Materials and reagents

N-(3-(dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), albumin from bovine serum (BSA), N-hydroxysuccinimide (NHS), HAuCl4, poly (diallyldimethylammonium chloride) (PDDA), and anti-LC3 antibody produced in rabbit (LC3 cAb) were purchased from Sigma-Aldrich. Anti-LC3 rabbit antibody (LC3 dAb) was purchased from Novus Biologicals. The remaining proteins and antibodies used in this study were purchased from Proteintech Group Inc. Methylene blue (MB) was purchased from Maya Reagent Co., Ltd. Sodium citrate and sodium acetate were purchased from Sangon Biotech (Shanghai, China). Ltd. Graphene oxide (GO) was obtained from Nanjing XFNANO Materials Tech Co. Ltd. FeCl3 and ethylene glycol were supplied by Sinopharm Chemical Reagent Co., Ltd. 4-mercaptobutyric acid (C4H8O2S) was purchased from Shanghai Biochempartner Co.,Ltd. 1-butanethiol (C4H10S) was purchased from Shanghai Aladdin Bio-Chem Tech Co., Ltd. Ultrapure water (18.2 MΩ·cm) was obtained using a Millipore water purification system. The HBcAg ELISA kit was purchased from Shanghai Kehua Bio-Engineering Co. Ltd. Human IL-6, IL-10 ELISA kits, fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Co., Ltd. RPMI 1640 medium was purchased from HyClone, Cytiva. Penicillin-streptomycin solution and BCA protein assay kits were obtained from Beyotime Biotechnology Co., Ltd. CD9, CD63, TSG101 were purchased from Proteintech Group, Inc. CEA, CA15-3 were purchased from Beijing Key-Bio Biotech Co., Ltd. HER2 was purchased from Novoprotein Scientific Inc. All cell lines were obtained from cell bank of Chinese Academy of Science. BALB/c mice were purchased from Qinglongshan Animal Center. Healthy human, benign breast disease patient and breast cancer patient peripheral blood samples were from Zhongda Hospital.

B16F10, HepG2, EL4, Hepa1-6, MDA-MB-231, and epirubicin-resistant 4T1 cell line (4T1/EPB) cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin and grown in a humidified atmosphere at 37 °C with 5% CO2.

Isolation and characterization of LC3+ EVs

LC3+ EVs from B16F10 cells were prepared as previous report [23]. Briefly, the B16F10 cell culture supernatant was centrifuged at 450 g to remove dead cells and debris. To harvest LC3+ EVs released by B16F10 cells, the supernatant was centrifuged at 12,000 g and the sediment was resuspended with PBS and then purified by utilizing LC3-labeled magnetic beads (CST, USA). The total protein concentration of LC3+ EVs was determined using a BCA protein assay kit, according to the manufacturer’s instructions. LC3+ EVs were then fixed in 1.6% paraformaldehyde, 0.1% ruthenium red, 2.5% glutaraldehyde, 0.064% picric acid, and 100 mM sodium cacodylate, post-fixed with osmium tetroxide and potassium ferricyanide, and embedded in Epon. TEM images were obtained using a JEM 2100 transmission electron microscope (JEOL, Tokyo, Japan). The proteins extracted from LC3+ EVs were incubated with the primary anti-LC3 rabbit antibody and exposed to horseradish peroxidase (HRP)-conjugated affinipure goat anti-rabbit IgG. Protein bands were visualized using a Tanon 5200 chemiluminescent imaging system (Tanon, China). LC3+ EVs were stained with anti-LC3 rabbit antibody and PE-conjugated goat anti-rabbit IgG (rIgG-PE) and then analyzed using a BD Calibur cytometer (BD, USA).

Screening for specific anti-LC3 matched antibody pair

Several anti-LC3 antibodies specific to LC3 were selected as candidates. The specificity of the antibody candidates was analyzed using western blot. Proteins extracted from LC3+ EVs were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk in TBS containing 1% Tween-20 (TBST) at room temperature for 1 h. Afterwards, the membranes were incubated with primary antibodies referred to antibody candidates diluted in 5% skim milk in TBST at 4 °C overnight and exposed to respective HRP-conjugated secondary antibodies at room temperature for 1 h after being washed five times with TBST. Protein bands were visualized using a chemiluminescence assay.

The match between specific anti-LC3 antibodies was verified by flow cytometry. The matching of antibodies indicated antibodies against different antigen epitopes. Briefly, LC3+ EVs were stained with rabbit anti-LC3 antibody and subsequently stained with the same or another rabbit anti-LC3 antibody and then incubated with the corresponding rIgG-PE. LC3+ EVs stained with rIgG-PE were used as the negative controls.

Synthesis of GH-MB

For the preparation of GH, 5 mL of a 5 mg mL− 1 GO suspension was sonicated for 30 min and then mixed with 2 mL of ethylene glycol. Afterwards, the mixture was sealed in a Teflon autoclave at 180 °C for 12 h, followed by cooling to room temperature. The resulting black hydrogel was rinsed with ultrapure water to obtain the GH.

To prepare GH-MB, GH was sonicated for 30 min and then dispersed in ultrapure water. Subsequently, 4 mL of 1 mg mL-1 MB was added to 5 mL of 1 mg mL-1 GH, and the mixture was stirred at room temperature for 2 h. Finally, the composite was centrifuged and re-dispersed in ultrapure water.

Preparation of Fe3O4-Au composites

The Fe3O4 nanoparticles were synthesized by a hydrothermal method [33]. Briefly, 0.65 g FeCl3 and 0.2 g sodium citrate were dissolvedin ethylenee glycol (20 mL), and 1.2 g sodium acetate was added to themixturey. The mixture was then transferred to a Teflon autoclave at 200 °C for 10 h after vigorous stirring for 30 min at room temperature. The precipitate was collected by centrifugation, washed with ultrapure water and ethanol, and dispersed in ethanol for further use.

Au nanoparticles (AuNPs) were first synthesized for the preparation of the Fe3O4-Au composites. The AuNPs were synthesized via reduction of HAuCl4 according to previous literature [34]. Briefly, 100 mL of 0.01% (w/v) HAuCl4 solution was heated to ebullition and 2 mL of 1% sodium citrate was added under stirring. The mixture was then heated for 15 min and cooled for further use.

To prepare Fe3O4-Au composites, the pH of the Fe3O4 nanoparticles was adjusted to 9.5, and 1 mL of 1 mg mL-1 Fe3O4 nanoparticles was mixed with 0.4 mL of 4% PDDA solution and then stirred for 30 min at room temperature. The obtained suspension was rinsed with ultrapure water and redispersed in 1 mL of ultrapure water. Finally, 10 mL of AuNPs (pH 7.0) was added to the above solution, followed by stirring at room temperature for 7 h and centrifugation to obtain the precipitate as Fe3O4-Au composites.

Preparation of cAb-beads (Fe3O4-Au-conjugated LC3 cAb)

The Fe3O4-Au-conjugated LC3 cAb was prepared by covalent binding of the amino groups of the LC3 cAb to the carboxyl groups of the AuNPs. Briefly, 0.1 µL of 4-mercaptobutyric acid was added to 1 mL of 1 mg mL− 1 Fe3O4-Au, and the mixture was stirred overnight at room temperature. After centrifugation, the precipitate was rinsed with ultrapure water and dispersed in ultrapure water. Then 0.1 µL 1-butanethiol was added to the above solution, followed by stirring for 1 h at room temperature. 4-mercaptobutyric acid and 1-butanethiol were used to block the nonspecific binding sites of AuNPs. Then, the mixture was washed with ultrapure water, redispersed into 0.1 M PBS, and mixed with an equal volume of EDC (20 mg mL− 1)/NHS (10 mg mL− 1) solution to activate the carboxyl groups on the AuNPs. After centrifugation and rinsing, 10 µL of LC3 cAb was added to 1 mL of the resulting solution and the mixture was stirred at 4 °C overnight. Finally, 10 mg of BSA was incubated in the solution at 4 °C for 1 h, and the obtained Fe3O4-Au-conjugated cAb solution was stored at 4 °C until further use.

Fabrication of immunosensor and detection of LC3+ EVs

Before immunosensor fabrication, the bare glassy carbon electrode (GCE) was polished with 0.3 and 0.05 μm alumina powder and washed ultrasonically in ethanol and ultrapure water. Subsequently, 10 µL GH-MB (1 mg mL− 1) was dropped onto the GCE and dried at room temperature for 24 h. Next, 10 µL of EDC (20 mg mL− 1)/NHS (10 mg mL− 1) solution was added to the electrode for 2 h as a covalent binding agent to link the amino group on LC3 dAb with the carboxyl group on GH. After rinsing with PBS, 10 µL of 10 µg mL− 1 LC3 dAb in 0.1 M PBS was dropped onto GCE and incubated at 4 °C overnight. After rinsing, 10 µL of 0.5% (w/v) BSA in 0.1 M PBS was incubated on the GCE at room temperature for 30 min to block nonspecific binding sites. At the same time, different concentrations of LC3+ EVs in 0.1 M PBS were incubated with the cAb-beads at 37 °C for 100 min followed by magnetic enrichment, which was denoted as LC3+ EVs/cAb-beads. Finally, the LC3+ EVs/cAb-beads were dropped onto the GCE and incubated at 37 °C for 60 min. The resulting immunosensor was rinsed with PBS and stored for future use.

Electrochemical measurements were performed in deoxidized 0.1 M PBS solution using a CHI 660E workstation (CHI, USA) with a conventional three-electrode system composed of the modified electrode as the working electrode, platinum wire as the counter electrode, and Ag/AgCl (3 M KCl) as the reference electrode. DPV measurements were carried out from − 0.6 to 0.1 V in deoxidized 0.1 M PBS solution.

Establishment and treatment of the 4T1/EPB TB mouse model

The 4T1/EPB TB mouse model was established and treated according to our previous study [35]. 4T1/EPB cells in the logarithmic growth phase were digested and resuspended in PBS, the cell concentration was adjusted to 5 × 105 mL-1 and the cell suspension was injected subcutaneously into the right breast pad of mice to establish a 4T1/EPB TB mouse model. For the treatment, 4T1/EPB TB mice were vaccinated by subcutaneous injection of normal saline or vaccine containing 30 µg ubiquitinated proteins (UPs) on days 8, 10, and 12 (3 mice per group).

Enzyme-linked immunosorbent assay (ELISA)

The specificity of the cAb-beads was verified using a human HBcAg ELISA kit, according to the manufacturer’s protocol. HBcAg was used as an interfering protein. Briefly, cAb-beads were incubated with HBcAg, centrifuged, washed, and resuspended in PBS. The above product, PBS, cAb-beads, and HBcAg were cultured in a 96-well plate pre-coated with the capture antibody of HBcAg. After washing off unbound proteins, an HRP-labeled detection antibody of HBcAg was added to the well, which catalyzed the colorimetric reaction with TMB as a substrate and was terminated by H2SO4. PBS and HBcAg were used as the negative and positive controls, respectively. A microplate reader (Thermo Fisher Scientific, USA) was used to measure the absorbance of the final product at 450 nm.

Confocal laser scanning microscopy (CLSM)

To confirm the conjugation of LC3 cAb to Fe3O4-Au, 100 µL of 4-mercaptobutyric acid- and 1-butanethiol-blocked Fe3O4-Au composites and 100 µL of cAb-beads were mixed with 1 µL of rIgG-PE and incubated for 2 h at room temperature. The precipitates were then removed by magnetic separation, washed three times with PBS, and resuspended in 100 µL of PBS. The above two substances and Fe3O4-Au composites, cAb-beads were added dropwise on slides and observed by CLSM.

To investigate the binding ability of the cAb-beads to LC3+ EVs, 50 µL of LC3+ EVs stained with PKH67 (PKH67-labeled LC3+ EVs) and 1 µL of LC3 cAb or PBS were incubated at 4 °C for 1 h. The suspensions were then centrifuged at high speed, and the precipitates were obtained after washing three times with PBS. Finally, the precipitates were respectively resuspended in 50 µL of PBS. The above substances were then added to 100 µL of cAb-beads and incubated at room temperature for 2 h. After magnetic separation and washing three times with PBS, the precipitates were resuspended in 100 µL of PBS. The above substances, cAb-beads, and PKH67-labeled LC3+ EVs were added dropwise onto the slides and observed using CLSM.

To evaluate the specificity of the cAb-beads, 1 µL of Alexa Fluor 594-conjugated goat anti-mouse IgG (mIgG-594) was added to 100 µL of cAb-beads, 100 µL of cAb-beads and 50 µL of PKH67-labeled LC3+ EVs, respectively, and then incubated for 2 h at room temperature. Precipitates were obtained by magnetic separation, washed three times with PBS, and resuspended in 100 µL of PBS. The above substances were added to the slides and observed using CLSM.

Surface plasmon resonance (SPR) assay

Experiments were performed at 25 ℃ on a BIAcore T200 instrument (GE Healthcare), employing CM5 sensor chips in accordance with the manufacturer’s instruction. Briefly, a total of 50 µL of LC3 protein was combined with a 10 mM sodium acetate solution (pH 5.0), subsequently immobilized onto the CM5 sensor chip surface using the amine-coupling procedure, followed by blocking the remaining activated groups with 1 M ethanolamine (pH 8.5). The adjacent aisle, serving as the reference, underwent a similar activation and blocking procedure, albeit being immobilized with PBS (pH 5.0). To compare the binding affinities of antibodies towards LC3 before and after conjugation through EDC/NHS, antibodies were injected into the flow cell at concentrations of 1, 0.5, 0.25, 0.125, and 0.000625 nM in phosphate buffered saline containing detergent (PBS-P + buffer) (pH 7.4) at a flow rate of 10 uL min− 1, allowing for 60 s of association and 90 s of dissociation. Data pertaining to the samples were acquired utilizing the BIAcore T200 Control software (v. 2.0, GE Healthcare). The dissociation constant KD was determined utilizing the BIAcore T200 Evaluation software (GE Healthcare) based on KD = Kd/Ka (where Ka is the association rate constant, and Kd is the dissociation rate constant), with the KD value being inversely proportional to the antibody affinity [36].