WO2019241794A1 - Identification et caractérisation de micro-organismes - Google Patents

Identification et caractérisation de micro-organismes Download PDF

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Publication number
WO2019241794A1
WO2019241794A1 PCT/US2019/037533 US2019037533W WO2019241794A1 WO 2019241794 A1 WO2019241794 A1 WO 2019241794A1 US 2019037533 W US2019037533 W US 2019037533W WO 2019241794 A1 WO2019241794 A1 WO 2019241794A1
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Prior art keywords
microbe
channels
ligand
polymer
channel
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PCT/US2019/037533
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English (en)
Inventor
Kevin Jay HACKER
Aldrich Lau
Robert Eason
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Hacker Kevin Jay
Aldrich Lau
Robert Eason
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Publication of WO2019241794A1 publication Critical patent/WO2019241794A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present technology relates generally to systems, devices, and methods for microorganism detection, identification, and/or characterization, and in particular provides systems, devices, and methods for quickly detecting and identifying infectious microorganisms.
  • Infections can be caused by a variety of microorganisms including bacteria, fungi, parasites, viruses, and the like.
  • infections can be serious and can require quick treatment to minimize associated risks.
  • one infection associated risk is sepsis, a life- threatening condition caused by an inflammatory immune response triggered by an infection.
  • the most common cause of sepsis is a bacterial infection.
  • the risk of death from sepsis is as high as 30%, from severe sepsis as high as 50%, and from septic shock as high as 80%.
  • the mortality risk associated with sepsis is correlated with time to treatment— the longer a patient is infected before beginning treatment, the more likely the patient will suffer serious effects or die from the infection. Accordingly, antibiotics are given as soon as the underlying infectious agent is identified.
  • Figures 1 A-C illustrate a microfluidic device in accordance with select embodiments of the present technology.
  • Figure 2 is a schematic illustration of a cross-sectional view of a microfluidic device in accordance with select embodiments of the present technology.
  • Figures 3A-C illustrate different microbes flowing through microfluidic device channels in accordance with select embodiments of the present technology.
  • Figures 4A-C illustrate a microbe flowing through three different microfluidic device channels in accordance with select embodiments of the present technology.
  • Figures 5A-C illustrate a microfluidic device having a plurality of distinct capture materials arranged in series in accordance with select embodiments of the present technology.
  • Figure 6 illustrates a microfluidic device channel in accordance with select embodiments of the present technology.
  • Figure 7 illustrates a microfluidic device channel with a plurality of ridges and recesses in accordance with select embodiments of the present technology.
  • Figure 8A illustrates a microfluidic device channel having a plurality of beads in accordance with select embodiments of the present technology.
  • Figure 8B illustrates a microfluidic device channel having a plurality of microposts in accordance with select embodiments of the present technology.
  • Figure 9 is a schematic illustration of a microfluidic device channel in accordance with select embodiments of the present technology.
  • Figure 10 is a schematic illustration of a microfluidic device channel in accordance with select embodiments of the present technology.
  • Figure 11 is a schematic illustration of a microfluidic device having six channels arranged in parallel in accordance with select embodiments of the present technology.
  • Figure 12 is a schematic illustration of a microfluidic device having a single channel with six different ligands arranged in series in accordance with select embodiments of the present technology.
  • Figure 13 is a schematic illustration of a microfluidic device having six channels each having their own distinct inlet in accordance with select embodiments of the present technology.
  • Figures 14A-B are schematic illustrations of a microfluidic device having two channels, with each channel having five ligands arranged in a series in accordance with select embodiments of the present technology.
  • Figures 15A-D illustrate flow results obtained by testing a microbe in a six-channel microfluidic device, and further illustrate identifying the microbe by comparing the flow results to a database of flow results for known microbes in accordance with select embodiments of the present technology.
  • Figure 16 is a schematic illustration of a chemical reaction used to prepare several saccharide-based ligands in accordance with select embodiments of the present technology.
  • Figure 17 is a flowchart of a method for fabricating microfluidic devices in accordance with select embodiments of the present technology.
  • Figure 18 is a schematic illustration of a workflow for fabricating microfluidic devices in accordance with select embodiments of the present technology.
  • Figure 19 is a schematic illustration of a workflow for fabricating microfluidic devices using oligonucleotide linkers in accordance with select embodiments of the present technology.
  • Figures 20A-B illustrate a sample collection and preparation device in accordance with select embodiments of the present technology.
  • Figures 21A-C illustrate methods of selectively recovering microbes following testing in accordance with select embodiments of the present technology.
  • the present technology provides systems, devices, and methods for identifying and/or characterizing microbes via affinity binding.
  • the present technology identifies microbes specifically, identifies microbes by Gram stain, and/or determines microbe susceptibility to specific antibiotics in minutes rather than many hours or days required by other methods.
  • infections and associated diseases such as sepsis and necrotizing soft tissue infections
  • small delays in receiving appropriate treatment can greatly increase the risks associated with the infection.
  • the present technology thus improves positive treatment outcomes by facilitating quicker and more targeted treatment of microbial infections.
  • the present technology reduces the time to identify one or more properties of an unknown microbe by utilizing affinity binding and microfluidic principles, negating the requirement to use traditional time-consuming microbial identification techniques such as microbe culturing, cell lysis clean up, DNA isolation, DNA hybridization, or DNA amplification.
  • the present technology enables clinicians to quickly and accurately select an appropriate treatment for an infection and increases positive patient outcomes.
  • the present technology provides microfluidic devices for identifying one or more properties of an unknown microbe.
  • the microfluidic devices include one or more channels having capture materials that selectively bind various microbes at various affinities.
  • the unidentified microbe Prior to being loaded into the device, the unidentified microbe is suspended in a buffer solution, which is then inserted into the device and flowed through one or more channels within the device. After the buffer solution has flowed through the one or more channels, the channels are imaged to determine the flow results, which indicate (1) whether the microbe was retained by the capture material, and (2) how far along the channel the microbe traveled before being retained by the capture material. From the flow results, the relative affinity of the microbe to the capture material can be determined and certain properties of the microbe can be determined.
  • the flow results can also be compared to reference images obtained by flowing known microbes through the device. Based on a comparison between the test images and the reference images, the microbe can be identified and/or further characterized.
  • Figure 1A is an isometric view of a microfluidic device 100 in accordance with an embodiment of the present technology.
  • the device 100 includes a body 102 and a cover 104. While Figure 1A illustrates the body 102 and the cover 104 having a generally rectangular shape, other shapes, such as generally oval, triangular, pentagonal, and the like are possible and included within the scope of the present technology. Regardless of the shape of the body 102 and the cover 104, the body 102 and the cover 104 combine to form the device 100.
  • the body 102 and cover 104 can be releasably secured together, fixedly secured together, and/or are integrally manufactured such that the body 102 and the cover 104 together comprise a single unitary substrate.
  • the body 102 and cover 104 can comprise plastic, glass, or other suitable substrates.
  • at least one of the body 102 or the cover 104 is an optically transparent plastic or glass material to facilitate imaging of the internal structures of the device 100.
  • the device 100 further includes pressure sensitive adhesive (PSA) tape (not shown) between the body 102 and the cover 104.
  • PSA tape at least partially spaces apart the cover 104 and the body 102 to define one or more channels within the device 100.
  • the device further includes an inlet 106 and an outlet 108.
  • the inlet 106 is configured to receive a buffer solution containing a microbe and provides access to a channel (not shown), which extends between the inlet 106 and the outlet 108.
  • Figures 1B and 1C are cross-sectional views of the device 100 taken along the lines indicated in Figure 1A.
  • the inlet 106 provides access to a channel 105.
  • the channel 105 is defined by an empty space between the body 102 and the cover 104.
  • the side walls of the channel 105 may be defined by the tape, the lower surface defined by the body 102, and the upper surface defined by the cover 104.
  • the channel 105 can be defined by an empty space within the body 102.
  • the channel 105 extends a longitudinal length of the device 100 between the inlet 106 and the outlet 108 and is configured such that a fluid solution introduced at the inlet 106 will flow along the length of the channel 105 to the outlet 108.
  • a pump (not shown) can be operably coupled to the channel 105 to regulate the fluid flow rate.
  • the channel 105 can be coated with capture material 110.
  • the capture material 110 includes ligands bound to reactive polymers, which are immobilized on the surfaces defining the channel 105.
  • the ligands can selectively bind and retain different microbes at different affinities.
  • any ligand that can bind to the polymer and selectively bind microbes at various affinities can be used in the present technology.
  • the ligands can include saccharides, saccharide derivatives, antibiotics, antibiotic derivatives, peptides, peptide derivatives, aptamers, and other molecules configured to selectively bind surface molecules on microbes. While the illustrated embodiments depict the capture materials as a coating on the surfaces of the device 100 defining the channel 105, other embodiments include a three-dimensional scaffolding of capture materials occupying substantially all of the internal volume of the channel 105.
  • the channel 105 can be fully enclosed by the body 102 and the polymer 110 along at least a portion of its length. In other embodiments, however, the channel 105 may only be bound on one, two, or three sides, with three, two, or one sides open to the ambient air (not shown). In some embodiments, the longitudinal cross-sectional geometry of the channel is a shape other than rectangular (e.g., circular, triangular, etc.).
  • the channel 105 has dimensions suitable for promoting flow through the channel. For example, the channel 105 can have the dimensions and associated characteristics set forth in Table 1 Table 1. Channel dimensions and turn around time
  • the device 100 can be positioned adjacent an imager 200, as illustrated in Figure 1B.
  • the imager 200 can be positioned adjacent the device such that the imager 200 can capture an image of the channel 105.
  • the imager 200 can be on a movable platform and move to image different locations along the channel 105.
  • the images of the channel 105 may illustrate (1) whether the microbe was retained by the capture material, and (2) how far along the channel the microbe traveled before being retained by the capture material. This information can be used to help identify and/or characterize the microbe.
  • unknown microbes are isolated from blood or another tissue sample obtained from a patient by a low speed spin to generate microbes in plasma, then pelleted by a high speed spin to form a pellet of microbes.
  • the microbes are resuspended in buffer, stained fluorescently, and loaded in inlet 106.
  • the buffer flows through the channel via hydrodynamic flow.
  • the device may be configured to initiate turbulent flow of the microbe-buffer solution to increase the interactions between microbes flowing through the channel and the capture material 110 (see, e.g., Figure 7).
  • the channel 105 may include features that disrupt flow of the solution to maximize collisions between the microbes and the capture molecules. Examples of such features may include forward, reverse, or forward and reverse herringbone ridges. By disrupting the flow of the microbe-buffer solution, microbe capture efficiency can be increased.
  • Additional buffer can be added to flow the microbe suspension completely through the channels to the outlet 108.
  • the channel 106 is imaged using imager 200 to determine the location of the bound microbes.
  • the information on the unknown microbe’s location is compared with a library of locations of known microbes that were run through the same channel under the same conditions and stored. The identity of the unknown microbe is displayed if the locations match that of a known microbe.
  • Figure 2 is a cross-sectional view of the device 100 and illustrates a portion of the channel 105 with a microbe 150 bound to the capture material 110.
  • capture material 110 can include a polymer scaffold 130 and a plurality of ligands 135 bound to the polymer scaffold 130.
  • the ligands 135 bind to and retain the microbe.
  • the polymer scaffold 130 is bound to the channel surfaces 102, 104 via attachment molecules 145.
  • Figures 3A-C illustrate microbes 150 in a buffer solution (not shown) flowing through the channel 105 of device 100. More specifically, Figures 3 A-C illustrates three different microbes— a first microbe l50a, a second microbe l50b, and a third microbe l50c (collectively referred to herein as the“microbes 150”)— flowing through the channel 105 from the inlet 106 towards the outlet 108, as indicated by the arrows. As illustrated in Figures 3 A, the first microbe l50a flowed through the full length of the channel 105 without being retained by the capture material 110.
  • the second microbe l50b flowed through a significant length of the channel 105 before being retained by the capture material 110.
  • the third microbe l50c was retained by the capture material 110 within the channel at a position proximate the inlet 106. Based on how far each of the microbes 150 traveled down the length of the channel 105 before being retained, the relative affinity of the microbes 150 for the capture material 110 can be ascertained. As will be discussed in detail below, the affinity of the microbes 150 for the capture material 110 can be used to help identify and/or characterize the microbes.
  • Figures 4A-C illustrate another embodiment of microfluidic devices in accordance with the present technology. Unlike in Figures 3 A-C, the devices illustrated in Figures 4A-C each have different capture materials.
  • device 400a includes a first capture material 110
  • device 400b includes a second capture material 111
  • device 400c includes a third capture material 112.
  • the devices 400 can be separate devices, or can be incorporated as multiple channels in a single device 400 (devices 400a, 400b, and 400c are collectively referred to herein as“devices 400”).
  • Figures 4A-C further illustrate an exemplary microbe 150 flowing through the channels 105.
  • the microbe 150 delivered to the devices 400 is the same. As illustrated in Figure 4A, the microbe 150 has little to no affinity to the capture material 110 because it flowed through the entire length of the channel l05a without being retained by the capture material. As illustrated in Figure 4B, the microbe 150 has relatively low affinity to the capture material 111 because it flowed through a substantial length of the channel l05b before being retained by the capture material. As illustrated in Figure 4C, the microbe 150 has relatively high affinity to the capture material 112 because it was retained by the capture materials 112 at a position adjacent the inlet l06c of channel l05c.
  • microbe 150 can be characterized in that it has little to no affinity to capture material 110, relatively low affinity to capture material 111, and relatively high affinity to capture material 112.
  • An imager (not shown) can be used to capture images of devices 400 to determine the position, and thus relative affinity, of the microbe to the capture materials.
  • FIGS 5A-C illustrate another embodiment of a microfluidic device 500 in accordance with the present technology.
  • device 500 includes a body 104 (e.g., a plastic substrate), a cover 102, PSA tape 103 between the body 104 and the cover 102, and a channel 105 defined at least partially by the PSA tape 103.
  • the device 500 includes discrete zones of different capture materials.
  • the device includes a first zone having a first capture material l lOa, a second zone having a second capture material l lOb, a third zone having a third capture material l lOc, and a fourth zone having a fourth capture material l lOd.
  • the capture materials 1 lOa-d have the same reactive polymer but different ligands. In other embodiments, the capture materials 1 lOa-d have different reactive polymers and different ligands. By having multiple capture materials arranged in a series along a channel 105, fewer cells are required to identify the microbes.
  • the microbe 150 is captured in the fourth zone having the fourth capture material 113. Accordingly, the microbe 150 has a relatively high affinity to the fourth capture material 113, and relatively low affinity for the first capture material 110, the second capture material 111, and the third capture material 112.
  • Figure 6 illustrates another embodiment of a microfluidic device 600 in accordance with the present technology.
  • a single capture material 110 occupies the full length of the channel 105.
  • the relative concentration of the ligands on the capture material 110 differ between a proximal end l05a of the channel 105 adjacent the inlet (relatively low concentration) and a distal end l05b of the channel 105 adjacent the outlet (relatively high concentration).
  • FIG. 7 illustrates a channel 105 of a microfluidic device 700 configured to initiate turbulent flow.
  • the top surface of the channel e.g., defined by a surface of the cover 104
  • the ridges 107 can be arranged in a forward, reverse, or forward and reverse herringbone pattern to facilitate turbulent flow.
  • the bottom surface of the channel (e.g., defined by a surface of the base 102) can include the immobilized capture material 110.
  • the ridges 107 and recesses 109 disrupt the flow of the solution to maximize collisions between the microbes 150 and the capture material 110.
  • Figures 8A-B illustrate additional embodiments of channels 105 that can be included in the microfluidic devices described herein.
  • Figure 8 A illustrates one embodiment of a channel 105 in a microfluidic device 800.
  • the channel 105 includes a plurality of beads 120.
  • the beads 120 are coated with the ligand that selectively captures and retains various microbes.
  • Figure 8B illustrates an embodiment of a channel 105 in a microfluidic device 850.
  • the channel 105 includes a plurality of microposts 125.
  • the microposts 125 can be coated with the ligand that selectively captures and retains various microbes.
  • Figure 9 is a schematic illustration of a microfluidic device channel 905 in accordance with embodiments of the present technology.
  • the channel 905 has a plurality of enlarged portions 905a, 905b, and 905c connected by passageways 905d and 905e.
  • channels 905 may include greater or fewer enlarged portions than explicitly discussed herein.
  • the enlarged portion 905a has a greater internal diameter than the enlarged portion 905b
  • the enlarged portion 905b has a greater internal diameter than the enlarged portion 905c.
  • the degree of interaction between a capture material coating the walls of the enlarged portions 905a-c and a microbe flowing through the channel 905 can be altered (not shown).
  • the passageways 905d and 905e are not coated with capture material such that the microbe will freely flow through passageways 905d and 905e.
  • FIG 10 is a schematic illustration of a microfluidic device channel 1005 in accordance with embodiments of the present technology.
  • the channel 1005 has a generally downward spiraling configuration that affects the flow velocity of microbes through the channel.
  • microbe binding to capture material can be altered, and additional information about the relative affinity of the microbe to the capture material can be obtained.
  • the devices, channels, and capture materials described with respect to Figures 1 A-10 may be combined in various arrangements to form multi-channel devices. Accordingly, the specific embodiments discussed herein do not limit the scope of configurations the microfluidic device may have.
  • FIG 11 is a schematic illustration of a microfluidic device 1100 having multiple channels 105.
  • Microfluidic device 1100 includes an inlet 106, a plurality of channels 105 arranged in parallel, and an outlet 108.
  • the plurality of channels includes a first channel l05a, a second channel l05b, a third channel l05c, a fourth channel l05d, a fifth channel l05e, and a sixth channel l05f.
  • a buffer solution containing a microbe is inserted into the device at inlet 106, the buffer solution will flow through each of the channels l05a-f in parallel.
  • a different ligand (not shown) can be used in each of the channels l05a-f.
  • each of the channels l05a-f can subsequently be imaged to determine the affinity of the microbe to at least six different ligands. While the illustrated embodiment depicts six channels, one skilled in the art will appreciate that device 1100 can have any number of channels. For example, device 1100 could have one, two, three, four, five, six, seven, eight, nine, ten, or more channels.
  • FIG 12 is a schematic illustration of a microfluidic device 1200 having a plurality of capture materials arranged in a single channel 105 in a series.
  • the device 1200 includes a first zone l05a having a first capture material l lOa, a second zone l05b having a second capture material l lOb, a third zone l05c having a third capture material l lOc, a fourth zone l05d having a fourth capture material 1 lOd, a fifth zone l05e having a fifth capture material 1 lOe, and a sixth capture zone l05f having a sixth capture material 1 lOf.
  • Each capture material 1 lOa-f has a different ligand, but can share the same polymer.
  • FIG 13 is a schematic illustration of yet another microfluidic device 1300 in accordance with the present technology.
  • Device 1300 is generally similar to device 1100, except that each of the plurality of channels l05a-f includes a corresponding inlet l06a-f and a corresponding outlet l08a-f. Each inlet l06a-f can receive the same microbe or different microbes.
  • Figure 14A illustrates a device 1400 having two channels l05a and l05b in parallel. Both channels l05a, l05b include five distinct capture materials 1 lOa-e arranged in a series. Microbe 150 can be identified by its relative binding affinity to each of the plurality of caputre materials 1 lOa- e.
  • the devcie 1400 can be used to test the relative affinity of multiple microbes (e.g., microbe l50a and microbe l50b) simultaneously.
  • a sample containing an unidentified microbe is obtained from a patient.
  • the sample can be a wound swab, a nasal swab, stool, saliva, water, or blood.
  • Microbes are isolated from the sample by a low speed spin or other suitable technique to generate microbes in plasma, then pelleted by a high-speed spin to generate a pellet of microbes.
  • the microbes further undergo a filtering process.
  • the microbes are resuspended in buffer and stained fluorescently. The fluorescently stained microbes are loaded in the inlet 106 for testing.
  • the one or more channels of the microfluidic devices are imaged after the buffer solution originally containing the microbes has flowed through the one or more channels.
  • the images depict (1) whether the microbe was retained by the capture material, and (2) how far along the channel the microbe traveled before being retained by the capture material.
  • the flow results from each individual channel can be plotted on a fluorescence vs. distance graph that displays the intensity of fluorescence as a function of distance traveled from the input.
  • the present technology further comprises a database storing flow results from a plurality of known microbes.
  • the database contains fluorescence vs. distance graphs for known microbes that have been previously tested using a comparable device under the same conditions.
  • the database may include flow results for at least six common bacteria ⁇ Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coir, commonly referred to as“ESKAPEc”). Flow results of the unknown microbe can be compared to flow results of the ESKAPEc microbes.
  • Figures 15 A-D illustrate examples of comparing flow results from an unknown microbe to flow results in the database to identify the unknown microbe.
  • Figure 15 A illustrates the flow results for an unknown microbe flowed through a microfluidic device having six channels l05a-f arranged in parallel. Each of the six channels l05a-f includes a different capture material.
  • channel l05a includes a polymer only and functions as a control channel.
  • Channels l05b-f includes, mannose, galactose, Gal (1-4) Gal, glycopeptide agent A, and Gram-negative peptide, respectively.
  • a sample containing an unknown microbe was prepared using the techniques detailed herein and delivered to each of the six channels l05a-f.
  • the flow results for each of the six channels l05a-f were then plotted in the fluorescence vs. distance graphs l60a-f.
  • the fluorescence vs. distance graphs l60a-f are then compared to a database storing flow results from a plurality of known microbes.
  • the flow results depicted in graphs l60a-f match the flow results of a known microbe, E. coli , depicted in stored graphs l70a-f. Accordingly, the unknown microbe is identified as A. coli , and appropriate treatment can be initiated.
  • Figure 15B illustrates another example of comparing flow results from an unknown microbe to flow results in the database to identify the unknown microbe.
  • the device in Figure 15B also includes 6 channels l05a-f, although channel l05e includes vancomycin as the capture material.
  • the flow results for each of the six channels l05a-f was plotted in the fluorescence vs. distance graphs l60a-f.
  • the flow results in the fluorescence vs. distance graphs l60a-f were compared to stored flow results of known microbes in the database. As illustrated, the flow results match the fluorescence vs. distance graphs l72a-f, thus identifying the unknown microbe as P. aeruginosa.
  • Figure 15C illustrates yet another example of identifying an unknown microbe using the present technology. In Figure 15C, the unknown microbe is identified as S. aureus.
  • Figure 15D illustrates an example of flow results not having a match in the database.
  • the flow results from the unknown microbe depicted in graphs l60a-f do not have an exact match to any of the flow results in the database (graphs l76a-f are returned as a close but inexact match). Nevertheless, the flow results provide useful information on the unknown microbes characteristics that can help a physician select an appropriate treatment.
  • graph l60e illustrates the unknown microbe is sensitive to vancomycin based on the relative affinity the microbe demonstrated toward vancomycin.
  • graph l60f illustrates that the unknown microbe is Gram-positive. This information can be used to select the appropriate treatment from the patient, even though the identity of the microbe is not determined.
  • the present technology rapidly identifies one or more properties of an unknown microbe. For example, in some embodiments, the present technology identifies one or more properties of an unknown microbe in less than one hour, less than 45 minutes, less than 30 minutes, less than 20 minutes, less than 15 minutes, and/or less than 5 minutes. Accordingly, the present technology enables clinicians to quickly and accurately identify properties of a microbe to facilitate quick treatment.
  • the microfluidic devices include capture materials that selectively capture various microbes based on relative binding affinities.
  • the capture materials generally include at least two components: a polymer immobilized on the channel surface, and a ligand that selectively binds surface molecules of the microbe.
  • the present technology includes reactive polymers that can be immobilized on activated surfaces of the microfluidic devices described herein.
  • the reactive polymers also bind the ligands configured to selectively retain microbes.
  • the following description includes several embodiments of reactive polymers included in the present technology, as well as methods for preparing the reactive polymers.
  • the present technology includes other polymers not explicitly discussed herein, as well as polymers described herein but prepared according to different techniques. Accordingly, the present technology is not limited by the following embodiments.
  • N,N-dimethylacrylamide-co-tetrafluorophenylacrylate at a molar feed ratio of 35% N,N-dimethylacrylamide, N,N-dimethyl acrylamide (DMA) and tetrafluoro- phenyl acrylate (TFPA) are purified by vacuum distillation.
  • the initiator, 2,2’-azobis(2,4- dimethylvaleronitrile), is used as received.
  • a solution including about 2.10 mmol of TFPA, about 11.30 mmol of DMA, and about 0.02 mmol of 2,2’-azobis(2,4-dimethylvaleronitrile) in 30 mL of anhydrous acetonitrile is bubbly purged with ultrapure argon for about 30 minutes at a flow rate of 60 mL/min and magnetically stirred with a l-inch stir bar at 200 rpm.
  • the reaction flask is then lowered into a 55°C oil bath while having the same 200 rpm stirring but reduced argon flow rate of 25 mL/min.
  • the polymerization is conducted under these conditions for 19 hours.
  • the reaction flask is then removed from the oil bath, cooled to ambient temperature, and exposed to ambient atmosphere prior to workup.
  • the acetonitrile is removed under reduced pressure in a Rota-Vap at 55 °C water bath temperature, and the residual monomers are removed in a vacuum oven at 0.1 millibar at 60 °C for 3 hours.
  • the polymer product is re-dissolved in 30 mL of anhydrous tetrahydrofuran (THF) at 55 °C. Concurrent with magnetic stirring, about 40 mL of anhydrous hexane is added dropwise until a cloudy suspension is obtained.
  • THF anhydrous tetrahydrofuran
  • the cloudy suspension is added to 1000 mL of anhydrous hexane in a 2-L poly(propylene) Erlenmeyer flask through a 22-gauge syringe needle in a fine stream while stirring at 150 rpm or higher with a 2-inch PTFE stirring blade.
  • the precipitated polymer is stirred for an additional period (e.g., 5 minutes).
  • the hexane is decanted and 500 mL of fresh anhydrous hexane is added and stirred gently for another period (e.g., 15 minutes).
  • the polymer product in coarse fibers is transferred into a large mouth 500-mL glass jar and dried under vacuum at 55 °C for 24 hours.
  • the molecular weight of the copolymer is expected in the range of 600 to 900 KDa.
  • the general structure of the copolymer can be schematically expressed as Structure A where the phenyl ring has 4 fluorine substituents at positions 2, 3, 5 and 6:
  • Poly(N ,N-dimethylacrylamide-co-pentafluorophenylacrylate) can be prepared according to the same protocol described above except the tetrafluorophenyl acrylate (TFPA) is replaced with pentafluoro-phenylacrylate (PFPA).
  • TFPA tetrafluorophenyl acrylate
  • PFPA pentafluoro-phenylacrylate
  • the present technology includes ligands that bind to various microbial surface molecules.
  • the relative affinity of an unidentified microbe to the ligand can be used to determine (1) the identity of the unknown microbe, and/or (2) additional characteristics of the microbe, such as Gram-stain and/or susceptibility to specific antibiotics.
  • a non-exhaustive list of exemplary ligands includes saccharides, saccharide derivatives, antibiotics, antibiotic derivatives, peptides, peptide derivatives, aptamers, and other molecules configures to selectively bind surface molecules on various microbes.
  • the ligands comprise monosaccharides such as galactose, glucose, and/or mannose, although other saccharides can be used as well.
  • the monosaccharides can be modified saccharides having an amine group that facilitates attachment to the immobilized polymer.
  • Figure 16 illustrates a general approach of adding an amine linker to saccharide substrates and subsequent attachment of the saccharides to a reactive polymer surface.
  • the illustrated input substrates are lactose, maltose, and mannobiose.
  • the terminal sugars bound to the immobilized polymer network are galactose 135a, glucose 135b, and mannose 135c, respectively.
  • the input saccharide e.g., lactose
  • the input saccharide is synthetically glycosylated at Cl with an amino-alcohol linker.
  • the amine group on the linker, as well as hydroxyl groups on the sugar, are either masked or protected during the glycosylation reaction to ensure the linker is properly attached.
  • the amine group can be masked as an azide group or protected as a benzyloxycarbonyl (Cbz) derivative or using another standard amine protecting group.
  • Cbz protection as illustrated in Figure 16
  • the amine group is subsequently conveniently revealed by catalytic hydrogenation in a synthetic step that requires no subsequent purification.
  • Synthetic intermediates and final product can be analyzed by NMR (1H and 13C) and by MS. The free amine group can then interact with and bind to the reactive polymer previously immobilized on the plastic substrate.
  • the ligands include antibiotics or antibiotic derivatives.
  • antibiotics or antibiotic derivatives as the ligand (e.g., the capture material) in one or more channels of the devices described herein, the relative susceptibility of the microbe to various antibiotics can be determined, even if the actual identify of the microbe cannot be determined (e.g., if the flow results do not match any of the flow results included in the database of flow results). This information can enable a physician to select the appropriate antibiotic for treating the infection.
  • the ligands include surface antibiotics such as tetracyclines (e.g., doxycyeline), cephalosporins (e.g., cefepime), quinolones (e.g., ciprofloxacin), macrolides (e.g., azithromycin), g!ycopeptides (e.g., vancomycin), aminoglycosides (e.g., amikacin), lincomycins (e.g., clindamycin), and carbapenems (e.g., doripenem).
  • tetracyclines e.g., doxycyeline
  • cephalosporins e.g., cefepime
  • quinolones e.g., ciprofloxacin
  • macrolides e.g., azithromycin
  • g!ycopeptides e.g., vancomycin
  • aminoglycosides e.
  • Antibiotics such as vancomycin can be conjugated onto Ti substrate surfaces through silane/PEG linkers and can bind onto pathogen cells, such as S. aureus and Staphylococcus aureus , to inhibit transpeptidation and glycosylation on the cell wall.
  • pathogen cells such as S. aureus and Staphylococcus aureus
  • the ligands include Gram-negative or Gram-positive peptides.
  • the relative affinity of the microbe to the Gram-negative or Gram-positive peptides can be used to determine whether the microbe is Gram-negative or Gram -positive. This information can further enable a physician to select an appropriate treatment for the infection caused by the microbe, even if the microbe is not specifically identified.
  • the present technology includes other ligands not explicitly discussed herein, as well as ligands described herein but prepared according to different techniques. Accordingly, the present technology is not limited by the foregoing embodiments.
  • FIG. 17 is a flowchart illustrating a method 1700 of fabricating microfluidic devices in accordance with the present technology.
  • Method 1700 includes fabricating inlets and outlets onto a first optically transparent plastic slide in step 1702.
  • the first optically transparent plastic slide can be combined with a second optically transparent plastic slide to form the general body and cover of the microfluidic device.
  • the surfaces of the plastic slides defining the channel are activated in step 1704.
  • Fabrication continues by cutting slots for channels in step 1706. In embodiments where the channels are cut directly into the plastic substrate, the slots can be cut before the plastic is activated.
  • the PSA tape can be arranged on the activated plastic substrate to form the one or more channels.
  • a reactive polymer is grafted onto the channels.
  • the reactive polymer can bind to surface reactive groups on the surface activated slides.
  • ligands are bound to the reactive polymer, and the device is ready for operation. If not fabricated for immediate use, the device can be stored in an aluminum pouch in the freezer to prevent degradation.
  • FIG 18 is a schematic overview of the workflow described in method 1700.
  • Fabrication begins with a native plastic substrate 102.
  • surface reactive groups 140 are bound to the surface.
  • the surfaces of the plastic substrate are exposed to an aqueous solution of an oxidant under inert atmosphere at elevated temperature with constant stirring for 2 to 4 hours.
  • the surfaces are then thoroughly rinsed with deionized water.
  • the progress of activation is monitored by measuring the water contact angel (WCA).
  • WCA water contact angel
  • the target WCA is ⁇ 40 degrees.
  • Additional analytical techniques such as XPS, can be employed to verify the surface density of the surface reactive groups 140.
  • the activated surface is exposed to a solution of the reactive polymer 130, synthesized separately, in an organic solvent in the presence of a catalyst.
  • the grafting takes 4 to 8 hours depending on the surface activity and the concentration of the reactive polymer 130.
  • the surfaces are then rinsed thoroughly with the same organic solvent and then water.
  • the progress of grafting can be monitored by measuring WCA.
  • the target WCA is >75 degrees.
  • step 1710 To conjugate the ligands onto the polymer scaffold (step 1710), disaccharides or other ligand pre-cursors are immobilized by reductive amination with mono-protected diamines, followed by deprotection and subsequent attachment to the reactive polymer 130 by amide bond formation, thereby generating the affinity ligand 135.
  • Dynal Dynabeads M- 270 manufactured by ThermoFisher
  • amine groups can be suspended and coated with a 3 : 1 copolymer of dimethyl acrylamide (DMA) and N-hydroxysuccinimidyl acrylate (NHS-acrylate) in aqueous buffered acetonitrile solution (pH 8). After allowing time for polymer attachment to microspheres (via amide bond formation), excess polymer in solution can be aspirated away from magnetically isolated beads.
  • DMA dimethyl acrylamide
  • NHS-acrylate N-hydroxysuccinimidyl acrylate
  • the beads can be briefly washed and then resuspended and dispersed in a buffered solution of amino-saccharide for 2 hours, then again magnetically isolated and excess amino-saccharide aspirated. After a second washing of the beads, any remaining unreacted NHS- ester groups on the surface can be capped with a solution of aqueous dimethylamine. After a final wash, the saccharide-functionalized microspheres can be stored in pH 7 buffer at 4°C until time of use.
  • the entire surface can be immersed in ammonia solution (50 mM in aqueous ethanol, pH 10) to cap any remaining reactive pentafluorophenyl ester groups and to maximize the hydrophilicity of the surface for enhanced performance in a microfluidic device.
  • ammonia solution 50 mM in aqueous ethanol, pH 10
  • preparing microfluidic devices as described herein includes activating the surface of the substrate, grafting a polymer onto the activated surface, and conjugating a ligand to the immobilized polymer.
  • Activating the substrate surface enables the surface to bind and retain the polymer.
  • the surfaces are activated by adding oxygen. Once oxygenated, reactive polymers are added to the substrate and immobilized on the oxygenated surface. Once immobilized, ligands are added and bind to the polymer.
  • the ligand polymer network forms the capture material configured to bind and selectively retain various microbes.
  • the surface of a silicon wafer or glass substrate is etched by exposing the surface to a solution of 1 : 1 :5 v/v of NH 4 OH (29%), H2O2 (30%) and water at 60 °C for 10 minutes.
  • the substrate surface is rinsed with water and then hydroxylated by exposing the surface to a solution of 1 : 1 :6 v/v of hydrochloric acid (37%), H2O2 (30%), and water at 60 °C for 10 minutes.
  • the surface is then rinsed with water and blow-dried with nitrogen.
  • the activate surfaces are used for immediate grafting.
  • the siliceous substrate surface is activated by aminosilylation.
  • aminosilylation immediately after the hydroxylation, the surface is exposed to a 2% solution of triethoxysilylpropylamine, (EtO)3Si(CH2)3NH2 in anhydrous acetonitrile for 30 minutes with constant stirring.
  • the aminosilylated surface is rinsed with acetonitrile, blow-dried, and heated in a convection oven at 50 °C for 5 minutes.
  • the aminated surfaces are used for immediate grafting.
  • the activated surface is exposed to a solution of 85 mg of the grafting polymer and 16 pL of benzyldimethylamine in 25 mL of anhydrous acetonitrile. It is tumbled in a rotisserie at ambient temperature for 18 hours. The polymer-grafted surface is rinsed with plenty of anhydrous acetonitrile and blow-dried with nitrogen.
  • the reactive polymers described herein can be grafted onto a variety of plastic substrate surfaces for incorporation into microfluidic devices.
  • the plastic surfaces can include one or more of polystyrene, poly(methyl methacrylate), Nylons, polyolefins, poly(ethylene terephalate (PET), polymers of cyclic olefins (COP), copolymers of cyclic olefins (COC), and the like.
  • Polyolefins, COC, COP, and other plastic substrates can be hydroxylated by oxygen plasma or by exposing the plastic substrate to a solution of ammonium persulfate (APS) under constant nitrogen bubbling at an elevated temperature.
  • APS ammonium persulfate
  • the hydroxylated surface is exposed to a solution of about 85 mg of the grafting polymer and about 16 pL of benzyldimethylamine in about 25 mL of anhydrous acetonitrile.
  • the plastic substrate is tumbled in a rotisserie at ambient temperature for 18 hours.
  • the polymer-grafted surface is rinsed with anhydrous acetonitrile and blow-dried with nitrogen.
  • amino and hydroxyl groups are introduced onto the surface of a plastic substrate by various chemical means. The amino and hydroxyl groups can be used to bind the grafting polymer.
  • the concentration of polymer added to the activated surface is higher than the critical entanglement concentration at which the polymer chains start to interpenetrate to form a polymer scaffold.
  • the polymer-grafted surface is exposed to a solution of 2.0 mg of the biomolecule and 5 pL of BzDMA in 5.0 mL of dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) for 18 hours at ambient temperature with constant tumbling in a rotisserie.
  • the conjugated surface is then rinsed with plenty of buffer.
  • the bioconjugated surface is stored under buffer at 0 to 4 °C.
  • Figure 19 illustrates attaching a ligand to the reactive polymer network using complimentary oligonucleotides.
  • the activated, hydroxylated surface is generated in reaction I by exposing the plastic surface to an aqueous solution of an oxidant under inert atmosphere at elevated temperature with constant stirring for 2 to 4 hours. The surfaces are then thoroughly rinsed with deionized water. The progress of activation is monitored by measuring the water contact angel (WCA). The target WCA is ⁇ 40 degrees. Additional analytical techniques, such as XPS, can be employed to verify the surface density of the surface reactive groups.
  • the activated surface is exposed to a solution of the reactive polymer in an organic solvent in the presence of a catalyst (e.g., BzDMA).
  • a catalyst e.g., BzDMA
  • the grafting can take 4 to 8 hours depending on the surface activity and the concentration of the reactive polymer.
  • the surfaces are then rinsed thoroughly with the same organic solvent and then water.
  • the progress of grafting can be monitored by measuring WCA.
  • the target WCA is >75 degrees.
  • reaction II amine containing oligonucleotides are immobilized by reductive amination with mono-protected diamines, followed by deprotection and subsequent attachment to reactive polymer surface by amide bond formation thereby generating the oligo complement attached to the copolymer.
  • Reaction III illustrates the chemistry in the preparation of a ligand-conjugated oligo. Saccharides or other ligands are attached to amino-oligos in a manner analogous to attachment of diamine linkers. For example, using mannobiose, the sugar is oxidized at the reducing end with sodium periodate. After destroying excess oxidant the resulting bis-aldehyde is reductively aminated with amino-oligo in the presence of sodium cyanoborohydride, then the oligo-saccharide hybrid is purified by reverse phase chromatography.
  • Reaction IV illustrates the hybridization of ligand-oligos on the grafted surface. Multiple spots of nanodrops may be made to make the desired size section of ligand.
  • the present technology further includes a sample preparation device.
  • Figures 20A and 20B illustrate an embodiment of a sample preparation device in a sample collection stage and a sample delivery stage, respectively.
  • a chamber 202 with a prefilter 204 and 0.45 um filter 206 is attached to a first valve 1 220a.
  • a 0.22 um filter 208, a first wash 230a, and channels conjugated with affinity ligands are also attached to the first valve 220a.
  • the 0.22 um filter 208 is also attached to a second valve 220b.
  • the second valve 220b is also connected to a second wash 230b and waste 240.
  • the first wash 230a is connected to a first pump 2l5a
  • the second wash 230b is connected to a second pump 2l5b
  • the waste 240 is connected to a vacuum 245.
  • the chamber 202 has a plunger (not shown).
  • the 0.45 um filter 206 can be other sizes suitable for permitting microbes to pass therethrough.
  • the 0.22 um filter 208 can be other sizes suitable to prevent microbes to pass therethrough. Using the dual filtering enable microbes to be washed and retained in a second chamber 225.
  • a sample containing the microbes 150 is loaded into the chamber 202.
  • the first valve 220a is open to the 0.22 um filter 208 and the second valve 220b is open to the waste 240.
  • the vacuum 245 is activated, and the sample is drawn through the filters.
  • the first wash 230a with stain is flowed into the second chamber 225 for retained microbes and the retained microbes 150 are stained. Following staining, flow of the second wash 230b is initiated to deliver the stained microbes 150 to the channels for affinity testing.
  • a plunger (not shown) is used to assist in filtering
  • the sample is loaded into the chamber 202.
  • the plunger is added to the chamber 202.
  • the first valve 220a is open to the 0.22 um filter 208 and the second valve 220b is open to the waste.
  • the plunger is pushed down and vacuum is applied via vacuum 245 to push the sample containing the microbes through the filters.
  • the filtered microbes 150 are retained in the second chamber 225.
  • the second valve 220b is opened to the waste and the first wash 230a with stain is delivered to the second chamber 225.
  • the fluorescent stain can be a SYTO dye (Molecular Probes), although other stains suitable for marking microbes can be sued.
  • the first valve 220a is opened to the second chamber 225 and the channels and the second valve 220b is opened for flow of the second wash 230b through the 0.22 um filter 208 and chamber 225 and into the channels, thereby pushing the washed microbes 150 into the channel.
  • FIG. 21A-B illustrate a first embodiment of recovering microbes when a plurality of microbes l50a-c are bound to a plurality of capture materials l lOa-c.
  • the capture materials l lOa-c include a polymer 130 linked to a first oligonucleotide and respective ligands l35a-c linked to a second oligonucleotide.
  • the first and second oligonucleotides are hybridized, thus retaining the ligands l35a-c to the polymer 130 in distinct zones.
  • the first and second oligonucleotides can have a restriction enzyme cutting site 132.
  • the restriction enzyme cutting site 132 for each capture material l lOa-c is the same.
  • a restriction enzyme 133 e.g., BamHl
  • An elution buffer can be flowed through the channel and the microbes l50a- c are freed from the capture materials l lOa-c ( Figure 21B).
  • Figure 21C illustrates another embodiment where the first and second oligonucleotides in each respective capture material l lOa-c are configured to be cut by different enzymes.
  • capture material 1 lOa includes a first enzyme cutting site l32a to be cut by a first enzyme (e.g., EcoRL).
  • Capture material l lOb includes a second enzyme cutting site l32b to be cut by a second enzyme (e.g., BamHl).
  • Capture material 1 lOc includes a third enzyme cutting site l32c to be cut by a third enzyme (e.g., Sal 1).
  • the microbes retained by capture materials 1 lOa-c can be selectively released by selecting the appropriate restriction enzyme.
  • the devices can be used to sort microbes.
  • the restriction site is only on the oligonucleotide attached to the ligand 135.
  • an elution buffer containing a polymerase, dNTP, and Mg++ is added to extend the target oligonucleotide attached to the polymer 130, and the restriction enzyme needed to elute the microbes bound to the specific ligand is added to the channels. The channel is incubated for the proper time and temperature. After oligonucleotide extension and digestion, the cell suspension is collected.
  • microfluidic devices described herein are also useful in a number of other applications. As one skilled in the art will appreciate, the following applications can utilize many of the above-recited features of the microfluidic devices.
  • microbiome field is growing exponentially. However, only 10% of the sequence data is from microbes that researchers are interested in. Sequence from low abundance microbes is difficult to obtain, and more functional studies are needed to understand the role of certain bacterial species.
  • researchers need a cheap and robust technology to enrich for microbes they want to study; the present technology provides devices for enrichment of microbes.
  • many of the ligands described herein are unique in the field of microbial enrichment because they are polyvalent and synthetic or semi-synthetic. Thus, the ligands can be manufactured at scale, and enrichment of microbes can occur quickly under a wide range of conditions.
  • a microfluidic device having one channel such as the device illustrated in Figure 1 is used to enrich microbes .
  • the captured microbes are eluted after capture by increasing the salt of the buffer to facilitate elution or by using a cleavable capture molecule such as the immobilized ligands described above with respect to Figures 21A-C.
  • the capture material could have capture materials coupled to the channel with oligonucleotides as described in Figure 19 and the microbes eluted by a nuclease as illustrated in Figure 21A.
  • the channel could have a plurality of distinct capture materials coupled to the channel with oligonucleotides and arranged in a series.
  • Capture materials with affinity to unwanted microbes could be closer to the inlet and the capture materials with affinity to the desired microbe at the end of the series. After flowing the sample through and washing the desired microbes could be released by using a restriction enzyme as illustrated in Figure 21B. After elution, the microbes can be sequenced directly or cultured and then sequenced.
  • a microfluidic device having multiple channels with the same ligand can be captured and eluted under the same or similar conditions.
  • the capture and elution can be similar to that described above with respect enriching microbes.
  • FMT sometimes referred to as stool transplant
  • a stool sample is obtained from healthy donors.
  • the present technology could make FMT more safe and robust by enriching or depleting desired microbes.
  • affinity ligands to desired bacteria can be immobilized in the channel. The microbes can be captured and eluted as described herein.
  • affinity ligands for the microbe could be immobilized on the channel and the flow-through of the channel collected, such that any undesired microbes are retained in the device, while the desired microbes flow through the device to the outlet. 4. Enrich Clostridium difficile for DNA sequencing
  • Clostridium difficile The diagnostic market for Clostridium difficile is $1.6 billion. By selectively binding Clostridium difficile with the affinity ligands, the present technology could be used in epidemiological studies and diagnostic testing.
  • the sample preparation device in Figure 20 can be used to isolate microbes from stool. Clostridium difficile can then be isolated using the microfluidic devices as described herein.
  • the present technology can be used to assist MALDI-TOF or other methods, such as Fourier Transform-Infrared Spectroscopy, in identifying microbes.
  • mixed samples can be separated using the present technology.
  • microbes are bound, eluted, and transferred to another device for detection by an additional method.
  • the microfluidic devices described herein are used to both enrich microbes and add additional information to assist MALDI-TOF or other methods in identifying strains.
  • a microfluidic device for identifying one or more properties of a microbe comprising:
  • the substrate includes:
  • one or more channels having immobilized capture material configured to selectively bind microbes
  • an inlet port configured to provide access to the one or more channels; wherein, when a solution containing an unknown microbe is released onto the device at the inlet—
  • a relative binding affinity between the immobilized capture material and the unknown microbe is determined based at least in part on whether the immobilized capture materials captured and retained the unknown microbe
  • the relative binding affinity is used to identify one or more properties of the unknown microbe.
  • the immobilized capture material comprises a polymer immobilized on the substrate and a ligand bound to the polymer.
  • the ligand includes a first oligonucleotide linker having a first nucleotide sequence
  • the polymer includes a second oligonucleotide linker having a second nucleotide sequence complimentary to the first nucleotide sequence
  • the ligand is bound to the polymer via hybridization of the first oligonucleotide linker to the second oligonucleotide linker.
  • the one or more channels comprise at least four individual channels arranged in parallel, and wherein each of the at least four individual channels includes a different ligand.
  • the one or more channels comprise at least one channel having a plurality of ligands, and wherein the plurality of ligands are arranged in a series in discrete capture zones.
  • a body portion housing the one or more channels
  • a cover portion configured to mate with the body portion and enclose the one or more channels, wherein the cover portion includes the inlet port.
  • a method of identifying one or more properties of a microbe comprising: releasing a solution containing a fluorescently stained unidentified microbe into the inlet of a microfluidic device, wherein the microfluidic device includes at least one channel operably coupled to the inlet, and wherein the channel includes immobilized ligands that selectively bind specific microbes;
  • identifying one or more properties of the microbe 17.
  • the method of example 16 further comprising comparing the relative binding affinity to a database of binding affinities for known microbes and the ligands.
  • ligand is a saccharide, saccharide derivative, antibiotic, antibiotic derivative, peptide, peptide derivative, or aptamer.
  • identifying the one or more properties of the microbe comprises identifying the Gram- stain of the unidentified microbe.
  • identifying the one or more properties of the microbe comprises identifying the Gram- stain of the unidentified microbe.
  • identifying the one or more properties of the microbe comprises identifying the susceptibility of the microbe to the antibiotic. 26. The method of any of examples 16-25 wherein the one or more properties are identified within 15 minutes of releasing the solution into the device.
  • a method of fabricating a microfluidic device comprising:
  • activating the surface of one or more channels comprises treating the surface with oxygen plasma.
  • the ligand includes a first oligonucleotide linker having a first nucleotide sequence
  • the polymer includes a second oligonucleotide linker having a second nucleotide sequence complimentary to the first nucleotide sequence
  • conjugating the ligand to the reactive polymer comprises hybridizing the first oligonucleotide linker to the second oligonucleotide linker.

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Abstract

La présente technologie concerne des dispositifs microfluidiques qui identifient une ou plusieurs propriétés d'un microbe inconnu. Les dispositifs microfluidiques comprennent un ou plusieurs canaux qui présentent des ligands immobilisés. Ledit un ou lesdits plusieurs canaux est/sont traversé(s) par une solution contenant un microbe coloré inconnu. Ledit un ou lesdits plusieurs canaux est/sont représenté(s) en image et, sur la base de l'affinité de liaison du microbe inconnu pour les ligands immobilisés, une ou plusieurs propriété(s) du microbe inconnu est/sont déterminée(s).
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