Isolation, characterisation and detection of breath-derived extracellular vesicles

The physical characterisation, capture and detection of extracellular vesicles (EVs) and exosomes derived from breath condensate is reported. Breath-derived EVs were isolated from breath condensate and captured on a gold substrate using two complimentary methods. The characterised and isolated EVs were detected using surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS). EIS was done using aptamers as a targeting moiety and showed a larger change in resistance between dilute concentrations of EVs (less than 7 μg/mL).This is the first report of EVs and exosomes isolated and characterised from breath. In addition, EVs from a non-invasive and easily available source such as breath opens up further avenues in the detection of pulmonary diseases.

Exhaled breath has been a key biological media used to assess human health, along with urine and blood. Each person has the capacity to exhale 10,000 L of breath each day, making exhaled breath condensate (EBC) an attractive matrix that contains accessible biomarkers for the diagnosis of diseases [1][2][3][4] . It has been shown that EVs from EBC are a novel and non-invasive avenue for pulmonary disease detection 5 . To date, however, the physical characterisation of isolated EVs and exosomes from EBC has not been done despite previous evidence of the presence. EVs, in particular exosomes, are nano-sized vesicles secreted by most living cells. Previously thought to be leftover debris, literature has demonstrated that they can play a vital role in cell-to-cell transport, cell function and intercellular communication due to their contents which are specific to the cell of origin 6,7 . They have shown massive potential as biomarkers for a wide range of diseases, for example, cancer and PRION-related diseases, as they are found in most bodily fluids, including blood 4 , saliva 8,9 , breast milk 10 , and urine 11 . EVs from EBC are, therefore, a feasible target to analyse when compared to small molecules (such as volatile organic compounds), as they are a larger size of 40-100 nm (making them easier to detect, capture and characterise), highly abundant and have accessible and specific target proteins on the surface. The detection of EVs using plasmonic and electrochemical sensors has been done previously using specific cell-line derived EVs 12,13 . Kilic et al. 13 showed the use of an electrochemical sensor to measure EVs isolated from a MCF-7 cell line. However, this is the first attempt at detecting of EVs isolated from EBC with low available concentrations. In this work, the presence was verified by isolating and physically characterising EBC-derived EVs according to the "minimal information for studies of EVs" (MISEV) requirements 14 . Sinha et al. 5 verified the presence of an exosome-specific biomarker collected from EBC (CD63) but did not characterise the physical properties which were achieved here using photoluminescence spectroscopy, transmission electron microscopy (TEM), cryo-scanning electron microscopy (cryo-SEM), western blot and dynamic light scattering (DLS). Then, the EVs were detected from EBC using different techniques such as SPR, using fluorescent InP/ZnS quantum dots (QD) conjugated to an EV-specific antibody (AB), and EIS, using an EV-specific aptamer. The above detection protocols can be translated to disease-specific EBC-derived EVs for the purposes of disease detection and a breathalyser.

Results and discussion
For the isolation of EBC-derived EVs, breath condensate was first isolated using a frozen column to condense the breath. A size-exclusion column was then used to produce highly pure EVs with minimal protein aggregates. To confirm the presences of EV, characterisation was done using TEM and Cryo-SEM to investigate their morphology and size. According to both the cryo-SEM and TEM images (shown in Fig. 1), the size of the EVs was varied and observed to be anywhere between 50 to 170 nm. The morphology was observed to be spherical and    and also the different concentrations of EVs. However, this is non-specific binding to the now available and reactive gold surface sites. As this binding was independent to the excess concentrations of EV, it is therefore an inaccurate representation of EV capture with high specificity. Supplementary figure S-5(d) further proves the high specificity of Apt1 to the CD63 proteins on the membrane of the breath-derived EVs. Apt2 was chosen as it had the same length as Apt1 but with a different sequence. Although there was a small response in R et between Apt2 and the different concentrations of EVs, this was not concentration-dependent and is trivial when compared to the response when using Apt1, especially when paralleling the highest concentration of EV added thereby emphasising the high specificity of Apt1 for the breath-derived EVs. These control results are further shown in Fig. 2D along with the calibration curve for three different protein concentrations of EVs. The significant increase in resistance values for the higher concentrations of EVs is clearly visible here when compared to each of the controls shown which have negligible changes.

Conclusions
In conclusion, EBC-derived EVs and exosomes were isolated using size-exclusion columns and physically characterised using imaging techniques such as TEM, Cryo-SEM and DLS. Their morphology was spherical, and their sizes were between 50-170 nm. The ability of methods such as SPR and EIS towards detection of dilute concentrations of breath-derived EVs was also demonstrated by targeting the CD63 protein that is known to exist on the membrane of exosomes (sub-population of EVs). EIS was able to show a larger change between three different concentrations of the EVs which makes this label-free method the most suitable for the detection of dilute concentrations. Further potential of this work can extend to detection of clinically relevant populations of these EVs that are easily isolated from a non-invasive source for the detection of pulmonary diseases.

Methods
Breath sampling. The breath sampling was done as a proof-of-concept by the researcher. Subject provided written informed consent prior to participation.

Chemicals and materials.
All chemicals and materials were purchased from Sigma Aldrich unless specified otherwise.
EVs isolation. Prior to collection of the condensate, the subject did not consume food or drink water for 2 h.
The condensate was then collected by breathing out through the mouth every 3 s and in through the nose every 10 s. The breath was collected in a Eppendorf tube that was attached to a 1 mL syringe. The Eppendorf tube and 1 mL syringe were wrapped in a frozen ice gel pack. The breathing process was done for approximately 30 min ensuring the entire apparatus stayed frozen and then aliquoted into small vials for EVs isolation. The EBC was collected into an Eppendorf tube and diluted with 1 × phosphate buffered saline (up to 1 mL of PBS; Thermo Fisher, cat. 10010023). This was then filtered using a 0.22 μm syringe filter after which the EVs were isolated using qEV size exclusion chromatography (Izon Science Ltd.). Fractions 7, 8, 9 and 10 were mixed and concentrated using a 30 kDa centrifugal filter and re-diluted in 200 μL PBS. Protein concentration in the EBC-derived EVs sample was measured by using absorbance at 280 nm at NanoDrop ® ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific) and consisted of 0.023 mg/mL. EV isolation for Cryo-SEM. Initially, the sample was centrifuged for 10 min at 300×g following next cycle of centrifugation at 3000×g for 15 min to get rid of debris. Supernatant was collected for future analysis. In were mixed with 5.0 mL of oleylamine. This mixture was heated to 120 • C under vacuum for 2 h. The atmosphere was switched to nitrogen and the temperature was set to 180 • C at which point, the tris-(diethylamino) phosphine (1.6 mmol) was added rapidly. The InP core growth was allowed to proceed for 20 min, after which sulfur (2.2 mol/L) in tri-octylphosphine (1 mL) was added to the cores for shelling over 10 min. At 60 min, the temperature was ramped to 200 • C . At 120 min, Zn(undecylenate)2 (1 g) in 4 mL of octadecene was slowly injected dropwise over a period of 10 min after which the temperature was increased to 220 • C . At 150 min, 0.7 mL of TOP-S was added over a period of 10 min and the temperature was further ramped to 240 • C . At 180 min, Zn(undecylenate)2 (0.5 g) in 2 mL of octadecene was added slowly and the temperature was ramped to 260 • C . At 210 min, the reaction was terminated with rapid cooling to room temperature and subsequent dilution in toluene. The QDs were then washed using ethanol for precipitation and re-dispersed in toluene. This method yielded red-emitting QDs. The method used below for ligand exchange was based on a previous procedure published by Dobhal et al. 12 0.30 g of Mercaptosuccinic acid (MSA) was stirred in 1 mL of toluene for 15 min after which 1 mL of 10 mg/mL QDs were added. 1 mL of ammonium hydroxide (30%) and 1 mL of Milli-Q water were added after 1 min. This was left to stir for 2 h. The coloured aqueous layer was purified by precipitating in ethanol and centrifuging. The clear supernatant was discarded and the pellet was re-dispersed in 1 mL of Milli-Q water. The water-soluble QDs were stored in the dark at 4 • C . For the conjugation of the QDs to an antibody, the following AB was used: CD63 Monoclonal AB (Ts63) from Thermo Fisher Scientific, catalog 10628D, RRID AB_2532983. 50 μL of 0.05 M of N-hydroxysuccinimide (NHS) and 0.02 M of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was mixed with 1 mL of 1 mg/mL QD-MSA for 10 min. Then, 10 μL of a 0.5 mg/mL AB-CD63 was added. This was left to stir for 2 h. The conjugate (InP/ZnS-AB) was washed using a 30 kDa Amicon centrifugal filter twice with water and re-dispersed in 1 mL of water.
Transmission electron microscopy. TEM2100F (JEOL Ltd.) was used for imaging purposes. EV samples were drop-casted on the formvar side of the grid which had been previously plasma cleaned. After 15 min, the sample was stained for 6 min using 4.5% uranyl acetate. After this, the grid was washed twice with Milli-Q water and left to dry for 30-45 min before loading into the microscope.
Cryo-scanning electron microscopy. For cryo-SEM imaging we used a JSM6500F (JEOL Ltd.), equipped with a 97 μA emission gun and a ALTO 2500 (GATAN UK) cold stage maintained below − 12 • C . Specimens were imaged at low acceleration voltages of 10 kV, and working distances of 8.4 mm. Specimens were cryo-fixed using a manual drop plunging where a 10 μL drop of a sample of EVs in PBS were set on top of a sample stage, maintaining its droplet shape. This was manually plunged into liquid nitrogen. The frozen droplet was transferred into a freeze fracture system. A cooled knife was used to fracture the droplet exposing the inner part of the drop. The sample was coated with 5-10 nm of platinum and transferred into the SEM.
UV-Vis spectroscopy. The Cary50 Bio (Agilent technologies) was used for absorbance measurements.
Photoluminescence spectroscopy. The FLS980 (Edinburgh Instruments) was used for all PL measurements. All the InP QDs samples were excited at 480 nm. Prior to making a QY measurement, it was ensured that the QD sample absorbance at the excitation wavelength of 480 nm was below 0.1 absorbance units to avoid any self-absorbance effects. All PLQY calculations were done using the integrating sphere and a direct excitation method.
Surface plasmon resonance. This protocol is based on a modified protocol used by Dobhal et al. 12  www.nature.com/scientificreports/ Electrochemical impedance spectroscopy. This protocol is based on a modified method used by Queirós et al. 16 EIS experiments were carried out using a three-electrode cell: working electrode is gold (Au), counter electrode is platinum (Pt), reference electrode is silver/silver chloride (Ag/AgCl). Measurements were carried out using a PalmSens3 ® potentiostat with a 5 mmol/L Fe(CN) 3− 6 /Fe(CN) 4− 6 electrolyte solution in PBS electrolyte buffer solution (10 mmol/L PBS with 150 mmol/L NaCl electrolyte solution at pH 7.4). Experiments were carried out at room temperature pressure and all solutions were purged under nitrogen gas for 15 min prior to measurements. Impedance spectra was collected between a frequency range of 0.1-10000.0 Hz range. Precleaning of each electrode was monitored using cyclic voltammetry (CV). Each electrode was pre-treated in 5 mL of a degassed solution 0.1 mol/L NaOH for 200 scans ( 8 min) before being rinsed with Milli-Q water and dried under nitrogen. The electrode was then polished using 0.3 μmol/L alumina for 2 min before being rinsed with Milli-Q water and dried under nitrogen. This process was repeated using 0.05 μmol/L alumina and further rinsed with Milli-Q water and ethanol. The electrode was sonicated in ethanol followed by sonication in Milli-Q water for 5 min each. The Au electrode was cleaned with 3 mL of 0.5 mmol/L H 2 SO 4 for 60 scans. After cleaning, the electrode was immobilised in MPA for 1 h and was washed with ethanol and Milli-Q water and dried with nitrogen. The Au-electrode was then immersed in an aqueous solution of 0.2 mol/L NHS and 0.05 mol/L EDC for 1 h before being washed with Milli-Q water and dried with nitrogen. The aptamer used for EIS was synthesised by Integrated DNA Technologies, named amino-modified CD63 aptamer (Apt1): 5-NH2-(CH2)6-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-3. The Au-electrode was then immersed overnight in a 5 μmol/L aptamer solution before being washed with Milli-Q ® water. The electrode was finally immobilised in 50 μL of different concentrations of breath-derived EVs in 10 mmol/L PBS solutions, starting from the lowest concentration, for 30 min each. Each experiment was run independently three times. Analysis of the Nyquist plots obtained in each measurement was carried out using the EIS Spectrum Analyser programme, using the Randles equivalent electric al circuit with an unweighted function and the Nelder-Mead (NM Simp) algorithm. This gave both impedance values and relative estimated errors of the calculated parameters.