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2. Excluding contaminants
Contaminants in a TRPS context could include:
— Non-target particles in the same size range (bacterial vesicles,
intracellular vesicles, lipoproteins)
— Particulates that could block pores (microorganisms, cells and debris,
protein aggregates, dust)
— Pore modification agents (free protein, charged coating agents)
— Destructive or disruptive agents (proteases and nucleases, cells or
microorganisms left in samples, harsh detergents that could disrupt
membrane particles or membrane-bound proteins).
CONTAMINANTS
EXCLUDED STEPS TAKEN TO CLEAN
SAMPLES OR REDUCE
Reagents and clean
calibration samples
(DI, electrolyte,
wetting and coating
solutions) Dust, pollen, etc. Use clean glassware rinsed twice with
deionised water, and new, dust-free tubes
and filters
Microorganisms and
their secretions/
products Make up electrolyte from powder weekly,
filter-sterilise daily, store solutions at 2-8 °C
Trace chemicals Use reagent-grade buffers and
electrolytes, fresh deionised water, clean
glassware
Precipitates/salt
crystals Filter all solutions that will come into contact
with the pore daily with a 0.22 µm filter
Biological samples
e.g. plasma Large particles
(cells, debris, etc.) Centrifuge samples at 2000 g for 10
minutes to remove cells, then spin
supernatant at 10000 g for 10 minutes to
remove debris and apoptotic bodies
Small particles and
solutes (destructive
enzymes, nutrients,
etc.) Remove proteins and solutes via a Size
Exclusion Chromatography (SEC) qEV
column. Exclude the buffy coat when
working with whole blood
Microorganisms and
their secretions/by-
products Store samples at -80 °C only after
centrifugation and SEC steps have been
completed, flush qEVs in an antimicrobial
agent before storage, do not use bench
PBS – always make up buffers from powder
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