BACKGROUND

Field of the Invention

The present invention relates to methods and systems for cell lysis. More specifically, embodiments of the present invention relate to methods and systems for rapid continuous flow pathogen cell lysis on a microfluidic device.

Discussion of the Background

Cell lysis has many different applications. In particular, the rapid processing of whole blood is useful for the diagnosis of bloodstream infections. While healthcare providers wait on current methods to process whole blood and diagnose a specific infection, it is necessary to use broad-range therapies to treat infected patients, resulting in higher healthcare costs and increasing antibiotic resistance of pathogenic bacteria. Similarly, healthcare providers may have a desire to process other types of biological samples to diagnose infections, including, but not limited to, sputum, urine, cerebrospinal fluid, and the like.

Conventional cell lysis, in which the lipid membrane and cell wall are disrupted chemically, cannot work for the purposes of rapidly processing pathogenic cells. In conventional chemical cell lysis, cells must be lysed in lysis buffer and complete cellular lysis may require incubation for a prolonged period of time as well as agitation of the sample by vortexing or shaking Cell lysis in the context of microfluidic devices is also problematic. If the enzyme and sample are simply mixed while flowing through a microfluidic channel the microfluidic device, there is not enough residence time in the channel for cell lysis to be effective. Further, the lysis buffer may interfere with downstream processing. Mechanical lysis using rigid microstructures has been used for continuous flow lysis of mammalian cells, but is extremely challenging for pathogen cells because of the small size of the pathogen cells.

Chemical and mechanical methods have been used to attempt rapid pathogen cell lysis. For instance, Mahalanabis et al. (Lab Chip, 2009, 9, 2811-2817) reported a microfluidic system involving a hybrid chemical/mechanical method for lysing pathogen cells. They reportedly achieved a 13.3-16.6 μL/min flow rate, and their process reportedly involved two 30-90 minute enzyme incubations and a combination of detergents.

There are several problems with the current technology of pathogen cell lysis. First, the conventional approach is slow. Usually, the conventional approach takes hours or days to finish. This is partly because the conventional approach requires multiple enzyme incubations and requires cells to sit in a lysis buffer. The conventional approach does not utilize a rapid, continuous flow. That is, cells must sit in the lysis buffer in order for the enzymatic reaction to be effective. Another problem is that such methods rely on detergents or chaotropic salts, which are also slow and can inhibit downstream PCR. The presence of these lysis buffer components can also necessitate additional steps to separate them from the DNA to be used in later applications.

Although various methods and devices exist to lyse pathogen cells, none of these provides or describes a single device that is capable of rapid continuous flow cell lysis of pathogen cells. Thus, there is a need to develop improved microfluidic systems and methods for rapid continuous flow cell lysis.

SUMMARY OF THE INVENTION

The present invention relates to methods and systems for rapid continuous flow cell lysis. More specifically, embodiments of the present invention relate to methods and systems for the isolation of pathogen cells on a microfluidic device.

In one aspect, the present invention provides a microfluidic device for rapid continuous flow pathogen cell lysis. In one embodiment, the microfluidic device comprises a microfluidic channel and a microporous structure located within the microfluidic channel. The microfluidic device further comprises an enzyme immobilized on a surface of the microporous structure which is configured to lyse pathogen cells in fluid flowing through the microfluidic channel.

In some embodiments, the microporous structure is a monolith, which could be, for example, a polymer monolith or a silica monolith. In other embodiments, the microporous structure is a packed column of beads. In one embodiment, the beads have bifunctional chemistry. In some embodiments, the beads are made from material having bifunctional chemistry. In other-embodiments, the beads are coated with a material having bifunctional chemistry. One example of a material having bifunctional chemistry is chitosan. Other examples are well known to the skilled artisan. In additional embodiments, the enzyme comprises one or more proteolytic enzymes, for example, lysozyme and/or proteinase K. In some embodiments, the enzyme is covalently immobilized on the surface of the microporous structure. In some embodiments, the pathogen cells are bacterial cells (which can include gram-positive and gram-negative bacteria). In other embodiments, the pathogen cells are other types of pathogens, such as, for example, fungal cells. In some embodiments, the enzyme is configured to lyse pathogen cells in fluid flowing through the microporous structure in the microfluidic channel to provide a sufficient residence time in the microporous structure to lyse the pathogen cells.

In another aspect, the present invention provides a method for rapid continuous flow pathogen cell lysis. In one embodiment, the method comprises introducing pathogen cells into a microfluidic channel of a microfluidic device, wherein the microfluidic channel comprises a microporous structure with a lysis-promoting enzyme immobilized on a surface of the microporous structure. The method further comprises flowing the pathogen cells through the microporous channel of the microfluidic device, whereby the pathogen cells are lysed by the enzymes immobilized on the microporous structure. In some embodiments, the microporous structure is a monolith, which could be, for example, a polymer monolith or a silica monolith. In other embodiments, the microporous structure is a packed column of beads, as described herein. In some embodiments, the pathogen cells are bacterial cells. In additional embodiments, flowing the pathogen cells through the microfluidic channel comprises flowing the pathogen cells at a flow rate to provide sufficient residence time in the microporous structure to lyse the pathogen cells. In some embodiments a flow rate of at least about 20 to about 2000 μL/min, or at least about 20 to about 1000 μL/min, or at least about 50 to 500 μL/min, or at least about 50 to about 200 μL/min, or at least about 100 μL/min is utilized.

In some embodiments, the method further comprises providing the microfluidic device, wherein said microfluidic device comprises a microporous structure located in a microfluidic channel, and immobilizing a lysis-promoting enzyme on the surface of the microporous structure within the microfluidic device, before introducing pathogen cells into the microfluidic channel.

In another aspect, the present invention provides a method for making a microfluidic device. In one embodiment, the method comprises providing a microfluidic device, wherein said microfluidic device comprises a microporous structure located in a microfluidic channel. The method further comprises immobilizing a lysis-promoting enzyme on the surface of the microporous structure within the microfluidic channel of the microfluidic device.

The above and other embodiments of the present invention are described below with reference to the accompanying drawings.

