Isolation of intact bacteria from blood by selective cell lysis in a microfluidic porous silica monolith

Abstract

Rapid and efficient isolation of bacteria from complex biological matrices is necessary for effective pathogen identification in emerging single-cell diagnostics. Here, we demonstrate the isolation of intact and viable bacteria from whole blood through the selective lysis of blood cells during flow through a porous silica monolith. Efficient mechanical hemolysis is achieved while providing passage of intact and viable bacteria through the monoliths, allowing size-based isolation of bacteria to be performed following selective lysis. A process for synthesizing large quantities of discrete capillary-bound monolith elements and millimeter-scale monolith bricks is described, together with the seamless integration of individual monoliths into microfluidic chips. The impact of monolith morphology, geometry, and flow conditions on cell lysis is explored, and flow regimes are identified wherein robust selective blood cell lysis and intact bacteria passage are achieved for multiple gram-negative and gram-positive bacteria. The technique is shown to enable rapid sample preparation and bacteria analysis by single-cell Raman spectrometry. The selective lysis technique presents a unique sample preparation step supporting rapid and culture-free analysis of bacteria for the point of care.

Keywords: Engineering; Materials science.

Fig. 1

a Synthesis of porous silica monolith in fused silica capillary and thermoplastic mold via sol–gel chemistry. b Competitive reactions during the synthesis of monolith. Silanol groups present on glass capillary form covalent bonds with the monolith during this step

Fig. 2

a SEM image of a silica monolith synthesized within a 100 μm ID fused silica capillary, revealing uniform porosity and excellent wall anchoring of the monolith. b Histogram of pore size. Critical diameter for RBC hemolysis (2r*) is marked with an arrow

Fig. 3

a Integration of a silica monolith brick into a thermoplastic chip. A circular tape is placed on a monolith inserted into a COP substrate, and solvated COP is applied to the exposed surface. After partial drying, the tape is removed, the device is enclosed by another COP substrate, and fluid ports are inserted into holes that provide a flow path through the monolith. b SEM image of a monolith brick cut by wafer dicing saw. c Image of a device during whole blood perfusion

Fig. 4

DLS measurement of a initial 25× diluted blood, chemically lysed blood, and blood lysed by perfusion through the monolith device, revealing a significant reduction in cell debris size for mechanical monolith lysis over chemical lysis. b DLS measurement of E. cloacae suspended in 1× PBS, and sample perfused through the monolith device, showing no change in bacteria size. c DLS measurement of 100× diluted blood spiked with E. cloacae, and sample collected from the outlet of a porous monolith. The broad peak in the inlet sample indicates a mixed population of blood cells and small bacterial cells, whereas the outlet sample showed significant reduction in large (>2 µm) cells, as confirmed in the optical images. Scale bars = 25 µm

Fig. 5

a Monolith length dependence of RBC hemolysis. Totally, 50× diluted blood in 1× PBS was perfused through capillary monoliths of various lengths at a flow rate of 10 μL/min. b Passage rate of RBC and viable bacteria at different flow rates and lengths of monolith-containing capillary. Scale bars = 50 µm. Error bars are ±1SD. Contrast of optical images was adjusted for visibility

Fig. 6

a RBC lysis efficiency of whole blood in high-throughput devices following perfusion at 10 μL/min (EC E. cloacae, LL L. lactis, BS B. subtilis. Error bars are ±SD. N = 3 for blood and B + EC, and N = 2 for B + LL, B + BS. b Blood cell lysis and bacterial separation following serial operation using two monoliths. Surfaces were passivated with BSA/Tween 20. Over 99.999% RBC lysis was obtained while preserving viability of L. lactis and E. cloacae. Scale bars = 100 µm. c Raman spectra of whole blood spiked with E. cloacae (upper) before and (under) after processing through porous silica monolith