Journal: bioRxiv

Article Title: Combining Anion Exchange and Size Exclusion Chromatography for Extracellular Vesicle Enrichment from Small Volumes of Human and Mouse Plasma for Quantitative Proteomics

doi: 10.64898/2026.03.11.711200

Figure Lengend Snippet: Workflow for EV enrichment from 4 ml (A) and 0.5 ml (B). EV markers (CD63 and Syntenin-1) and plasma contaminant proteins (Albumin and ApoA1(shown in red)) by western blot analysis (panel C). Starting plasma volume, final volume post concentration with the centrifugal filter and gel loading volume were used to calculate the concentration factor and equivalent plasma starting amounts indicated above figure.

Article Snippet: Membranes were blocked in 5% skim milk in PBS at room temperature for 1 hour before overnight incubation with primary antibodies Syntenin (Abcam, cat# ab133267), CD63 (Abcam, cat#ab134045), CD63 (Abcam, cat#ab8219), Albumin (Cell Signalling Technology, cat#4929S), Calnexin (Cell Signalling Technology, cat#2679S) ApoA1 (Santa Cruz, cat# SC-376818).

Techniques: Clinical Proteomics, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Combining Anion Exchange and Size Exclusion Chromatography for Extracellular Vesicle Enrichment from Small Volumes of Human and Mouse Plasma for Quantitative Proteomics

doi: 10.64898/2026.03.11.711200

Figure Lengend Snippet: SEC Void optimisation. Pooled SEC fractions 1-4 collected using a 2.7 mL void volume (1-4A) and a 2.8 mL void volume (1-4B) were prepared in triplicate from 0.5 mL plasma. Western blot analysis of contaminant proteins Albumin (green) and lipoprotein ApoA1 (red) (A) with 0.5 µl plasma included as a positive control (+). EV markers CD63 and syntenin-1 and small EV exclusion marker Calnexin (B). Samples (0.5 ml and 0.4 ml) from Experiment 3.2 included as a western blot control and 2µl platelet lysate included as a calnexin positive control (+). Electron microscopy (C) visualisation and size and concentration histogram (D) of EV representative SEC sample (Void 2.7 ml).

Article Snippet: Membranes were blocked in 5% skim milk in PBS at room temperature for 1 hour before overnight incubation with primary antibodies Syntenin (Abcam, cat# ab133267), CD63 (Abcam, cat#ab134045), CD63 (Abcam, cat#ab8219), Albumin (Cell Signalling Technology, cat#4929S), Calnexin (Cell Signalling Technology, cat#2679S) ApoA1 (Santa Cruz, cat# SC-376818).

Techniques: Clinical Proteomics, Western Blot, Positive Control, Marker, Control, Electron Microscopy, Concentration Assay

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Selective Lipoprotein Removal Enables High‐Purity EV Isolation from Plasma via Aptamer‐Based Mesh Filtration

doi: 10.1002/smll.202514724

Figure Lengend Snippet: Aptamer‑based affinity filtration enables rapid, selective removal of lipoproteins from plasma EV preparations. (a) Density–size plots of plasma particles illustrate the substantial biophysical overlap between lipoproteins (LDL, VLDL, HDL) and extracellular vesicles (EVs) under conventional ultracentrifugation (UC) and size‐exclusion chromatography (SEC), motivating the need for an affinity‐based separation axis; (b) Fluorescence microscopy shows successful immobilization of FAM‐labeled aptamers (green) onto the nylon mesh and specific binding of Cy5‐labeled (V)LDL and HDL (red), confirming target‐selective molecular recognition. Bare nylon mesh processed without aptamer immobilization (blank control) exhibited negligible fluorescence signals under identical imaging conditions. Green‐only (FAM‐aptamer), red‐only (Cy5‐labeled lipoprotein), and merged images were acquired from the same field of view. (c) Schematic workflow of the sequential capture process: (V)LDL particles are first retained by ApoB100‐specific aptamers in the upper mesh layer, followed by HDL capture via ApoA1 aptamers in the lower layer, yielding a permeate enriched in EVs with lipoproteins efficiently removed.

Article Snippet: Aptamers targeting ApoB‐100 and ApoA‐1 were synthesized with a 3′‐terminal C6‐NH 2 modification (Bioneer, Daejeon, Korea).

Techniques: Filtration, Clinical Proteomics, Size-exclusion Chromatography, Fluorescence, Microscopy, Labeling, Binding Assay, Control, Imaging

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Selective Lipoprotein Removal Enables High‐Purity EV Isolation from Plasma via Aptamer‐Based Mesh Filtration

doi: 10.1002/smll.202514724

Figure Lengend Snippet: Functional validation of the ApoFilter platform for differential and sequential capture of (V)LDL and HDL (a) Schematic of the hybrid ApoFilter system validated using compositionally pure (V)LDL and HDL preparations; (b–d) Comparative capture efficiency of ApoFilter modules, showing NTA‐quantified retained fractions and residual particles in the permeate for (b) pure (V)LDL, (c) pure HDL, and (d) a mixed (V)LDL+HDL sample; (e–g) ELISA‐based quantification of ApoA1‐ and ApoB100‐positive lipoproteins, illustrating retention efficiency in the capture fractions and residual levels in permeate for (e) pure (V)LDL, (f) pure HDL, and (g) a mixed (V)LDL+HDL sample; (h) Western blot analyses of key protein markers (ApoA1, ApoB100, albumin, CD9, and CD63) in negative control, ApoFilter A, ApoFilter B, and ApoFilter H fractions, demonstrating molecular specificity and integrity of captured and permeate samples.

Article Snippet: Aptamers targeting ApoB‐100 and ApoA‐1 were synthesized with a 3′‐terminal C6‐NH 2 modification (Bioneer, Daejeon, Korea).

Techniques: Functional Assay, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Selective Lipoprotein Removal Enables High‐Purity EV Isolation from Plasma via Aptamer‐Based Mesh Filtration

doi: 10.1002/smll.202514724

Figure Lengend Snippet: Specificity and performance validation of the ApoFilter system for extracellular vesicle isolation from complex human plasma. (a) Schematic illustration of the ApoFilter workflow, depicting sequential gravity‐driven capture of (V)LDL and HDL and collection of EV‐enriched permeate; (b,c) SEM and TEM imaging analyses, revealing nanoscale aggregation states and morphological heterogeneity of plasma‐derived lipoproteins, highlighting their relevance for downstream fluorescence‐based assays; (d) Optimization of dispersion conditions, showing that extended incubation induces lipoprotein aggregation, while brief sonication restores expected nanoparticle size distributions; (e) Fluorescence‐NTA analysis of plasma spiked with Alexa Fluor–labeled anti‐ApoB100 and anti‐ApoA1 antibodies confirms near‐complete removal of fluorescently labeled lipoproteins following ApoFilter processing, with dotted traces indicating the permeate fractions; (f) ELISA quantification of ApoA1 and ApoB100, demonstrating efficient depletion of HDL and (V)LDL, with preservation of EV markers (CD9, CD63, CD81) and albumin recovery.

Article Snippet: Aptamers targeting ApoB‐100 and ApoA‐1 were synthesized with a 3′‐terminal C6‐NH 2 modification (Bioneer, Daejeon, Korea).

Techniques: Biomarker Discovery, Isolation, Clinical Proteomics, Imaging, Derivative Assay, Fluorescence, Dispersion, Incubation, Sonication, Labeling, Enzyme-linked Immunosorbent Assay, Preserving

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Selective Lipoprotein Removal Enables High‐Purity EV Isolation from Plasma via Aptamer‐Based Mesh Filtration

doi: 10.1002/smll.202514724

Figure Lengend Snippet: Synergistic removal of lipoproteins and preservation of EV yield by integrating ApoFilter with standard EV isolation workflows. (a) Western blot analysis of ApoA1 and ApoB100 (lipoprotein markers), CD9 and CD63 (EV markers), and albumin across samples isolated by ultracentrifugation (UC), size‐exclusion chromatography (SEC), or ExoTFF, with (+) or without (–) ApoFilter pretreatment; (b–d) ELISA‐based quantification of ApoA1, ApoB100, and CD9 demonstrating enhanced lipoprotein depletion and preserved EV markers across each workflow following ApoFilter pretreatment; (e) Nanoparticle tracking analysis (NTA) showing particle concentrations for each isolation method, highlighting the reduction of non‐EV particles upon ApoFilter integration; (f) Total protein concentration measured by BCA assay, showing reduced protein loads across workflows incorporating ApoFilter, consistent with efficient lipoprotein removal rather than albumin depletion.

Article Snippet: Aptamers targeting ApoB‐100 and ApoA‐1 were synthesized with a 3′‐terminal C6‐NH 2 modification (Bioneer, Daejeon, Korea).

Techniques: Preserving, Isolation, Western Blot, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Protein Concentration, BIA-KA

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Selective Lipoprotein Removal Enables High‐Purity EV Isolation from Plasma via Aptamer‐Based Mesh Filtration

doi: 10.1002/smll.202514724

Figure Lengend Snippet: Performance comparison of EV isolation workflows with and without ApoFilter pretreatment. Radar plots depict percentage recovery of EV markers (CD9, CD63, CD81) and removal of contaminant proteins (ApoA1, ApoB100, albumin) for each method: (a) UC, (b) ApoFilter→UC, (c) SEC, (d) ApoFilter→SEC, (e) ExoTFF, and (f) ApoFilter→ExoTFF. Integration of ApoFilter markedly increases lipoprotein (ApoA1/ApoB100) depletion while maintaining or enhancing EV marker recovery across all workflows.

Article Snippet: Aptamers targeting ApoB‐100 and ApoA‐1 were synthesized with a 3′‐terminal C6‐NH 2 modification (Bioneer, Daejeon, Korea).

Techniques: Comparison, Isolation, Marker