Exosomes have been involved in a variety of fields, including drug delivery and disease diagnosis, yet there is still a shortage of exosome detection tools (Huda et al. 2021; Zhu et al. 2022). Exosomes are naturally cell-secreted signaling molecules with a size range of 20–150 nm comprising a diverse array of biomolecules like as DNA, RNA, proteins, mRNA, microRNA, long noncoding RNA, circular RNA (Zhang et al. 2019; Gurunathan et al. 2021; Gurung et al. 2021). There are several technologies available for isolating exosomes from various biological fluids, including ultracentrifugation, chromatography, and ultrafiltration, as well as a few commercial kits, but each has its own limitations (Pammi et al. 2022). The current work describes a gold nanoparticle (AuNPs) based rapid and successful strategy for exosome detection in human serum utilizing anti-exosomal tetraspanin antibodies. Exosomes, have various surface tetraspanin markers, but CD9, CD63, and CD81 are reported as dominantly expressed and mostly utilized for exosome isolation and purifications (Kowal et al. 2016; Khushman et al. 2017). However, their expression varies depending on the parent cell type, with CD63 being the most addressed as a highly expressed one (Khushman et al. 2017). As a result, three tetraspanins, CD9, CD63, and CD81, have been chosen to raise antibodies for exosome capture. The extracellular region of all three tetraspanin surface markers was adopted for antibody production. Correspondingly, the sequence was codon-optimized (Table.2), cloned in the PET 28a plasmid by Gene Universal, and expressed in the BL21 codon plus strain of E. coli. In a study, it was described a variety of expression strains as well as how BL21-Codon Plus (DE3) produced the highest protein yield (Assadi et al. 2008; Robichon et al. 2011).
The extracellular region of CD9, CD63, and CD81 tetraspanin has been retrieved online from NCBI, which provides a wide range of online resources for true biological information and data from 38 distinct databases, including a gene bank, a nucleic acid sequence, a PubMed database, Bio Project, BLAST databases, and so on (Wheeler et al. 2005). Furthermore, the purity of three tetraspanins- exosomes surface marker as well as its targeted antibodies was characterized through SDS-PAGE. The following SDS-PAGE for antibodies revealed 80–90% purity and two distinct bands of roughly 68kDa and 28kDa, whereas refolded antigenic extracellular protein revealed 70–80% purity.
Moreover, IgY concentration in egg yolk elevated throughout the immunization span until week 6, when it started to rise rapidly at 2 weeks and stagnated at 4 weeks. As a result, it is possible to conclude that the chicken's immune system takes approximately 2 weeks to give a boost in raising antibodies (Sudjarwo et al. 2017). However, a decrease in titer has been reported in the case of chickens about after 6 weeks of immunization. Even so, a significant decline in titer has been revealed in chickens approximately 6 weeks after immunization (Sudjarwo et al. 2017). Additionally, it was demonstrated that chickens immunized intramuscularly with the antigen, can raise targeted antibodies for more than 200 days, and also provides a high titer and tenfold greater specificity than subcutaneous injection (Sudjarwo et al. 2017). In this article, we demonstrate that a stagnant phase of antibodies titration has arrived after the third immunization as measured by indirect ELISA (Fig. 3). For indirect-ELISA anti- chicken HRP-secondary antibody has been internally conjugated in accordance with a recent article technique and yielded a titer of 1:6000 (Pavliuchenko et al. 2019). Although, along with the antibody kinetics, titration of all three antibodies was optimized using Indirect-ELISA and resulted in a remarkable titer (Fig. 3).
The antibodies were conjugated to AuNPs, which were synthesized following the previously mentioned, traditional Turkevich method for citrate-stabilised gold nanoparticles (Muhammad et al. 2018). Optimization of particles morphology, size, and surface distribution were conducted by Transmission electron microscope and spectrophotometer. As previously reported in several studies, TEM images demonstrate that decreasing the temperature results in a larger diameter of gold nanoparticles (Dong et al. 2020). The nanoparticles were determined spherical in morphology and monodispersed in nature through TEM. The UV-vis absorption spectra of both AuNPs synthesized at 99 ˚C and 80 ˚C show well-defined surface plasmon resonance peaks at 520 nm and 526 nm, respectively (Fig. 4). Furthermore, a shift in the maximum absorbance wavelength of the UV-Vis spectra in contrast to naked AuNPs was interpreted as evidence of antibody conjugation on the AuNPs (Fig. 4). Two different types of nanoparticles, each coupled with three anti-tetraspanins antibodies, were synthesized and characterized here. In comparison to naked nanoparticles, the peak shift was clearly noticed at a difference of 3–4 nm for all six conjugated complexes for each type of AuNP. Hence, this distinct difference in the absorption spectra peaks indicated a successful conjugation and was utilized as a tool for exosome capturing.
In this study PEG was used to stabilize of IgY@AuNPs complex and the stability of IgY@AuNPs was analyzed using 0.1 M NaCl solution. Similarly, in a study, Gold nanoparticles introduced to 0.1 M NaCl immediately resulted in a decrease in the absorbance peak, demonstrating their inability to be used in buffer solutions (Christau et al. 2017). Moreover, gold nanoparticles are highly unstable, and various environmental changes, such as pH changes or excessive centrifugation, cause immediate NP aggregation, as evidenced in several studies by the visible colour change (from red to purple) of the AuNPs solution, indicating an increase in particle size (Christau et al. 2017; Polte et al. 2010). Consequently, PEG is considered one of the best polymers for increasing the stability of nanoparticles even after numerous centrifugations during the process (Dallari et al. 2021). Here, the non-covalent immobilization technique was used to conjugate the antibody with nanoparticles, in which antibody was spontaneously absorbed onto the surface of citrate-stabilised nanoparticles with three types of interactions i.e., ionic, hydrophobic, and dative interactions. The attraction between the hydrophobic parts of the antibody and the metal surface results in the formation of a non-covalent bond (Ljungblad et al. 2009). Ionic attraction exists between negatively charged groups on the surface of gold nanoparticles and positively charged groups in antibodies caused by positively charged amino acids and their N-terminal region (Ljungblad et al. 2009; Jazayeri et al. 2016). Furthermore, the dative interactions (coordinate covalent bond) are responsible to form a covalent bond between the AuNPs surface and free sulfhydryl groups of the antibody (Ljungblad et al. 2009; Jazayeri et al. 2016). Before treating with PEG, the antibody and AuNPs complex were stabilized and blocked using BSA due to its nature to prevent the non-specific binding of molecules and so enhance the assay sensitivity. Similarly, in a recent study, BSA was reported to be effective in preventing nanoparticle aggregation (Leopold et al. 2017).
In this article, we provide a more efficient and successful approach for isolating exosomes from human serum, which could potentially be employed for exosome isolation or exosomal cargo exploration. Anti-CD63 antibodies were applied to better understand and characterize the exosomes in human serum. Additionally, the expression of exosomal surface protein receptors was investigated, and observed that CD63 is prevalent in serum, but CD81 appears to be the rarest population (Karimi et al. 2022). However, they came to the conclusion that although CD9-enriched exosomes are also present, they are less numerous than CD63 (Karimi et al. 2022). Additionally, various groups used anti-CD63 antibodies to isolate the population of tiny EVs (Karimi et al. 2022; Pammi et al. 2022). Considering this, the TEM findings demonstrated that both conjugates, which were CD63 conjugated to both the 99 ˚C and 80 ˚C AuNPs, had efficiently captured the exosomes (Fig. 5).
In a nutshell, the primary objective of this study was to establish a rapid, cost-effective, and efficient method of isolating exosomes from biological fluids by simply adding extra weight to them. This procedure begins with the identification of extracellular sites, then moves on to the production of antigen in bacterial cells, which is then introduced into chickens to produce anti-exosomal antibodies. ELISA and UV-vis spectrophotometry were employed to characterize antibody titration and synthesis of AuNPs respectively. Furthermore, antibodies were bound to AuNPs to isolate exosomes, and this complex was used to pull down the exosomes from fluids using the antibodies' specificity. The isolated exosomes were successfully captured by TEM and could be used for a variety of purposes, including disease diagnosis, drug delivery, and miRNA extraction. We anticipate that the proposed approach will be valuable in more in-depth research on exosomal subcategories, and it can be applied to different sized small extracellular vesicles.