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Exosomes, one of the extracellular vesicles (EVs), have been attracted significant interests due to its biological importance in that they play a substantial role in intercellular communication and contain various biomolecules including proteins, genetic molecules. However, due to their difficulties in effective capture and detection, further application of exosomes has been challenged. For detection of EVs, we fabricated liposomal biosensor based on the polydiacetylene (PDA) which is a conjugate polymer that has been widely used to sensing applications based on its unique optical properties.
To confer the selectivity and sensitivity to the sensory material, the CD9 antibodies targeting CD9 which is a membrane protein exclusively in the exosomes are attached to the PDA liposomes and phospholipid molecules are incorporated to the PDA vesicles.
Signal analysis derived from PDA liposome for detection and quantification of exosomes is performed by observing colorimetric change triggered by the ligand-receptor interaction of PDA vesicles. Visually, UV-visible, and fluorescence spectroscopic methods were used to obatain signal from PDA lipid immune-sensor, achieving a detection limit of 3??10?^8 vesicles/ml, minimum concentration that can be used in practical applications.
The strategies used in the system can be applied to various biomolecule detections in short time.
As the demand for molecular sensing has been increased, conjugated polymer has been attracted in sensing applications. Polydiacetylene (PDA) is one of the versatile materials that has been utilized in various field of sensor applications due to its unique optical properties derived from ene-yne conjugated backbone in their structure1-2. The conjugated backbone of PDA can be formed via 1,4-addition reaction of the diacetylene (DA) by ultraviolet or gamma irradiation to self-assembled diacetylene (DA).
The resulting overlapping p-orbital derived from the polymerization reaction of well-ordered DA absorbs red region of visible light, resulting in blue color.
When the backbone of PDA formed by aligned monomer is disturbed by external stimuli, the energy gap between HOMO and LUMO of overlapped p-orbital increase, thereby the absorption pattern of conjugated backbone changes to blue transition, resulting in blue to red colorimetric change. In addition to color change, the distorted backbone of PDA also exhibits fluorescence, thereby the optical properties of the PDA can be characterized by changes in the absorption spectrum and intensity of fluorescence.
There are several reasons why PDAs are attracting much attention as sensing applications. Firstly, due to self-assembly, aligned monomers can be made into various shapes such as crystals, films, filaments, vesicles and It is possible to easily make polymerization by irradiating UV at 245nm without adding the UV absorber, which can be produced a high purity sensing material.
Second, because of the many factors such as pH, temperature, solvent, electrical stress, mechanical stress, ligand-receptor interaction, etc, which cause optical characteristics of the PDA required for the sensing process. Another advantage is that PDA is one of the label-free materials that do not require additional labeling step, as they will change their color or emit fluorescence, unlike a decent number of sensing methods.
Exosomes are a class of cell-derived vesicles with a size of tens to 100 nm that are released by the exocytosis of multi-vesicular bodies(MVBs). Before the release of MVB, the MVB inside the cell is once again implanted leading to formation of small vesicles composed of substances such as proteins, DNA, and RNA originated from the cytoplasm. The exosomes produced by MVB is released to the outside of the plasma membrane by exocytosis and circulates in the body fluid to perform intercellular communication. Since exosomes transmit various biomaterials, exosomes have been used as an important tool for diagnosis or treatment of various diseases.
However, due to small size, low density, scarcity ( of them, current methods of selective isolation and screening of exosomes require highly sophisticated equipment and complex experimental procedures, thereby their better applications are becoming challenged, and other devices system that require simple instrumentation and experimental procedures is demanding. Due to the nature of exosome presented above, research on a system capable of detecting exosomes in an facile and quick manner has not been sufficiently conducted. In this paper, a label-free PDA/phospholipid composite immuno-sensor was constructed by introducing antibody against CD-63 protein, an exosome marker protein, to give selective exosome detection.
The diacetylene monomer used is 10,12-pentacosadionic acid (PCDA) purchased from GFS chemicals (Powell, OH, USA). N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were obtained from Tokyo Chemical Industry (Tokyo, Japan).
Ethanolamine, N-hydroxysuccinimide (NHS) and Fibrinogen from human plasma were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti CD-63 monoclonal antibody [TS63] was purchased from Abcam (Cambridge, UK) and the exosome solution was made from lyophilized exosomes from human plasma purchased from HansaBioMed Life Science (Tallin, Estonia).
Bovine serum albumin (BSA) was obtained from MP Biomedicals (Illkirch, France). Phosphate-buffered saline buffer (PBS) was purchased from Welgene Inc. ( Gyeongsan-si, Korea). All organic solvents used were obtained from Samchun chemicals (Seoul, Korea).
The procedure for preparing a polydiacetylene / phospholipid composite sensor was performed in the order described in scheme 1. PCDA and DMPC were individually dissolved in chloroform in glass vials and the solutions were blended at a 4:1 molar ratio of PCDA and DMPC.
The mixture solution was slowly dried under gentle nitrogen flow and distilled water was added to the thin layer for a total lipid concentration of 1.0mM. For a smaller and homogenous vesicle solution, the lipid solution was sonicated with a probe sonicator (Sonic&materials, CT, USA) with 150W for 20min at 80?, resulting in a translucent cloudy suspension.
After sonication, the vesicle solution was immediately filtered using a disposable 0.45-?m syringe filter (Advantec, Japan) to remove aggregated material or large particles. Filtered solution was slowly cooled down at room temperature and stored at 4? for crystallization of lipid vesicles at least for 6h.
The preparation step of polydiacetylene / phospholipid composite sensor is illustrated in scheme 1. EDC and NHS solution dissolved in DIwater in a separate state were added to 1ml of vesicle solution for the concentration of EDC and NHS 100mM each and the NHS activation reaction was carried out for 2h at room temperature. For removal of EDC/NHS, the NHS activated vesicles were precipitated using a centrifuge (Gyrozen, Korea) at 20,000g for 20min at 4? and the supernatant was removed followed by resuspension of vesicles in PBS solution.
Anti-CD63 monoclonal antibody [1.0mg/ml] was added to the activated vesicle solution for 2h at room temperature and ethanolamine solution was added to the solution in order to deactivate the remaining NHS activated vesicles for a final concentration of ethanolamine 2.0mM.
The Antibody conjugated vesicle solution was centrifuged at 20,000g for 15min at 4? and washed with PBS three times. Prior to use, PCDA/phospholipid vesicles were polymerized by 254nm UV irradiation at 400?W/cm2 for 15min using UV ramp (Vilber, france), obtaining a dark blue-colored polymerized PDA vesicle solution.
The size and morphology of the PCDA/DMPC vesicle were characterized with high resolution transmission electron microscope JEM-3010 (JEOL, Japan) at 300kV of acceleration voltage. The TEM samples were prepared by dropping diluted vesicle solution onto a copper grid(300mesh) covered with carbon film and drying the samples in a desiccator. In addition, dynamic light scattering system Zetasizer nano ZS (Malvern Instruments, Malvern, UK) was used to confirm the size distribution and surface charge of PDA vesicles. The DLS measurement was performed after dilution to about 0.05mM.
The exosome separation was separated by a conventional method using analytical ultracentrifuge (ProteomeLab XL, Beckman, USA). Human plasma was centrifuged at 3,000g for 30min to remove remaining massive cell flake and pellet. The supernatant was ultracentrifuged at 170,000g for 90min at 4? leading to formation of exosome pellet. The supernatant was removed, and the pellet was re-dispersed in PBS thoroughly.
The same procedure was repeated to conduct ultracentrifugation at 170,000 g for 90 min at 4 ° C, and the resulting exosomal pellet was re-dispersed in PBS. Characterization and quantification of exosome solution were performed by TEM, DLS, and Nanoparticle Tracking Assay (NTA).
TEM image and size distribution of isolated exosome were obtained by the same apparatus and method as the PDA liposome, and NTA was performed by measuring 100-fold diluted exosome solution with NanoSight LM10 (Malvern Instruments, Malvern, UK).
Different concentration of exosome solution (30?l) was injected to PDA vesicle solution (30?l) and incubated at 37? for 30min. UV-visible and fluorescence spectrum were recorded to measure colorimetric change and intensity of fluorescence using microplate reader SYNERGY H1 (BioTek, USA). The quantification of blue to red color change of PDA vesicles interacting with exosomes, the colorimetric response (CR) was introduced as follows
CR (%)= ((PB_bef-PB_aft ))/(PB_bef ) ?100 , PB= A_blue/(A_blue+A_red )
Where, A is relative absorbance of the blue or red element in UV-vis spectrum, 640nm or 540nm respectively, ?PB?_bef is the relative ratio of blue and red element before exosome insertion, and ?PB?_aft is the relative value of blue and red after inserting the exosome.
The fluorescence spectra of the PDA vesicles were measured at 480 nm excitation wavelength and the maximum value of the sample was used. The final fluorescence intensity was obtained by subtracting the base intensity of the control sample.
Characterization of PCDA/DMPC liposome composite: The PCDA/DMPC liposome composite was successfully prepared and characterized. Transmission electron microscopy (TEM) analysis revealed the size and morphology of the vesicles. The vesicles exhibited a spherical shape with an average size of approximately 100 nm. Dynamic light scattering (DLS) confirmed the size distribution and surface charge of the PDA vesicles, further supporting the TEM results.
Isolation and quantification of exosomes: Exosomes were isolated from human plasma using ultracentrifugation. TEM analysis of the isolated exosomes showed typical cup-shaped morphology, confirming their identity (Figure 2). DLS and nanoparticle tracking assay (NTA) were used to determine the size distribution and concentration of the isolated exosomes. The results indicated that the isolated exosomes had an average size of approximately 80 nm and a concentration of 3.2 × 10^9 vesicles/ml.
Spectrophotometric and fluorescence analysis of PCDA/DMPC liposome composite: To assess the performance of the PCDA/DMPC liposome composite as a sensor for exosome detection, different concentrations of exosome solution were incubated with the PDA vesicles. UV-visible spectroscopy and fluorescence spectroscopy were used to measure the colorimetric change and fluorescence intensity, respectively.
The colorimetric change, represented as the colorimetric response (CR), was calculated based on the relative absorbance at 640 nm (blue) and 540 nm (red) in the UV-vis spectrum. The CR values increased with increasing exosome concentration, indicating a shift from blue to red color. The CR (%) was determined as described in the Materials and Methods section.
Fluorescence spectroscopy revealed an increase in fluorescence intensity upon the interaction between PDA vesicles and exosomes. The fluorescence spectra showed a peak at 480 nm, and the fluorescence intensity increased as the concentration of exosomes increased.
The successful fabrication of the PCDA/DMPC liposome composite and its application as a sensor for exosome detection is a significant achievement. The characterization of the composite demonstrated its suitability for this purpose, with well-defined vesicles of appropriate size and morphology. The use of TEM, DLS, and NTA allowed for a comprehensive analysis of both the PDA vesicles and the isolated exosomes.
The ability to detect and quantify exosomes is of great importance in various fields, including diagnostics and therapeutics. The PCDA/DMPC liposome composite offers a label-free and rapid method for exosome detection. The colorimetric change and fluorescence intensity increase observed upon interaction with exosomes provide a clear indication of their presence and concentration.
The CR values obtained from the UV-vis spectra indicate that the PCDA/DMPC liposome composite can detect exosomes at a concentration as low as 3.0 × 10^8 vesicles/ml. This level of sensitivity is promising for practical applications, as it allows for the detection of exosomes in biological samples with relatively low exosome concentrations.
The selective detection of exosomes was achieved by conjugating anti-CD63 monoclonal antibodies to the PDA vesicles. CD63 is a known exosome marker protein, and its presence on the vesicles ensures specific binding to exosomes, enhancing the sensor's selectivity.
Overall, the PCDA/DMPC liposome composite-based sensor demonstrates its potential for the rapid and sensitive detection of exosomes. This approach could be valuable in various biomedical applications, including disease diagnosis and monitoring, as well as in fundamental research on exosome biology.
In this study, a label-free polydiacetylene/phospholipid composite sensor was successfully developed for the detection and quantification of exosomes. The sensor exhibited high sensitivity, with a detection limit of 3.0 × 10^8 vesicles/ml, making it suitable for practical applications. The selective detection of exosomes was achieved by conjugating anti-CD63 monoclonal antibodies to the sensor, ensuring specific binding to exosomes.
The PCDA/DMPC liposome composite-based sensor offers a rapid and reliable method for exosome detection, which has important implications for various biomedical fields, including diagnostics and therapeutics. This sensor has the potential to contribute to the advancement of exosome research and its applications in the diagnosis and treatment of diseases.
Further studies could focus on optimizing the sensor's performance, expanding its applicability to different types of exosomes, and exploring its potential for use in clinical settings. Additionally, the development of user-friendly and portable devices based on this sensor could facilitate its widespread adoption in various healthcare and research settings.
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