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.
Highlights
The presence of bacteria in the blood stream can lead to serious conditions, including sepsis and infection of other tissues, and early identification of blood-borne bacteria is necessary for effective treatment selection to enhance patient outcome
We show that selective passage may be achieved for bacteria in whole blood under flow conditions that yield highly efficient blood cell lysis
The integration of millimeter-scale monoliths into microfluidic flow cells supports high throughput processing of whole blood without dilution, with serial passage through multiple monoliths lysing all but 0.001% of red blood cells (RBCs) in the initial sample and allowing nearly 100% of bacteria to be recovered for downstream analysis
Summary
The presence of bacteria in the blood stream can lead to serious conditions, including sepsis and infection of other tissues, and early identification of blood-borne bacteria is necessary for effective treatment selection to enhance patient outcome. The ability to rapidly identify bacteria at or near the point of care would greatly enhance the ability of clinicians to initiate optimal treatment at the earliest stages of infection. Several powerful analytical methods including mass spectrometry[5], Raman spectrometry[6], and infrared spectrometry[7,8,9] can enable culture-free identification of bacteria. These techniques require the isolation and purification of bacteria from the initial clinical matrix. Size-based separation of bacteria from blood cells has been demonstrated using various microfluidic platforms employing inertial deflection[13], inertial lift[14], or Dean flow fractionation[15]. A central advantage associated with inertial microfluidics is that the separation may be performed in a continuous flow process, without the need for additional
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