Simultaneous isolation and preconcentration of exosomes by ion concentration polarization

Abstract

Exosomes carry microRNA biomarkers, occur in higher abundance in cancerous patients than in healthy ones, and because they are present in most biofluids, including blood and urine, these can be obtained noninvasively. Standard laboratory techniques to isolate exosomes are expensive, time consuming, provide poor purity, and recover on the order of 25% of the available exosomes. We present a new microfluidic technique to simultaneously isolate exosomes and preconcentrate them by electrophoresis using a high transverse local electric field generated by ion-depleting ion-selective membrane. We use pressure-driven flow to deliver an exosome sample to a microfluidic chip such that the transverse electric field forces them out of the cross flow and into an agarose gel which filters out unwanted cellular debris while the ion-selective membrane concentrates the exosomes through an enrichment effect. We efficiently isolated exosomes from 1× PBS buffer, cell culture media, and blood serum. Using flow rates from 150 to 200 μL/h and field strengths of 100 V/cm, we consistently captured between 60 and 80% of exosomes from buffer, cell culture media, and blood serum as confirmed by both fluorescence spectroscopy and nanoparticle tracking analysis. Our microfluidic chip maintained this recovery rate for more than 20 min with a concentration factor of 15 for 10 min of isolation.

Keywords: Exosomes; Ion concentration polarization; Microfluidics.

Figure 1.

a) Schematic of the overall set-up where a syringe pump drives sample into the microchip. b) A zoomed view of the channels; as the exosomes pass through the intersection of the perpendicular channels, an electric field drives them into the gel where they concentrate at the membrane. c) A view from below the chip of the exosome isolation process with fluorescently-labeled exosomes.

Figure 2.

Exosomes isolated from a sample of 1 X PBS after 10 min using a flow rate of 150 µL/hr and a field strength of 100 V/cm. a) The exosome size distribution before isolation experiments. b) Size distribution of the exosomes collected from the gel. c) Size distribution of the exosomes in the effluent. Error bars represent the standard error with n = 5. d) Quantitative exosome recovery as measured by the fluorescence of exosomes labeled with CFSE dye. The effluent was collected at 10 min intervals for two different flow rates using a constant field strength of 100 V/cm. The error bars are standard deviations with n = 3.

Figure 3.

Exosomes isolated directly from cell culture media using our microfluidic chip after 10 min with a flow rate of 150 µL/hr and an electric field of 100 V/cm. a) Single capture from NanoSight of cell culture media before isolation, b) Average size distribution of cell culture media before isolation. c) Single capture from NanoSight of exosomes isolated in gel. d) Average size distribution of exosomes isolated in gel. Error bars represent the standard error with n = 5.

Figure 4.

Size distributions for exosomes from blood serum. The flow rate was 150 µL/hr, and the field strength was 100 V/cm. a) Inlet single capture. b) Inlet average. c) Gel single capture. d) Gel average. e) Effluent single capture. f) Effluent average. Error bars represent the standard error with n = 5.