Virus purification and enrichment by hydroxyapatite chromatography on a chip

Abstract

The spread of infectious diseases has become a global health concern. In order to diagnose infectious diseases quickly and accurately, next-generation DNA sequencing techniques for genetic analysis of infectious viruses have been developed rapidly. However, it takes a very long time to pretreat clinical samples for genetic analysis using next-generation sequencers. We have therefore developed a microfluidic chromatography chip that can purify and enrich viruses in a sample using hydroxyapatite particles packed in a micro-column. We demonstrated the purification of virus from a mixture of virus and FBS protein, and enrichment of the virus using this novel microfluidic chip.

Keywords: Hydroxyapatite chromatography; Infectious disease; Microfluidic chip; Virus enrichment; Virus purification.

Fig. 1

(a) Concept of the microfluidic chip for purification and enrichment of virus using hydroxyapatite chromatography. The microfluidic chip has a column for hydroxyapatite chromatography and a pair of switching valves. Hydroxyapatite particles are introduced into the column through inlet 1. The sample is introduced into the column through inlet 2 and both viruses and impurities in the sample are adsorbed onto the surface of hydroxyapatite particles. Then, an elution buffer is introduced into the column to elute the impurities such as proteins. Then, another elution buffer is introduced into the column to elute the viruses. (b) The fabricated microfluidic chip. Upstream and downstream of the column, 50-μm-diameter cylindrical micropillars placed at 20-μm intervals hold the hydroxyapatite particles in the column. The diameter of the hydroxyapatite particles is 40 μm.

Fig. 2

Fabrication process of the proposed microfluidic chip. The chip consists of a PDMS microchannel and a PDMS substrate. The PDMS microchannel was produced by replica molding using a master mold fabricated by photolithography.

Fig. 3

Fabrication process of a pair of switching valves. Two 8-mm-diameter holes were punched in the microfluidic chip using a biopsy punch. The PDMS disks in the biopsy punch were reinserted in the holes in the microfluidic chip to provide a pair of switching valves. The valves are opened or closed by rotation of the PDMS disks. This switching valve has the important advantage of being very easy to fabricate.

Fig. 4

Fabricated switching valve. The proposed valves enable the microchannels to be switched by rotation of the valves. The switching valve can be easily rotated by hand.

Fig. 5

Negative control (a) and positive control (b) for the hemagglutination reaction. The negative result appears as a red dot in the center of the round-bottomed plate because the red blood cells settle out. The positive result forms a uniform reddish color across the well because each agglutinating molecule on the virus coat binds to multiple red blood cells to form a bridged structure.

Fig. 6

Schematic of microfluidic chips for virus enrichment process. The width and the length of the microcolumns were (a) 2 mm and 10 mm, (b) 1 mm and 10 mm, (c) 1 mm and 20 mm, (d) 1 mm and 40 mm, (e) 0.5 mm and 40 mm.

Fig. 7

Results of the virus purification process from virus suspensions which included 5%, 10%, 15%, 20% FBS protein. Before chromatography, the hemagglutination reaction gave negative results even though the suspensions contained virus because FBS protein inhibits hemagglutination. After chromatography, the hemagglutination reaction gave positive results because the FBS proteins in the samples were removed by chromatography.

Fig. 8

Results of virus enrichment. (a) Microcolumns B, C and D are 1 mm wide. (b) Microcolumns A, C and E are equal in volume. The most effective enrichment was achieved with the microcolumn C, which is 1 mm wide and 20 mm long, providing an enrichment efficiency of 7.26. N = 4, *P < 0.05.