MICROFLUIDIC PLATFORM FOR THE CONCENTRATION AND DETECTION OF BACTERIAL POPULATIONS IN LIQUID

A microfluidic device for concentrating and detecting bacteria in liquids, and related methods are described. The device includes a first filter chamber for capturing bacteria and performing incubations of the bacteria with one or more reagents, and a second filter chamber for capturing and concentrating a detectable material, with little or no binding of detectable material by the first filter. In an aspect, bacteria are incubated with growth media and engineered phage that cause the bacteria to produce an enzyme. In an aspect, the enzyme is capture in the second filter chamber and exposed to a substrate to produce a detectable signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority Applications

All subject matter of the Priority Applications and of any and all applications related to the Priority Applications by priority claims (directly or indirectly), including any priority claims made and subject matter incorporated by reference therein as of the filing date of the instant application, is incorporated herein by reference to the extent such subject matter is not inconsistent herewith.

SUMMARY

In an aspect, a microfluidic device includes, but is not limited to, a sample inlet port adapted to receive a fluid sample containing bacteria of interest; a first filter chamber located downstream from the sample inlet port, the first filter chamber containing a first filter having a first area and formed from a first porous material having a pore size adapted to capture the bacteria of interest; a sample inlet channel connecting the sample inlet port to an upstream end of the first filter chamber; a sample control valve in the sample inlet channel, the sample control valve adapted to control a flow of the sample fluid from the sample inlet port to the upstream end of the first filter chamber; at least one first reagent inlet port located upstream of the first filter chamber and in fluid communication with the upstream end of the first filter chamber, the at least one first reagent inlet port adapted to deliver to the first filter chamber a first reagent containing a bacteriophage specific to the bacteria of interest and adapted to cause the bacteria of interest to release a reporter enzyme; at least one first reagent control valve adapted to control a flow of the first reagent from the first reagent inlet port to the upstream end of the first filter chamber; and a second filter chamber located downstream from the first filter chamber, the second filter chamber containing a second filter having a second area and formed from a second porous material adapted to specifically bind the reporter enzyme, wherein the second area is smaller than the first area; and a detection chamber control valve located downstream of the first filter chamber and adapted to control a flow of fluid to the second filter chamber; wherein the first filter is adapted to not bind the reporter enzyme. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a method of concentrating bacteria for detection includes, but is not limited to, introducing a fluid sample containing bacteria of interest in a carrier fluid to a sample inlet port of a microfluidic device; drawing the carrier fluid through a first filter in a first filter chamber of the microfluidic device and through a waste port downstream of the first filter chamber while the bacteria of interest are captured by the first filter; drawing a first reagent including growth media for the bacteria of interest from a first reagent inlet port into the first filter chamber; incubating the bacteria of interest captured by the first filter with the first reagent in the first filter chamber for a first incubation period sufficient to increase at least one of the metabolic activity or the number of cells of the bacteria of interest; drawing the first reagent through the first filter and through the waste port while the bacteria of interest remain captured by the first filter; drawing a second reagent including a bacteriophage specific to the bacteria of interest from a second reagent inlet port into the first filter chamber; incubating the bacteria of interest captured by the first filter with the second reagent in the first filter chamber for a second incubation period sufficient to produce expression of a reporter enzyme by the bacteria of interest; drawing a fluid containing the expressed reporter enzyme through the first filter, through a second filter in a second filter chamber of the microfluidic device, and through the waste port while the expressed reporter enzyme is captured by the second filter; and incubating the expressed reporter enzyme captured by the second filter with a third reagent in the second filter chamber for a third incubation period sufficient to produce a detectable signal in the detection chamber. In addition to the foregoing, other method aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a microfluidic device for bacteria detection includes, but is not limited to, a sample inlet port for receiving a fluid sample containing bacteria of interest; a first filter chamber containing a first filter adapted for capturing bacteria of interest from the fluid sample; first microfluidic means for introducing bacterial growth media to the first filter chamber; second microfluidic means for introducing phage specific to the bacteria of interest to the first filter chamber, the phage adapted to cause the bacteria of interest to produce a reactive material capable of reacting to produce a detectable signal; third microfluidic means for flushing reactive material from the first filter chamber, the reactive material released from the bacteria of interest responsive to introduction of the phage; and a second filter chamber containing a second filter for specifically capturing the reactive material flushed from the first filter chamber, wherein the second filter is smaller than the first filter to amplify the detectable signal; wherein the first filter is adapted to not capture the reactive material. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

DETAILED DESCRIPTION

The present invention relates to methods and system for detecting the presence of contaminants such as bacteria in liquids. In particular, the present invention relates to microfluidic devices for concentration and detection of bacteria in liquids.

FIGS. 1A to 1Hillustrate in simplified form a process for concentrating and detecting bacteria, suitable for performance in a microfluidic device. InFIG. 1A, a sample100containing bacteria102in fluid104is added to a first filter106. For example, it is of interest to detect the presence ofEscherichia coli(E. coli) in drinking water. InFIG. 1B, fluid104passes through first filter106, while bacteria102are captured by first filter106. InFIG. 1C, growth media110are added, and bacteria102are incubated in growth media110on first filter106, during a first incubation. In an aspect, bacteria present in an environmental sample are in a stationary growth phase. During the first incubation, the metabolic rate of the bacteria increases as the bacteria are exposed to growth media. Recovery of metabolic rate may take about 2 hours, for example. In an aspect, bacteria are allowed to replicate following metabolic recovery, to increase their numbers. For example, in cases where low bacterial concentrations are expected, bacteria may be allowed to replicate to produce a larger detectable signal. Bacterial replication can be obtained by incubating the bacteria in growth media for a sufficiently long amount of time after their metabolic rate has recovered (e.g., depending on the type of bacteria, about 20 minutes may be enough time for the bacterial population to double after metabolic rate has recovered). InFIG. 1D, growth media110are removed from first filter106, while bacteria102are captured by filter106. InFIG. 1E, a reagent112containing an engineered phage is added to first filter106. The engineered phage causes bacteria102to produce an enzyme114as well as replicate the phage. In an aspect, lytic protein released by the phage causes lysis of the bacteria, releasing phage and enzyme during a second incubation. InFIG. 1F, following the second incubation, enzyme114is flushed through first filter106to second filter116, carried by reagent112. Additional fluid (e.g. an additional wash of growth media) may be used to ensure complete transfer. Lysed bacteria122remain in first filter106. As shown inFIG. 1G, during a third incubation, enzyme114captured in second filter116is incubated with an enzyme substrate124. In an aspect, enzyme substrate124is added to the second filter116just prior to the third incubation. Following the third incubation, as shown inFIG. 1H, a detectable signal126produced by reaction of enzyme114with enzyme substrate124is detected from second filter116with a detector128.

Important aspects of the process illustrated inFIGS. 1A to 1Hare that first filter106captures the bacteria102, but not enzyme114, and that second filter116captures enzyme114. First filter106captures and concentrates bacteria102from liquid sample100. Second filter116has a smaller area than first filter106, in order to concentrate enzyme114to produce a greater detectable signal126. In an aspect, the “area” of the first filter or the second filter is a “binding area” or “effective filtering area” of the filter, which is related to the surface area of the filter but is not necessarily identical to the surface area of the filter. The first filter and the second filter are independently optimized for their respective functions.

FIG. 2is a schematic diagram of a microfluidic device200for performing a process as outlined inFIGS. 1A-1H. Microfluidic device200includes a sample inlet port202adapted to receive a fluid sample containing bacteria of interest, and a first filter chamber204located downstream from the sample inlet port202. For example, in an aspect, microfluidic device200is adapted to process a fluid sample having a volume of at least about 100 ml. First filter chamber204contains a first filter206having a first area and formed from a first porous material having a pore size adapted to capture the bacteria of interest. For example, in an aspect the first porous material has a pore size of about 0.45 μm. In an aspect, the first porous material has a pore size of less than about 0.45 μm. In an aspect, the first filter functions to filter bacteria from the sample fluid, which may be, for example, an environmental sample. In an aspect, the first porous material is a non-cellulose material. For example, in various aspects, the first porous material is formed from polyvinyilidene fluoride (PVDF), polycarbonate (PC), tracked-etched polycarbonate (PCTE), polyethersulfone (PES), and tracked-etched polyester. Use of non-cellulose material in the first filter prevents or minimizes binding of reporter enzyme to the first filter when the reporter enzyme includes a cellulose binding region (as discussed elsewhere herein). In general, the first filter material is selected such it captures the bacteria of interest without significantly binding the reporter enzyme (or other reporter molecules or materials). In an aspect, the first porous material has low protein binding activity.

Sample inlet channel208connects sample inlet port202to an upstream end210of first filter chamber204, and sample control valve212in sample inlet channel208is adapted to control a flow of sample fluid from sample inlet port202to upstream end210of first filter chamber204. Microfluidic device200includes at least one first reagent inlet port214located upstream of first filter chamber204and in fluid communication with the upstream end210of first filter chamber204. First reagent inlet port214is adapted to deliver to first filter chamber204a first reagent containing a bacteriophage specific to the bacteria of interest and adapted to cause the bacteria of interest to release a reporter enzyme. First filter206is adapted to bind the bacteria of interest, but not bind the reporter enzyme. At least one first reagent control valve216is adapted to control a flow of the first reagent from first reagent inlet port214to the upstream end210of first filter chamber204. A second filter chamber220, which functions as a detection chamber (from which a detectable signal can be detected) is located downstream from first filter chamber204. Second filter chamber220contains a second filter222having a second area and formed from a second porous material adapted to specifically bind the reporter enzyme. In an aspect, the second area is smaller than the first area. For example, in an aspect, the first area is about 315 mm2and the second area is about 3.14 mm2.

The function of the second membrane is to capture the enzyme, which in an aspect contains a cellulose binding domain. Accordingly, the second porous material includes a cellulose-based material such as regenerated cellulose, cellulose acetate, cellulose ester, and nitrocellulose. The size of the membrane is selected to concentrate the chemiluminescence reaction onto a smaller surface area for increased output signal.

In an aspect, the second porous material has a pore size of about 0.2 μm, for example. However, cellulose based porous materials are available with a variety of pore sizes, and materials with other pore sizes may be used, as appropriate for specific applications. In an aspect, second filter chamber220includes a detection region224configured to allow detection of a signal resulting from the reporter enzyme from outside the microfluidic device. In an aspect, detection region224includes a window formed from a clear material in microfluidic device200, allowing a signal resulting from reaction of the reporter enzyme with an enzyme substrate to be detected from outside microfluidic device200.

A detection chamber control valve226is located downstream of first filter chamber204and adapted to control a flow of fluid to second filter chamber220.

In general, fluid channels connecting components of microfluidic device200have dimensions on the order of a 100 μm high and a millimeter or two wide. For example, in various aspects, two or more of sample inlet port202, the at least one first reagent inlet port214, first filter chamber204, and second filter chamber220are fluidically connected by at least one fluid channel having a width of about 2 mm and height of about 100 μm. In some aspects, fluid channels may be between about 1 mm wide and about 3 mm wide and up to about 200 μm high. Different channel geometries may be used, depending upon the volume and types of fluids being handled.

In an aspect, the various valves in microfluidic device200(including, but not limited to first reagent control valve216and detection chamber control valve226) include pneumatically controlled valves. In this case, microfluidic device200also includes at least one air channel (for example as illustrated herein below inFIG. 18) for connecting at least one pneumatic pressure source to each such pneumatically controlled valve. In an aspect, air channels used to control pneumatically controlled valves have dimensions of about 1 mm wide and 100 μm high. In an aspect, microvalves are diaphragm valves. Pneumatically controlled diaphragm valves may be, for example, as described in U.S. Pat. No. 7,607,641 to Yuan or U.S. Pat. No. 6,431,212 to Hayenga et al, both of which are incorporated herein by reference. Other types of microvalves may be used, as well, and microfluidic devices as described herein are not limited to use with any specific type of microvalve.

In an aspect, microfluidic device200includes at least one air port230fluidically connected to the upstream end210of first filter chamber204and adapted for connection to a negative pressure (vacuum) source (not shown), e.g. to draw fluid into first filter chamber204. As used herein, the “upstream end” of first filter chamber204refers to upstream of first filter206, but not upstream of an inlet to the filter chamber. Further detail regarding the configuration of first filter chamber204is provided herein below. In an aspect, vent control valve232controls the flow of air through air port230. In some aspects, air port230may be vented to the atmosphere to release excess pressure within first filter chamber204. Alternatively, a positive pressure source may be attached to air port230to increase a pressure within first filter chamber204and/or drive fluid out of first filter chamber204. The same approach for venting and/or modifying pressure can be used with the second filter chamber, though not specifically depicted or described herein.

In an aspect, microfluidic device200includes at least one waste port234located downstream of first filter chamber204and adapted to receive fluid waste from the downstream end236of the first filter chamber204, and at least one waste control valve238adapted to control a flow of fluid waste from downstream end236of first filter chamber204to at least one waste port234. For example, in an aspect the at least one waste port234is adapted for connection to at least one negative pressure source (not shown).

In an aspect, microfluidic device200includes at least one at least one waste port234located downstream of second filter chamber220and adapted to receive fluid waste from the downstream end240of second filter chamber220. As depicted inFIG. 2, the waste port can be the same one used to receive waste fluid from first filter chamber204(i.e., waste port234). Alternatively, a separate waste port may be used. In an aspect, such a waste port is adapted for connection to a negative pressure source for drawing waste fluid into the waste port.

In an aspect, first reagent inlet port214is adapted to receive the first reagent from a reagent source, which may be, for example, a reservoir of liquid reagent external to the microfluidic device. In an aspect, microfluidic device200includes a reservoir containing lyophilized reagent in fluid communication with the at least one reagent inlet port (e.g. reservoir242depicted inFIG. 2), wherein the at least one first reagent inlet port214is adapted to receive a fluid adapted to rehydrate the lyophilized reagent to produce the first reagent for delivery to the first filter chamber.

In an aspect, microfluidic device200includes at least one second reagent inlet port250located upstream of first filter chamber204and in fluid communication with upstream end210of first filter chamber204, the at least one said second reagent inlet port250adapted to deliver to the first filter chamber a second reagent, and at least one second reagent control valve252adapted to control a flow of the second reagent from second reagent inlet port250to upstream end210of the first filter chamber204.

In an aspect, microfluidic device200includes a reservoir (not shown, but like reservoir242) containing lyophilized second reagent in fluid communication with second reagent inlet port250, where second reagent inlet port250is adapted to receive a fluid adapted to rehydrate the lyophilized second reagent to produce the second reagent for delivery to first filter chamber204.

In an aspect, microfluidic device200includes at least one third reagent inlet port256located upstream of first filter chamber204and in fluid communication with the upstream end210of first filter chamber204, the at least one said third reagent inlet port256adapted to deliver to first filter chamber204a third reagent, and at least one third reagent control valve258adapted to control a flow of the third reagent from third reagent inlet port256to the upstream end210of first filter chamber204. In an aspect, microfluidic device200includes a reservoir (not shown, but like reservoir242) containing lyophilized third reagent in fluid communication with the at least one third reagent inlet port256, wherein the at least one third reagent inlet port256is adapted to receive a fluid capable of rehydrating the lyophilized third reagent to produce the third reagent for delivery to first filter chamber204.

In an aspect, microfluidic device200also includes a bypass channel258fluidically connecting third reagent inlet port256to the downstream end236of first filter chamber204and the upstream end262of second filter chamber220, and a bypass valve264adapted to control a flow of the third reagent from the third reagent inlet port256to the downstream end236of first filter chamber204and upstream end262of second filter chamber220.

In an alternative configuration, the third reagent inlet port is in fluid communication with the downstream end of the first filter chamber and the upstream end of the second filter chamber, so that the third reagent can be delivered from the third reagent inlet port to the second filter chamber, and the third reagent control valve is adapted to control a flow of the third reagent from the third reagent inlet port to the upstream end of the second filter chamber. This is circuit configuration is obtained by modifying the fluid circuity depicted inFIG. 2by removing third reagent control valve and the fluid channel connecting third reagent inlet port256to the upstream end210of first filter chamber204. Examples of such configurations can be seen, e.g. in the devices depicted inFIGS. 18 and 21.

FIG. 3is a flow diagram of a method300of concentrating bacteria for detection, comprising, which can be performed using a microfluidic device as depicted inFIG. 2. Method300includes introducing a fluid sample containing bacteria of interest in a carrier fluid to a sample inlet port of a microfluidic device, at302; drawing the carrier fluid through a first filter in a first filter chamber of the microfluidic device and through a waste port downstream of the first filter chamber while the bacteria of interest are captured by the first filter, at304; drawing a first reagent including growth media for the bacteria of interest from a first reagent inlet port into the first filter chamber, as indicated at306; incubating the bacteria of interest captured by the first filter with the first reagent in the first filter chamber for a first incubation period sufficient to increase at least one of the metabolic activity or the number of cells of the bacteria of interest, at308; drawing the first reagent through the first filter and through the waste port while the bacteria of interest remain captured by the first filter, at310; drawing a second reagent including a bacteriophage specific to the bacteria of interest from a second reagent inlet port into the first filter chamber, at312; incubating the bacteria of interest captured by the first filter with the second reagent in the first filter chamber for a second incubation period sufficient to produce expression of a reporter enzyme by the bacteria of interest, at314; drawing a fluid containing the expressed reporter enzyme through the first filter, through a second filter in a second filter chamber of the microfluidic device, and through the waste port while the expressed reporter enzyme is captured by the second filter, at316; and incubating the expressed reporter enzyme captured by the second filter with a third reagent in the second filter chamber for a third incubation period sufficient to produce a detectable signal in the detection chamber, at318.

Further method aspects are shown inFIGS. 4-9. In these figures, steps302-318are as described in connection withFIG. 3. Optional and alternative steps are outlined with dashed lines.

FIG. 4depicts a method400, including further aspects relating to the bacterial sample and first incubation. In an aspect, the fluid sample is a water sample, as indicated at402. In various aspects, bacteria of interest areEscherichia coli, as indicated at404, or more generally, coliform bacteria, as indicated at406. In an aspect, the first reagent includes Luria-Bertani media, as indicated at408. Various other bacterial growth media may be used, as known to those having ordinary skill in the art. The first incubation period lasts about 2 hours at a temperature of about 37 degrees Celsius, for example, as indicated at410, and412, respectively. More generally, the first incubation period may last between about 1.5 hours and about 2.5 hours, as indicated at414, and be between about 25 degrees Celsius and about 45 degrees Celsius, as indicated at416.

FIG. 5depicts a method500, including further aspects relating to the second reagent and incubation period. In various aspects, the bacteriophage includes an engineered reporter bacteriophage, as indicated at502and/or a reporter bacteriophage specific to the bacteria of interest, as indicated at504. In an aspect, the bacteriophage is adapted to lyse the bacteria of interest to release a reporter enzyme, as indicated at506. In another aspect, the second reagent includes a fluid containing a cocktail of reporter bacteriophages, as indicated at508. In some aspects, the second reagent includes a fluid containing a reporter enzyme, as indicated at510. As an example, the second reagent includes T7-NanoLuc®-CBM (Cellulose Binding Module), as indicated at512.

Method500includes incubating the bacteria of interest captured by the first filter with the second reagent in the first filter chamber for a second incubation period sufficient to produce expression of a reporter enzyme by the bacteria of interest314, as discussed herein above. In an aspect, the reporter enzyme has a cellulose-binding domain, as indicated at514. In an aspect, the second incubation period lasts about 1 hours, as indicated at520, and is performed at about 37 degrees Celsius, as indicated at522. More generally, the second incubation period may last between about 0.5 and about 2.0 hours, as indicated at524, and may be performed at between about 25 degrees Celsius and about 45 degrees Celsius, as indicated at526.

FIG. 6depicts a method600, including further aspects relating the third incubation. In various aspect, incubating the expressed reporter enzyme with the third reagent generates a chemiluminescent signal, as indicated at602, a fluorescent signal, as indicated at604, or a colorimetric signal, as indicated at606. In an aspect, the detectable signal corresponds to the amount of the expressed reporter enzyme captured by the second filter, as indicated at608. The detectable signal can be detected with a luminometer, as indicated at610, or with other equipment capable of detecting an optical signal. In an aspect, the detectable signal may be in a non-visible portion of the electromagnetic spectrum, and equipment suitable for detecting other electromagnetic signals may be used.

FIG. 6also includes steps relating to handling of excess fluids after they have passed through the waste port. In some aspects, method600includes drawing at least one of the first reagent, the second reagent, and the third reagent through the waste port and into a waste reservoir, as indicated at612. In other aspects, method600includes drawing at least one of the first reagent, the second reagent, and the third reagent through the waste port and into a reagent reservoir, as indicated at614. As discussed herein above, waste reagents can be collected in a reagent reservoir and reused. In particular, in an aspect, a water sample which has previously passed through the first filter can be used to rehydrate lyophilized reagent to produce a second reagent for introduction into the first filter. Alternatively, rather than recycling the solvent (water) component of the reagent, the solute component of the reagent may be collected, either for reuse or to prevent release into the environment in the case that it includes a hazardous material.

FIG. 7depicts a method700providing further detail of aspects of fluid handling in the microfluidic device. Performance of method700with microfluidic device200is illustrated inFIG. 2. InFIG. 2andFIGS. 10-17, which are discussed herein below, fluid flow is indicated by heavy black lines, air flow is indicated by heavy dashed lines, open valves are indicated in black, and closed valves are indicated in white. Components identified by reference numbers inFIGS. 10-17are as described above in connection withFIG. 2. As indicated at702inFIG. 7, and illustrated inFIG. 2, drawing the carrier fluid from202through the first filter206in the first filter chamber204of the microfluidic device and through the waste port234downstream of the first filter chamber204while the bacteria of interest are captured by the first filter206includes opening a sample control valve212between the sample inlet port202and the first filter chamber204, opening a waste control valve238downstream of the first filter chamber204, and applying a negative pressure at the waste port234downstream of the filter chamber, as indicated at702inFIG. 7.

In addition, as shown inFIG. 7at704, and illustrated inFIG. 10, in an aspect, drawing the first reagent including growth media for the bacteria of interest from the first reagent inlet port214into the first filter chamber204includes closing the sample control valve212and waste control valve238, opening a first reagent control valve216between the first reagent inlet port214and the first filter chamber204, opening a vent control valve232between the filter chamber204and a vent outlet (air port230), and applying a negative pressure to the vent outlet (air port230).

In a further aspect, as shown inFIG. 7at706, and illustrated inFIG. 11, incubating the bacteria of interest captured by the first filter206with the first reagent in the first filter chamber204for the first incubation period sufficient to increase at least one of the metabolic activity or the number of cells of the bacteria of interest includes closing a first reagent control valve216and a vent control valve232.

FIG. 8is a flow diagram of a method800relating to further fluid handling aspects. In a further aspect, as shown inFIG. 8at802and illustrated inFIG. 12, drawing the first reagent through the first filter206and through the waste port234while the bacteria of interest remain captured by the first filter206includes opening a vent control valve232and a waste control valve238and applying a negative pressure at the waste port234.

In a further aspect, as shown inFIG. 8at804and illustrated inFIG. 13, drawing the second reagent including the bacteriophage specific to the bacteria of interest from the second reagent inlet port250into the first filter chamber204includes closing waste control valve238, opening a second reagent control valve252between the second reagent inlet port250and the first filter chamber204, and applying a negative pressure to the vent outlet (air port230).

In a further aspect, as shown inFIG. 8at806and illustrated inFIG. 14, incubating the bacteria of interest captured by the first filter206with the second reagent in the first filter chamber204includes closing a second reagent control valve252and a vent control valve232.

FIG. 9is a flow diagram showing further aspects of a method900of concentrating bacteria for detection. In an aspect, as shown inFIG. 9at902, and illustrated inFIG. 15, the fluid containing the expressed reporter enzyme includes the third reagent, wherein the third reagent is drawn from a third reagent inlet port256into the first filter chamber204, as indicated at902. For example, in an aspect, drawing the fluid containing the expressed reporter enzyme through the first filter206, through the second filter222in the second filter chamber220of the microfluidic device, and through the waste port234while the expressed reporter enzyme is captured by the second filter222includes opening a third reagent control valve258between a third reagent inlet port256and the first filter chamber204, opening a detection chamber control valve226downstream of the first filter chamber204, and applying a negative pressure at the waste port234, wherein the second filter chamber220is fluidically connected between the detection chamber control valve226and the waste port234, as indicated at904inFIG. 9.

Alternatively, as shown inFIG. 9at906, the fluid containing the expressed reporter enzyme includes the second reagent (here, the fluid remaining in the first filter chamber following the second incubation), and wherein the third reagent is drawn from a third reagent inlet port256into the second filter chamber220. For example, as shown inFIG. 9at908, in an aspect this can be accomplished by drawing the fluid containing the expressed reporter enzyme through the first filter, through the second filter in the second filter chamber of the microfluidic device, and through the waste port while the expressed reporter enzyme is captured by the second filter. This could be done by opening vent control valve232upstream of first filter chamber204, opening detection chamber control valve226fluidically connected between the downstream end236of the first filter chamber204and an upstream end262of the second filter chamber220, and applying a negative pressure at waste port234. As shown inFIG. 9at908, and illustrated inFIG. 16, the third reagent is drawn from a third reagent inlet port256into the second filter chamber220prior to the third incubation period by closing the vent control valve232, opening a third reagent control valve (here, bypass valve264) fluidically connected between a third reagent inlet port256and a downstream end236of the first filter chamber204, opening a detection chamber control valve226, and applying a negative pressure at the waste port234, wherein the second filter chamber220is fluidically connected between the detection chamber control valve226and the waste port234.

In a further aspect, as shown inFIG. 8at808and illustrated inFIG. 17, incubating the expressed reporter enzyme captured by the second filter222with the third reagent in the second filter chamber220for the third incubation period includes closing the third reagent control valve258and the detection chamber control valve226. Following the incubation period, a detectable signal is detected from second filter chamber220.

FIG. 18is a photograph of an example of a microfluidic device1800containing fluid circuitry for performing a method as described in connection withFIGS. 2andFIG. 4-9. In an aspect, microfluidic device1800is used for detectingE. coliin a water sample.FIG. 18is top view of microfluidic device1800. In an aspect, in use, microfluidic device1800is placed on a horizontal surface, with the surface visible inFIG. 18facing upward. Alternatively, in some aspects microfluidic devices as described herein may be oriented vertically, e.g. to reduce footprint and/or to process more samples in parallel. Microfluidic device1800is formed from a laminated polymeric substrate1802. In the example ofFIG. 18, microfluidic device1800is formed of polycarbonate sandwiched between layers of acrylic. Layers are adhered together by a pressure sensitive adhesive. Layers are aligned and adhered together. Construction of microfluidic device1800is described in greater detail herein below.

Sample inlet port1804includes an attached Luer lock that permits a syringe filter or cup containing sample fluid to be interfaced with microfluidic device1800. Sample fluid travels from sample inlet port1804through fluid channel1806to first filter chamber1808. Flow of sample fluid is controlled by sample control valve1810, which is a pneumatically controlled valve. Air channel1812connects to air port1814which is configured for connection with a pneumatic pressure source for controlling sample control valve1810. In microfluidic device1800, air port1814includes a hose barb that can be connected to a line leading to a pneumatic pressure source. Alternatively, air ports can be configured for connection to a pneumatic pressure source by having a smooth surface around the air port, to which an o-ring or other seal-forming element can be pressed or clamped to form a sealed connection.

First reagent inlet port1816includes a Luer lock. First reagent inlet port1816is connected to fluid channel1806by channel1818. First reagent control valve1820is controlled via air channel1822connected to air port1824. Second reagent inlet port1830also includes a Luer lock. Second reagent inlet port1830is connected to fluid channel1806by channel1832. Second reagent control valve1834is controlled via air channel1836connected to air port1838. First reagent inlet port1816and second reagent inlet port1830are fluidically connected to the upstream end1840of first filter chamber1808. Third reagent inlet port1850is fluidically connected to the downstream end1852of first filter chamber1808. This is allows for delivery of third reagent in the manner depicted inFIG. 16. Third reagent control valve1854is controlled via air channel1856leading to air port1858. From downstream end1852of first filter chamber1808, fluid can be delivered to waste port1860under control of waste control valve1862, or second filter chamber1864under control of detection chamber control valve1866. Waste control valve1862is controlled via air channel1870to air port1872, and detection chamber control valve1866is controlled via air channel1874to air port1876. Channel1878provides for waste fluid and/or air to be drawn from the downstream end of second filter chamber1864to waste port1860.

Air ports1824,1838,1858,1872, and1876include hose barbs for connecting to a pneumatic pressure source for controlling valve operation. Waste port1860also includes a hose barb, for connection to a negative pressure source. As noted above, a fluid reservoir (not shown; external to microfluidic device1800) may be associated with waste port1860, to collect fluid exiting waste port1860. Sample inlet port1804and reagent inlet ports1816,1830and1850include Luer locks for interfacing with fluid sources.

First filter chamber1808has flattened cylinder shape to accommodate filter1890, which is disk shaped with a central hole1892. Filter1890is formed from polyvinyldifluoride, with a thickness of 110-150 μm and pore size of about 0.45 μm (available from Sterlitech Corporation, Kent, Wash.). Filter1890capturesE. colifrom the fluid sample. A spiral channel1894in the upper surface of first filter chamber1808distributes fluid rapidly over the top surface of filter1890, within the spiral channel1894, before it spreads laterally and downward through filter1890. The function of the first filter is to filter the bacteria from the environmental sample. In an aspect, it is desired to process at least 100 mL within a relatively short period of time (e.g., few minutes).

Filtration time is influenced by membrane pore size (here, 0.45 μm or smaller), channel aspect ratio, channel length-membrane size, and effective filtering area, which is depends upon spiral channel geometry. At the same time, it is desired to reducing adverse protein interactions (enzyme binding) and minimizing device footprint, to enhance portability of the device.

In an aspect, the first filter has an area of 315 mm2. The area of the spiral channel above the first filter is 200 mm2. The channel is 200 μm high, giving a channel volume of 40 μl. Hypothetically, the channel area above the filter can accommodate, in a single layer, about 0.2 mm3or 0.2 mg of bacteria (assuming bacteria areE. coli, each having dimensions of 0.5 μm×2 μm and mass of 1 pg).

The construction of first filter chamber1808can be understood with reference toFIG. 19A, which is a cross-sectional side view of first filter chamber1808, taken at section line A-A inFIG. 18. The top surface of the microfluidic device1800is indicated at1900, and the bottom surface is indicated at1902. Fluid enters at the top of first filter chamber1808from fluid channel1806at upstream end1840from fluid channel1806, and exits at downstream end1852. The direction of fluid flow is indicated by arrows inFIG. 19A. As can be seen, fluid channel1806is formed in a second layer of microfluidic device1800. Fluid travels through via1906from fluid channel1806to spiral channel1894. InFIG. 19A, fluid flow out of the plane of the page is indicated by a circle containing a dot, and fluid flow into the plane of the page is indicated by a circle containing an X. Fluid flows in spiral channel1894sequentially through segments1894a,1894b,1894cand1894d.At the same time, fluid penetrates through filter1890to a corresponding channel1908on the lower surface of filter chamber1808, where it flows through segments1908a,1908b,1908c,1908d,and1908e.Channel1908collects fluid that has passed through filter1890. Fluid then passes through central hole1892to channel1912that exits downstream of the filter at the center of first filter chamber1808. As can be seen inFIG. 19A, although channel1912exits first filter chamber1808in a layer above filter1890, fluid enters channel1912only after it has passed through filter1890.

FIG. 19Bis a cross-sectional side view of second filter chamber1864, taken at section line B-B inFIG. 18. The top surface of the microfluidic device1800is indicated at1900, and the bottom surface is indicated at1902. Fluid enters at the top of second filter chamber1864from at inlet1920, which is fluidically connected to the downstream end1852of first filter chamber1808, as shown inFIG. 18. It passes through second filter1922and exits via channel1878, which as discussed herein above leads to waste port1860, as shown inFIG. 18. Second filter1922is formed from nitrocellulose having a pore size of about 0.2 μm and thickness of between about 101.6 and about 190.5 μm (manufactured by Pall Industries, Port Washington, N.Y.). Second filter1922binds the cellulose binding module tag on the enzyme. Second filter1922can have different pore sizes providing it captures the reporter enzyme, e.g. by binding the cellulose binding module tag. The material forming the structure of microfluidic device1800is substantially transparent, hence a detectable signal produced by material in second filter chamber1864and/or captured by second filter1922can be detected through top surface1900. In embodiments in which the main structure of the microfluidic device is formed from a material that does not transmit the detectable signal, at least one surface of the second filter chamber can be formed from a material transparent to the detectable signal, to permit detection of the detectable signal from the exterior of the microfluidic device.

FIG. 20depicts an alternative layout for a microfluidic device2000for performing fluid handling steps substantially similar to those performed by the microfluidic device ofFIG. 18. Microfluidic device2000includes sample inlet port2002, first reagent inlet port2004, and second reagent inlet port2006, connected to channel2008leading to inlet2010of first filter chamber2012. Sample control valve2014, first reagent control valve2016and second reagent control valve2018are controlled via air ports2020,2022, and2024, respectively. Spiral channel2026runs from inlet2010to outlet2030. As described in connection withFIG. 18, spiral channel2026is on the upstream side of the first filter chamber2012(i.e., on a first side of the filter, which is not depicted inFIG. 20, but as described in connection withFIG. 18). A corresponding spiral channel (not shown) is on the downstream side of the first filter chamber (i.e., on a second side of the filter). Outlet2030is located on the downstream side of the first filter chamber2012, and receives fluid that has passed through the first filter and entered the spiral channel on the downstream side of the filter. Vent2032is located on the first (upstream) side of the first filter chamber, at a distal end of spiral channel2026, such that a vacuum applied to vent2032(via air port2034) causes fluid to flow into spiral channel2026, as described in connection with step806ofFIG. 8. In addition, air port2034can be opened to permit fluid to be drawn through the first filter and into the second filter chamber, e.g. as described in connection with step908ofFIG. 9. Microfluidic device2000also includes third reagent inlet port2040, second filter chamber2042, outlet port2044, and vent2046. Fluid flow downstream of first filter chamber2012is controlled by third reagent control valve2050, detection chamber control valve2052, and waste control valve2054, controlled via air ports2060,2062, and2064, respectively. Air port2066is connected to vent2046. It will be appreciated that the microfluidic devices depicted inFIGS. 18 and 20provide two different layouts for performing substantially the same fluid handling functions. The devices differ in the arrangements of air ports and fluid inlets and outlets on the device, and differ slightly in venting arrangement. For example, other configurations may be used to optimize particular aspects of device performance or reduce device footprint.

Microfluidic devices as described herein can be attached to fluid sources supplying sample and reagent fluids, to pneumatic control lines for controlling operation of pneumatic valves, and one or more negative pressure source with associated waste or reagent reservoir for collecting fluid that has passed through the device. In an aspect, a microfluidic device includes attached hose barbs and/or Luer locks for connecting to air or fluid sources, as shown inFIG. 18. In other aspects, air or fluid sources include o-rings or other seal-forming elements that are pressed or clamped against the microfluidic device to form a sealed connection with respective air or fluid inlet openings in the device. Air or fluid sources may be connected individually to a microfluidic device, or multiple air and/or fluid sources may be connected to a microfluidic device via a manifold device that provides connection to multiple air or fluid inlet openings at the same time. Fluid waste or air vent lines may be connected to a microfluidic device in the same manner.

Pneumatic microvalves can be controlled, for example, by an ADEPT (ALine Development Platform) 12 Channel Pneumatic Controller from ALine, Inc., Rancho Dominguez, Calif., USA). The ADEPT is a programmable microfluidic controller that can operate up to 16 independent pneumatic valves under software control with programming from a computer interface, or, alternatively, by manual switches.

Incubation steps as described herein may be performed by placing the microfluidic device into an incubator. Alternatively, in an aspect, the microfluidic device may include one or more onboard heating element (e.g. a resistive element). In another aspect, the microfluidic device may be locally heated by application of energy via a laser, focused RF or ultrasonic energy, or the like.

In an aspect, multiple microfluidic devices can be processed in parallel by using a custom-built device that is adapted to interface with multiple microfluidic devices at the same time. Such a device could include, for example, positive and negative pressure sources for controlling valves and driving the flow of fluid through the device, reagent sources, and reservoirs for capturing (and optionally recycling) waste fluid. In an aspect, a reagent source could include a reservoir or liquid reagent.

As noted above, microfluidic devices as described herein can be formed from a laminated polymeric substrate. For example, in some aspects, microfluidic devices are formed from layers of polycarbonate sandwiched between layers of acrylic. Materials for use in microfluidic devices as described herein may be selected for various properties, including biocompatibility, optical clarity (for detection area) and low protein binding. In some aspects, channels and chambers are formed by laser etching; alternatively, channels and chambers can be die cut or formed by other manufacturing methods. In an aspect, layers are aligned and adhered together with a pressure sensitive adhesive (such as silicone plus tackifiers). Alternatively, other adhesive materials, such as thermally sensitive adhesives can be used. Microfluidic devices as described herein can be formed with different numbers and types of layers.

Microfluidic devices as described herein can be manufactured by various processes, for example as described in Levine, Leanna, M. “Developing Diagnostic Products Using Polymer Laminate Technology,” Aline, Inc., Redondo Beach, Calif.; and Fiorini, Gina S., Chiu, Daniel T., 2005, “Disposable microfluidic devices: fabrication, function, and application,” BioTechniques 38: 429-446, March 2005, each of which is incorporated herein by reference. In an aspect, a microfluidic device can be manufactured from cast plastic material (e.g. polydimethylsiloxane (PDMS)), e.g. as described in Friend, James and Yeo, Leslie (2010) “Fabrication of microfluidic devices using polydimethylsiloxane,” BIOMICROFLUIDICS 4, 026502, doi: 10.1063/1.3259624, which is incorporated herein by reference. For example, a device can be manufactured from laminated polymeric sheet materials by a reel-to-reel process of the type described, for example, in U.S. Published Patent Application No. 2009/0173428 to Klingbeil et al. and U.S. Pat. No. 6,375,871 to Bentsen et al., both of which are incorporated herein by reference. Devices can be made through injection molding processes, as well.

Detection of bacteria in contaminated fluid samples can be performed with different combinations of reagents. In the examples described herein, an engineered phage causes bacteria to produce an enzyme that produces luminescence when it interacts with substrate. In an aspect, the phage can be engineered to cause production of a NanoLuc® Reporter enzyme that includes a cellulose binding module tag that causes it to bind to the nitrocellulose material of the second filter. The NanoLuc® Reporter enzyme is used in combination with Nano-Glo® Luciferase Assay Reagent (the third reagent) (both obtained from Promega Corporation, Madison, Wis.) to produce a detectable signal at λ=460 nm. The luminescence can be detected with a luminometer. It will be appreciated that microfluidic devices as described herein can be configured (through appropriate selection of filter materials) to work in combination with bacteria and assay reagents other than those described specifically herein.

In the example provided herein, bacteria are lysed by the engineered phage used to induce production of the reporter enzyme. Alternatively, the microfluidic device could be modified to produce lysis of the bacteria through some other mechanism. For example, means for lysing the bacteria can include, but are not limited to, reagents such as enzymes, changing device temperature, sonication, or pressure. In an aspect, the microfluidic device includes lysing means for lysing the bacteria of interest to release the reactive material. For example, in various aspects, a lysing means includes heating means, acoustic means (e.g., a sonicator), a pressure source, a reagent source, or an enzyme source. In an aspect, the microfluidic device is configured to cooperate with an external lysing means, such as an external heat source or external acoustic source for providing sonication.

Microfluidic devices described herein utilize microfluidic means such as various combinations of microchannels, microvalves, filters, fluid or air ports, associated fluid sources, reagent reservoirs (containing liquid or lyophilized reagent materials), and positive and negative pressure sources, to perform a variety of functions, including, but not limited to, capturing bacteria of interest from the fluid sample, introducing bacterial growth media, introducing phage specific to the bacteria of interest, flushing reactive material (e.g., an enzyme) released from the bacteria of interest responsive to introduction of the phage, capturing the reactive material flushed from the first filter chamber, and performing readout of the detectable signal, It will be appreciated that various different microfluidic circuit configurations can provide equivalent functionality, and the invention is not limited to the specific fluid circuitry configurations depicted herein.

Aspects of the subject matter described herein are set out in the following numbered clauses:

Clause 1. A microfluidic device comprising:

a sample inlet port adapted to receive a fluid sample containing bacteria of interest;

a first filter chamber located downstream from the sample inlet port, the first filter chamber containing a first filter having a first area and formed from a first porous material having a pore size adapted to capture the bacteria of interest;

a sample inlet channel connecting the sample inlet port to an upstream end of the first filter chamber;

a sample control valve in the sample inlet channel, the sample control valve adapted to control a flow of the sample fluid from the sample inlet port to the upstream end of the first filter chamber;

at least one first reagent inlet port located upstream of the first filter chamber and in fluid communication with the upstream end of the first filter chamber, the at least one first reagent inlet port adapted to deliver to the first filter chamber a first reagent containing a bacteriophage specific to the bacteria of interest and adapted to cause the bacteria of interest to release a reporter enzyme;

at least one first reagent control valve adapted to control a flow of the first reagent from the first reagent inlet port to the upstream end of the first filter chamber; and

a second filter chamber located downstream from the first filter chamber, the second filter chamber containing a second filter having a second area and formed from a second porous material adapted to specifically bind the reporter enzyme, wherein the second area is smaller than the first area; and

a detection chamber control valve located downstream of the first filter chamber and adapted to control a flow of fluid to the second filter chamber;

wherein the first filter is adapted to not bind the reporter enzyme.

Clause 2. The microfluidic device of clause 1, wherein the microfluidic device is adapted to process a fluid sample having a volume of at least about 100 ml.

Clause 3. The microfluidic device of clause 1, wherein two or more of the sample inlet port, the at least one reagent inlet port, the first filter chamber, and the second filter chamber are fluidically connected by at least one fluid channel having a width of about 2 mm and height of about 100 μm.

Clause 4. The microfluidic device of clause 1, wherein the first porous material includes at least one of polyvinyilidene fluoride (PVDF), polycarbonate (PC), tracked-etched polycarbonate (PCTE), polyethersulfone (PES), and tracked-etched polyester.

Clause 5. The microfluidic device of clause 1, wherein the first porous material has low protein binding activity.

Clause 6. The microfluidic device of clause 1, wherein the first porous material is a non-cellulose material.

Clause 7. The microfluidic device of clause 1, wherein the first porous material has a pore size of about 0.45 μm.

Clause 8. The microfluidic device of clause 1, wherein the first porous material has a pore size of less than about 0.45 μm.

Clause 9. The microfluidic device of clause 1, wherein the second porous material includes a cellulose-based material.

Clause 10. The microfluidic device of clause 1, wherein the second porous material includes at least one of regenerated cellulose, cellulose acetate, cellulose ester, and nitrocellulose.

Clause 11. The microfluidic device of clause 1, wherein the second porous material has a pore size of about 0.2 μm.

Clause 12. The microfluidic device of clause 1, wherein the second filter chamber includes a detection region configured to allow detection of a signal resulting from the reporter enzyme from outside the microfluidic device.

Clause 13. The microfluidic device of clause 1, wherein the first area is about 315 mm2and the second area is about 3.14 mm2.

Clause 14. The microfluidic device of clause 1, wherein at least one of the sample control valve, the first reagent control valve, and detection chamber control valve includes a diaphragm valve.

Clause 15. The microfluidic device of clause 1, wherein at least one of the sample control valve, the first reagent control valve, and the detection chamber control valve includes a pneumatically controlled valve.

Clause 16. The microfluidic device of clause 15, including at least one air channel for connecting at least one pneumatic pressure source to the pneumatically controlled valve.

Clause 17. The microfluidic device of clause 1, including at least one air port fluidically connected to the upstream end of said first filter chamber and adapted for connection to a negative pressure source.

Clause 18. The microfluidic device of clause 1, including

at least one at least one waste port located downstream of the first filter chamber and adapted to receive fluid waste from the downstream end of the first filter chamber; and

at least one waste control valve adapted to control a flow of fluid waste from the downstream end of the first filter chamber to the at least one waste port.

Clause 19. The microfluidic device of clause 18, wherein the at least one waste port is adapted for connection to at least one negative pressure source.

Clause 20. The microfluidic device of clause 1, including

at least one at least one waste port located downstream of the second filter chamber and adapted to receive fluid waste from the downstream end of the second filter chamber.

Clause 21. The microfluidic device of clause 20, wherein the at least one waste port is adapted for connection to at least one negative pressure source.

Clause 22. The microfluidic device of clause 1, wherein the at least one first reagent inlet port is adapted to receive the first reagent from a reagent source.

Clause 23. The microfluidic device of clause 1, including a reservoir containing lyophilized reagent in fluid communication with the at least one reagent inlet port, wherein the at least one first reagent inlet port is adapted to receive a fluid adapted to rehydrate the lyophilized reagent to produce the first reagent for delivery to the first filter chamber.

Clause 24. The microfluidic device of clause 1, including

at least one second reagent inlet port located upstream of the first filter chamber and in fluid communication with the upstream end of the first filter chamber, the at least one said second reagent inlet port adapted to deliver to the first filter chamber a second reagent;

at least one second reagent control valve adapted to control a flow of the second reagent from the second reagent inlet port to the upstream end of the first filter chamber.

Clause 25. The microfluidic device of clause 24, including a reservoir containing lyophilized second reagent in fluid communication with the at least one second reagent inlet port, wherein the at least one second reagent inlet port is adapted to receive a fluid adapted to rehydrate the lyophilized second reagent to produce the second reagent for delivery to the first filter chamber.

Clause 26. The microfluidic device of clause 24, including

at least one third reagent inlet port located upstream of the first filter chamber and in fluid communication with the upstream end of the first filter chamber, the at least one said third reagent inlet port adapted to deliver to the first filter chamber a third reagent; and

at least one third reagent control valve adapted to control a flow of the third reagent from the third reagent inlet port to the upstream end of the first filter chamber.

Clause 27. The microfluidic device of clause 26, including a reservoir containing lyophilized third reagent in fluid communication with the at least one third reagent inlet port, wherein the at least one third reagent inlet port is adapted to receive a fluid capable of rehydrating the lyophilized third reagent to produce the third reagent for delivery to the first filter chamber.

Clause 28. The microfluidic device of clause 26, including

a bypass channel fluidically connecting the third reagent inlet port to the downstream end of the first filter chamber and the upstream end of the second filter chamber, and

a bypass valve adapted to control a flow of the third reagent from the third reagent inlet port to the downstream end of the first filter chamber and the upstream end of the second filter chamber.

Clause 29. The microfluidic device of clause 24, including

at least one third reagent inlet port in fluid communication with the downstream end of the first filter chamber and the upstream end of the second filter chamber, the at least one said third reagent inlet port adapted to deliver a third reagent to the second filter chamber; and

at least one third reagent control valve adapted to control a flow of the third reagent from the third reagent inlet port to the upstream end of the second filter chamber.

Clause 30. The microfluidic device of clause 1, formed from laminated polymeric sheet materials by a reel-to-reel process.

Clause 31. The microfluidic device of clause 1, formed from cast polymeric material.

Clause 32. The microfluidic device of clause 1, formed by injection molding.

Clause 33. A method of concentrating bacteria for detection, comprising:

introducing a fluid sample containing bacteria of interest in a carrier fluid to a sample inlet port of a microfluidic device;

drawing the carrier fluid through a first filter in a first filter chamber of the microfluidic device and through a waste port downstream of the first filter chamber while the bacteria of interest are captured by the first filter;

drawing a first reagent including growth media for the bacteria of interest from a first reagent inlet port into the first filter chamber;

incubating the bacteria of interest captured by the first filter with the first reagent in the first filter chamber for a first incubation period sufficient to increase at least one of the metabolic activity or the number of cells of the bacteria of interest;

drawing the first reagent through the first filter and through the waste port while the bacteria of interest remain captured by the first filter;

drawing a second reagent including a bacteriophage specific to the bacteria of interest from a second reagent inlet port into the first filter chamber;

incubating the bacteria of interest captured by the first filter with the second reagent in the first filter chamber for a second incubation period sufficient to produce expression of a reporter enzyme by the bacteria of interest;

drawing a fluid containing the expressed reporter enzyme through the first filter, through a second filter in a second filter chamber of the microfluidic device, and through the waste port while the expressed reporter enzyme is captured by the second filter; and

incubating the expressed reporter enzyme captured by the second filter with a third reagent in the second filter chamber for a third incubation period sufficient to produce a detectable signal in the detection chamber.

Clause 34. The method of clause 32, wherein the fluid containing the expressed reporter enzyme includes the third reagent, wherein the third reagent is drawn from a third reagent inlet port into the first filter chamber.

Clause 35. The method of clause 32, wherein the fluid containing the expressed reporter enzyme includes the second reagent, and wherein the third reagent is drawn from a third reagent inlet port into the second filter chamber.

Clause 36. The method of clause 32, including detecting the detectable signal with a luminometer.

Clause 37. The method of clause 32, wherein the fluid sample is a water sample.

Clause 38. The method of clause 32, wherein the bacteria of interest areEscherichia coli.

Clause 39. The method of clause 32, wherein the bacteria of interest are coliform bacteria.

Clause 40. The method of clause 32, wherein incubating the expressed reporter enzyme with the third reagent generates a chemiluminescent signal.

Clause 41. The method of clause 32, wherein incubating the expressed reporter enzyme with the third reagent generates a fluorescent signal.

Clause 42. The method of clause 32, wherein incubating the expressed reporter enzyme with the third reagent generates a colorimetric signal.

Clause 43. The method of clause 32, wherein the reporter enzyme has a cellulose-binding domain.

Clause 44. The method of clause 32, wherein the detectable signal corresponds to the amount of the expressed reporter enzyme captured by the second filter.

Clause 45. The method of clause 32, wherein the first incubation period lasts about 2 hours.

Clause 46. The method of clause 32, wherein the first incubation period lasts between about 1.5 hours and about 2.5 hours.

Clause 47. The method of clause 32, wherein the first incubation period is performed at about 37 degrees Celsius.

Clause 48. The method of clause 32, wherein the first incubation period is performed at between about 25 degrees Celsius and about 45 degrees Celsius.

Clause 49. The method of clause 32, wherein the second incubation period lasts about 1 hour.

Clause 50. The method of clause 32, wherein the second incubation period lasts between about 0.5 hours and about 2 hours.

Clause 51. The method of clause 32, wherein the second incubation period is performed at about 37 degrees Celsius.

Clause 52. The method of clause 32, wherein the second incubation period is performed at between about 25 degrees Celsius and about 45 degrees Celsius.

Clause 53. The method of clause 32, wherein drawing the carrier fluid through the first filter in the first filter chamber of the microfluidic device and through the waste port downstream of the first filter chamber while the bacteria of interest are captured by the first filter includes opening a sample control valve between the sample inlet port and the filter chamber, opening a waste control valve downstream of the filter chamber, and applying a negative pressure at the waste port downstream of the filter chamber.

Clause 54. The method of clause 32, including drawing at least one of the first reagent, the second reagent, and the third reagent through the waste port and into a waste reservoir.

Clause 55. The method of clause 32, including drawing at least one of the first reagent, the second reagent, and the third reagent through the waste port and into a reagent reservoir.

Clause 56. The method of clause 32, wherein drawing the first reagent including growth media for the bacteria of interest from the first reagent inlet port into the filter chamber includes closing the sample control valve and waste control valve, opening a first reagent control valve between the first reagent inlet port and the filter chamber, opening a vent control valve between the filter chamber and a vent outlet, and applying a negative pressure to the vent outlet.

Clause 57. The method of clause 32, wherein the first reagent includes Luria-Bertani media.

Clause 58. The method of clause 32, wherein incubating the bacteria of interest captured by the first filter with the first reagent in the filter chamber for the first incubation period sufficient to increase at least one of the metabolic activity or the number of cells of the bacteria of interest includes closing a first reagent control valve and a vent control valve.

Clause 59. The method of clause 32, wherein drawing the first reagent through the first filter and through the waste port while the bacteria of interest remain captured by the first filter includes opening a vent control valve and a waste control valve and applying a negative pressure at the waste port.

Clause 60. The method of clause 32, wherein the bacteriophage includes an engineered reporter bacteriophage.

Clause 61. The method of clause 32, wherein the bacteriophage includes a reporter bacteriophage specific to the bacteria of interest.

Clause 62. The method of clause 32, wherein the bacteriophage is adapted to lyse the bacteria of interest to release a reporter enzyme.

Clause 63. The method of clause 32, wherein the second reagent includes a fluid containing a cocktail of reporter bacteriophages.

Clause 64. The method of clause 32, wherein the second reagent includes a fluid containing a reporter enzyme.

Clause 65. The method of clause 32, wherein the second reagent includes T7-NanoLuc®-Cellulose Binding Module.

Clause 66. The method of clause 32, wherein drawing the second reagent including the bacteriophage specific to the bacteria of interest from the second reagent inlet port into the first filter chamber includes closing a waste control valve, opening a second reagent control valve between the second reagent inlet port and the first filter chamber, and applying a negative pressure to the vent outlet.

Clause 67. The method of clause 32, wherein incubating the bacteria of interest captured by the first filter with the second reagent in the first filter chamber includes closing a second reagent control valve and a vent control valve.

Clause 68. The method of clause 33, wherein drawing the fluid containing the expressed reporter enzyme through the first filter, through the second filter in the second filter chamber of the microfluidic device, and through the waste port while the expressed reporter enzyme is captured by the second filter includes opening a third reagent control valve between a third reagent inlet port and the first filter chamber, opening a detection chamber control valve downstream of the first filter chamber, and applying a negative pressure at the waste port, wherein the second filter chamber is fluidically connected between the detection chamber control valve and the waste port.

Clause 69. The method of clause 32, wherein incubating the expressed reporter enzyme captured by the second filter with the third reagent in the second filter chamber for the third incubation period includes closing the third reagent control valve and the detection chamber control valve.

Clause 70. The method of clause 34, including

drawing the fluid containing the expressed reporter enzyme through the first filter, through the second filter in the second filter chamber of the microfluidic device, and through the waste port while the expressed reporter enzyme is captured by the second filter by opening a vent control valve upstream of the first filter chamber, opening a detection chamber control valve fluidically connected between the downstream end of the first filter chamber and an upstream end of the second filter chamber and applying a negative pressure at the waste port, wherein the second filter chamber is fluidically connected between the detection chamber control valve and the waste port; and

drawing the third reagent into the second filter chamber prior to the third incubation period by closing the vent upstream of the first filter chamber, opening a third reagent control valve fluidically connected between a third reagent inlet port and a downstream end of the first filter chamber, opening a detection chamber control valve, and applying a negative pressure at the waste port.

Clause 71. A microfluidic device for bacteria detection, comprising:

a sample inlet port for receiving a fluid sample containing bacteria of interest;

a first filter chamber containing a first filter adapted for capturing bacteria of interest from the fluid sample;

first microfluidic means for introducing bacterial growth media to the first filter chamber;

second microfluidic means for introducing phage specific to the bacteria of interest to the first filter chamber, the phage adapted to cause the bacteria of interest to produce a reactive material capable of reacting to produce a detectable signal;

third microfluidic means for flushing reactive material from the first filter chamber, the reactive material released from the bacteria of interest responsive to introduction of the phage; and

a second filter chamber containing a second filter for specifically capturing the reactive material flushed from the first filter chamber, wherein the second filter is smaller than the first filter to amplify the detectable signal;

wherein the first filter is adapted to not capture the reactive material.

Clause 72. The microfluidic device of clause 70, including lysing means for lysing the bacteria of interest to release the reactive material.

Clause 73. The microfluidic device of clause 71, wherein the lysing means includes heating means.

Clause 74. The microfluidic device of clause 71, wherein the lysing means includes acoustic means.

Clause 75. The microfluidic device of clause 71, wherein the lysing means includes a pressure source.

Clause 76. The microfluidic device of clause 71, wherein the lysing means includes a reagent source.

Clause 77. The microfluidic device of clause 71, wherein the lysing means includes an enzyme source.

Clause 78. The microfluidic device of clause 71, wherein at least one of the first microfluidic means, the second microfluidic means, and the third microfluidic means includes a reservoir containing lyophilized reagent.

Clause 79. The microfluidic device of clause 71, wherein at least one of the first microfluidic means, the second microfluidic means, and the third microfluidic means includes a port for interfacing with an external fluid source.

Clause 80. The microfluidic device of clause 71, wherein at least one of the first microfluidic means, the second microfluidic means, and the third microfluidic means includes at least one microchannel and at least one valve.

Clause 81. The microfluidic device of clause 79, wherein the at least one valve includes at least one pneumatically actuated valve and at least one air channel adapted for connection to a pressure source.

Clause 82. The microfluidic device of clause 79, including at least one negative pressure source located downstream of the at least one valve.

Clause 83. The microfluidic device of clause 81, wherein the at least one negative pressure source is located downstream of first filter chamber.

Clause 84. The microfluidic device of clause 70, wherein the first filter includes a porous non-cellulose material having a pore size of about 0.45 μm, and wherein the second filter includes a cellulose-based material.

Clause 85. The microfluidic device of clause 70, wherein the second filter chamber includes a detection region configured to allow detection of the detectable signal from outside the microfluidic device.