Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-μL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (<i>n</i> = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with <i>r</i>² value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (<i>n</i> = 5). The detection limit was estimated to be 2.9 × 10⁷ exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h

Effect of multiple individual lipid species in diagnosis of prostate cancer.

<p>The points indicated by the two head arrows are the predictive powers of top 15 plasma apparent lipid species when they are used together in diagnosis of prostate cancer. Using top 15 plasma apparent lipid species has the highest sensitivity (93.6%), the highest specificity (90.1%), and higher accuracy (ROC Area, 97.3%) in the diagnosis of prostate cancer as compared with using any other combination of different numbers.</p

Lipid classes in differentiation of prostate cancer (Concentration: nmol/µl).

<p> <b><i>Sens. = Sensitivity, Spec. = Specificity, Prec. = Precision, F-Meas. = F-measure.</i></b></p

Comparison of subject distributions and patient characteristics between upper and lower halves in the cluster of top 15 apparent lipid species.

<p>Comparison of subject distributions and patient characteristics between upper and lower halves in the cluster of top 15 apparent lipid species.</p

Comparison of predictive values (%) of the top 15 plasma lipid biomarkers in diagnosis of prostate cancer in training set and testing set.

<p> <b><i>Sens. = Sensitivity, Spec. = Specificity, Prec. = Precision, F-m. = F-measure, AUC = Area under (ROC) curve.</i></b></p

Comparison of predictive powers among groups with different numbers of identified plasma lipid biomarkers.

<p>Comparison of predictive powers among groups with different numbers of identified plasma lipid biomarkers.</p

Top 35 individual plasma apparent lipid species as candidate biomarkers for prostate cancer (Concentration: pmol/µl).

*<p>Apparent lipid species identities are based on the mass/charge ratio of the intact lipid ion and one characteristic fragment. Sens. = Sensitivity, Spec. = Specificity, Prec. = Precision, F-Meas. = F-measure.</p

Comparison of Principal Component Analysis (PCA) with 390 and 15 selected plasma apparent lipid species.

<p>A: The first component in PCA cross all 390 detected plasma apparent lipid species accounts for 28.3% of the overall variance; B: The first component in PCA cross 15 selected plasma apparent lipid biomarkers accounts for 86.9% of the overall variance.</p

Mass spectra of phosphocholine-containing lipids (Pre-184 positive mode, including biomarker species.

<p>A: Spectra of 15 selected apparent lipid species in a representative patient with prostate cancer. B: Spectra of 15 selected apparent lipid species in a representative male control. Spectral intensities were normalized to that of internal standard LPC(13∶0). The intensities of phosphocholine-containing internal standards (I.S.) are indicated in green. The intensities of the identified biomarkers are shown in red. Internal standards and biomarkers (15 selected apparent lipid species) are labeled.</p