Cartridge with a mixing area for endotoxin detection

Abstract

The present invention is directed to methods, compositions, and devices useful for detecting and / or quantifying microbial contaminants, including endotoxins. In embodiments, for absorbance-based assays, cartridges containing dry compositions useful in combination with handheld readers / devices are provided.

Description

[Technical Field]

[0001] The present invention is directed to methods, compositions, and devices useful for the detection and / or quantification of microbial contaminants, including endotoxins. In embodiments, for absorbance-based assays, cartridges are provided that contain dry compositions suitable for use in conjunction with handheld readers / devices. [Background technology]

[0002] Microbial contamination, including contamination with gram-positive bacteria, gram-negative bacteria, yeast, fungi, and mold, can cause serious illness and even death in humans. The pharmaceutical, medical device, and food industries often require frequent, accurate, and sensitive testing for the presence of such microbial contaminants to meet specific standards, such as those imposed by the United States Food and Drug Administration (USFDA) or the Environmental Protection Agency.

[0003] There is a need for a convenient and rapid method of analyzing samples for the presence of microbial contaminants, preferably with a small, portable device that can be easily used in a variety of settings. The present invention meets these needs. Summary of the Invention

[0004] In embodiments, provided herein is a cartridge for determining the presence and / or amount of microbial contaminants in a sample, the cartridge comprising: a housing having an optical sample well, a fluid inlet port, and a conduit fluidly connecting the fluid inlet port and the optical sample well; a pump mechanism associated with the housing and fluidly connected to the fluid inlet port, the conduit, and the optical sample well; and a mixing region disposed along the conduit configured to contain a dry composition comprising a hemocyte lysate.

[0005] In a further embodiment, provided herein is a cartridge for determining the presence and / or amount of microbial contaminants in a sample, the cartridge comprising: a housing comprising a cover section, a lower section mechanically connected to the cover section, and a manifold section mechanically connected to the lower section, the lower section comprising two optical sample wells, a fluid inlet port, and a conduit fluidly connecting the fluid inlet port and the optical sample wells, the conduit comprising an aspiration region, a fluid restrictor, and a mixing region, the manifold section comprising a mixing portion configured to contain a dry composition comprising a hemocyte lysate, the mixing portion corresponding to the mixing region of the conduit; and a pump mechanism associated with the housing and fluidly connected to the fluid inlet port, the conduit, and the two optical sample wells.

[0006] Further provided herein is a method for detecting the presence of a microbial contaminant in a sample, the method comprising introducing a sample into a fluid inlet port of a cartridge described herein, conveying the sample to a conduit, conveying the sample into a mixing area of ​​the conduit, mixing the sample with a hemocyte lysate and a chromogenic substrate to produce a mixed sample, conveying the mixed sample from the mixing area to a sample well, and measuring optical properties of the mixed sample in the optical sample well, wherein a change in the optical properties indicates the presence of a microbial contaminant in the sample.

[0007] In a further embodiment, provided herein is a method for detecting the presence of a microbial contaminant in a sample, the method comprising introducing a sample into a fluid inlet port of a cartridge described herein, transferring the sample to an aspiration region, transferring the sample from the aspiration region through a fluid restrictor to a mixing region, mixing the sample with a hemocyte lysate and a chromogenic substrate to generate a mixed sample, transferring the mixed sample to an optical sample well, and measuring optical properties of the mixed sample in the optical sample well, wherein a change in optical properties indicates the presence of a microbial contaminant in the sample.

[0008] Further embodiments, features, and advantages of the embodiments, as well as the structure and operation of various embodiments, are described in detail below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0009] [Figure 1A] 1A-1G show various views and components of a cartridge for endotoxin detection according to embodiments herein. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 2A] 2A-2D show various views and components of a cartridge for endotoxin detection according to embodiments herein. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 3A] 3A and 3B show various views and components of a lower section for use in a cartridge for endotoxin detection, according to embodiments herein. [Figure 3B] Same as above. [Figure 4A] 4A and 4B show various views and components of a cover section for use in a cartridge for endotoxin detection according to embodiments herein. [Figure 4B] Same as above. [Figure 5A] FIG. 5A shows a manifold section for use in a cartridge for endotoxin detection, according to an embodiment herein. [Figure 5B] 5B and 5C show additional manifold sections for use in cartridges for endotoxin detection, according to embodiments herein. [Figure 5C] Same as above. [Figure 6] FIG. 6 shows an exemplary reader for use with the cartridges described herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] It should be understood that the specific implementations shown and described herein are examples and are not intended to otherwise limit the scope of the present application in any way.

[0011] Published patents, patent applications, websites, company names, and scientific literature referenced herein are incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between a reference cited herein and a specific teaching of this specification shall be resolved in favor of the latter. Similarly, any conflict between an art-understood definition of a word or phrase and a specifically set forth definition of a word or phrase herein shall be resolved in favor of the latter.

[0012] As used herein, the singular forms "a," "an," and "the" specifically include the plural of the term they refer to, unless the context clearly dictates otherwise. As used herein, the term "about" is used to mean approximately, in the range of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical value. Generally, as used herein, the term "about" is used to modify a numerical value within a variance of 20% above and below the stated numerical value.

[0013] Unless otherwise defined, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. Reference is made herein to various methodologies and materials known to those skilled in the art.

[0014] Testing for microbial contaminants Various assays have been developed to detect the presence and / or amount of microbial contaminants in test samples. Often, hemocyte lysates prepared from the hemolymph of crustaceans, such as horseshoe crabs, are utilized. These assays typically utilize, in various ways, the coagulation cascade that occurs when the hemocyte lysate is exposed to a microbial contaminant. Examples of hemocyte lysates include amebocyte lysates (AL) generated from the hemolymph of horseshoe crabs Limulus polyphemus, Tachypleus gigas, Tachypleus tridentatus, and Carcinoscorpius rotundicauda. Amebocyte lysates generated from the hemolymph of Limulus, Tachypleus, and Carcinoscorpius species are referred to as Limulus amebocyte lysate (LAL), Tachypleus amebocyte lysate (TAL), and Carcinoscorpius amebocyte lysate (CAL), respectively.

[0015] Assays that use LAL include, for example, gel clot assays, end-point turbidimetric assays, kinetic turbidimetric assays, and end-point chromogenic assays (Prior (1990), "Clinical Applications of the Limulus Amoebocyte Lysate Test," CRC PRESS 28-34). However, these assays suffer from one or more drawbacks, including the cost of the reagents, the speed of the assay, and limited sensitivity range. Furthermore, these assays typically require that the sample sent to the testing facility be removed from the source of the sample being tested.

[0016] Cartridges for endotoxin detection In embodiments, provided herein are cartridges for determining the presence and / or amount of microbial contaminants in a sample. Samples that can be tested using the various cartridges, devices, and methods described herein include liquid samples from biological processes, pharmaceutical preparations, etc., and can be from large-scale or small-scale processes.

[0017] 1A, in embodiments, cartridge 100 comprises a housing 102 that provides structural support for the cartridge. As used herein, "cartridge" means a freestanding element for receiving and holding a sample to be tested for the presence and / or amount of microbial contaminants.

[0018] The housing 102 can be made from any suitable material, including various plastics or glasses. The housing 102 can be made from a single piece of material, or can comprise separate pieces or sections that are joined together to create the housing 102, and thus the entire cartridge 100, as described herein.

[0019] The housing 102 includes an optical sample well 104. FIG. 1F shows a side view of the optical sample well 104, which is in the shape of a container or ampoule between the top and bottom surfaces of the housing 102. The optical sample well 104 is preferably constructed from a plastic or glass material and is preferably optically clear or transmissive, allowing light to pass through the bottom and top of the optical sample well, thereby allowing the light to contact the sample contained within the optical sample well. In embodiments, the optical sample well 104, as well as the remainder of the housing 102, can be made from a yellow-tinted glass or polymer, preferably blocking light between about 400 nm and about 450 nm (i.e., 405 nm) from passing through, while allowing other wavelengths to pass. Such embodiments increase the sensitivity of the methods described herein by reducing the amount of light passing through the optical sample well 104, reducing crosstalk, and reducing stray light. The optical well 104 is preferably cylindrical in shape with an outer wall, and upper and lower surfaces, preferably formed from various elements of the housing 102, as are the upper and lower portions of the well.

[0020] The housing 102 of the cartridge 100 also includes a fluid inlet port 106 and a conduit 108. The fluid inlet port 106 is preferably a tube, slit, hole, or other suitable opening at the end of a long or narrow section 119 of the housing 102. The conduit 108 is housed inside the fluid inlet port 106 and is used to contact a sample. The fluid inlet port 106 is designed to allow a sample to be drawn upward into the fluid inlet port in a sample introduction direction 114 (FIG. 1F). The conduit 108 fluidly connects the fluid inlet port 106 and the optical sample well 104. That is, the conduit 108 provides a tubular or microfluidic connection between the tip of the fluid inlet port 106 and the optical sample well 104, allowing a liquid sample to be transferred through the conduit 108 into the optical sample well 104 after being drawn into the port. In embodiments, the conduits 108 can be formed as channels cut into the surface of the housing 102, or can be formed as tubing or microtubing from various polymers such as poly(styrene).

[0021] In embodiments, conduit 108 preferably includes a first section that can be used to hold a fluid sample after introduction into fluid inlet 106. As shown in FIGS. 1F and 1G, upon introduction of a sample into fluid inlet port 106 in sample introduction direction 114, the sample advances to aspiration region 140, an initial location within conduit 108, where the sample is held and maintained before further transmission into conduit 108. Preferably, aspiration region 140 has a volume of about 0.05 μL to about 500 μL and is used as an initial volume reservoir for the sample after introduction into the cartridge. This aspiration region 140 is engageable by a cartridge user when the cartridge is inserted into a test sample and a desired volume is initially drawn into sample inlet port 106 for subsequent testing. In typical embodiments, the volume of the suction region 140 is between about 10 μL and about 200 μL, more preferably between about 30 μL and about 100 μL, between about 30 μL and about 80 μL, between about 30 μL and about 50 μL, or about 30 μL, about 40 μL, or about 50 μL.

[0022] The cartridge 100 preferably further comprises a mixing region 142 disposed along the conduit 108. The mixing region 142 is preferably separated from the sample inlet port 106 and the aspiration region 140 (if provided) by a fluid restrictor 144. The mixing region 142 is configured to contain (and, in an exemplary embodiment, preferably does contain) a dry composition including a hemocyte lysate. As described herein, the mixing region provides a portion of the conduit 108 into which a sample suspected of containing a microbial contaminant is introduced so that the sample can be mixed with the dry composition including the hemocyte lysate. As described herein, the mixing region 142 also further comprises a chromogenic substrate. Thus, the mixing region 142 provides an area in which a test sample can contact both the hemocyte lysate and the chromogenic substrate, preferably both dried (either dried as separate components or dried together in the same dry composition) and then mixed with the hemocyte lysate and the chromogenic substrate. Various mechanisms can be used to mix the liquid test sample, hemocyte lysate, and chromogenic substrate, and can include, for example, a magnetic stirring element 146 (see FIG. 1G), such as a stir bar, stir rod, or small magnetic beads or spheres. For example, when cartridge 100 is installed in a reader or other suitable mechanism, a magnet can be placed below, above, or around mixing region 142, and magnetic stirring element 146 can be rotated, translated, or otherwise moved within the mixing region to agitate and move the test sample and mix it with components that have been dried (or otherwise added to the mixing region). Other mixing mechanisms can include a variety of agitators, either external or internal, including tapping mechanisms, stirring or vibrating mechanisms, ultrasonic mechanisms, etc. In a typical embodiment, the volume of mixing region 142 is from about 10 μL to about 200 μL, more preferably from about 30 μL to about 100 μL, from about 30 μL to about 80 μL, from about 30 μL to about 50 μL, or about 30 μL, about 40 μL, or about 50 μL.

[0023] In an exemplary embodiment, the fluid restrictor 144 is disposed between the aspiration region 140 and the mixing region 142 and may be any suitable mechanism or structure that reduces flow between the sample inlet port 106 / aspiration region 140 and the mixing region 142 further downstream. In embodiments, the fluid restrictor 144 may be a plastic section or element installed within the conduit 108, or may be a portion of the conduit 108, or in other embodiments, may simply be a restriction in the conduit 108 that slows fluid entering the sample inlet port 106 from traveling further along the conduit 108, instead acting as a way for the fluid sample to be collected in the aspiration region 140. In embodiments, the fluid restrictor 144 acts as a buffer between the aspiration region and the mixing region. In embodiments, the fluid restrictor 144 is not a valve because it cannot be switched on or off, but rather provides a mechanism for slowing fluid movement to allow the various components to properly fill along the conduit. However, in further embodiments, valve structures can be added to the conduit 108 to control the flow of fluid between any of the various components along the length of the conduit.

[0024] In embodiments, cartridge 100 further includes a pump mechanism 110 associated with housing 102. Pump mechanism 110 is preferably a three-position syringe (one-, two-, four-, five-position, etc. syringes can also be used) including a barrel 118 (see FIGS. 1G and 3A) and a plunger 116 slidable within the barrel. Pump mechanism 110 can be attached directly to housing 102 via a suitable mechanism (e.g., glue, adhesive, mechanical bands, wraps, staples, etc.) or can be an element fabricated as an integral part of housing 102 (e.g., as part of lower section 186 of the housing). Pump mechanism 110 is fluidly connected to fluid inlet port 106, conduit 108, and optical sample well 104, such that actuation of pump mechanism 110 can draw sample into fluid inlet port 106, then into conduit 108, and into sample well 104, as described herein, for example, as shown in FIG. 1F, where 114 indicates the direction of sample introduction.

[0025] In an exemplary embodiment, the pumping mechanism 110, preferably a three-position syringe, moves to a first position that transfers the sample to the fluid inlet port 106 and the conduit 108, preferably to the aspiration region 140, but does not pass through, over, or around the fluid restrictor 144. For example, a three-position syringe can create a vacuum to draw or draw the sample into the fluid inlet port 106 and then into the conduit 108 and aspiration region 140. The pumping mechanism 110 preferably moves to a second position that provides transport of the sample from the aspiration region 140, through the fluid restrictor 144, and to the mixing region 142. After mixing the sample with the various dry components, the pumping mechanism 110 preferably moves to a third position where the mixed sample is transported to the optical sample well 104 (again, through another section of the conduit 108).

[0026] By introducing or drawing the sample into fluid inlet port 106 and thereafter into conduit 108, and preferably maintaining the sample within suction region 140 with pump mechanism 110 in a first position, cartridge 100 with the sample can be prepared at the sampling location (e.g., an assembly line, factory, facility, sample reservoir, or storage tank) and then maintained in a ready state prior to detection or measurement. An advantage of this design is that the sample can be held within cartridge 100 for a period of time (preferably from a few minutes (e.g., 10-30 minutes) up to about 1-2 hours, or more) prior to measurement, thereby allowing multiple different samples to be obtained without risk of compromising sample quality. Furthermore, if further operations are required after the sample is obtained, these operations can be performed and the sample can then be analyzed.

[0027] In embodiments, the length of the conduit 108, which may include the length from the tip of the fluid inlet port 106 to the sample well 104, is about 1 cm to about 15 cm, more preferably about 1 cm to about 10 cm or about 1 cm to about 5 cm. The conduit 108 can include an aspiration region 140 and a mixing region 142 (as well as other regions or segments, if desired) that are about 0.2 mm to about 10 mm in length, with the total length of the conduit 108 being about 5 mm to about 50 mm. The diameter or cross-sectional width of the conduit 108 is preferably about 0.1 mm to about 1 mm, about 0.5 mm to about 1 mm, about 0.5 mm to about 0.8 mm, or about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, or about 0.8 mm. Using a cross-sectional width of the conduit 108 on the order of about 0.5 mm helps reduce wicking and prevent sample loss. In embodiments, the fluid restrictor 144 preferably allows fluid to pass over or through a diameter of about 0.05 mm to about 1 mm. In embodiments, the conduit 108 can include a bent or folded pattern (or other similar orientation that minimizes surface area while maximizing the length of the conduit 108), while in other embodiments, the conduit 108 can be a generally straight channel from the tip of the fluid inlet port to the sample well. In other embodiments, there can be two or more conduits, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more conduits. Using a section of the conduit 108 having a length on the order of about 0.2 mm to about 1 mm helps reduce wicking, air pocket formation, and sample loss while the sample is being read for analysis, including with the devices / readers described herein.

[0028] The sample size that can be maintained within conduit 108 prior to measurement and analysis is preferably between about 25 μl and about 200 μl, more preferably between about 50 μl and about 150 μl, between about 50 μl and about 100 μl, between about 50 μl and about 80 μl, or about 40 μl, about 50 μl, about 60 μl, about 65 μl, about 70 μl, about 75 μl, about 80 μl, about 90 μl, or about 100 μl. In an embodiment, the total volume that can be accommodated in cartridge 100 is preferably about 50 μl to about 150 μl, about 50 μl to about 100 μl, about 50 μl to about 80 μl, or about 40 μl, about 50 μl, about 60 μl, about 65 μl, about 70 μl, about 75 μl, about 80 μl, about 90 μl, or about 100 μl.

[0029] In an embodiment, the housing 102 preferably comprises four optical sample wells 104, two of which are configured to contain (and preferably contain) an agent representative of a microbial contaminant dried within the optical sample well. In another embodiment, the housing 102 may comprise two optical sample wells 104, one of which is configured to contain (and preferably contain) an agent representative of a microbial contaminant dried within the optical sample well.

[0030] The size of the optical sample well 104, and therefore preferably the volume the well can hold, is determined by the height of the well walls and the diameter of the top and bottom of the well. Preferably, the optical sample well 104 has a height on the order of about 100 μm to about 20 mm, more preferably about 100 μm to about 10 mm or about 100 μm to about 5 mm, and a diameter on the order of about 100 μm to about 20 mm, more preferably about 100 μm to about 10 mm or about 100 μm to about 5 mm. In embodiments, the optical sample well 104 suitably holds about 1 μl to about 5 mL of sample, or about 1 μl to about 1 mL, or about 1 μl to about 500 μl, about 1 μl to about 50 μl, about 1 μl to about 20 μl, about 1 μl to about 10 μl, or about 1 μl, about 2 μl, about 3 μl, about 4 μl, about 5 μl, about 6 μl, about 7 μl, about 8 μl, about 9 μl, about 10 μl, about 11 μl, about 12 μl, about 13 μl, about 14 μl, or about 15 μl.

[0031] As described herein, mixing region 142 preferably contains a dry composition comprising a hemocyte lysate. As used herein, "dry composition" includes a material that is lyophilized, freeze-dried, or vitrified to form a dry cake, powder, crystal, or film. Methods of freeze-drying or vitrification are known in the art. The dry composition is preferably dried onto (i.e., on the side of) mixing region 142, or can be provided as dried onto manifold section 188 as described herein, or can be in the form of a free pellet included with / within the mixing region, or can be added to the mixing region as desired / needed.

[0032] As used herein, the term "hemocyte lysate" refers to any lysate, or portion or component thereof, produced by lysis and / or membrane permeabilization of hemocytes, e.g., amebocytes and hemolymphocytes, obtained by (i) extraction from crustaceans or insects, and / or (ii) in vitro culture after extraction from a host. 2+Hemocyte cellular material extruded from hemolymph cells by contact with a membrane permeabilizing agent such as an ionophore (i.e., extruded other than by lysis), or otherwise extracted without cell lysis, is also considered to be a hemolymph lysate.

[0033] A typical amebocyte lysate is an amebocyte lysate prepared from the blood of crustaceans, such as horseshoe crabs or Dungeness crabs. As used herein, the term "amebocyte lysate" is understood to mean any lysate, or portion or component thereof, produced by lysis, extrusion, or extraction of cellular contents from amebocytes extracted from crustaceans, such as horseshoe crabs. Amebocyte lysates contain at least one component of an enzyme cascade and / or cause coagulation in the presence of endotoxins, such as gram-negative bacterial endotoxins, and / or glucans, such as (1→3)-β-D glucans, produced by yeast or molds. Typical amebocyte lysates can be derived from horseshoe crabs, including crabs belonging to the genus Limulus, e.g., Limulus polyphemus; Tachypleus, e.g., Tachypleus gigas and Tachypleus tridentatus; and Carcinoscorpius, e.g., Carcinoscorpius rotundicauda.

[0034] Limulus amebocyte lysate (LAL) is used as the amebocyte lysate of choice in many bacterial endotoxin assays due to its sensitivity, specificity, and relative ease of avoiding interference from other components that may be present in the sample. When combined with a sample containing bacterial endotoxin, and optionally with a specific LAL substrate, LAL reacts with the endotoxin in the sample to produce a detectable product, such as a gel, increased turbidity, or, in the case of a synthetic chromogenic substrate, a colored or luminescent product. The product can be detected, for example, either visually or by use of a photodetector.

[0035] When bacterial endotoxin contacts LAL, it initiates a series of enzymatic reactions known in the art as the factor C pathway, which can involve three serine protease enzyme precursors, termed factor C, factor B, and procoagulant enzymes. Upon exposure to endotoxin, the endotoxin-sensitive factor, factor C, is activated. Activated factor C then hydrolyzes and activates factor B, which in turn activates procoagulant enzymes to produce clotting enzymes. Clotting enzymes then bind to specific sites, e.g., Arg, on coagulogens, fibrinogen-like clotting proteins in invertebrates. 18 -Thr 19 and Arg 46 -Gly 47 to produce a coagulin gel. See, e.g., U.S. Patent No. 5,605,806.

[0036] Methods for increasing the sensitivity of hemocyte lysates to endotoxins include, for example, without limitation, aging the crude hemocyte lysate, adjusting the pH, adjusting the concentration of divalent cations, adjusting the concentration of coagulogens, chloroform extraction, and adding serum albumin, biocompatible buffers, and / or biological detergents.

[0037] For example, in embodiments, a hemocyte lysate for use in the dry compositions described herein can be a purified hemocyte lysate that is substantially free of coagulogen. In another embodiment, the substantially coagulogen-free hemocyte lysate is a substantially coagulogen-free LAL. Those skilled in the art will understand, upon interpreting the present disclosure, that various amounts of reduced coagulogen result in increased speed, sensitivity, and / or resolution in a chromogenic assay, such as an LAL assay. In some embodiments, the term "substantially free" refers to a hemocyte lysate having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, or less than 0.5% (wt / wt) of coagulogen relative to the total protein in the hemocyte lysate, as measured by SDS-PAGE with protein staining and confirmed by Western blot. In some embodiments, the term "substantially free" refers to LAL having less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or 0.5% (wt / wt) coagulogen relative to the total protein in the LAL as measured by SDS-PAGE with protein staining and confirmed by Western blot. In some embodiments, the term "substantially free" refers to purified LAL having less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or 0.5% (wt / wt) coagulogen relative to the total protein in the LAL as measured by SDS-PAGE with protein staining and confirmed by Western blot.

[0038] In some embodiments, the term "substantially free" refers to a hemocyte lysate having less than 10% or less than 5% (wt / wt) coagulogen relative to the total protein in the hemocyte lysate as determined by SDS-PAGE with protein staining and confirmed by Western blot. In some embodiments, the term "substantially free" refers to an LAL having less than 10% or less than 5% (wt / wt) coagulogen relative to the total protein in the LAL as determined by SDS-PAGE with protein staining and confirmed by Western blot. In some embodiments, the term "substantially free" refers to a purified LAL having less than 10% or less than 5% (wt / wt) coagulogen relative to the total protein in the LAL as determined by SDS-PAGE with protein staining and confirmed by Western blot.

[0039] In some embodiments, the term "substantially free" refers to a hemocyte lysate having a coagulogen concentration of less than about 20 μg / μL, less than about 15 μg / μL, less than about 10 μg / μL, less than about 5 μg / μL, less than about 4 μg / μL, less than about 3 μg / μL, less than about 2 μg / μL, or less than about 1 μg / μL. In some embodiments, the term "substantially free" refers to an LAL having a coagulogen concentration of less than about 20 μg / μL, less than about 15 μg / μL, less than about 10 μg / μL, less than about 5 μg / μL, less than about 4 μg / μL, less than about 3 μg / μL, less than about 2 μg / μL, or less than about 1 μg / μL. In some embodiments, the term "substantially free" refers to purified LAL having a coagulogen concentration of less than about 20 μg / μL, less than about 15 μg / μL, less than about 10 μg / μL, less than about 5 μg / μL, less than about 4 μg / μL, less than about 3 μg / μL, less than about 2 μg / μL, or less than about 1 μg / μL.

[0040] Exemplary LAL that is substantially free of coagulogens is described in U.S. Patent Application Nos. 2018 / 0208964 and 2018 / 0038864, the disclosures of both of which are incorporated herein by reference in their entireties.

[0041] Those skilled in the art will understand that different methods can be used to remove coagulogens from hemocyte lysates, e.g., LAL. These methods may vary in efficiency, purification speed, cost, and effort, but are within the knowledge of those skilled in the art. In some embodiments, the hemocyte lysate, e.g., LAL, is substantially free of coagulogens, and the composition is made by a method comprising: (a) obtaining a solution derived from lysed amebocytes from Limulus polyphemus; (b) combining the solution from (a) with a buffer; (c) subjecting the combination from (b) to continuous tangential flow filtration (TFF) using a 20 kDa to 50 kDa membrane filter to produce a retentate; and (d) centrifuging the retentate from (c) at greater than 20,000 × g for greater than 25 minutes to produce a supernatant, wherein the supernatant is purified LAL that is substantially free of coagulogens.

[0042] In some embodiments, the hemocyte lysate is purified Limulus amebocyte lysate. The term "purified Limulus amebocyte lysate" (or "purified LAL"), which is substantially free of coagulogen, refers to LAL, as described above, that is substantially free of coagulogen and that has been further processed to remove components that cloud the appearance of the LAL. In some embodiments, purified LAL is produced by centrifuging LAL that is substantially free of coagulogen. In some embodiments, the term "purified LAL" refers to LAL that has been centrifuged at greater than 1800 g (i.e., 1800×gravity), greater than 2200 g, greater than 2600 g, greater than 3000 g, greater than 3400 g, greater than 3800 g, greater than 4200 g, greater than 4600 g, greater than 5000 g, greater than 5400 g, greater than 5800 g, greater than 6000 g, greater than 6100 g, or greater than 6200 g for a period of time sufficient to visibly clarify the LAL without damaging the enzymes. In some embodiments, the term "purified LAL" refers to LAL that has been centrifuged at 1800-8000 g, 2200 g-7600 g, 2600 g-7200 g, 3000 g-7200 g, 3400 g-7200 g, 3800 g-7200 g, 4200 g-7200 g, 4600 g-7200 g, 5000 g-7200 g, 5400 g-7200 g, 5800 g-7200 g, or 6100 g-7200 g for a period of time sufficient to visibly clarify the LAL without damaging the enzyme.

[0043] In some embodiments, the term "purified Limulus amebocyte lysate" (or "purified LAL") that is substantially free of coagulogen refers to the substantially free coagulogen LAL described above that has been further processed to remove components that cloud the appearance of the LAL by centrifuging the substantially free coagulogen LAL at greater than 20,000 x g, greater than 22,000 x g, greater than 24,000 x g, greater than 25,000 x g, greater than 26,000 x g, greater than 28,000 x g, greater than 30,000 x g, greater than 35,000 x g, greater than 40,000 x g, greater than 45,000 x g, or greater than 50,000 x g. In some embodiments, the substantially coagulogen-free LAL is centrifuged at greater than 20,000-50,000 x g, 20,000-40,000 x g, 25,000-50,000 x g, 25,000-40,000 x g, or 30,000-40,000 x g. In some embodiments, the substantially coagulogen-free LAL is centrifuged for greater than 20 minutes, greater than 30 minutes, greater than 40 minutes, or greater than 60 minutes. In some embodiments, the substantially coagulogen-free LAL is centrifuged for 20-120 minutes, 20-90 minutes, 20-60 minutes, 20-40 minutes, or about 30 minutes.

[0044] In some embodiments, the term "purified LAL" refers to LAL that has been centrifuged for more than 3 minutes, more than 4 minutes, more than 5 minutes, more than 6 minutes, more than 7 minutes, more than 8 minutes, more than 9 minutes, or more than 10 minutes. In some embodiments, the term "purified LAL" refers to LAL that has been centrifuged for 3 to 30 minutes, 4 to 25 minutes, 4 to 20 minutes, 5 to 15 minutes, or 5 to 10 minutes. Those skilled in the art will appreciate that slower centrifugation speeds may require longer centrifugation times, and adjusting the time and / or speed accordingly will reduce the visual cloudiness of the LAL. In some embodiments, the term "purified LAL" refers to LAL that is substantially free of coagulogen that has been centrifuged at about 5000 g to about 7000 g for about 3 to about 10 minutes, or at about 6120 g for 5 minutes. In embodiments, purified LAL substantially free of coagulogen is produced by centrifuging a solution derived from lysed amebocytes from Limulus polyphemus at 2,000 rpm (980 g) for 8 minutes at 4° C. Purified LAL is identified in the supernatant after centrifugation. In some embodiments, the resulting supernatant is then combined with a buffer, and the resulting combination of supernatant and buffer is then subjected to tangential flow filtration using a 30 kDa membrane filter to produce a retentate, which is then centrifuged at 5,000 rpm (6120 g) for 5 minutes at 4° C. to produce a supernatant, which is purified LAL substantially free of coagulogen. In embodiments, the solution derived from lysed amebocytes from Limulus polyphemus is a pool of multiple Limulus polyphemus lysed amebocytes.

[0045] In some embodiments, the hemocyte lysate is obtained by obtaining a solution derived from lysed amebocytes from Limulus polyphemus. In some embodiments, the solution is then combined with a buffer, and the resulting combination of solution and buffer is then subjected to continuous tangential flow filtration (TFF) using a 20 kDa to 50 kDa membrane filter to produce a retentate, which is then centrifuged at greater than 20,000 x g for greater than 25 minutes at 4°C to produce a supernatant, which is purified LAL substantially free of coagulogen. In embodiments, the lysate is derived from lysed amebocytes from Limulus polyphemus or a pool of multiple Limulus polyphemus lysed amebocytes. In some embodiments, the continuous TFF comprises at least four diafiltration volumes (DV). In some embodiments, the continuous TFF comprises at least five diafiltration volumes. In some embodiments, the continuous TFF comprises at least six diafiltration volumes. In some embodiments, the continuous TFF comprises at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 diafiltration volumes.

[0046] The cartridges, methods, and compositions described herein can be used with any chromogenic substrate that is cleaved by the clotting enzyme in a hemocyte lysate. U.S. Patent No. 5,310,657, for example, describes an exemplary chromogenic substrate having the formula R1-A1-A2-A3-A4-B-R2, where R1 represents hydrogen, a blocking aromatic hydrocarbon, or an acyl group; A1 represents an L- or D-amino acid selected from Ile, Val, or Leu; A2 represents Glu or Asp; A3 represents Ala or Cys; A4 represents Arg; B represents a linkage selected from ester and amide; and R2 represents a chromogenic or luminogenic group covalently attached to the C-carboxyl terminus of the arginine via the B linkage, where the luminogenic or chromogenic moiety can be cleaved from the remainder of the chromogenic substrate to generate a chromogen or fluorogen. A typical chromogenic substrate has the consensus sequence acetate-Ile-Glu-Ala-Arg-pNA, where pNA represents a paranitroaniline group. U.S. Pat. No. 4,188,264 describes peptide substrates with a structure consisting of L-amino acids of the sequence R1-Gly-Arg-R2, where R1 represents an N-terminally modified amino acid and R2 is a group that can be released by enzymatic hydrolysis to generate the colored compound HR2. U.S. Pat. No. 4,510,241 discloses chromogenic peptide substrates that differ from previous substrates because the Gly moiety in the sequence is replaced by Ala or Cys. Alternatively, chromogenic substrates can contain fluorophores, such as 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin, and 4-methoxy-2-naphthylamine.

[0047] Various concentrations of the chromogenic substrate can be used, hi some embodiments, the concentration of the chromogenic substrate is 0.1 g / L to 0.5 g / L, 0.1 g / L to 0.4 g / L, 0.2 g / L to 0.4 g / L, 0.2 g / L to 0.3 g / L, or 0.2 g / L to 0.25 g / L.

[0048] Assay inhibition or acceleration occurs when a substance in the test sample interferes with the hemocyte lysate reaction. Inhibition results in a longer reaction time, indicating a lower level of microbial contamination than may actually be present in the test sample. Acceleration results in a shorter reaction time, indicating a higher level of microbial contamination than may actually be present in the test sample.

[0049] Exemplary amounts of hemocyte lysate, chromogenic substrate, and / or agents representative of microbial contaminants that can be dried into various dry compositions are described herein or can be easily determined by one of ordinary skill in the art. In some embodiments, the dry composition contains components in amounts that provide a ratio of about 30% to 50% hemocyte lysate and a ratio of 10% to 30% chromogenic substrate (v / v). In some embodiments, the dry composition contains components in amounts that provide a ratio of about 35% to about 45% hemocyte lysate and a ratio of 15% to 25% chromogenic substrate, or a ratio of about 40% hemocyte lysate and a ratio of 20% chromogenic substrate (wt / wt). In other embodiments, the dry composition contains components in amounts that provide a ratio of about 30% to 50% substantially coagulogen-free LAL and a ratio of 10% to 30% chromogenic substrate (v / v). In some embodiments, the dry composition contains components in amounts such that a ratio of substantially coagulogen-free LAL and chromogenic substrate of about 35% to about 45%, or a ratio of substantially coagulogen-free LAL and chromogenic substrate of about 15% to 25%, or a ratio of substantially coagulogen-free LAL and 20% (wt / wt) is provided. In other embodiments, the dry composition contains components in amounts such that a ratio of purified LAL and chromogenic substrate of about 30% to 50% (v / v) is provided. In some embodiments, the dry composition contains components in amounts such that a ratio of purified LAL and chromogenic substrate of about 35% to about 45% is provided, or a ratio of substantially coagulogen-free LAL and 20% (wt / wt) is provided.

[0050] In some embodiments, the dry composition comprises about 1 μg to about 50 μg of hemocyte lysate and about 0.1 μg to 5 μg of chromogenic substrate, about 1 μg to about 30 μg of hemocyte lysate and about 0.5 μg to 4.0 μg of chromogenic substrate, about 2 μg to about 20 μg of hemocyte lysate and about 1.0 μg to about 3.0 μg of chromogenic substrate, or about 4 μg to about 25 μg of hemocyte lysate and about 1.0 μg to about 2 μg of chromogenic substrate. In some embodiments, the dry composition comprises about 1 μg to about 50 μg of LAL substantially free of coagulogen and about 0.1 μg to about 5 μg of chromogenic substrate, or about 1 μg to about 30 μg of LAL substantially free of coagulogen and about 0.5 μg to about 5 μg of chromogenic substrate, or about 2 μg to about 20 μg of LAL substantially free of coagulogen and about 1.0 μg to about 3.0 μg of chromogenic substrate, or about 1 μg to about 30 μg of LAL substantially free of coagulogen and about 1.0 μg to about 2.0 μg of chromogenic substrate, wherein the chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA. In some embodiments, the dry composition comprises about 4 μg to about 25 μg of purified LAL substantially free of coagulogen and about 1 μg to about 1.5 μg of chromogenic substrate, where the chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA. In some embodiments, the dry composition comprises about 1 μg to about 50 μg of purified LAL and about 0.1 μg to about 5 μg of chromogenic substrate, or about 1 μg to about 30 μg of purified LAL and about 0.5 μg to about 5 μg of chromogenic substrate, or about 2 μg to about 20 μg of purified LAL and about 1.0 μg to about 3.0 μg of chromogenic substrate, or about 1 μg to about 30 μg of purified LAL and about 1.0 μg to about 2.0 μg of chromogenic substrate, where the chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA. In some embodiments, the dry composition comprises about 4 μg to about 25 μg of purified LAL and about 1 μg to about 1.5 μg of a chromogenic substrate, wherein the chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA.

[0051] In some embodiments, the microbial contaminant control is between about 0.1 EU / ml and 1 EU / ml. In some embodiments, the microbial contaminant is bacterial endotoxin at a concentration between about 0.1 EU / ml and 1 EU / ml, and 1 μl to 10 μl is used.

[0052] In a preferred embodiment, a formulation containing about 10% to about 60% hemocyte lysate, about 20% to about 50% hemocyte lysate, or about 30% to about 40% hemocyte lysate, or about 10% to about 60% LAL substantially free of coagulogen, about 20% to about 50% LAL substantially free of coagulogen, or about 30% to about 40% LAL substantially free of coagulogen, is freeze-dried to obtain a dry composition. In some embodiments, a formulation containing about 10% to about 60% purified LAL, about 20% to about 50% purified LAL, or about 30% to about 40% purified LAL is freeze-dried. The volume of the freeze-dried hemocyte lysate, LAL formulation substantially free of coagulogen, or purified LAL formulation is generally about 1 μL to about 10 μL. In further embodiments, the formulation can contain about 20% to about 30% or about 25% to about 30% LAL substantially free of hemocyte lysate or coagulogen, with a deposition volume of about 3 μL to about 10 μL, or about 3 μL to about 8 μL, or about 3.5 μL to about 7 μL. In further embodiments, the formulation can contain about 20% to about 30% or about 25% to about 30% purified LAL, with a deposition volume of about 3 μL to about 10 μL, or about 3 μL to about 8 μL, or about 3.5 μL to about 7 μL.

[0053] Cartridge 100 can also include a pH indicator, such as pH paper or other suitable compound or composition that can be used to directly determine the pH of a sample. The pH indicator can be directly associated with optical sample well 104, or, depending on the orientation of cartridge 100, can also be associated with conduit 108. In either embodiment, the pH indicator is readily usable to measure the pH of a sample. Cartridge 100 can also include a bar code useful for identifying the cartridge to facilitate storage, automated data collection, etc.

[0054] 1A and 1B, housing 102 includes four optical sample wells, although other or additional numbers of optical sample wells, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., can be utilized. As described herein, in embodiments, two of the four optical sample wells further include an agent representative of microbial contaminants dried onto the optical sample well, which can act as a control to verify that the methods described herein are functioning correctly, as well as various substrates, lysates, etc. In other embodiments, housing 102 can include two optical sample wells, one sample well acting as a control and the other as a test well, or can include four, six, eight, ten, twelve, fourteen, etc. optical sample wells, where a portion of the optical sample wells (e.g., two, four, six, eight, etc.) act as controls and the other optical sample wells act as test wells.

[0055] To verify the lack of inhibition or enhancement, the control optical sample wells are preferably "spiked" with a known amount of an agent representative of the microbial contaminant to be measured. Preferably, the microbial contaminant spike results in a final microbial contaminant concentration in the sample that, on a log basis, approaches the midpoint between the highest and lowest microbial contaminant concentrations in the calibration curve. For example, in an assay with a calibration curve ranging from 50 endotoxin units (EU) / mL to 0.005 EU / mL, the sample can be spiked to contain a final microbial contaminant concentration of approximately 0.5 EU / mL. In an assay with a calibration curve ranging from 1 EU / mL to 0.01 EU / mL, the microbial contaminant spike results in a final microbial contaminant concentration of approximately 0.1 EU / mL.

[0056] Preferably, a "spike," or known amount of agent representing the microbial contaminant to be measured, is dried onto the optical sample well. The spiked sample(s) are analyzed in parallel with the unspiked or test sample. The resulting microbial contaminant concentration in the unspiked sample and the microbial contaminant recovered in the spiked sample are then calculated and compared. The recovered microbial contaminant is preferably equal to within about 50% to 200% of the known spike concentration. If a sample (or diluent) is found to inhibit or enhance the reaction, the sample may require further dilution until the inhibition or enhancement is resolved. It may be desirable to initially test a 10-fold dilution of the sample to test for inhibition or enhancement.

[0057] In additional embodiments, the dry composition in mixing area 142 can include a variety of other reagents / reactants, allowing the cartridges described herein to be used for additional reactions. In such embodiments, the ability to introduce a sample into mixing area 142 containing predetermined amounts of desired reactants (e.g., buffers, enzymes, stabilizers, etc.) as a freeze-dried or vitrified dry composition provides a platform for a variety of additional testing opportunities beyond endotoxin detection.

[0058] In an embodiment, as shown in FIG. 1F , the housing 102 includes a sample shutoff mechanism, preferably a liquid-impermeable membrane 121, that aids in filling and maintaining the sample volume within the optical sample wells 104. As used herein, a “liquid-impermeable membrane” refers to a substrate that allows air to pass through but is largely impermeable to liquid, preferably preventing any liquid from passing through the membrane. Examples of such membranes include various rubbers and polymers, including, for example, poly(propylene) membranes, poly(tetrafluoroethylene) (PTFE) membranes, other fluoropolymers, and the like. Preferably, the liquid-impermeable membrane 121 is fluidly connected to the optical sample wells 104, such that the liquid sample filling the optical sample wells contacts the liquid-impermeable membrane and stops flowing upon striking the liquid-impermeable membrane, thereby maintaining the sample volume(s) in each of the optical sample wells to be the same (or within about 1-10% of the same volume), allowing samples to be compared to one another.

[0059] 1C-1E, cartridge 100 comprises housing 102 and is preferably fabricated from three individual sections, e.g., cover section 184, bottom section 186, and manifold section 188, that are mechanically connected together to form cartridge 100. Utilizing three sections that are connected together allows for easier fabrication and assembly of the various portions of cartridge 100. For example, the channels, microchannels, or tubing that make up conduits 108, fluid inlet ports 106, and optical sample wells 104 can be pre-formed or placed in bottom section 186 and then connected together with cover section 184 and manifold section 188 to form the completed cartridge 100.

[0060] Methods for mechanically connecting the sections of cartridge 100 include various adhesives or glues, laser welding, ultrasonic welding, mechanical screws, or self-mating connectors, as well as bands or clips. In additional embodiments, the mechanical connection between two sections can be achieved by heat fusion or thermal bonding. In embodiments, the various sections (184, 186, 188) of housing 102 can be injection molded and then heat or fusion bonded to one another, thereby preferably sealing off conduits 108, optical sample wells 104, and fluid inlet ports 106. In further embodiments, one or all of cover section 184, bottom section 186, and manifold section 188 of housing 102 can be made using opaque plastic, thereby reducing unwanted light passing through the housing during measurements and reducing crosstalk.

[0061] In an embodiment, as shown in Figures 1A-1E, provided herein is a cartridge 100 for determining the presence and / or amount of a microbial contaminant in a sample, the cartridge comprising a housing 102 having a cover section 184, a lower section 186 mechanically connected to the cover section 184, and a manifold section 188 mechanically connected to the lower section 186.

[0062] 1A-1B show views of cartridge 100 with cover section 184 visible (FIG. 1A), and with bottom section 186 and manifold section 188 visible (FIG. 1B).

[0063] Cover section 184 preferably includes various notches and connection points (see FIG. 1C) for connecting various components and is preferably constructed from a clear plastic or polymeric composition.

[0064] The lower section 186 is preferably a plastic element comprising four optical sample wells 104, a fluid inlet port 106, and a conduit 108 fluidly connecting the fluid inlet port and the optical sample wells. See FIG. 1F. The conduit 108 is preferably a notch or channel formed within the structure of the lower section 186, but in other embodiments, may be a microfluidic channel or tubing added to the lower section. FIGS. 1F-1G show one embodiment of the lower section 186 in which the conduit 108 passes continuously along one side of the lower section 186 and includes within the conduit aspiration region 140, fluid restrictor 144, and mixing region 142. This single-planar embodiment of the conduit 108 can be further illustrated in FIG. 1G, in which all of the components of the conduit 108 reside in the same plane. 2A and 2B also show this planar embodiment, in which the conduit 108 passes through the entire length of the lower section 186, from the fluid inlet port 106 to the sample well 104. FIG. 2B shows a cross section of the lower section 186, passing through the conduit 108. The sample well 104 shown in FIG. 2A is essentially the sidewall of the sample well, with the top and bottom of the well provided by the cover section 184 and manifold section 188, respectively. FIG. 2A shows typical locations of the aspiration region 140, fluid restrictor 144, and mixing region 142 along the length of the conduit 108.

[0065] In another embodiment, as shown in FIG. 1D , the lower section 186 can include a conduit 108 that starts on one side of the lower section and then traverses through the lower section 186 to the other side. For example, as shown in FIGS. 2C and 2D , the conduit starts at the “top” of the lower section 186, connected to the sample inlet port 106. The conduit 108 then traverses through the lower section 186 at a first through-hole 206 to the “bottom” side of the lower section 186, and then backs out at a second through-hole 206 and traverses to the “top” side of the lower section before connecting to the sample well 104. This “traverse” can also be seen in FIGS. 1A , 1B , and 1D . FIG. 2C shows the location of the suction region 140 and fluid restrictor 144 at the “top” of the lower section 186. The mixing region 142 can be seen below the lower section 186 in the cross-section shown in FIG. 2D .

[0066] Manifold section 188 of cartridge 100 preferably includes a mixing portion 520 configured to contain a dry composition including a hemocyte lysate, where mixing portion 520 corresponds to mixing region 142 of conduit 108. That is, mixing portion 520 of manifold section 188 coincides with mixing region 142 of conduit 108 in lower section 186 when the sections are connected to form a cartridge. In a preferred embodiment, manifold section 188 also includes sample well portion 504.

[0067] FIG. 3A shows the lower section 186, with the conduit 108 being a single channel that runs along one side of the lower section 186, the length of the long or narrow section 119 of the housing 102, from the sample inlet port 106 to the sample well 104. FIG. 3A shows a view of the lower section 186, with the conduit 108 being a channel formed within the lower section 186. FIG. 5A shows an embodiment of a manifold section 188 that would fit within the lower section 186 at the cutout 302 in FIG. 3A. The manifold section 188 shown in FIG. 5A preferably includes a sample well portion 504 that, when connected together with the cover section 184 and lower section 186, forms the "lower part" of the sample well 104.

[0068] FIG. 3B shows another embodiment of the lower section 186 in which the conduit 108 traverses through the lower section via first and second through-holes 206. As shown in FIG. 3B, the conduit 108 begins as a channel formed in the lower section 186 and then passes through the first through-hole 206. FIG. 3B shows a notch 304 into which the manifold section 188 shown in FIG. 5B can be placed. The manifold section 188 of FIG. 5B preferably includes a sample well portion 504 that, when connected with the cover section 184 and the lower section 186 and a mixing portion 520 that corresponds to (i.e., matches) the mixing region 142 of the lower section 186, forms the "lower portion" of the sample well 104 (see FIGS. 1B, 1E, 2D, and 3B). FIG. 1E also shows the manifold 188 from FIG. 5B before it is connected to the cover section 184 and the bottom section 186 to form the complete cartridge 100 (see also FIG. 1B, which shows a view of the "bottom" of the cartridge into which the manifold section 188 preferably fits).

[0069] Figures 4A and 4B show exemplary cover sections 184, with the cover section in Figure 4A mating with a single channel conduit 108 and the cover section in Figure 5B mating with a conduit 108 passing through the lower section. Typical areas where laser welds 402 may occur are shown.

[0070] As described herein, cartridge 100 preferably includes a pump mechanism 110 associated with housing 102 and fluidly connected to fluid inlet port 106, conduit 108, and (two) optical sample wells 104. With either design, pump mechanism 110 is preferably integral to (i.e., part of) lower section 186, as shown in Figures 3A and 3B. In an embodiment, the pump mechanism is a three-position syringe, with a first position creating a vacuum to draw sample into aspiration region 140, a second position providing transport from aspiration region 140 through fluid restrictor 144 to mixing region 142, and a third position providing transport from mixing region 142 to sample well 104.

[0071] Preferably, as shown in the exemplary embodiment, lower section 186 includes four optical sample wells, two of which are configured to contain an agent representative of a microbial contaminant dried within the optical sample well. In an embodiment, manifold section 188 preferably contains an agent representative of a microbial contaminant dried onto sample well portion 504 of the manifold section (FIG. 5A or 5B). In an embodiment, manifold section 188 further includes a chromogenic substrate dried onto mixing portion 520 of manifold section 188 such that when cartridge elements are connected, the manifold provides both components to mixing region 142. In a preferred embodiment, the hemocyte lysate is Limulus amebocyte lysate and the agent in the sample well is bacterial endotoxin.

[0072] Thus, in embodiments, manifold section 188 is preferably fabricated with all of the dried components useful for the assays described herein. Preferably, sample well portion 504 of manifold 188 contains an agent representative of a microbial contaminant dried thereon, and mixing portion 520 of manifold section 188 contains both the hemocyte lysate and chromogenic substrate provided to mixing region 142 dried thereon. Manifold section 188 may thus be a separately provided element of the cartridge, removable and replaceable, allowing for reuse of the cartridge, if desired.

[0073] The manifold section 188 may also include a lens 502 to allow monitoring of the sample fluid entering and / or exiting the mixing region 142. This lens 502 may be optically monitored by a laser or other light-based mechanism to verify the location and placement of the fluid sample during the analytical procedure.

[0074] Detection Method Also provided herein is a method for detecting the presence of microbial contaminants in a sample. In an embodiment, the method preferably includes introducing a sample into a fluid inlet port of a cartridge described herein. For example, the sample may be held in a container or may be drawn directly from a reaction process or batch, or a pharmaceutical solution. Preferably, the sample is introduced via sample introduction direction 114 by creating a vacuum via a pumping mechanism 110, preferably a three-position syringe, to introduce the sample into fluid inlet port 106. The sample is also preferably transferred to conduit 108 during this initial process by the vacuum created by the pumping mechanism. Preferably, the sample is transferred to suction region 140 of conduit 108.

[0075] Pump mechanism 110, preferably a three-position syringe, is then placed in a second position to transfer the sample from conduit 108 (preferably, aspiration region 140) to mixing region 142 of conduit 108. In embodiments, this transfer occurs from aspiration region 140 through fluid restrictor 144 to mixing region 142.

[0076] The sample is then preferably mixed with a hemocyte lysate and a chromogenic substrate to produce a mixed sample. As described herein, this mixing preferably occurs with a magnetic mixing element 146, for example, by rotating or translating an element within the mixing region with a magnet. The mixed sample is then transferred from the mixing region to the optical sample wells 104. Preferably, the transfer or transport of the sample is stopped by a liquid-impermeable membrane 121 fluidly connected to the optical sample wells. As described herein, stopping the transport allows each of the optical sample wells to be filled to a substantially uniform volume (such that the volumes vary within about 10%, preferably about 5%, or about 1% of each other), allowing comparison between the optical sample wells.

[0077] The method further includes measuring an optical property of the sample in the optical sample well. In the method for detecting the presence of a microbial contaminant, a change in the optical property indicates the presence of a microbial contaminant in the sample.

[0078] As described herein, the optical property is preferably the absorbance of the sample at a preselected wavelength of light, and the change in the optical property is a change in absorbance. The measured optical property can be absorbance at a specific wavelength, transmittance at a specific wavelength, fluorescence at a specific wavelength, or a change (e.g., increase or decrease) in optical density. For example, the optical property can be a change in absorbance or transmittance at a wavelength in the range of about 200 nm to about 700 nm, more preferably in the range of about 350 nm to about 450 nm, or about 400 nm to about 410 nm, or about 405 nm.

[0079] As described herein, typical mechanisms for sealing or connecting the cover section 184, bottom section 186, and manifold section 188 of the cartridge 100 include laser welding and ultrasonic welding. Preferably, one or more of these sections are made from a polymeric material, such as polystyrene, containing a small amount of carbon black (e.g., 0.5-10% by weight, preferably 2-5% by weight). The inclusion of carbon black in one or more of the sections allows the sections to be laser welded together. Methods for performing laser welding are known in the art, as disclosed, for example, in Klien, "Laser Welding of Plastics," Wiley-VCH, Germany (2012), the disclosure of which is incorporated herein by reference in its entirety. The use of laser welding mitigates problems associated with heat-induced degradation of liquid-impermeable membranes.

[0080] Methods of ultrasonic welding are known in the art, as disclosed, for example, in Shoh, "Welding of thermoplastics by ultrasound," Ultrasonics 14:209-217 (1976), the disclosure of which is incorporated herein by reference in its entirety. The use of ultrasonic welding mitigates problems associated with heat-induced degradation of liquid-impermeable membranes.

[0081] In an exemplary embodiment, a cartridge 100 described herein containing a sample in an optical sample well 104 can be inserted into a measurement or reader device 600, such as shown in FIG. 6 . Note that the reader device 600 of FIG. 6 is provided for illustrative purposes only and does not limit the scope of the present invention, including the methods of reading or analyzing the cartridge described herein. The cartridge 100 is preferably insertable into the reader device 600 so that measurements of the optical properties of the sample can be performed. As shown, the reader device 600 is preferably a handheld or easily portable device that can be employed in a variety of laboratory or clinical environments and can be easily operated and controlled via simple touchscreen commands. Exemplary components of the reader device 600 include a light source, preferably an LED light source, capable of generating light at wavelengths of about 350 nm to about 450 nm, or about 400 nm to about 410 nm, or about 405 nm. Additionally, a light panel (e.g., light guides, mirrors, prisms, etc.) is included within the reader device 600 to provide illumination to each of the optical sample wells 104, providing a signal channel that can be read by a detector, preferably an array of photodiodes. An additional reference channel is provided as a control to determine whether the correct light intensity and wavelength are being provided. The reader device 600 can also include a heater, for example, to maintain the temperature of the sample, preferably between about 25°C and about 40°C, to facilitate the enzymatic reaction. Exemplary heaters can be fabricated from flexible films of polyimide, e.g., DUPONT KAPTON®, and silicone rubber. Various other components of the reader device 600, such as computer circuitry for determining and / or quantifying the amount of absorption, are known in the art and can be readily incorporated into such devices.

[0082] When the cartridge 100 is inserted into the reader device 600, heating can be performed by a heater to facilitate the desired enzymatic reaction. Light is provided through the optical sample wells 104 containing the sample, and the absorbance is read by a detector, preferably a photodiode. The absorbances from the various optical sample wells are then compared, preferably with one or more of the optical sample wells serving as controls containing microbial contaminants. The presence and / or amount of endotoxin in the sample can then be determined, for example, by comparing the amount in the sample to the amount in the control against a standard calibration curve. One of skill in the art can readily create such a standard calibration curve using the absorbance of known amounts of endotoxin. In additional embodiments, the reader device 600 can also be provided with a stored (i.e., retained in the reader device and initially provided) or predetermined calibration curve that the operator can use to determine the amount of endotoxin in a test sample. Such stored or predetermined calibration curves can be provided for various endotoxins and can be updated by the user as needed, for example, by downloading calibration curves from a maintained database. Multiple cartridges 100 can be inserted into the reader device 600 and read simultaneously, allowing the presence and / or quantity of microbial contaminants in many different samples to be determined simultaneously. Additional Exemplary Embodiments

[0083] An embodiment is a cartridge for determining the presence and / or amount of microbial contaminants in a sample, comprising: a housing having an optical sample well, a fluid inlet port, and a conduit fluidly connecting the fluid inlet port and the optical sample well; a pump mechanism associated with the housing and fluidly connected to the fluid inlet port, the conduit, and the optical sample well; and a mixing region positioned along the conduit configured to contain a dry composition including a blood cell lysate.

[0084] Embodiment 2 includes a cartridge as described in embodiment 1, wherein the housing includes four optical sample wells, two of the four optical sample wells configured to contain an agent representative of a microbial contaminant dried within the optical sample well.

[0085] Embodiment 3 comprises a cartridge according to embodiment 1 or 2, further comprising a chromogenic substrate dried within the mixing region.

[0086] Embodiment 4 comprises the cartridge of any of Embodiments 1-3, wherein the housing comprises a cover section and a bottom section mechanically connected to one another.

[0087] Embodiment 5 comprises the cartridge of embodiment 4, wherein the housing further comprises a manifold section mechanically connected to the lower section, and wherein the dry composition is dried on a mixing portion of the manifold section.

[0088] Embodiment 6 comprises the cartridge of embodiment 5, wherein the mixing portion further comprises a chromogenic substrate dried thereon.

[0089] Embodiment 7 comprises the cartridge of embodiment 5 or 6, wherein the manifold section further comprises an agent representative of a microbial contaminant dried onto the sample well portion of the manifold section.

[0090] Embodiment 8 comprises the cartridge of any of embodiments 1-7, wherein the mixing region comprises a magnetic stirring element contained therein.

[0091] Embodiment 9 comprises the cartridge of any of embodiments 1-8, wherein the conduit comprises an aspiration region and a fluid restrictor disposed between the fluid inlet port and the mixing region.

[0092] Embodiment 10 comprises the cartridge of any one of Embodiments 1 to 9, wherein the hemocyte lysate is a limulus amebocyte lysate.

[0093] Embodiment 11 comprises the cartridge of embodiment 2, wherein the agent representative of a microbial contaminant is a bacterial endotoxin.

[0094] Embodiment 12 comprises a cartridge according to any of embodiments 9 to 11, wherein the pump mechanism is a three-position syringe, the first position creating a vacuum to introduce the sample into the aspiration region, the second position providing transport from the aspiration region through the fluid restrictor to the mixing region, and the third position providing transport from the mixing region to the sample well.

[0095] Embodiment 13 is a cartridge for determining the presence and / or amount of microbial contaminants in a sample, comprising: a housing having a cover section, a lower section mechanically connected to the cover section, and a manifold section mechanically connected to the lower section, wherein the lower section has two optical sample wells, a fluid inlet port, and a conduit fluidly connecting the fluid inlet port and the optical sample wells, the conduit having an aspiration region, a fluid restrictor, and a mixing region, the manifold section having a mixing portion configured to contain a dry composition including a hemocyte lysate, the mixing portion corresponding to the mixing region of the conduit; and a pump mechanism associated with the housing and fluidly connected to the fluid inlet port, the conduit, and the two optical sample wells.

[0096] Embodiment 14 includes a cartridge as described in embodiment 13, wherein the lower section includes four optical sample wells, two of the four optical sample wells configured to contain an agent representative of a microbial contaminant dried within the optical sample well.

[0097] Embodiment 15 comprises a cartridge according to embodiment 14, wherein the manifold section further comprises an agent representative of a microbial contaminant dried onto the sample well portion of the manifold section.

[0098] Embodiment 16 comprises a cartridge according to any one of claims 13 to 15, further comprising a chromogenic substrate dried onto the mixing portion of the manifold section.

[0099] Embodiment 17 comprises a cartridge according to any one of claims 13 to 16, wherein a pump mechanism is integrated into the lower section, the pump mechanism being a three-position syringe, the first position generating a vacuum to introduce the sample into the aspiration region, the second position providing transport from the aspiration region through the fluid restrictor to the mixing region, and the third position providing transport from the mixing region to the sample well.

[0100] Embodiment 18 comprises the cartridge of any one of embodiments 13 to 17, wherein the hemocyte lysate is a limulus amebocyte lysate.

[0101] Embodiment 19 comprises the cartridge of any of embodiments 14-18, wherein the agent is a bacterial endotoxin.

[0102] Embodiment 20 is a method for detecting the presence of a microbial contaminant in a sample, comprising introducing the sample into a fluid inlet port of the cartridge described in embodiment 3, conveying the sample to a conduit, conveying the sample into a mixing area of ​​the conduit, mixing the sample with a hemocyte lysate and a chromogenic substrate to produce a mixed sample, conveying the mixed sample from the mixing area to a sample well, and measuring optical properties of the mixed sample in the optical sample well, wherein a change in the optical properties indicates the presence of a microbial contaminant in the sample.

[0103] Example 21 includes the method of example 20, wherein measuring the optical property is a change in absorbance of light at a preselected wavelength.

[0104] Embodiment 22 includes the method of embodiment 21, wherein the change in absorbance of light at the preselected wavelength is compared to a calibration curve.

[0105] Embodiment 23 includes the method of embodiment 22, wherein the calibration curve is a stored calibration curve.

[0106] Embodiment 24 is the method of any of embodiments 20 to 23, wherein introducing the sample comprises generating a vacuum via a pumping mechanism to introduce the sample into the fluid inlet port and the conduit.

[0107] Embodiment 25 is the method of any of embodiments 20 to 24, wherein the transferring comprises transporting the sample from the mixing region to the optical sample well via a pump mechanism.

[0108] Embodiment 26 is a method for detecting the presence of a microbial contaminant in a sample, comprising introducing the sample into a fluid inlet port of the cartridge described in embodiment 16, transferring the sample to an aspiration region, transferring the sample from the aspiration region through a fluid restrictor to a mixing region, mixing the sample with a hemocyte lysate and a chromogenic substrate to generate a mixed sample, transferring the mixed sample to an optical sample well, and measuring optical properties of the mixed sample in the optical sample well, wherein a change in optical properties indicates the presence of a microbial contaminant in the sample.

[0109] Example 27 includes the method of example 26, wherein measuring the optical property is a change in absorbance of light at a preselected wavelength.

[0110] Embodiment 28 includes the method of embodiment 27, wherein the change in absorbance of light at the preselected wavelength is compared to a calibration curve.

[0111] Embodiment 29 includes the method of embodiment 28, wherein the calibration curve is a stored calibration curve.

[0112] Embodiment 30 includes a method according to any one of embodiments 26 to 29, wherein introducing the sample includes generating a vacuum via a pumping mechanism to introduce the sample into the fluid inlet port and the suction region; communicating in b) includes transporting the sample from the suction region to the mixing region via a pumping mechanism; and communicating in d) includes transporting the mixed sample from the mixing region to the optical sample well via a pumping mechanism.

[0113] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations can be made to the methods and applications described herein without departing from the scope of any of the present embodiments.

[0114] Although specific embodiments have been illustrated and described herein, it is to be understood that the claims or clauses are not limited to the specific forms or arrangements of parts described and illustrated. Although exemplary embodiments have been disclosed herein and specific terms have been used, these terms are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0115] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A cartridge for determining the presence and / or amount of a microbial contaminant in a sample, comprising: a. A housing comprising a cover section, a lower section mechanically connected to said cover section, and a manifold section mechanically connected to said lower section, i. the lower section comprises four optical sample wells, a fluid inlet port, and conduits fluidly connecting the fluid inlet port and the optical sample wells, wherein two of the four optical sample wells are configured to contain an agent representative of the microbial contaminant dried within the optical sample well; ii. the conduit comprises a suction region, a fluid restrictor, and a mixing region; iii. The manifold section includes a housing having a mixing portion configured to contain a dry composition including a hemocyte lysate, the mixing portion corresponding to the mixing region of the conduit; b. a pumping mechanism associated with said housing and fluidly connected to said fluid inlet port, said conduit, and said four optical sample wells, said pumping mechanism being a three-position syringe.

2. The cartridge of claim 1 , wherein the manifold section further comprises the agent representative of the microbial contaminant dried onto a sample well portion of the manifold section.

3. 3. The cartridge of claim 1 or 2, further comprising a chromogenic substrate dried onto the mixing portion of the manifold section.

4. 4. The cartridge of claim 1, wherein the pump mechanism is integrated into the lower section, and wherein a first position generates a vacuum to introduce the sample into the aspiration region, a second position provides transport from the aspiration region through the fluid restrictor to the mixing region, and a third position provides transport from the mixing region to the optical sample well.

5. The cartridge according to any one of claims 1 to 4, wherein the hemocyte lysate is a Limulus amebocyte lysate.

6. The cartridge of any one of claims 1 to 5, wherein the agent is a bacterial endotoxin.

7. 1. A method for detecting the presence of a microbial contaminant in a sample, comprising: a. introducing the sample into the fluid inlet port of the cartridge of claim 3 and transmitting the sample to the conduit; b. conveying the sample into the mixing region of the conduit; c. mixing the sample with the hemocyte lysate and the chromogenic substrate to produce a mixed sample; d. transferring the mixed sample from the mixing region to the four optical sample wells; and e. measuring optical properties of said combined sample in said four optical sample wells, wherein a change in said optical properties indicates the presence of said microbial contaminant in said sample.

8. The method of claim 7 , wherein said measuring said optical property is a change in absorbance of light at a preselected wavelength.

9. The method of claim 8 , wherein the change in absorbance of light at the preselected wavelength is compared to a calibration curve.

10. 10. The method of claim 9, wherein the calibration curve is a stored calibration curve.

11. 11. The method of any one of claims 7 to 10, wherein the introducing the sample comprises creating a vacuum via the pump mechanism to introduce the sample into the fluid inlet port and the conduit.

12. The method of any one of claims 7 to 11, wherein the transferring comprises transporting the sample from the mixing region to the four optical sample wells via the pump mechanism.

13. 1. A method for detecting the presence of a microbial contaminant in a sample, comprising: a. introducing the sample into the fluid inlet port of the cartridge of claim 3 and conveying the sample to the aspiration region; b. transferring the sample from the aspiration region through the fluid restrictor to the mixing region; c. mixing the sample with the hemocyte lysate and the chromogenic substrate to produce a mixed sample; d. transferring the mixed sample to the four optical sample wells; and e. measuring optical properties of said combined sample in said four optical sample wells, wherein a change in said optical properties indicates the presence of said microbial contaminant in said sample.

14. The method of claim 13 , wherein the measuring of the optical property is a change in absorbance of light at a preselected wavelength.

15. The method of claim 14, wherein the change in absorbance of light at the preselected wavelength is compared to a calibration curve.

16. 16. The method of claim 15, wherein the calibration curve is a stored calibration curve.

17. 17. The method of any one of claims 13 to 16, wherein the introducing the sample comprises generating a vacuum via the pumping mechanism to introduce the sample into the fluid inlet port and the suction region; the transferring in b) comprises transporting the sample from the suction region to the mixing region via the pumping mechanism; and the transferring in d) comprises transporting the mixed sample from the mixing region to the four optical sample wells via the pumping mechanism.

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