Concentration and purification of human immunodeficiency virus type 1 virions by microfluidic separation of superparamagnetic nanoparticles

Abstract

The low concentration and complex sample matrix of many clinical and environmental viral samples presents a significant challenge in the development of low cost, point-of-care viral assays. To address this problem, we investigated the use of a microfluidic passive magnetic separator combined with on-chip mixer to both purify and concentrate whole-particle human immunodeficiency virus type 1 (HIV-1) virions. Virus-containing plasma samples are first mixed to allow specific binding of the viral particles with antibody-conjugated superparamagnetic nanoparticles, and several passive mixer geometries were assessed for their mixing efficiencies. The virus-nanoparticle complexes are then separated from the plasma in a novel magnetic separation chamber, where packed micrometer-sized ferromagnetic particles serve as high magnetic gradient concentrators for an externally applied magnetic field. Thereafter, a viral lysis buffer was flowed through the chip and the released HIV proteins were assayed off-chip. Viral protein extraction efficiencies of 62% and 45% were achieved at 10 and 30 muL/min throughputs, respectively. More importantly, an 80-fold concentration was observed for an initial sample volume of 1 mL and a 44-fold concentration for an initial sample volume of 0.5 mL. The system is broadly applicable to microscale sample preparation of any viral sample and can be used for nucleic acid extraction as well as 40-80-fold enrichment of target viruses.

Fig. 1

a) Schematic of the device. Viral plasma sample is mixed with functionalized magnetic nanoparticles, then trapped in the magnetic separation chamber. b) Schematic of the magnetic separator, The microfluidic trapping chamber is 5mm long × 4mm wide × 120μm high. 25–75μm iron particles are physically trapped before the 20μm high outlet channel and are compacted into random close packing by the force of the flow. c) Suspension of polydisperse iron particles

Fig. 2

(a) Integrated mixer and separator device, with mixing channel on the left, and magnetic separation chamber on the right. (b) Microscope pictures of SU8 channel molds for the different mixer designs. (b-1) modified 2D Tesla structure. Channels are 100um high and 200um wide in the straight sections. (b-2) Staggered herringbone device. Main channels are 100um high and 300um wide. Herringbone features are 20um high and 50um wide. (b-3) Combined tesla + herringbone. Channels are 100um high and 200um widein the straight sections. Herringbone features are 20um high and 50um wide. (b-4) Straight channel device. Channels are 100um high and 300um wide.

Fig. 3

Separation efficiencies of the magnetic separator unit alone at varying flow rates (open circles). Comparison with the Miltenyi MACS μ-column and separator (filled circle). Control with no iron particle gradient concentrators (open triangle).

Fig. 4

(a) Viral capture efficiency of different mixers combined with the magnetic separator. 250 cycles of each mixer at 30μL/min. (b) Herringbone mixers of different lengths (c) Herringbone mixer (250 cycles) at different flow rates.

Fig. 5

(a) Viral protein recovered in first 5μL of eluted fluid. (b) Concentration factor versus initial sample volume and processing time, predicted and actual values for 30μL/min flow rate.