the present disclosure can generate a signal by simple mixing without a complicated procedure because detection is possible on an aqueous medium. In particular, the amount of poly-diacetylene liposome can be appropriately adjusted according to the purpose of use and the amount of sample, and the signal derived from poly-diacetylene can be conveniently measured using universally used UV-Vis spectroscopy, fluorescence spectroscopy, etc. can Moreover, it has the advantage of providing a wide range of use compared to the surface plasmon resonance and electrical measurement devices required in the existing chip method. Therefore, it is expected to be utilized in various diagnostic fields in the future.

1 is a diagram exemplarily showing the exosome detection principle and its implementation procedure based on the label-free polydiacetylene liposome according to an embodiment of the present disclosure;
2 is a diagram schematically illustrating a procedure for preparing polydiacetylene liposomes modified with multiple receptors according to an embodiment and a sequence for detecting exosomes in a label-free manner using the same;
3 is a scanning electron microscope (SEM) photograph of PCDA/DMPC liposomes prepared in Examples;
4 is a scanning electron microscope (SEM) photograph of the diacetylene liposome into which the exosome biomarker receptor is introduced in the Example;
5 is a scanning electron microscope (SEM) photograph of an aggregate of poly-diacetylene liposomes and exosomes into which receptors are introduced in Examples;
6 is a graph showing changes in the UV-Vis absorption spectrum of poly-diacetylene liposomes according to the concentration of exosomes in the sample in Examples;
7 is a transmission electron microscope (TEM) photograph after adding exosomes to polydiacetylene liposomes modified with multiple receptors in Examples;
8 is a transmission electron microscope (TEM) photograph after adding albumin protein and fibrinogen to polydiacetylene liposomes modified with multiple receptors in Examples;
9 is a photograph showing the color change before and after adding the exosomes to the polydiacetylene liposomes modified with multiple receptors in Examples;
10 is a fluorescence photograph taken before and after the addition of the exosome solution to the polydiacetylene vesicles attached to the substrate; And
11 is a graph showing the change in fluorescence intensity with time when the poly-diacetylene/phospholipid vesicle according to the receptor introduced into the poly-diacetylene ER is in contact with the exosome-containing sample.

The present invention can all be achieved by the following description with reference to the accompanying drawings. It is to be understood that the following description is to be understood as describing preferred embodiments of the present invention, and the present invention is not necessarily limited thereto.

In addition, the accompanying drawings may be expressed somewhat exaggerated compared to the thickness (or height) of the actual layer or the ratio with other layers to help understanding, and the meaning will be properly understood by the specific purpose of the related description to be described later. can

Terms used herein may be defined as follows.

"Binding or binding" may mean binding or linking to a surface in a covalent or non-covalent manner.

"Sample" is not limited to a specific type or form as long as it can contain the target pathogen to be detected. Illustratively, the sample may be a biological sample, for example, a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. A biological tissue is a collection of cells, usually a specific kind of collection of intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures, including connective tissue, epithelial tissue, muscle tissue, and nerve. This may be an organization or the like. Examples of biological tissue may also include organs, tumors, lymph nodes, arteries, and individual cell(s).

It may be understood that the expressions “on” and “on” are used to refer to the concept of relative position. Accordingly, not only the case where another component or layer is directly present in the mentioned layer, but also another layer (intermediate layer) or component may be interposed or present therebetween. Similarly, the expressions "under", "under" and "below" and the expressions "between" may also be understood as relative concepts with respect to the position. Also, the expression “sequentially” may be understood as a relative position concept.

Full disclosure

1A and 1B respectively illustrate the detection principle and implementation procedure of the exosome detection system based on the label-free polydiacetylene liposome according to one embodiment.

Referring to the drawings, as a detection platform for exosomes in a sample, poly-die acetylene liposomes that cause a color change in response to an external stimulus are generally used. In this case, poly- die acetylene is a polymer formed in the polymerization reaction of die acetylene, specifically conjugated. As a polymer, it is a material whose optical properties can be changed without a separate label. Polydiacetylene may be prepared in various shapes and structures such as crystals, films, and liposomes depending on the purpose of use. Poly die acetylene forms a stable structure having an inner space isolated by a lipid layer membrane (specifically a bilayer) in an aqueous solution state, which may be referred to as "poly die acetylene liposome".

The diacetylene monomer used for polydiacetylene synthesis maintains a sufficiently close distance in an aqueous solution by a non-polar bond. At this time, the triple bond is converted to a double bond under UV irradiation at 254 nm, and a specific site of the polymer backbone with other monomers. is polymerized in such a way that the It does not express color before irradiation with ultraviolet rays, but expresses blue with a maximum absorption wavelength of 640 nm after polymerization. Thereafter, the absorption spectrum is changed by external stimuli such as temperature, pH, solvent, and molecular recognition, so that color transition to red having a maximum absorption wavelength of 540 nm may occur.

In this embodiment, a receptor is introduced into polydiacetylene to impart specific binding properties or selectivity to a specific molecule (specifically, a biomarker present on the surface of an exosome, which is a mediator of diagnosis or drug delivery). Such a receptor may be a molecular group or a receptor capable of recognizing a specific substance, and by introducing it, it is possible to induce a change in unique optical properties due to the conjugated structure of polydiacetylene.

As such, the change in the optical properties of the polydiacetylene liposome can be measured with the naked eye or a measurement means or device commonly used in the art to qualitatively and/or quantitatively detect the exosomes in the sample.

poly die acetylene liposome Produce

1A and 1B , first, a lipid (specifically, a phospholipid) with a diacetylene monomer is combined in a solution (eg, an organic solvent such as chloroform, tetrahydrofuran, etc.) in a bar, self-assembly (self-assembly) ) can form lipid vesicles with an inner space isolated by a lipid layer membrane. As such, the charge stability and membrane flexibility of polydiacetylene (PDA) liposomes prepared in a subsequent step can be enhanced by combining phospholipids with diacetylene monomers.

According to an exemplary embodiment, the diacetylene monomer may be a compound containing a diacetylene group, for example, consisting of pentacosadiyonic acid (PCDA), trocosadiyonic acid (TCDA), heneicosadiynoic acid (HCDA) and heptadecadiynoic acid (HDDA). It may be at least one selected from the group. Specifically, the diacetylene monomer may be PCDA and/or TCDA, and more specifically PCDA. In this regard, in an exemplary embodiment, it may contain a terminal selected from at least one selected from a carboxyl group, an amine group, a sulfone group, and the like that allows a specific receptor to bind to PCDA, and may also contain an EDC/NHS coupling reaction, a sulfone- It may be advantageous to be able to easily attach the receptor to the diacetylene monomer through a maleimide binding reaction, an avidin-biotin binding reaction, or the like.

In addition, phospholipids are, for example, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl Glycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), Palmitoylstearoylphosphatidylcholine (PSPC), Palmitoylstearolphosphatidylglycerol (PSPG), Mono-oleoyl-phosphatidylethanolamine (MOPE), Dilauroyl Ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleate) Oiloxy)-3-(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylic acid (DMPA), dipalmitoylphosphatidylic acid (DPPA), distearoylphosphatidyl Acid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine ( DPPS), and may be at least one selected from the group consisting of distearoylphosphatidylserine (DSPS). Specifically, it may be at least one selected from the group consisting of DMPC and DMPA, and more specifically, may be DMPC.

In this regard, when inserting a phospholipid, it may affect the sensitivity of polydiacetylene (PDA), and it may be advantageous to combine it in an appropriate ratio in consideration of this. Illustratively, the molar ratio of diacetylene monomer to phospholipid may range, for example, from about 2 to 10:1, specifically from about 3 to 8:1, and more specifically from about 4 to 6:1. Illustratively, the temperature when the diacetylene monomer and the phospholipid are combined may be in the range of, for example, about 20 to 40° C., specifically, about 25 to 37° C., but this may be understood as illustrative.

According to an exemplary embodiment, after formation of the lipid vesicles, for example, a drying step (eg, under an inert gas atmosphere) may be performed, followed by addition of water and a solution (or dispersion) containing the vesicles. wherein the concentration (total concentration) of the lipid in the ER-containing solution is, for example, within the range of about 0.1 to 2 mM, specifically about 0.4 to 1.5 mM, more specifically about 0.5 to 1.2 mM. Adjustable. In addition, it is possible to induce the formation of uniform vesicles by optionally performing ultrasonic irradiation prior to the subsequent polymerization reaction. In this regard, when preparing a uniform endoplasmic reticulum-containing solution (or dispersion), the temperature may be controlled, for example, in the range of about 60 to 90° C., specifically about 70 to 80° C., but this is understood as illustrative can be

According to an exemplary embodiment, the size of the formed lipid vesicles (measured by dynamic light scattering (DLS) method) is, for example, about 50 to 500 nm, specifically about 100 to 300 nm, more specifically about 200 to 250 nm. range, which may be understood as illustrative.

Next, to modify the lipid ER by attaching (fixing) or conjugating a receptor having a specific binding ability to an exosome (specifically a target exosome), specifically, a biomarker on the surface of the exosome to the lipid ER. can

Alternatively, the surface of the lipid ER may be activated using a chemical functional group prior to attachment of the receptor to the lipid ER. Such a chemical functional group may be selected from various types capable of binding or coupling (eg, covalent bonding, etc.) to or binding to a receptor to be attached or bound to the surface of the lipid ER. Representatively, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC) can be used as the above-described chemical functional groups, and the receptor is immobilized on the lipid endoplasmic reticulum. or useful for bonding.

According to an exemplary embodiment, the receptor is typically at least selected from the whole antibody or a partial antibody containing an antigen binding region (F _ab , F _(ab′)2 , scFv, etc.), aptamers, and the like. can be one In this case, the aptamer can function as a nucleic acid-based receptor capable of receiving proteins such as CD-9, CD-63, CD-81, CD-82, which are biomarkers of exosomes. More typically, it may be an antibody and/or an aptamer.

Meanwhile, according to an exemplary embodiment, the receptor may be a single receptor including a biomarker receptor specific or corresponding to a specific biomarker among exosomes.

According to an alternative embodiment, the receptor may be a multi-receptor comprising two or more various biomarker receptors that correspond to or are specific for a plurality of biomarkers in exosomes. To this end, it may include attaching any one of the multiple receptors to the lipid endoplasmic reticulum, and then attaching the others sequentially. As such, when multiple receptors are introduced, polydiacetylene liposomes modified with multiple receptors prepared in a subsequent procedure not only exhibit specificity for exosomes in a mixed solution, but also induce a more efficient ligand-receptor response to measure signal Because it can amplify , it may be advantageous compared to the case where a single receptor is applied. However, the scope of receptors applicable to embodiments of the present disclosure may be understood as a concept including both single receptors and multiple receptors.

According to an exemplary embodiment, the modified or fixed amount of the receptor (i.e., single receptor or multiple receptor) is from about 0.0001 to 0.05, specifically from about 0.0005 to 0.01, or more, of the total molar concentration of diacetylene and phospholipids constituting the lipid endoplasmic reticulum. Specifically, it may be determined in the range of about 0.001 to 0.005, but this may be understood as an example.

According to an exemplary embodiment, after formation of the modified lipid vesicles, a polymerization reaction may be performed to form a modified polydiacetylene liposome (ie, a polydiacetylene (PDA) liposome complex). In this case, the polymerization reaction may be a photopolymerization reaction by ultraviolet irradiation or a polymerization reaction by gamma-ray irradiation, and more specifically, a polymerization reaction using ultraviolet irradiation.

According to a specific embodiment, the polymerization reaction may be a photopolymerization reaction by ultraviolet irradiation, the irradiated ultraviolet wavelength is, for example, about 100 to 400 nm, specifically about 150 to 350 nm, more specifically about 200 to It may have a wavelength in the range of 300 nm. In addition, the UV intensity may be controlled within, for example, about 200 to 600 μW/cm 2 , specifically about 300 to 500 μW/cm 2 , and more specifically about 350 to 450 μW/cm 2 . In addition, the ultraviolet irradiation time, for example, may be selected within the range of about 10 to 60 minutes, specifically about 20 to 50 minutes, more specifically about 25 to 40 minutes. However, the polymerization conditions by such ultraviolet irradiation may be understood as illustrative.

The size (measured by a dynamic light scattering (DLS) method) of the polydiacetylene (or modified polydiacetylene) liposome thus prepared is, for example, about 100 to 300 nm, specifically about 120 to 200 nm, more specifically It may be in the range of about 130 to 160 nm, but this may be understood as an example. As an example, the size of the polydiacetylene liposomes modified with the receptor and formed through polymerization is, for example, at least about 10%, specifically at least about 12%, more specifically about 15 to 20% compared to the lipid vesicles before modification. It may have an increased size.

In addition, the poly-die acetylene liposome modified by the receptor may exhibit a different charge than that of the unmodified case, and when it is not modified, the poly-die acetylene liposome is relatively free of charge by a functional group (eg, a carboxyl group) attached to the monomer of the poly- die acetylene. It may exhibit a large negative charge (eg, about -30 to -20 mV), while in its acceptor-modified form it may exhibit a relatively small negative charge (eg, about -10 to 0 mV).

poly die acetylene liposome used exosomes detection

According to one embodiment of the present disclosure, it is possible to detect exosomes contained in a sample, specifically, an aqueous sample using a polydiacetylene liposome.

Although the present disclosure is not bound by any particular theory, the above-described optical property changes can be explained as follows.

As the biomarker present on the surface of the exosome and the receptor immobilized on polydiacetylene specifically bind or bind (ie, ligand-receptor interaction), a physical stimulus can be applied to the surface of the polydiacetylene polymer. This physical stimulus is transmitted to the main chain of the solid-liquid polymer, and the conjugated chain is twisted to cause a change in the absorption spectrum and fluorescence. In this case, larger clusters can be formed by inducing a chain reaction through ligand-receptor interaction.

According to an exemplary embodiment, the size of the cluster formed by the binding of the exosome and the modified polydiacetylene liposome is, for example, about 200 to 3000 nm, specifically about 300 to 1500 nm, more specifically about 500 to 1000 It may be in the nm range, and compared to the size of the PDA liposome before binding to the exosome, for example, it may be at least about 50%, specifically about 100 to 1000%, more specifically about 300 to 500% more increased. . However, the present disclosure is not limited to this numerical range, and may be changed according to the properties, types, etc. of the exosomes.

According to an exemplary embodiment, the biomarker on the exosome may typically be a tetraspanin protein, the tetraspanin protein being selected from the group consisting of CD-9, CD-63, CD-81 and CD-82. It may be at least one selected from These biomarkers correspond to ligands, and can be specifically bound to a receptor immobilized on the modified polydiacetylene liposome through a ligand-receptor reaction.

As described above, the receptor may be a single receptor or multiple receptors, and when fixed in the form of multiple receptors, more ligands between polydiacetylene liposomes and exosomes by inducing binding to a plurality of biomarkers present in exosomes It can provide the effect of amplifying the signal by inducing the receptor response to occur.

This specific reaction can typically be made in an aqueous medium, and as an example, a modified polydiacetylene liposome aqueous solution and a sample (ie, an exosome-containing aqueous solution) can be prepared and combined, respectively. According to an exemplary embodiment, the concentration of the modified polydiacetylene liposome aqueous solution is, for example, in the range of about 0.05 to 0.5 mg/mL, specifically about 0.1 to 0.4 mg/mL, more specifically about 0.2 to 0.3 mg/mL. can be adjusted within In addition, the temperature and time can be appropriately adjusted so that the ligans-receptor reaction can occur effectively, for example, the temperature is about 25 to 50° C., specifically about 30 to 45° C., more specifically about 35 to 40° C. The time may be adjusted within the range, for example, about 10 to 60 minutes, specifically about 15 to 50 minutes, more specifically about 20 to 40 minutes. However, the above-described temperature and time conditions are provided by way of example, and the present disclosure is not limited thereto.

However, when the sample contains a significant amount of residues (debris, flakes, etc.) in the sample, such as when the sample is human fluid, specifically blood, only exosomes can be selectively isolated prior to detection. This separation process may be performed, for example, by centrifugation, filter, or the like.

As described above, in the exosome detection method or system according to this embodiment, exosomes present in the sample can be detected or detected with high selectivity by adding the modified polydiacetylene solution to the sample body fluid, liquid mixture solution, etc. . Alternatively, the sample may be an exosome solution in which the separation process has been performed.

In addition, it is useful as an exosome sensor because it can provide a simple and intuitive signal compared to a chip-based system, which is a conventional exosome detection technology. In particular, the presence or absence of exosomes (qualitative analysis) can be confirmed by measuring the color change and/or fluorescence expressed by simply mixing in aqueous solution, and furthermore, the concentration of exosomes in the sample (quantitative analysis) can be predicted, etc. , it is simple and provides good reliability. As an example, in applying the above-described exosome detection method, the detection limit of exosomes in the sample (ie, exosome-containing solution) is, for example, about 1×10 ¹² vesicles/mL or less, specifically about 1× It may be 10 ¹¹ vesicles/mL or less, and more specifically, about 3×10 ⁸ vesicles/mL or less.

According to an alternative embodiment, qualitative/quantitative analysis of the sample may be performed by contacting the sample (or exosome-containing solution) while the modified polydiacetylene ER is attached or fixed to the substrate. In this regard, the substrate is not particularly limited as long as it can detect a signal generated from a ligand-receptor reaction, and may be, for example, a material such as silicon, quartz, glass, ceramic, transparent plastic, etc., specifically It may be a slide glass.

As such, the prepared poly-diacetylene liposome is advantageous in terms of expandability as it can be applied in various forms by attaching to a substrate of various materials and structures depending on the use.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are only provided for easier understanding of the present invention, and the present invention is not limited thereto.

Example

The materials used in this example are as follows:

- 10,12-pentacosadiyonic acid (PCDA) was purchased from GFS Chemicals (Powell, OH).

- 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were purchased from Tokyo Chemical Industry (Tokyo, Japan).

- N-hydroxysuccinimide (NHS) and ethanolamine were purchased from Sigma-Aldrich (St. Louis, MO).

- Monoclonal antibodies (Anti CD-9, CD-63, CD-81 and CD-82) were purchased from Abcam (Cambridge, U.K.).

-PBS buffer (pH 7.4) was manufactured by Welgene, Inc. (Gyeongsan-si, Korea).

Example 1

Detection of polydiacetylene exosomes with a single receptor

By following the procedure shown in FIG. 1b, poly-diacetylene liposomes introduced with a single receptor were prepared, and exosomes were detected using a poly-diacetylene/phospholipid vesicle (liposome) exosome sensor dispersed in a solution.

Preparation of PCDA/DMPC lipid vesicles (liposomes)

A solution obtained by dissolving PCDA and DMPC in chloroform was put into a glass vial and mixed well to adjust the molar ratio of PCDA:DMPC to 4:1, and then a thin lipid layer was formed by evaporating chloroform in a nitrogen atmosphere flow. (lipid endoplasmic reticulum).

After the lipid layer was completely dried, distilled water was added to adjust the lipid concentration to 1.0 mM, and the temperature was raised by bathing at a temperature of 80° C. for 5 minutes. Using a tip sonicator, ultrasonic waves were irradiated with an output of 150 W for 20 minutes. By doing so, the lipid in the form of a film was uniformly dispersed in the aqueous medium. The lipid vesicle solution, which was dispersed using ultrasound, was immediately filtered with a syringe filter having a size of 0.45 μm. After cooling the filtered aqueous solution at room temperature, it was left in a refrigerator at 4° C. for at least 6 hours to form PCDA/DMPC lipid vesicles (liposomes).

The SEM photograph of the prepared PCDA/DMPC endoplasmic reticulum (liposome) is shown in FIG. 3 . According to the figure, it can be seen that the lipid vesicles form a crystalline phase.

- Modified PCDA/DMPC lipid vesicles (liposomes) preparation

The EDC/NHS concentration was adjusted to 100 mM by adding EDC/NHS to the chilled PCDA/DMPC lipid vesicles, and then reacted at room temperature for 2 hours to NHS-activated the lipid vesicles. The activated liposome was centrifuged at 25000 g at 4 °C for 15 minutes to remove the supernatant, and distilled water was again injected in the same amount to the precipitate to redisperse. The same procedure was repeated 3 times, and the third solution was filled with 5 volumes of PBS instead of distilled water. 100 uL of anti-CD-63 antibody at a concentration of 1 mg/mL was added to 1 mL of lipid vesicle solution to adjust the antibody concentration to 0.1 mg/mL.

Antibodies were introduced into NHS-activated lipid vesicles while stirring in a refrigerator at 4°C for 4 hours.

The antibody-introduced lipid ER solution was adjusted to a concentration of 2 mM by adding an ethanolamine-PBS solution, followed by stirring at 4° C. for 2 hours to inactivate the remaining NHS-activated lipid ER.

The solution thus obtained was centrifuged at 20000 g at 4 °C for 15 minutes to remove the supernatant, and the remaining antibody and ethanolamine were repeated three times by adding PBS in the same amount to the precipitate and redispersing again. was removed. The SEM photograph of the lipid vesicle into which the antibody was introduced is shown in FIG. As shown in the figure, it can be confirmed that the receptor, the antibody, was uniformly fixed on the lipid endoplasmic reticulum.

- Preparation of polydiacetylene (or modified polydiacetylene) liposomes

On the other hand, before adding the exosomes, PDA liposomes were prepared by irradiating the modified lipid endoplasmic reticulum with ultraviolet light at a wavelength of 254 nm with an intensity of 400 μW/cm 2 for 30 minutes until it turned blue.

- Detection of exosomes in samples

The blue PDA liposome solution (concentration: 0.4 mg/mL, 1.0 mM) and the exosome solution to be measured (concentration: 3×10 ⁸ vesicles/mL or more) were mixed in the same volume, and then left at 37° C. for 30 minutes. The ligand-receptor reaction was maximized between the exosome and the polydiacetylene liposome. The SEM image of the aggregate formed by the reaction between the PDA liposome and the exosome is shown in FIG. 5 . As shown, the exosomes are firmly fixed between the PDA liposome particles.

- Absorbance analysis

Using UV-Vis spectroscopy, the absorbance at 540 nm and 640 nm of the liposome solution to which PBS was added and the liposome solution to which the exosome solution was added were measured and compared.

The color change (%) was calculated by substituting the absorbance at 540 nm and 640 nm using Equation 1 below.

[Equation 1]

In addition, the result of measuring the UV-Vis absorption spectrum while changing the exosome concentration is shown in FIG. 6 . According to the figure, it can be seen that the absorbance in the long wavelength band (red band) increases as the concentration of exosomes in the sample increases.

The above results suggest that a PDA ER cluster was formed by ligand-receptor interaction between an antibody, which is a receptor conjugated on PDA, and an exosome, and as a result, a color change of the PDA ER was induced.

- Fluorescence intensity analysis

The fluorescence intensity of polydiacetylene was measured using a fluorescence microscope from ZEISS. Specifically, the fluorescence intensity of the liposome solution to which PBS was added and the liposome mixed solution to which the exosome solution was added was measured at an excitation wavelength of 500 nm and fluorescence intensity around 640 nm, and fluorescence intensity was subtracted from the control (PBS solution). The change in intensity was measured. As a result, it was confirmed that the fluorescence intensity in the liposome solution to which the exosome solution was added was significantly higher than that of the control.

Example 2

Exosomes were detected by preparing polydiacetylene liposomes into which multiple receptors were introduced according to the procedure shown in FIG. 2 .

Preparation of polydiacetylene (PDA) liposomes

- PCDA/DMPC lipid vesicles (liposomes) preparation

A solution obtained by dissolving PCDA and DMPC in tetrahydrofuran was injected into a vial containing distilled water with a syringe to prepare a diacetylene suspension (PCDA:DMPC molar ratio was adjusted to 4:1). At this time, the total concentration of PCDA and phospholipid was adjusted to be 1.0 mM.

The prepared lipid vesicle suspension was irradiated with ultrasound at an output of 150W for 15 minutes to form lipid vesicles (liposomes) and homogenization was performed. Immediately after the ultrasonic dispersion was completed, the lipid vesicle solution was filtered with a syringe filter having a size of 0.45 μm. After cooling the filtered aqueous solution at room temperature, it was left in a refrigerator at 4° C. for at least 6 hours to form PCDA/DMPC lipid vesicles (liposomes).

Modified PCDA/DMPC lipid vesicles (liposomes) preparation

EDC/NHS was added to cold-cooled PCDA/DMPC lipid vesicles to adjust the total concentration to 100 mM, and then reacted at room temperature for 2 hours to NHS-activated lipid vesicles. The activated liposome was centrifuged at 4 °C at 25000 x g for 15 minutes to remove the supernatant, and distilled water was again injected in the same amount to the precipitate to redisperse. The same procedure was repeated 3 times, and the third solution was filled with 5 volumes of PBS instead of distilled water. An antibody solution having specific binding ability to CD-9, CD-63, CD-81 and CD-82 was added to 1 mL of the activated lipid endoplasmic reticulum solution. At this time, the total concentration of the added antibody was adjusted to be 10 uM. The concentration ratio of each antibody was equally added according to the number of types of the antibody solution to be added.

Residual NHS-activated lipid vesicles were inactivated by adding 2 mM ethanolamine-PBS to the antibody-introduced lipid ER solution or by stirring at 1 mg/mL BSA at 4° C. for 2 hours.

The solution thus obtained was centrifuged at 20000 g at 4 °C for 15 minutes to remove the supernatant, and the remaining antibody and ethanolamine were repeated three times by adding PBS in the same amount to the precipitate and redispersing again. was removed.

Thereafter, polydiacetylene (or modified polydiacetylene) liposomes were prepared according to the same procedure as in Example 1.

Detection of exosomes in samples

After mixing the solution containing the PDA liposome prepared above and the exosome solution used in Example 1 in the same volume, it was left at 37° C. for 30 minutes to maximize the ligand-receptor reaction between the exosome and the polydiacetylene liposome. did. After adding the exosomes, a transmission electron microscope (JEOL product name: JEM-3010) photograph of the PDA liposome is shown in FIG. 7 .

According to the figure, as the exosome was added to the PDA liposome, an aggregate was formed by mutual binding due to the interaction between tetraspanin and multiple receptors on the surface of the exosome.

Meanwhile, in order to confirm the selectivity of the receptor-introduced PDA liposome to the exosome, the same experiment was performed for each albumin protein and fibrinogen, which are abundantly present in body fluid or plasma, and analyzed using a transmission electron microscope. The results are shown in FIG. 8 (left figure: albumin protein, right figure: fibrinogen). According to the figure, it was confirmed that, unlike the case of adding exosomes, no aggregates were formed.

In addition, when the exosome solution was added to the solution containing the PDA liposome, the color change of the solution was visually observed. The results are shown in FIG. 9 .

As can be seen from the figure, the color of the solution changed from blue to pale purple, which is a ligand-receptor interaction between multiple receptors conjugated on PDA liposomes and tetraspanin on the surface of exosomes in PDA. This indicates that a structural change occurred and affected the solution color.

Example 3

In this embodiment, the exosomes were detected using a sensor modified with a single receptor and multiple receptors based on a sensor in which a polydiacetylene/phospholipid endoplasmic reticulum (liposome) exosome was immobilized on a substrate.

Specifically, a solution containing an antibody having specific binding ability to CD-63 as a receptor (single receptor), an antibody having specific binding ability to CD-81 (single receptor), and a combination thereof (multi receptor), respectively Except for use, a modified PCDA/DMPC lipid vesicle (liposome) was prepared according to the same procedure as in the previous example. Then, a modified liposome array of about 0.3 mm (using Labnext's manual microarrayer) was formed on a slide glass (substrate) coated with amine in a thermo-hygrostat where humidity of 70% or more was maintained at room temperature. After the formation of the array, the reaction was carried out at room temperature for about 4 hours. The substrate to which the polydiacetylene endoplasmic reticulum was attached was washed with 0.1% TWEEN 20 solution, washed twice with distilled water, dried to remove moisture from the surface, and the exosome solution was dripped onto each array part. did. After 1 hour, the fluorescence intensity of the polydiacetylene ER was measured with a fluorescence microscope. The results are shown in FIGS. 10 and 11, respectively.

10 is a fluorescence photograph taken before and after the addition of the exosome solution to the poly-diacetylene vesicles attached to the substrate.

According to the figure, the modified poly-die acetylene ER shows fluorescence due to ligand-receptor interaction when the exosome-containing sample is contacted while attached to the substrate, and shows low fluorescence before injection. It was confirmed that the fluorescence gradually increased by interaction with the exosomes.

11 is a graph showing the change in fluorescence intensity with time when the poly-diacetylene/phospholipid vesicle according to the receptor introduced into the poly-diacetylene ER is in contact with the exosome-containing sample.

Referring to the figure, rather than introducing a single receptor (antibodies having specific binding ability to CD-63 or CD-81), a plurality of receptors (antibodies having specific binding ability to CD-63 and CD-81) It was confirmed that a higher fluorescence signal can be obtained from the poly-diacetylene endoplasmic reticulum introduced (a combination of antibodies having a specific binding ability with respect to). This indicates that more receptor binding sites can be secured in the sample of the same exosome concentration through the introduction of a plurality of receptors.

Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (15)

a) providing a poly-diacetylene liposome modified with a receptor having a specific binding ability with respect to a biomarker present on the surface of the exosome; and
b) measuring the change in optical properties induced by the ligand-receptor reaction between the exosome and the modified polydiacetylene liposome in the sample as the modified polydiacetylene liposome is in contact with the exosome-containing sample;
A method of detecting exosomes comprising a. According to claim 1, wherein the step a),
a1) self-combining diacetylene monomer and phospholipid to form lipid vesicles having an interior space isolated by a lipid layer membrane;
a2) attaching the receptor to the lipid ER to form a modified lipid ER; and
a3) polymerizing the modified lipid endoplasmic reticulum to form a modified polydiacetylene liposome;
A method of detecting exosomes comprising a. The method of claim 2, wherein at least one biomarker is present on the surface of the exosome, and the receptor is a single receptor specific for the at least one biomarker. The method of claim 2, wherein a plurality of biomarkers are present on the surface of the exosome, and the receptor is a multiple receptor specific for the plurality of biomarkers. The method of claim 4, wherein the receptor is a multi-receptor, wherein step a2) comprises attaching any one of the multi-receptors to the lipid endoplasmic reticulum and then attaching the rest sequentially. detection method. The method according to claim 2, wherein the diacetylene monomer is at least one selected from the group consisting of pentacosadiyonic acid (PCDA), trocosadiyonic acid (TCDA), heneicosadiynoic acid (HCDA), and heptadecadiynoic acid (HDDA), and
The phospholipids include dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroyl ethylphosphocholine ( DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleoyloxy)-3 -(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylic acid (DMPA), dipalmitoylphosphatidylic acid (DPPA), distearoylphosphatidylic acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylinositol (DMPS), dipalmitoylphosphatidylserine (DPPS), and dis A method of detecting exosomes, characterized in that at least one selected from the group consisting of thearoylphosphatidylserine (DSPS). The method according to claim 2, wherein the molar ratio of diacetylene monomer to phospholipid in step a1) is in the range of 2 to 10: 1. The method of claim 2, further comprising activating the lipid ER with a chemical functional group prior to step a2). The method according to claim 1, wherein the receptor is at least one selected from the group consisting of the whole antibody or a partial antibody containing an antigen binding region, and an aptamer. The method of claim 1, wherein the biomarker is a tetraspanin protein. The method of claim 10, wherein the tetraspanin protein is at least one selected from the group consisting of CD-9, CD-63, CD-81 and CD-82. The method according to claim 1, wherein the exosome-containing aqueous sample is a body fluid or a liquid from which the residue is previously separated. The method of claim 1, wherein the optical property change is measured by the naked eye or a measuring device. The method of claim 13, wherein the measuring device for the change in optical properties is a UV-Vis spectrometer or a fluorescence spectrometer. The method of claim 1, wherein the method is performed without labeling.

Images