Preparation of output sample including defined concentration of infectious agent for downstream testing

Abstract

To provide preparation of a diagnostic sample.SOLUTION: An output sample can be used for downstream tests such as downstream anti-infective or antibiotic susceptibility testing (AST). The method can include: diluting an aliquot of a source sample including an infectious agent to yield a diluted sample; exposing one or more sensors to the diluted sample, wherein at least part of each of the one or more sensors is in fluid communication with the diluted sample; incubating the diluted sample at an incubation temperature; monitoring a change in the solution characteristics of the diluted sample by using a parameter analyzer or a computing device coupled to the one or more sensors; and cooling the diluted sample to a cooling temperature when the solution characteristics of the diluted sample change by a threshold amount to yield the output sample of the defined concentration.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 597,657, filed December 12, 2017, and U.S. Provisional Patent Application No. 62 / 594,838, filed December 5, 2017, which are incorporated by reference in their entireties herein.

[0002] Technical Field

[0002] The present disclosure relates generally to diagnostic sample preparation, and more specifically to devices, systems, and methods for preparing an output sample containing a defined concentration of an infectious agent for downstream testing. [Background technology]

[0003] background

[0003] Infections caused by anti-infective resistant microorganisms or infectious agents are a significant problem for health care professionals in hospitals, nursing homes, and other health care environments. Rapid detection of the susceptibility of such infectious agents to antibiotics or other anti-infectives is crucial to prevent the spread of their resistance profile. Although new techniques (e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), rapid polymerase chain reaction (rapid PCR), etc.) have been developed to identify infectious agents in samples such as positive blood cultures, the first step of most testing protocols still involves the preparation of an output sample containing a defined concentration of the infectious agent. For example, most anti-infective or antibiotic susceptibility testing (AST) protocols require the preparation of an output sample or inoculum with a concentration matching the McFarland standard.

[0004]

[0004] Existing methods and equipment used to prepare such output samples include costly, time-consuming (e.g., up to 24 hours), and labor-intensive microbial culture techniques. However, such methods often require manual interpretation by skilled personnel and are prone to technical or clinician errors. In addition, certain biological samples that carry infectious agents, such as samples containing animal or human blood, are often difficult to evaluate using current optical techniques given the opacity of the samples. Moreover, such optical techniques often require bulky and expensive equipment. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] As a result of the above limitations and restrictions, a need exists for improved devices, systems, and methods for quickly and effectively preparing an output sample or standardized inoculum containing a defined concentration of an infectious agent for downstream testing. [Means for solving the problem]

[0006] overview

[0006] Various methods, devices, and systems for preparing an output sample at a defined concentration are disclosed. In one embodiment, a method for preparing an output sample at a defined concentration is disclosed. The method includes diluting an aliquot of a source sample containing an infectious agent at a dilution factor to generate a diluted sample, and exposing one or more sensors to the diluted sample. At least a portion of each of the one or more sensors is capable of fluid communication with the diluted sample when exposed to the diluted sample. The method may further include incubating the diluted sample at an incubation temperature. The diluted sample is capable of incubation when the one or more sensors are exposed to the diluted sample. The incubation temperature may be from about 33°C to about 37°C.

[0007] The method may also include monitoring a change in a solution property of the diluted sample using a parameter analyzer or computing device coupled to the one or more sensors. The method may further include cooling the diluted sample to a cooling temperature when the solution property of the diluted sample changes by a threshold amount to generate an output sample of a defined concentration. In some embodiments, the cooling temperature may be from about 4° C. to about 25° C.

[0008]

[0008] The method may also include, prior to monitoring the change in solution properties of the diluted sample, retrieving a universal lookup table from a database using one or more processors of a computing device coupled to the one or more sensors. The one or more processors of the computing device may set a threshold amount based on a defined concentration, concentration data obtained from the universal lookup table, and solution property data obtained from the universal lookup table. The universal lookup table may be created from a plurality of strain-specific lookup tables representing data measured from a plurality of reference samples monitored over time. At least one of the plurality of reference samples may include a reference infectious agent of a different species than the infectious agent in the original sample.

[0009]

[0009] In some embodiments, each of the plurality of strain-specific lookup tables can be created by monitoring the change in solution property of the reference sample over a time range, performing a sample enumeration assay of the reference sample over the same time range, converting the result of the sample enumeration assay to a reference sample concentration using a conversion factor, and relating the reference sample concentration to the change in solution property of the reference sample. The universal lookup table can be created by averaging all solution property changes from the plurality of strain-specific lookup tables for each reference sample concentration, and relating each reference sample concentration to the averaged solution property change. The sample enumeration assay can include an optical density measurement, a plate count assay, a flow cytometry assay, or a combination thereof.

[0010]

[0010] The method may further include retrieving a species-specific lookup table from a database based on the species of the infectious agent in the original sample before monitoring the change in solution properties of the diluted sample, and setting a threshold amount based on a defined concentration, concentration data obtained from the species-specific lookup table, and solution property data obtained from the species-specific lookup table. The species-specific lookup table can be generated from a plurality of strain-specific lookup tables representing data obtained from a plurality of reference samples monitored over time. Each of the plurality of reference samples may include a reference infectious agent of the same species as the infectious agent in the original sample.

[0011]

[0011] Each of the plurality of strain-specific lookup tables can be created by monitoring the change in solution property of the reference sample over a time range, performing a sample enumeration assay of the reference sample over the same time range, converting the result of the sample enumeration assay to a reference sample concentration using a conversion factor, and relating the reference sample concentration to the change in solution property of the reference sample. The species-specific lookup table can be created by averaging all solution property changes from the plurality of strain-specific lookup tables for each of the reference sample concentrations, and relating each of the reference sample concentrations to the averaged solution property change. In these embodiments, the sample enumeration assay can include an optical density measurement, a plate count assay, a flow cytometry assay, or a combination thereof.

[0012]

[0012] The method may further include diluting the output sample at another dilution factor to produce a further diluted sample, which may contain a concentration of the infectious agent required for downstream testing.

[0013] In some embodiments, the solution property can be oxidation-reduction potential (ORP) and the one or more sensors can be ORP sensors. The ORP can be monitored without the presence of any added reporter molecule in the diluted sample. Each of the one or more ORP sensors can include a redox active layer. Each of the one or more ORP sensors can include at least one of an active electrode and a reference electrode. In some embodiments, the redox active layer can include a gold layer, a platinum layer, a metal oxide layer, a carbon layer, or a combination thereof.

[0014]

[0014] In other embodiments, the solution property can be pH and the one or more sensors can be pH sensors. Each of the one or more pH sensors can include a pH sensitive layer. The pH can be monitored without the presence of any added reporter molecule in the diluted sample. Each of the one or more pH sensors can include at least one of an active electrode and a reference electrode. In some embodiments, the pH sensitive layer can include an oxide layer, a silane layer, a self-assembled mono layer (SAM), a hydrogel layer, a protein layer, a polymer layer, or a combination thereof.

[0015]

[0015] The raw sample may include body fluids, wound swabs or samples, rectal swabs or samples, other types of biological samples, cultures derived therefrom, or combinations thereof. Body fluids may include urine, blood, sputum, saliva, breast milk, spinal fluid, semen, vaginal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, amniotic fluid, cultures of body fluids that have tested positive for growth of an infectious agent, or combinations thereof. Infectious agents may include bacteria, fungi, molds, or combinations thereof.

[0016]

[0016] In another embodiment, a system for preparing an output sample of a defined concentration is disclosed. The system may include one or more fluid delivery or metering conduits configured to dilute an aliquot of an original sample containing an infectious agent at a dilution factor to generate a diluted sample. The system may also include one or more sensors. In some embodiments, the diluted sample is deliverable or otherwise introduceable to the one or more sensors. In other embodiments, the one or more sensors are exposed to the diluted sample by being positioned in fluid communication with the diluted sample.

[0017]

[0017] The system may further include an incubation component configured to incubate the diluted sample at an incubation temperature. The diluted sample may be incubated when the one or more sensors are exposed to the diluted sample. The incubation temperature may be about 33°C to about 37°C.

[0018] The system may also include at least one of a parameter analyzer and a computing device coupled to the one or more sensors, wherein the one or more processors of the parameter analyzer or computing device are capable of monitoring changes in solution properties of the diluted sample using the parameter analyzer or computing device coupled to the one or more sensors.

[0019] The system may further include a cooling component configured to cool the diluted sample to a cooling temperature when a solution property of the diluted sample changes by a threshold amount to generate an output sample of a predetermined concentration. In some embodiments, the cooling temperature may be from about 4° C. to about 25° C.

[0020]

[0020] The system may also include retrieving a universal lookup table from a database using one or more processors of a computing device coupled to one or more sensors before monitoring the change in solution properties of the diluted sample. The one or more processors of the computing device may set a threshold amount based on a defined concentration, concentration data obtained from the universal lookup table, and solution property data obtained from the universal lookup table. The universal lookup table may be created from a plurality of strain-specific lookup tables representing data measured from a plurality of reference samples monitored over time. At least one of the plurality of reference samples may include a reference infectious agent of a different species than the infectious agent in the original sample.

[0021] In some embodiments, each of the plurality of strain-specific lookup tables can be created by monitoring the change in solution property of the reference sample over a time range, performing a sample enumeration assay of the reference sample over the same time range, converting the result of the sample enumeration assay to a reference sample concentration using a conversion factor, and relating the reference sample concentration to the change in solution property of the reference sample. The universal lookup table can be created by one or more processors of the computing device by averaging all solution property changes from the plurality of strain-specific lookup tables for each reference sample concentration, and relating each reference sample concentration to the averaged solution property change. The sample enumeration assay can include an optical density measurement, a plate count assay, a flow cytometry assay, or a combination thereof.

[0022]

[0022] The one or more processors of the computing device can also retrieve a species-specific lookup table from the database based on the species of the infectious agent in the original sample before monitoring the change in solution properties of the diluted sample, and set the threshold amount based on the defined concentration, the concentration data obtained from the species-specific lookup table, and the solution property data obtained from the species-specific lookup table. The species-specific lookup table can be created from a plurality of strain-specific lookup tables representing data obtained from a plurality of reference samples monitored over time. Each of the plurality of reference samples can include a reference infectious agent of the same species as the infectious agent in the original sample.

[0023]

[0023] Each of the plurality of strain-specific lookup tables can be created by monitoring the change in solution property of the reference sample over a time range, performing a sample enumeration assay of the reference sample over the same time range, converting the result of the sample enumeration assay to a reference sample concentration using a conversion factor, and relating the reference sample concentration to the change in solution property of the reference sample. The species-specific lookup table can be created by averaging all solution property changes from the plurality of strain-specific lookup tables for each of the reference sample concentrations, and relating each of the reference sample concentrations to the averaged solution property change. In these embodiments, the sample enumeration assay can include an optical density measurement, a plate count assay, a flow cytometry assay, or a combination thereof.

[0024]

[0024] The system may further include diluting the output sample with one or more fluid delivery or metering conduits at other dilution factors to produce further diluted samples that may contain the infectious agent concentration required for downstream testing.

[0025] In some embodiments, the solution property can be oxidation-reduction potential (ORP) and the one or more sensors can be ORP sensors. The ORP can be monitored without the presence of any added reporter molecule in the diluted sample. Each of the one or more ORP sensors can include a redox active layer. Each of the one or more ORP sensors can include at least one of an active electrode and a reference electrode. In some embodiments, the redox active layer can include a gold layer, a platinum layer, a metal oxide layer, a carbon layer, or a combination thereof.

[0026]

[0026] In other embodiments, the solution property can be pH and the one or more sensors can be pH sensors. Each of the one or more pH sensors can include a pH-sensitive layer. The pH can be monitored without the presence of any added reporter molecule in the diluted sample. Each of the one or more pH sensors can include at least one of an active electrode and a reference electrode. In some embodiments, the pH-sensitive layer can include an oxide layer, a silane layer, a self-assembled monolayer (SAM), a hydrogel layer, a protein layer, a polymer layer, or a combination thereof.

[0027]

[0027] The raw sample may include body fluids, wound swabs or samples, rectal swabs or samples, other types of biological samples, cultures derived therefrom, or combinations thereof. Body fluids may include urine, blood, sputum, saliva, breast milk, spinal fluid, semen, vaginal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, amniotic fluid, cultures of body fluids that have tested positive for growth of an infectious agent, or combinations thereof. Infectious agents may include bacteria, fungi, molds, or combinations thereof. [Brief description of the drawings]

[0028] [Figure 1]

[0028] Certain steps of an example method for preparing an output sample for downstream testing are illustrated. [Diagram 2]

[0029] 1 illustrates additional steps of an example method for preparing an output sample for downstream testing. [Figure 3A]

[0030] 1 illustrates an example strain-specific lookup table used to generate a species-specific lookup table. [Figure 3B]

[0031] Illustrates pH growth curves and reference concentration curves of reference samples containing infectious agents of specific strains incubated over a range of time. [Figure 4A]

[0032] 1 illustrates one embodiment of a system for preparing an output sample for downstream testing. [Figure 4B]

[0033] 1 illustrates one embodiment of a test cartridge for use in certain devices and systems disclosed herein. [Figure 5A]

[0034] 1 illustrates a schematic diagram of one embodiment of a pH sensor for use as part of the methods and systems described herein. [Figure 5B]

[0035] 1 illustrates a schematic diagram of another embodiment of a pH sensor for use as part of the methods and systems described herein. [Figure 6A]

[0036] 1 illustrates a schematic diagram of one embodiment of an ORP sensor for use as part of the methods and systems described herein. [Figure 6B]

[0037] 1 illustrates a schematic diagram of another embodiment of an ORP sensor for use as part of the methods and systems described herein. [Figure 7A]

[0038] FIG. 1 illustrates a schematic diagram of one embodiment of a combination ORP·pH sensor for use as part of the methods and systems described herein. [Figure 7B]

[0039] FIG. 1 illustrates a schematic diagram of another embodiment of a combination ORP·pH sensor for use as part of the methods and systems described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description

[0040] The variations of the devices, systems, and methods described herein are best understood from the detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings may not be drawn to scale. To the contrary, dimensions of the various features may be arbitrarily expanded or reduced for clarity, and not all features may be seen or noted in all drawings. The drawings are included for illustrative purposes only and are not intended to define or limit the scope of the claims to those shown.

[0030]

[0041] 1 illustrates one embodiment of a method 100 for preparing an output sample 102 from an original sample 104 that includes an infectious agent 106. More specifically, the method 100 can provide an output sample 102 that includes a defined concentration 105 of the infectious agent 106.

[0031]

[0042] The raw sample 104 may include at least one of a biological sample, a body fluid, a wound swab or sample, a rectal swab or sample, and an infectious agent culture derived from a biological sample, a body fluid, a wound swab or sample, or a rectal swab or sample. The body fluid may include urine, blood, serum, plasma, saliva, sputum, semen, breast milk, joint fluid, spinal fluid, such as cerebrospinal fluid, wound material, mucus, fluid-associated feces, resuspended rectal or wound swab, vaginal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, amniotic fluid, an infectious agent or a body fluid or sample culture that has tested positive for infectious agent growth, such as a blood culture that has tested positive for infectious agent growth (i.e., a positive blood culture), or a combination thereof.

[0032]

[0043] The output sample 102 containing a defined concentration 105 of infectious agent 106 can be used as an inoculum for downstream testing, such as a downstream anti-infective or antibiotic susceptibility test (AST) to determine the susceptibility of the infectious agent 106 to one or more anti-infective agents or antibiotics.

[0033]

[0044] The infectious agent 106 that can be assayed using the methods or systems disclosed herein can be any metabolic unicellular or multicellular organism, including bacteria and fungi. In certain embodiments, the infectious agent 106 is selected from the group consisting of Acinetobacter, Acetobacter, Actinomyces, Aerococcus, Aeromonas, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, and the like. llus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Citrobacter Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella erella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Morganella,Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pandoraea, Pasteurella, Peptostreptococcus, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Ralstonia, Raoultella, Rhizobium, Rickettsia tsia, Rochalimaea, Rothia, Salmonella, Serratia, Shewanella, Shigella, Spirillum, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Treponema, Vibrio, Wolbachia, Yersinia, or combinations thereof. In other embodiments, the infectious agent 106 may be one or more fungi selected from the genera Candida or Cryptococcus or molds.

[0034]

[0045] Other specific bacteria that can be quantified using the methods and systems disclosed herein include Staphylococcus aureus, Staphylococcus lugdunensis, coagulase-negative Staphylococcus species (including, but not limited to, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, undifferentiated), Enterococcus faecalis, Enterococcus faecium ... faecium (including but not limited to Enterococcus faecium and other Enterococcus species, including undifferentiated species, excluding Enterococcus faecalis), Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus species (including but not limited to Streptococcus mitis, Streptococcus pyogenes, Streptococcus gallolyticus, Streptococcus faecium, Streptococcus faecalis, ... faecium, Streptococcus faecalis, Streptococcus faecium, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Streptococcus faecalis, Strept gallolyticus, Streptococcus agalactiae, Streptococcus pneumoniae, undifferentiated, Pseudomonas aeruginosaaeruginosa, Acinetobacter baumannii, Klebsiella spp. (including but not limited to Klebsiella pneumoniae, Klebsiella oxytoca, undifferentiated), Escherichia coli, Enterobacter spp. (including but not limited to Enterobacter cloacae, Enterobacter aerogenes, undifferentiated), Proteus spp. (including but not limited to Proteus mirabilis, Proteus vulgaris, Proteus spp. vulgaris, undifferentiated), Citrobacter species (including but not limited to Citrobacter freundii, Citrobacter koseri, undifferentiated), Serratia marcescens, Candida albicans, Candida glabrata, and Candida tropicalis.

[0035]

[0046] Other more specific bacteria that can be quantified include Acinetobacter baumannii, Actinobacillus species, Actinomycetes, Actinomyces species (including but not limited to Actinomyces israelii and Actinomyces naeslundii), Aeromonas species (including but not limited to Aeromonas hydrophila, Aeromonas veronii biovar sobria, and Aeromonas kaviae). caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus species (including, but not limited to, Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides species (including, but not limited to, Bacteroides fragilis, Bacteroides purpura, Bacteroides natto ... fragilis), Bartonella species (including but not limited to Bartonella bacilliformis and Bartonella henselae),Bifidobacterium species, including B. henselae; Bordetella species (including, but not limited to, Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica); Borrelia species (including, but not limited to, Borrelia recurrentis and Borrelia burgdorferi); Brucella species (including, but not limited to, Brucella abortus, Brucella canis, Brucella melitensis, and Brucella suis); suis), Burkholderia species (including, but not limited to, Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter species (including, but not limited to, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus), Capnocytophaga species, Cardiobacterium hominis, Chlamydia trachomatis, trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter species Coxiella burnetiiburnetii, Corynebacterium species (including, but not limited to, Corynebacterium diphtheriae, Corynebacterium jeikeum, and Corynebacterium), Clostridium species (including, but not limited to, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, and Clostridium tetani), Eikenella corrodens, Enterobacter species (including but not limited to, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, and Escherichia coli, e.g., opportunistic Escherichia coli, including but not limited to, enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and uropathogenic E. coli), Enterococcus species (including but not limited to, Enterococcus faecalis, faecalis and Enterococcus faecium), Ehrlichia species (including but not limited to Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiaerhusiopathiae, Eubacterium spp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus spp. (including but not limited to Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus) Helicobacter species (including, but not limited to, Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae), including Helicobacter parahaemolyticus, Kingella kingii, Klebsiella species (including, but not limited to, Klebsiella pneumoniae, Klebsiella granulomatis, and Klebsiella oxytoca), Lactobacillus species, Listeria monocytogenes, Leptospira interrogans, interrogans, Legionella pneumophila, Leptospira interrogansinterrogans, Peptostreptococcus spp., Moraxella catarrhalis, Morganella spp., Mobiluncus spp., Micrococcus spp., Mycobacterium spp. (including but not limited to, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum) marinum), Mycoplasma species (including, but not limited to, Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia species (including, but not limited to, Nocardia asteroides, Nocardia cyriacigeorgica, and Nocardia brasiliensis), Neisseria species (including, but not limited to, Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, multocida, Plesiomonas shigelloidesshigelloides, Prevotella spp., Porphyromonas spp., Prevotella melaninogenica, Proteus spp. (including, but not limited to, Proteus vulgaris and Proteus mirabilis), Providencia spp. (including, but not limited to, Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, acnes, Rhodococcus equi, Rickettsia species (including but not limited to Rickettsia rickettsii, Rickettsia akari, and Rickettsia prowazekii, Orientia tsutsugamushi (formerly Rickettsia tsutsugamushi, and Rickettsia typhi), Rhodococcus species, Serratia marcescens, Stenotrophomonas maltophilia, maltophilia, Salmonella species (including but not limited to Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis,enteritidis, Salmonella choleraesuis, and Salmonella typhimurium), Serratia species (including, but not limited to, Serratia marcesans and Serratia liquifaciens), Shigella species (including, but not limited to, Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Staphylococcus species (including, but not limited to, Staphylococcus aureus, Staphylococcus ketone, Staphylococcus spp., and Staphylococcus spp. aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus), Streptococcus species (including, but not limited to, Streptococcus pneumoniae (e.g., chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes pyogenes, Group B Streptococcus, Streptococcus agalactiae, Group C Streptococcus, Streptococcus anginosus, Streptococcus equismilis, Group D Streptococcus, Streptococcus bovisbovis, Group F Streptococcus, and Streptococcus anginosus (Group G Streptococcus), Spirillum minus, Streptobacillus moniliformi, Treponema species (including but not limited to Treponema carateum, Treponema petenue, Treponema pallidum, and Treponema endemicum), Tropheryma whippelii, Ureaplasma urealyticum, urealyticum, Veillonella spp., Vibrio spp. (including but not limited to, Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damselae, Vibrio damsela, and Vibrio furnisii), Yersinia species (including, but not limited to, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis),In particular, these may include Bacillus subtilis, Bacillus anguinea pigs, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus pseudotuberculosis, and Xanthomonas maltophilia.

[0036]

[0047] Additionally, other infectious agents 106 that may be quantified include, but are not limited to, Candida species (including, but not limited to, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), Aspergillus species (including, but not limited to, Aspergillus fumigatous, Aspergillus flavus, and Aspergillus clavatus), Cryptococcus species (including, but not limited to, Cryptococcus neoformans, Cryptococcus cereus, and Cryptococcus cereus), and / or other infectious agents that may be quantified include, but are not limited to, Candida species (including, but not limited to, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), Aspergillus species (including, but not limited to, Aspergillus fumigatous, Aspergillus flavus, and Aspergillus clavatus), Cryptococcus species (including, but not limited to, Cryptococcus neoformans, Cryptococcus gatti, Cryptococcus laurentii, and Cryptococcus albidus, Fusarium species (including, but not limited to, Fusarium oxysporum, Fusarium solani, Fusarium verticillioides, and Fusarium proliferatum), Rhizopus oryzae, Penicillium marneffei, Coccidiodes immitis, The fungi may include fungi or molds, including Bacillus subtilis, Bacillus anguicus, Bacillus subtilis ...

[0037]

[0048] The method 100 may include, in step 1A, introducing an aliquot of the raw sample 104 into a reaction vessel 108. The reaction vessel 108 may refer to one or more test tubes, reaction tubes, wells of a high throughput assay plate or well plate, e.g., a 96-well plate, a 192-well plate, or a 384-well plate, a culture plate or dish, a microfluidic conduit, or other container suitable for containing a biological sample.

[0038]

[0049] In other embodiments not shown in FIG. 1 , a stimulus solution can be added to the raw sample 104 before metering an aliquot of the raw sample 104 into the reaction vessel 108. The stimulus solution can be a nutrient solution or a growth solution. In these and other embodiments, the raw sample 104 can also be filtered prior to step 1A. This filtering step can include filtering the raw sample 104 using an instance of a filter, a microfluidic filter, or a combination thereof to filter out debris, inorganic matter, and larger cellular components, including blood cells or epithelial cells, derived from the raw sample 104.

[0039]

[0050] One or more fluid delivery conduits 110 can inject, deliver, or introduce aliquots of the original sample 104 into the reaction vessel 108. The fluid delivery conduits 110 can include tubes, pumps, containers, or microfluidic channels that deliver buffers, reagents, fluid samples including the original sample 104, or combinations thereof, to and between devices, apparatus, or containers within the system. For example, as shown in FIG. 1, the fluid delivery conduit 110 can represent a portion of a pump, such as a syringe pump. In other embodiments, the fluid delivery conduit 110 can include or represent at least a portion of a hydraulic pump, a pneumatic pump, a peristaltic pump, a vacuum pump or a positive pressure pump, a manual or mechanical pump, or combinations thereof. In other embodiments, the fluid delivery conduit 110 can include or represent at least a portion of an injection cartridge, a pipette, a capillary, a dispenser bottle, or combinations thereof. The fluid delivery conduit 110 may also be part of a vacuum system configured to draw fluid into or through a channel, tube, or passageway under vacuum. Additionally, the fluid delivery conduit 110 may include or represent at least a portion of a multi-channel delivery system or pipette.

[0040]

[0051] The method 100 may include diluting an aliquot of the original sample 104 in step 1B. For example, an aliquot of the original sample 104 may be diluted at a dilution factor or ratio to generate a diluted sample 112. The dilution factor may be from about 1:1 to about 1:10. The dilution factor may also be from about 1:10 to about 1:100. In some embodiments, the dilution factor may be from about 1:100 to about 1:10. 3 In other embodiments, the dilution ratio may be about 1:10. 3 ~about 1:10 7 In a further embodiment, the dilution factor may be 1:10 7 It could be super.

[0041]

[0052] An aliquot of the raw sample 104 may be diluted with a diluent solution 114. In one embodiment, the diluent solution 114 may include a growth medium or a growth inducer. In these and other embodiments, the diluent solution 114 may be a solution containing bacto-tryptone, soybean tryptic digest, yeast extract, beef extract, cation-adjusted Mueller Hinton Broth (CAMHB), Mueller Hinton Growth Medium (MHG), starch, acid hydrolysate of casein, calcium chloride, magnesium chloride, sodium chloride, blood or lysed blood (including lysed horse blood (LHB)), CAMHB-LHB, glucose or other carbohydrates, or combinations thereof. The growth inducer may include a carbon-based inducer, a nitrogen-based inducer, a mineral, a trace element, a biological growth factor, or any combination thereof. For example, the growth inducer may include, but is not limited to, a carbohydrate such as glucose or starch, ammonia, magnesium, an amino acid, a casamino acid, a vitamin, a peptide, blood, or a combination thereof. In one example embodiment, the diluent solution 114 may include tryptone, yeast extract, sodium chloride, starch, water and glucose.

[0042]

[0053] 1 illustrates one aliquot of original sample 104 being diluted in step 1B, it is contemplated by the present disclosure that additional aliquots of original sample 104 can be diluted to the same or different dilution ratios to generate additional diluted samples (e.g., a second diluted sample, a third diluted sample, a fourth diluted sample, etc.) The additional diluted samples can be used to create internal controls or duplicate samples.

[0043]

[0054] The method 100 may further include the optional step of identifying the species or other taxonomic type or characteristic of the infectious agent 106 in the original sample 104 in step 1C. In addition to species, other taxonomic types may include the genus, family, order, class, phylum, kingdom, and domain of the infectious agent 106 in the original sample 104.

[0044]

[0055] In some embodiments, identifying the species or other taxonomic type of the infectious agent 106 may include receiving such information from a user via an input device (e.g., a keyboard or touch screen) coupled to the computing device 116. In other embodiments, identifying the species or other taxonomic type of the infectious agent 106 may include receiving such information from another computing device communicatively coupled to the computing device 116 or retrieving such information from a database. The taxonomic type (e.g., species, genus, family, etc.) or characteristics of the infectious agent 106 may be stored in a memory of the computing device 116, a computing cloud, or a remote server accessible to the computing device 116 over a network.

[0045]

[0056] In some embodiments, identifying the species of the infectious agent 106 in the raw sample 104 may include determining the species 106 using a biochemical test (e.g., a metabolic test or a specific enzyme test), mass spectrometry, genotyping, phenotyping by culture plate, a test kit including phage, or a combination thereof. In some embodiments, the characteristic of the infectious agent 106 may be the response of the infectious agent 106 to a Gram staining test. For example, step 1C may include performing a Gram staining test to identify the infectious agent 106 as a Gram-positive or Gram-negative bacterium.

[0046]

[0057] In certain embodiments, the species of the infectious agent 106 in the original sample 104 may be identifiable, but the particular strain of the infectious agent 106 may remain unknown. In other embodiments, the taxonomic type or characteristics (e.g., species or gram type) of the infectious agent 106 in the original sample 104 need not be identified prior to proceeding to other steps of the method 100.

[0047]

[0058] The method 100 may further include selecting and retrieving a look-up table (LUT) from a database using the computing device 116 or other device in step 1D. The LUT can be selected based on information about the taxonomic type or characteristics of the infectious agent 106 in the original sample 104, or the lack of such information. For example, a species-specific LUT 210 (see FIG. 2) for the bacterial species Serratia marcescens (SMa) can be selected and retrieved when the species of the infectious agent 106 in the original sample 104 is identified as SMa. Also, by way of example, a universal LUT 212 (see FIG. 2) can be selected and retrieved when the species of the infectious agent 106 in the original sample 104 is unidentified or unknown. As other examples, LUTs organized by genus, family, order, class, phylum, kingdom, or domain can also be selected and retrieved. Additionally, LUTs organized by microbial characteristics, such as gram type, or functional capabilities, such as the ability to hydrolyze a particular protein or molecule, can also be selected or retrieved.

[0048]

[0059] The LUTs can be stored as part of the database software program in memory of the computing device 116. In other embodiments, the LUTs can be stored as part of the database software program on a computing cloud or a remote server accessible to the computing device 116 on a network. The computing device 116, or one or more processors thereof, can search through hundreds or thousands of stored LUTs to select an appropriate LUT based on information regarding the classification type (e.g., species) or characteristics of the infectious agent 106 in the original sample 104.

[0049]

[0060] As discussed in more detail in the following sections, species-specific LUTs 210, universal LUTs 212, and other LUTs organized by classification or characteristics can be created from a plurality of strain-specific LUTs 204 (see FIG. 2) that represent data measured from a plurality of reference samples 208 (see FIG. 2) monitored over time. If the LUT is a species-specific LUT 210, each of the plurality of reference samples 208 can include a reference infectious agent 214 (see FIG. 2) of the same species as the infectious agent 106 in the original sample 104. If the LUT is a universal LUT 212 or other type of inter-species LUT, at least one of the plurality of reference samples 208 can include a reference infectious agent 214 of a different species than the infectious agent 106 in the original sample 104.

[0050]

[0061] The method 100 may further include setting a threshold amount 118 at step 1E using the computing device 116 or another device communicatively coupled to the computing device 116. The threshold amount 118 may represent a target amount by which the solution properties of the diluted sample 112 need to be changed so that the concentration of the infectious agent 106 in the diluted sample 112 reaches the specified concentration 105. The threshold amount 118 may also represent a limit or maximum amount by which the solution properties of the diluted sample 112 may be changed before the concentration of the infectious agent 106 in the diluted sample 112 exceeds the specified concentration 105 (e.g., a ΔpH of -0.20). In some embodiments, the threshold amount 118 may be a threshold range (e.g., a ΔpH of about -0.15 to -0.25).

[0051]

[0062] The threshold amount 118 can be set using a computing device 116 (or other device, such as a parameter analyzer 120) communicatively coupled to one or more sensors 122 used to monitor solution properties of the diluted sample 112. The threshold amount 118 can be set based on the nominal concentration 105, concentration data obtained from a retrieved LUT (e.g., species-specific LUT 210 or universal LUT 212), and solution property data obtained from a retrieved LUT (e.g., species-specific LUT 210 or universal LUT 212). For example, the nominal concentration 105 of the output sample 102 can be 3×108 It can be set to colony forming units per milliliter (CFU / mL) or 3e8 CFU / mL. Also, for example, the nominal concentration 105 of the output sample 102 can be set to 5×10 5 The computing device 116 (or its processor) can select and retrieve an appropriate LUT based on the classification or characteristics of the infectious agent 106 in the original sample 104, and then the computing device 116 (or its processor) can set the ΔpH-0.20 threshold amount 118 based on the concentration and solution property data obtained from the LUT.

[0052]

[0063] The method 100 may further include exposing the sensor 122 to the diluted sample 112 or introducing the diluted sample 112 to the sensor 122 in step 1F such that at least a portion of the sensor 122 is in fluid communication with the diluted sample 112. The portion of the sensor 122 that is in fluid communication with the diluted sample 112 may include a functionalization (or pH active) layer (see Figures 5A, 5B, 7A, and 7B) or a redox active layer (see Figures 6A, 6B, 7A, and 7B) of the sensor 122.

[0053]

[0064] The sensor 122 can be configured to respond to changes in solution properties of the diluted sample 112. In some embodiments, the sensor 122 can be a pH sensor configured to respond to changes in pH of the diluted sample 112. In other embodiments, the sensor 122 can be an oxidation-reduction potential (ORP) sensor configured to respond to changes in ORP of the diluted sample 112. In still other embodiments, the sensor 122 can be a combination pH·ORP sensor configured to respond to changes in pH and ORP of the diluted sample 112.

[0054]

[0065] Step 1F may also include incubating the diluted sample 112 at an incubation temperature 124 for a period of time. The diluted sample 112 can be incubated while exposing the sensor 122 to the diluted sample 112. The diluted sample 112 can be incubated in the same reaction vessel 108 or can be transferred to a different reaction vessel 108 or container.

[0055]

[0066] The incubation temperature 124 can be between about 30° C. and 40° C. In some embodiments, the incubation temperature 124 can be between about 33° C. and 37° C. (or about 35° C.±2° C.). The diluted sample 112 can be incubated at the incubation temperature 124 for as long as necessary for the concentration of the infectious agent 106 in the diluted sample 112 to reach the specified concentration 105. In some embodiments, the incubation time can be between about 15 minutes and 60 minutes. In other embodiments, the incubation time can be between about 60 minutes and 120 minutes. In still other embodiments, the incubation time can be less than 15 minutes or more than 120 minutes.

[0056]

[0067] 1, exposing the sensor 122 to the diluted sample 112 may include directly immersing at least a portion of a handheld or probe instance of the sensor 122 in the diluted sample. In this embodiment, the handheld or probe instance of the sensor 122 may be a handheld pH sensor or a handheld ORP sensor coupled to a stand-alone parameter analyzer 120, such as a voltmeter or multimeter.

[0057]

[0068] In another example embodiment contemplated by the present disclosure, introducing the diluted sample 112 to the sensor 122 may include injecting, delivering, or otherwise introducing the diluted sample into a well or container containing the sensor 122 fabricated on a substrate. In yet another example embodiment shown in Figure 4A, introducing the diluted sample 112 to the sensor 122 may include placing or positioning a reaction vessel 404 containing the diluted sample 112 in a sample preparation device 402 (see Figure 4A) that includes a built-in sensor 122 having contacts or electrodes that are accessible or in fluid communication with the diluted sample 112 (see Figure 4A). The sensor 122 is discussed in more detail in the following section.

[0058]

[0069] Step 1F may further include monitoring changes in solution properties of the diluted sample 112 using a parameter analyzer 120 coupled to a sensor 122 or a computing device 116 coupled to the parameter analyzer 120 or the sensor 122. The solution properties of the diluted sample 112 can be monitored without the presence of any exogenous reporter molecule added to the diluted sample 112.

[0059]

[0070] 1 depicts the parameter analyzer 120 as a standalone device separate from the computing device 116, it is contemplated by the present disclosure that the parameter analyzer 120 and the computing device 116 can be integrated into one device, as should be understood by one of ordinary skill in the art. As illustrated in FIG. 1, the computing device 116 can be a mobile device, a handheld device, a tablet device, a laptop computer, or a desktop computer. In some embodiments, the parameter analyzer 120 can wirelessly communicate signals or results with the computing device 116.

[0060]

[0071] The solution properties of the diluted sample 112 can change with changes in the amount of ions or electroactive redox species in solution due to energy use, oxygen uptake or release, growth, or metabolism of the infectious agents 106 in the diluted sample 112. For example, the amount of electroactive redox species in the diluted sample 112 can change as a result of cellular activity (e.g., aerobic or anaerobic respiration of microorganisms) performed by the infectious agents 106 in the diluted sample 112. Also, by way of example, the amount of H in the diluted sample 112 can change. + The amount of ions can change as a result of cellular activity carried out by the infectious agent 106 in the diluted sample 112 .

[0061]

[0072] As a more specific example, the amount of electron donor (e.g., the amount of energy carrier such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2)) in the diluted sample 112 can change due to growth of the infectious agent 106 in the diluted sample 112. As another more specific example, the amount of depleted oxygen in the diluted sample 112 due to aerobic respiration can change due to growth of the infectious agent 106 in the diluted sample 112.

[0062] [Table 1]

[0063]

[0073] In the case where the monitored solution property is pH, the threshold amount 118 can be about ΔpH 0.01 to ΔpH 3.0. As a more specific example, the threshold amount 118 can be set to about ΔpH 0.20 (i.e., when the starting pH is normalized to pH 7.0, pH 6.8 is set as the pH threshold level (pH th )). When the solution property being monitored is ORP, the threshold amount 118 can be about Δ100 mV to Δ700 mV. As a more specific example, the threshold amount 118 can be set to about Δ100 mV (i.e., an ORP threshold level (V) of −100 mV when the starting ORP is normalized to 0 mV). th ) can be set.

[0064]

[0074] The method 100 may further include cooling the diluted sample 112 to a cooling temperature 126 when a solution property of the diluted sample 112 changes a threshold amount 118 in step 1G. The diluted sample 112 may be cooled when the concentration of the infectious agent 106 in the diluted sample 112 reaches a specified concentration 105 (i.e., when the threshold amount 118 is reached). Once the concentration of the infectious agent 106 in the diluted sample 112 reaches the specified concentration 105, the diluted sample 112 may be considered or represented as the output sample 102.

[0065]

[0075] The cooling temperature 126 can be between about 4° C. and 25° C. In some embodiments, the cooling temperature 126 can be between about 4° C. and 15° C. In other embodiments, the cooling temperature 126 can be less than 4° C. and greater than 25° C.

[0066]

[0076] In one embodiment, the computing device 116 or the parameter analyzer 120 can alert a user that the solution properties of the diluted sample 112 have changed by the threshold amount 118. For example, the computing device 116 or the parameter analyzer 120 can generate an audible, visual or graphic, tactile or kinetic alert, or a combination thereof, when the solution properties of the diluted sample 112 have changed by the threshold amount 118. As a more specific example, the computing device 116 or the parameter analyzer 120 can generate a message informing a user that the solution properties of the diluted sample 112 have changed by the threshold amount 118.

[0067]

[0077] The diluted sample 112 can be cooled to a cooling temperature 126 by placing it in an ice bath. The diluted sample 112 can also be cooled to a cooling temperature 126 by placing it in a refrigerator or freezer, a cold compartment, a cooling device, or a combination thereof. When implementing the method 100 using an integrated system, such as the system 400 shown in Figure 4A, the diluted sample 112 can be cooled by cooling components integrated within the sample preparation apparatus 402 (see Figure 4A).

[0068]

[0078] The method 100 may further include diluting the output sample 102 with another dilution factor to generate a further diluted sample. The further diluted sample may contain an infectious agent concentration required for a downstream test, such as a downstream AST assay. In this case, the further diluted sample may serve as an input sample for the downstream test. The diluent in the further diluted sample may be at any temperature and therefore may act as a coolant to cool the further diluted sample.

[0069]

[0079] Using the methods 100 and systems described herein, a laboratory or hospital can prepare an output sample 102 at a defined concentration 105 from an original sample 104 in a reduced preparation time 128. Using the methods 100 and systems described herein, the preparation time 128 can be between about 60 minutes and 120 minutes. In other embodiments, the preparation time 128 can be between about 1 minute and 60 minutes. In other embodiments, the preparation time 128 can be between about 120 minutes and 240 minutes. In some embodiments, the preparation time 128 can be greater than 240 minutes.

[0070]

[0080] One or more of the above-described steps of the method 100 can be stored as machine-executable instructions or logical commands in a non-transitory machine-readable medium (e.g., a memory or storage unit) of the computing device 116 or other device communicatively or electrically coupled to the computing device 116. Any of the parameter analyzer 120, the computing device 116, or other devices coupled to the parameter analyzer 120 or the computing device 116 may include one or more processors or controllers configured to execute the above-described instructions or logical commands.

[0071]

[0081] The steps depicted in Figure 1 do not require the particular order depicted in order to achieve the desired results. Moreover, certain steps or processes may be omitted or performed in parallel to achieve the desired results. Additionally, any of the systems or devices disclosed herein may be used in place of the devices or systems depicted in the steps of Figure 1.

[0072]

[0082] 2 illustrates an example method 200 for creating a composite or averaged lookup table (LUT) 202 from multiple strain-specific LUTs 204. The multiple strain-specific LUTs 204 may represent experimental data 206 obtained from monitoring multiple reference samples 208 over time. The averaged LUT 202 may be either a species-specific LUT 210, a universal LUT 212, or other LUTs organized by classification type or characteristic. For example, the averaged LUT 202 may also be a Gram-positive LUT or a Gram-negative LUT. Each of the reference samples 208 may include a reference infectious agent 214 of a known strain and known species.

[0073]

[0083] A species-specific LUT 210 can be generated from a plurality of strain-specific LUTs 204 that represent experimental data 206 obtained from monitoring a plurality of reference samples 208, where each of the reference samples 208 includes a reference infectious agent 214 of the same species as the infectious agent 106 in the original sample 104. For example, a species-specific LUT 210 can be generated for SMa from a plurality of strain-specific LUTs 204, including LUTs representing CDC-27 strain of SMa (SMa_CDC-27), CDC-91 strain of SMa (SMa_CDC-91), CDC-99 strain of SMa (SMa_CDC-99), CDC-121 strain of SMa (SMa_CDC-121), CDC-122 strain of SMa (SMa_CDC-122), CDC-130 strain of SMa (SMa_CDC-130), or a combination thereof. As another example, a species-specific LUT 210 can also be generated for Staphylococcus aureus (SAu) from multiple strain-specific LUTs 204 including LUTs for wild type strain of SAu (SAu_WT), CDC-483 strain of SAu (SAu_CDC-483), CDC-475 strain of SAu (SAu_CDC-475), ATCC 43300 strain of SAu (SAu-ATCC 43300), or combinations thereof.

[0074]

[0084] A universal LUT 212 can be created from multiple strain-specific LUTs 204 that represent experimental data 206 obtained from monitoring multiple reference samples 208 that include different species of reference infectious agents 214. For example, a universal LUT 212 can be created from multiple strain-specific LUTs 204 across several species, including strain-specific LUTs 204 representing the species SMa, SAu, Escherichia coli (ECo), Enterobacter cloacae (ECl), Enterobacter aerogenes (EAe), Klebsiella pneumoniae (KPn), or any combination thereof.

[0075]

[0085] The reference sample 208 can be prepared by resuspending an infectious agent colony from an infectious agent culture plate in growth medium to reach an initial concentration. As a more specific example, the reference sample can be a liquid bacterial culture prepared by inoculating a bacterial colony from a bacterial culture plate into growth medium. For example, the initial concentration of the reference infectious agent 214 is about 1×10 7 (or 1e7) CFU / mL.

[0076]

[0086] The method 200 may include monitoring changes in solution properties of the reference sample 208 over a time range in step 2A. The solution properties can be monitored by introducing the reference sample 208 to the sensor 122 or by otherwise exposing the sensor 122 to the reference sample 208 such that the functionalized layer (see FIGS. 5A, 5B, 7A, and 7B) or the redox active layer (see FIGS. 6A, 6B, 7A, and 7B) of the sensor 122 is in fluid communication with the reference sample 208. If the sensor 122 is a pH sensor, the solution property monitored can be the pH of the solution. If the sensor 122 is an ORP sensor, the solution property monitored can be the ORP of the solution. The solution properties of the reference sample 208 can be monitored without the presence of any exogenous reporter molecule added to the reference sample 208. The reference sample 208 can also be incubated at an incubation temperature 124 while the solution properties of the reference sample 208 are monitored by the sensor 122.

[0077]

[0087] In some embodiments, the sensor 122 can be coupled to a parameter analyzer 120 that is communicatively coupled to the computing device 116, or the sensor 122 can be directly coupled to the computing device 116. The computing device 116 can record and store data regarding changes in solution properties of the reference sample 208 at specific time intervals 216. For example, the computing device 116 can store pH changes of the reference sample 208 every 5 minutes, every 10 minutes, or every 15 minutes. In some embodiments, the time interval 216 can be between about 30 seconds and 20 minutes. In other embodiments, the time interval 216 can be between about 1 second and 30 seconds or more than 20 minutes.

[0078]

[0088] The method 200 may further include performing a sample enumeration assay 218 of the reference sample 208 over the same time period. The sample enumeration assay 218 may be a test or measurement performed to determine the concentration of the reference infectious agent 214 in the reference sample 208 at a particular time point. The concentration of the reference infectious agent 214 in the reference sample 208 may increase over a time period as the reference sample 208 is incubated and the growth medium provides nutrients to the reference infectious agent 214.

[0079]

[0089] In some embodiments, the sample enumeration assay 218 may include an optical density (OD) measurement, a plate count assay, or a flow cytometry assay. In other embodiments, the sample enumeration assay 218 may be other tests or measurements that determine the concentration of the reference infectious agent 214 in the reference sample 208. For example, the sample enumeration assay 218 may be an OD measurement performed at a wavelength of 600 nm (OD600 measurement) using a spectrophotometric device or system. The sample enumeration assay 218 may be performed by a device or system (e.g., a detector) communicatively coupled to the computing device 116, either directly or indirectly. The computing device 116 may record and store the results of the sample enumeration assay 218 in one or more databases stored in memory of the computing device 116, a computing cloud, or a remote server accessible to the computing device 116.

[0080]

[0090] The sample enumeration assay 218 can be performed simultaneously with the monitoring and recording of changes in solution properties of the reference sample 208. For example, an OD measurement can be performed at the same time interval 216 as the measurement of changes in solution properties of the reference sample 208. As a more specific example, a sample enumeration assay 218 (e.g., an OD measurement) can be performed on a reference sample 208, and changes in solution properties of the same reference sample 208 can be recorded every 5 minutes. In other embodiments, the sample enumeration assay 218 can be performed immediately before or after recording changes in solution properties of the reference sample 208.

[0081]

[0091] The method 200 may also include converting the results of the sample enumeration assay 218 to a reference sample concentration 220 using a conversion factor. For example, the results of an OD measurement may be converted to a numeric conversion factor (e.g., OD×(1.76×10 9) to a reference sample concentration 220 (expressed as CFU / mL). The conversion factor is typically instrument dependent and will vary from instrument to instrument. The computing device 116 (or its processor) or other device communicatively coupled to the computing device 116 can be programmed to convert the results of the sample enumeration assay 218 to a reference sample concentration 220 and store such reference sample concentration 220 in one or more databases stored in memory of the computing device 116, a computing cloud, or a remote server accessible to the computing device 116.

[0082]

[0092] The method 200 may further include creating each of the multiple strain-specific LUTs 204 by relating the calculated reference sample concentration 220 to a change in a solution property of a particular reference sample 208 at a particular time interval 216 in step 2B. For example, a strain-specific LUT 204 for SMa_CDC-27 can be created by relating the calculated reference sample concentration 220 of this particular reference sample 208 to a change in a solution property of the reference sample 208 measured every 5 minutes or every 10 minutes.

[0083]

[0093] The method 200 may further include creating an averaged LUT 202 using data from multiple strain-specific LUTs 204 in step 2C. As discussed above, the averaged LUT 202 may refer to either a species-specific LUT 210, a universal LUT 212, or other LUTs organized by classification type or characteristic. For example, the averaged LUT 202 may also be a Gram-positive LUT or a Gram-negative LUT. If the averaged LUT 202 is a species-specific LUT 210, the strain-specific LUTs 204 used to create the species-specific LUT 210 may include different strains of the same species of infectious agent. If the averaged LUT 202 is a universal LUT 212, the strain-specific LUTs 204 used to create the universal LUT 212 may include different strains of infectious agents across different species.

[0084]

[0094] In one embodiment, the averaged LUT 202 can be created by averaging all solution property variations 222 across multiple strain-specific LUTs 204 to generate several averaged solution property variations 224. The averaged solution property variations 224 can be calculated for each predefined reference sample concentration 220. For example, the predefined reference sample concentration 220 can be 1×10 8 CFU / mL, 2×10 8 CFU / mL, 3×10 8 CFU / mL, etc. In this example, the averaged LUT 202 can be created by calculating each averaged solution property variation 224 from the solution property variations 222 obtained from the multiple strain-specific LUTs 204.

[0085]

[0095] Step 2C may further include associating each of the predetermined reference sample concentrations 220 with an averaging calculation result. For example, the averaging LUT 202 may be created by associating the predetermined reference sample concentrations 220 with an average solution property variation 224.

[0086]

[0096] The averaged LUTs 202 can be selected and searched based on information about the taxonomic type or characteristics of the infectious agent 106 in the original sample 104 (see FIG. 1 ), or lack of such information. For example, the SMa species-specific LUT 210 can be selected and searched when the species of the infectious agent 106 in the original sample 104 is identified as SMa. Also, by way of example, the universal LUT 212 can be selected and searched when the species of the infectious agent 106 in the original sample 104 is unidentified or unknown. As other examples, averaged LUTs 202 organized by genus, family, order, class, phylum, kingdom, or domain can also be selected and searched. Additionally, averaged LUTs 202 organized by microbial characteristics, such as gram type, or functional capabilities, such as the ability to hydrolyze a particular protein or molecule, can also be selected or searched.

[0087]

[0097] As discussed above, the LUTs can be stored as part of a database software program in memory of the computing device 116. In other embodiments, the LUTs can be stored as part of a database software program on a computing cloud or a remote server accessible to the computing device 116 on a network. The computing device 116, or one or more processors thereof, can search through hundreds or thousands of stored LUTs to select an appropriate LUT based on information regarding the classification type (e.g., species) or characteristics of the infectious agent 106 in the original sample 104.

[0088]

[0098] Although method 200 is shown separately from method 100, it is contemplated by the present disclosure that method 200 may be considered a sub-step or pre-step of method 100, as should be understood by one of ordinary skill in the art. Additionally, the steps shown in FIG. 2 do not require the particular order shown to achieve the desired results. Additionally, certain steps or processes may be omitted or performed in parallel to achieve the desired results. Additionally, any of the systems or devices disclosed herein may be used in place of the devices or systems shown in the steps of FIG. 2.

[0089]

[0099] 3A illustrates example strain-specific LUTs 204 for various strains of SMa used to create a species-specific LUT 210 for SMa. As shown in FIG. 3A, the strain-specific LUTs 204 may include an SMa_CDC-27LUT, an SMa_CDC-91LUT, an SMa_CDC-99LUT, an SMaCDC-121LUT, an SMaCDC-122LUT, and an SMaCDC-130LUT. Although six strain-specific LUTs 204 are shown in the example embodiment of FIG. 3A, it is contemplated by the present disclosure that an averaging LUT 202, such as a species-specific LUT 210 or a universal LUT 212, may be created from at least three strain-specific LUTs 204. Increasing the number of strain-specific LUTs 204 may improve the accuracy of the averaging LUT 202.

[0090]

[0100] The strain-specific LUT 204 can include a reference sample concentration 220 and a solution property change 222. The solution property change 222 can be obtained from monitoring the change in solution property of a reference sample 208 containing a strain of SMa over a period of time. For example, Figure 3B illustrates a pH growth curve 300 representing the pH change of a reference sample 208 containing SMa_CDC-27.

[0091]

[0101] The reference sample concentration 220 can be obtained by converting the results of a sample enumeration assay 218 (see FIG. 2) performed on the reference sample 208 while simultaneously monitoring the change in solution properties of the reference sample 208. For example, FIG. 3B illustrates a reference sample concentration curve 302 that represents the increase in the concentration of an infectious agent in the reference sample 208 as measured by a sample enumeration assay 218 performed on the reference sample 208 over time. As shown in FIG. 3B, the pH growth curve 300 and the reference sample concentration curve 302 can be plotted using the same x-axis (time in minutes).

[0092]

[0102] 3A also illustrates that a species-specific LUT 210 for SMa can be created by averaging the solution property variation 222 across the various strain-specific LUTs 204 for SMa. For example, for a reference sample concentration 220 of 1e8 CFU / mL, the averaged solution property variation 224 can be calculated as a ΔpH of 6.96 or -0.04.

[0093]

[0103] 4A illustrates an example of a system 400 for preparing an output sample 102 from an original sample 104 that includes an infectious agent 106. More specifically, the system 400 can provide an output sample 102 that includes a defined concentration 105 of the infectious agent 106.

[0094]

[0104] The system 400 may include a sample preparation device 402 that integrates the functions of the computing device 116, the parameter analyzer 120, and the sensor 122. The system 400 may perform any of the steps of the method 100 of Figure 1. For example, one or more processors in the sample preparation device 402 may perform any of steps 1C, 1D, or 1E of the method 100.

[0095]

[0105] Additionally, the system 400 can include one or more reaction vessels 404 that can be fitted to the sample preparation device 402. For example, the original sample 104 can be introduced into the reaction vessel 404, and the reaction vessel 404 can be placed or positioned within a vessel receiving space 406 of the sample preparation device 402.

[0096]

[0106] In another embodiment shown in FIG. 4B, a test cartridge 408 can be used in place of or in addition to the reaction vessel 404. The test cartridge 408 can include a sample receiving surface 410 or space and a sample output surface 412 or space. The sample receiving surface 410 or space of the test cartridge 408 can receive an aliquot of the raw sample 104, and the output sample 102 can be injected, pumped, or otherwise introduced into the sample output surface 412 or space. The test cartridge 408 can interface with the sample preparation device 402 via a cartridge interface 414. The cartridge interface 414 can be an electronic interface configured to allow the sample preparation device 402 to read or retrieve information from the test cartridge 408, such as a universal serial bus (USB) interface, a high-speed serial computer expansion bus interface, or a combination thereof. In an embodiment in which the test cartridge 408 is used in place of the reaction vessel 404, the vessel receiving space 406 can be configured as a cartridge receiving slot or space.

[0097]

[0107] In one embodiment, a user can initiate the process of obtaining an output sample 102 from an original sample 104 by placing a reaction vessel 404 (or test cartridge 408) containing the original sample 104 into a vessel receiving space 406 (or cartridge receiving slot) of the sample preparation device 402 and inputting a specified concentration 105 into the sample preparation device 402. For example, the specified concentration 105 can be a concentration of 3e8 or 5e5 CFU / mL. The specified concentration 105 can be input via an input component of the sample preparation device 402, such as a keyboard, a touch screen, or a combination thereof. In other embodiments, the specified concentration 105 can be input via a computing device (e.g., a mobile device or a laptop) communicatively coupled to the sample preparation device 402, and the specified concentration 105 can be transmitted to the sample preparation device 402.

[0098]

[0108] In another embodiment, a user can initiate the process of obtaining an output sample 102 from an original sample 104 by inputting or otherwise providing information regarding the classification (e.g., species, family, order, etc.) or characteristics (Gram-positive or Gram-negative) of the infectious agent 106 in the original sample 106, and the system 400 can determine a specified concentration 105 of the output sample 102 required for any downstream tests.

[0099]

[0109] In some embodiments, one or more metering conduits in the sample preparation device 402 can then dilute the original sample 104 by introducing a diluent solution 114 into the original sample 104 to generate a diluted sample 112. A metering conduit may refer to a channel, passage, capillary, tube, or combination thereof in the sample preparation device 402. A metering conduit may also refer to a channel (e.g., a microfluidic channel), passage, capillary, or tube that functions as part of a hydraulic pump, a pneumatic pump, a peristaltic pump, a vacuum or positive pressure pump, a manual or mechanical pump, a syringe pump, or combination thereof in the sample preparation device 402.

[0100]

[0110] In one embodiment, the original sample 104 can be aspirated or pumped (e.g., via vacuum pressure) from the reaction vessel 404 into a channel, passage, capillary, tube, or combination thereof in the sample preparation device 402. In this embodiment, the diluent solution 114 can be introduced to the original sample 104 after the original sample 104 is aspirated or pumped from the reaction vessel 404. In other embodiments, the test cartridge 408 can include channels, conduits, or capillaries that pump or deliver the diluted sample 112, the diluent solution 114, the original sample 104, the output sample 102, or combinations thereof.

[0101]

[0111] In other embodiments, the original sample 104 can remain in the reaction vessel 404 (or test cartridge 408) and the diluent solution 114 can be introduced into the reaction vessel 404 (or test cartridge 408) through one or more ports along the base, top, or sides of the reaction vessel 404. In other embodiments, the diluent solution 114 can be introduced into the reaction vessel 404 (or test cartridge 408) through one or more ports defined in a cap or cover of the reaction vessel 404.

[0102]

[0112] In other embodiments, the original sample 104 can be diluted with a diluent solution 114 before being placed in the sample preparation device 402 .

[0103]

[0113] In some embodiments, at least a portion of the sensor 122 can extend into or be in fluid communication with the diluted sample 112 in the reaction vessel 402 (or in or on the test cartridge 408). For example, the electrodes (e.g., active and reference electrodes) can extend into or be in fluid communication with the diluted sample 112 through ports along the base of the reaction vessel 404 (or test cartridge 408). In other embodiments in which the original sample 104 or diluted sample 112 is aspirated or directed into the sample preparation device 402, the diluted sample 112 can be introduced to the sensor 122 in the sample preparation device 402 via one or more fluid delivery conduits, e.g., channels (e.g., microfluidic channels), passages, capillaries, or tubes in the sample preparation device 402.

[0104]

[0114] In some embodiments, the sensor 122 of the sample preparation device 402 may be a pH sensor that includes a reference electrode and an active electrode. In these embodiments, the active electrode may include a functionalized (or pH-active) layer 508 (see FIGS. 5A and 5B).

[0105]

[0115] In other embodiments, the sensor 122 of the sample preparation device 402 may be an ORP sensor that includes a reference electrode and an active electrode. In these embodiments, the active electrode may include a redox active layer 610 (see FIGS. 6A and 6B). As will be discussed in more detail, the redox active layer 610 may include a metal layer or a metal electrode.

[0106]

[0116] In yet other embodiments, the sensor 122 of the sample preparation device 402 may be a combination pH·ORP sensor that includes multiple reference and active electrodes. In these embodiments, at least one active electrode may include a redox active layer 712 and at least one other active electrode may include a functionalization layer 716 (see FIGS. 7A and 7B).

[0107]

[0117] In some embodiments not shown, the sensor 122 (or a portion thereof) can be integrated into the test cartridge 408. For example, the diluted sample 112 or the original sample 104 can be placed in fluid communication with the functionalized (or pH active) layer or redox active layer and reference electrode of the sensor 122 by introducing the diluted sample 112 or the original sample 104 onto the sample-receiving surface 410. In other examples, at least a portion of the test cartridge 408 can be immersed in the diluted sample 112.

[0108]

[0118] One or more processors in the sample preparation device 402 can be programmed to look up an averaging LUT 202, such as the species-specific LUT 210, based on information about the species of infectious agent 106 in the original sample 104. In other embodiments, one or more processors in the sample preparation device 402 can be programmed to look up a universal LUT 212 or other type of inter-species LUT based on a lack of information about the infectious agent 106 in the original sample 104.

[0109]

[0119] The LUTs can be stored as part of a database software program in a memory of the sample preparation device 402. In other embodiments, the LUTs can be stored as part of a database software program in a computing cloud or a remote server accessible to the sample preparation device 402 on a network. One or more processors of the sample preparation device 402 can be programmed to search through hundreds or thousands of stored LUTs to select an appropriate LUT based on the classification type (e.g., species) or characteristics of the infectious agent 106 in the original sample 104.

[0110]

[0120] The one or more processors of the sample preparation device 402 can be programmed to set the threshold amount 118 based on the nominal concentration 105 and the data in the averaging LUT 202. For example, the nominal concentration 105 can be entered by a user into the sample preparation device 402 as 3e8 or 5e5 CFU / mL. The one or more processors of the sample preparation device 402 can be programmed to select an averaged solution property change 224 of ΔpH of −0.20 as the threshold amount 118 because that solution property change is associated with a reference sample concentration 220 of 3e8 or 5e5 CFU / mL.

[0111]

[0121] The sample preparation device 402 may also include an incubation component that incubates the diluted sample 112 up to an incubation temperature 124 of about 30° C.-40° C. (or about 35° C.±2° C.). For example, the incubation component may incubate the diluted sample 112 in a reaction vessel 404 or may incubate a diluted sample 112 that has been aspirated or pumped into the sample preparation device 402. In other embodiments, the incubation component may incubate all or a portion of the test cartridge 408 containing the diluted sample 112.

[0112]

[0122] The sample preparation device 402 can monitor changes in solution properties (e.g., pH or ORP) of the diluted sample 112 while incubating the diluted sample 112. The sensor 122 of the sample preparation device 402 can be configured to respond to changes in solution properties (e.g., pH or ORP) of the diluted sample 112. The solution properties of the diluted sample 112 can be monitored without the presence of any exogenous reporter molecule added to the diluted sample 112.

[0113]

[0123] The sample preparation device 402 can include a cooling component to cool the diluted sample 112. The cooling component can cool the diluted sample 112 to a cooling temperature 126 when a solution property of the diluted sample 112 changes a threshold amount 118. The diluted sample 112 can be cooled when a concentration of an infectious agent 106 in the diluted sample 112 reaches a specified concentration 105.

[0114]

[0124] The cooling temperature 126 can be between about 4° C. and 25° C. In some embodiments, the cooling temperature 126 can be between about 4° C. and 15° C.

[0115]

[0125] In some embodiments, the sample preparation device 402 can cool the same reaction vessel 404 containing the diluted sample 112. In these embodiments, the now cooled diluted sample 112 in the same reaction vessel 404 can be considered the output sample 102 when the concentration of the infectious agent 106 in the reaction vessel 404 reaches the specified concentration 105.

[0116]

[0126] In other embodiments, the cooling component can cool all or a portion of the test cartridge 408 including the diluted sample 112. In these embodiments, the cooled diluted sample 112 at this point in the test cartridge 408 may be considered the output sample 102 when the concentration of the infectious agent 106 in the test cartridge 408 reaches the specified concentration 105.

[0117]

[0127] In other embodiments, the diluted sample 112 can be introduced, pumped, or otherwise transferred to a different reaction vessel 404 (or a different portion of the test cartridge 408) that is cooled. In these embodiments, the cooled diluted sample 112 in the different reaction vessel 404 (or a different portion of the test cartridge 408, e.g., the sample output surface 412 or space) may be considered the output sample 102.

[0118]

[0128] In one embodiment, the sample preparation device 402 can alert a user that the solution properties of the diluted sample 112 have changed by a threshold amount 118 and that the infectious agent 106 in the reaction vessel 404 (or in the test cartridge 408) has reached a specified concentration 105. For example, the sample preparation device 402 can generate an audible, visual or graphic, tactile or kinetic alert, or a combination thereof. As a more specific example, the sample preparation device 402 can generate a message on a display component informing the user that the output sample 104 is ready for retrieval or use in downstream testing.

[0119]

[0129] Although FIG. 4A illustrates one embodiment of the sample preparation device 402 as a benchtop device, it is contemplated by the present disclosure that the sample preparation device 402 or components thereof may be embodied as a cartridge, a test strip, a microelectromechanical system (MEMS) device, a lab-on-a-chip (LOC) device, a microfluidic chip, or combinations thereof, as should be understood by one of ordinary skill in the art.

[0120]

[0130] Additionally, while FIG. 4A illustrates a sample preparation device 402 having two vessel receiving spaces 406 (or two cartridge receiving slots), it is contemplated by the present disclosure that the sample preparation device 402 may have one vessel receiving space 406 (or one cartridge receiving slot) or three or more vessel receiving spaces 406 (or three or more cartridge receiving slots), and one of ordinary skill in the art should understand this. In embodiments in which the sample preparation device 402 includes multiple vessel receiving spaces 406 (or multiple cartridge receiving slots), the sample preparation device 402 may be considered a multiplex device. Additionally, while FIG. 4B illustrates a test cartridge 408 including one sample receiving surface 410 or space and one sample output surface 412 or space, it is contemplated by the present disclosure that the test cartridge 408 may include two or more sample receiving surfaces 410 or spaces (receiving two or more original samples 104) and two or more sample output surfaces 412 or spaces (accommodating two or more output samples 102).

[0121]

[0131] FIG. 5A illustrates a schematic diagram of one embodiment of a pH sensor 500 used as part of the methods and systems described herein. The sensor 500 of FIG. 5A can be or can refer to any of the sensors 122 shown in FIGS. 1, 2, and 4. The sensor 500 can be or can include an electrochemical cell including an active electrode 502 and an external reference electrode 506 positioned on a container wall 504 and a substrate layer 512. The active electrode 502 can include a functionalization layer 508 and a conductor layer 510. The sensor 500 can be configured to receive or be in fluid contact with a solution 514. For example, the sensor 500 can receive and hold the solution 514 within the container wall 504 as shown in FIG. 5A. In other embodiments not shown but contemplated by the present disclosure, one or more layers of the sensor 500 may be in fluid contact with the solution 514 even if the solution 514 is not held within the container walls 504 of the sensor 500 or even if the sensor 500 does not have a container wall 504.

[0122]

[0132] In all such embodiments, the solution 514 can be either the diluted sample 112 or the reference sample 208 or an aliquot thereof. The sensor 500 can be connected or coupled to a parameter analyzer 120. In one embodiment, the parameter analyzer 120 can be coupled to both the external reference electrode 506 and the active electrode 510. In other embodiments, the parameter analyzer 120 can be coupled to the external reference electrode 506, the conductor layer 510, or even other layers. As shown in FIG. 5A, the external reference electrode 506 can extend into the solution 514.

[0123]

[0133] When the parameter analyzer 120 is coupled to the external reference electrode 506, the conductor layer 510, or other layer, the parameter analyzer 120 can measure differences in electrical properties of the solution 514. The external reference electrode 506 can have a stable and known internal reference potential and can also act as a differential noise filter to remove electrical noise from measurements made by the sensor 500. Using this configuration, an operator or clinician can determine or record relative changes in the electrical properties of the sensor 500, without having to ascertain absolute changes. An operator or clinician can also determine or record relative differences in the electrical properties of multiple sensors 500 using the external reference electrode 506. In one embodiment, the external reference electrode 506 can be a stand-alone probe or electrode. In other embodiments, the external reference electrode 506 can be coupled to the parameter analyzer 120 or a computing device 116 (not shown) connected to the parameter analyzer 120. The parameter analyzer 120 can also be used to apply a voltage or current to the active electrode and the external reference electrode 506.

[0124]

[0134] In one embodiment, the external reference electrode 506 can be a silver / silver chloride (Ag / AgCl) electrode. In other embodiments, the external reference electrode 506 can be, but is not limited to, a saturated calomel reference electrode (SCE) or a copper-copper(II) sulfate electrode (CSE).

[0125]

[0135] The substrate layer 512 can be composed of any non-conductive material, such as, but not limited to, a polymer, an oxide, a ceramic, or a composite thereof. As shown in FIG. 5A, the conductor layer 510 can be disposed on or cover the substrate layer 512.

[0126]

[0136] The conductor layer 510 can be composed of, but is not limited to, a metal, a semiconductor material, a metal / metal salt, or a combination thereof. For example, but not limited to, the conductor layer 510 can be composed of silicon, gold, silver, aluminum, platinum, or a composite thereof. The conductor layer 510 can also be an organic semiconductor, a carbon nanotube, graphene, an organic conductor, such as those derived from polyacetylene, polyaniline, quinacridone, poly(3,4-ethylenedioxythiophene) or PEDOT, PEDOT:polystyrenesulfonate (PSS), or a combination thereof. The conductor layer 510 can be composed of any conductive material that allows for the measurement of a change in an electrical property, such as, but not limited to, a voltage change, a capacitance change, a conductance change, and / or a current change that is measured through the conductor layer 510, the functionalization layer 508, and the solution 514 to the external reference electrode 506.

[0127]

[0137] 5A, the functionalization layer 508 can be disposed on or cover the conductor layer 510. The functionalization layer 508 can include oxides, silanes, DNA, proteins, antibodies, self-assembled monolayers (SAMs), oxides, buffered hydrogels, PVC, parylene, polyACE, or any other biochemically active material. The functionalization layer 508 can be configured to facilitate interaction of the sensor 500 with ions, analytes, or other molecules or byproducts in the solution 514. For example, the functionalization layer 508 can be a pH-sensitive layer or a pH-active layer.

[0128]

[0138] In one example, the functionalization layer 508 is configured to react with hydrogen ions (H +), which can produce a change in an electrical property between the sensor 500 and the external reference electrode 506 that is detected by the parameter analyzer 120. In one embodiment, this interaction can produce a measurable change in an electrical property of the sensor 500 at the interface between the solution 514 and the functionalization layer 508 or the interface between the solution 514 and the conductor layer 510.

[0129]

[0139] For example, the parameter analyzer 120 can be a voltmeter, and the voltmeter can detect a voltage (potential) change (ΔV) at or near the functionalization layer 508 between the active electrode 502 and an external reference electrode 506 exposed to the solution 514. The voltage change can be determined relative to the external reference electrode 506 extending into or in contact with the solution 514. In this embodiment, the functionalization layer 508 and the conductor layer 510 can be considered part of the working or active electrode 502.

[0130]

[0140] 5A, the solution 514, the functionalization layer 508, and the conductor layer 510 may be surrounded by a container wall 504. The container wall 504 may be made of an inert or non-conductive material. The container wall 504 may include, but is not limited to, a polymeric material, such as polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), ceramic, glass, or a combination thereof.

[0131]

[0141] 5B illustrates a schematic diagram of another embodiment of a pH sensor 500 for use as part of the methods and systems described herein. Sensor 500 can be or represent any of sensors 122 shown in FIGS. 1, 2, and 4.

[0132]

[0142] In this embodiment, the sensor 500 can include an active electrode 516 or indicator electrode and an on-chip reference electrode 518. In this embodiment, the active electrode 516 (i.e., the active electrode) and the on-chip reference electrode 518 can be disposed on the same backing layer 512. The backing layer 512 can be constructed of the same materials as the backing layer 512 shown in FIG.

[0133]

[0143] The solution 514 can be simultaneously flowed over or exposed to both the active electrode 516 and the on-chip reference electrode 518. In this embodiment, the active electrode 516 and the on-chip reference electrode 518 can be separated by a vessel wall 504 or a vessel divider.

[0134]

[0144] The active electrode 516 may include a functionalization layer 508 disposed on or covering the conductor layer 510. The functionalization layer 508 may include an oxide, a silane, DNA, a protein, a hydroxyl group, an antibody, an oxide, a self-assembled monolayer (SAM), a buffered hydrogel, PVC, parylene, polyACE, or any other biochemically active material.

[0135]

[0145] 5B, a passivation layer 520 can be disposed on or cover the conductor layer 510. The passivation layer 520 can be configured to prevent interaction between the on-chip reference electrode 518 and the analyte, ions, or other molecules or by-products in the solution 514. For example, the passivation layer 520 can be a pH-insensitive layer. The passivation layer 520 can include a silane, a self-assembled monolayer (SAM), a buffered hydrogel, parylene, polyACE, or any other biochemically inert material.

[0136]

[0146] In this embodiment, the parameter analyzer 120 can have a lead connection wire, such as a copper wire, connected to the conductor layer 510 of the active electrode 516 and another lead connection wire connected to the conductor layer 510 of the on-chip reference electrode 518. The parameter analyzer 120 can also be used to apply a voltage or current to the active electrode and the on-chip reference electrode 518.

[0137]

[0147] In this and other embodiments, the sensor 500 shown in FIG. 5B miniaturizes the sensor configuration shown in FIG. 5A. The on-chip reference electrode 518 avoids the need for an external reference electrode, such as the external reference electrode 506. The on-chip reference electrode 518 can also be a silver / silver chloride (Ag / AgCl) electrode. In other embodiments, the on-chip reference electrode 518 can be, but is not limited to, a saturated calomel reference electrode (SCE) or a copper-copper(II) sulfate electrode (CSE). The on-chip reference electrode 518 provides a similar function to the external reference electrode 506 in this embodiment of the sensor 500. The passivation layer 520 of the on-chip reference electrode 518 prevents interaction between the conductor layer 510 covered by the passivation layer 520 and ions, analytes, or other molecules or by-products in the solution 514. This allows a reader or other device to distinguish the electrical signals obtained by the parameter analyzer 120. In some embodiments, the passivation layer 520 may represent an on-chip reference electrode 518 with a well-defined potential. In other embodiments, the on-chip reference electrode 518 may be without the passivation layer 520.

[0138]

[0148] In one embodiment where the conductor layer 510 is used as a reference electrode, the conductor layer 510 can be a metal covered with a metal salt such as a metal chloride. In another embodiment, the conductor layer 510 can also be covered with an oxide. For example, the conductor layer 510 can be a silver / silver chloride contact. In this embodiment, the conductor layer 510 can be covered with a passivation layer 520, such as, but not limited to, a KCL electrolyte gel or KCL solution, to prevent interaction of the conductor layer 510 with the analyte, ions, or other molecules or by-products in the solution 514 and to act as a reference electrode. In another embodiment, the on-chip reference electrode 518 can be covered with a small cell that stabilizes a small internal potential similar to a calomel electrode or an Ag / AgCl electrode.

[0139]

[0149] FIG 6A illustrates a schematic diagram of one embodiment of an ORP sensor 600 for use as part of the methods and systems described herein. The sensor 600 of FIG 6A may be or may represent any of the sensors 122 shown in FIGS. 1, 2, and 4. The sensor 600 may be an electrochemical cell that includes an active electrode 602 and an external reference electrode 604. In some embodiments of the sensor 600, the only electrodes of the sensor 600 are the active electrode 602 and the external reference electrode 604.

[0140]

[0150] The active electrode 602 can extend from or be disposed on a substrate layer 606. The substrate layer 606 can be constructed of any non-conductive material, such as, but not limited to, a polymer, an oxide, a ceramic, or a composite thereof. The electrochemical cell can be surrounded by or contained within a wall 608 configured to hold a sample solution 612. The wall 608 can be made of an inert or non-conductive material.

[0141]

[0151] The sample solution 612 may refer to either the diluted sample 112 or the reference sample 208 or an aliquot thereof. At least a portion of the external reference electrode 604 and the active electrode 602 may be in fluid communication or fluid contact with the sample solution 612. For example, the external reference electrode 604 may extend or be submerged in the sample solution 612. The external reference electrode 604 may also have a stable or known internal voltage, and the sensor 600 may use the external reference electrode 604 to determine or measure a relative change in the potential of the active electrode 602. In an embodiment, the external reference electrode 604 may be a stand-alone probe or electrode. In other embodiments, the external reference electrode 604 may be coupled to the parameter analyzer 120. In some embodiments, multiple sensors (including but not limited to a first sensor and a second sensor) may share and use the same external reference electrode 604.

[0142]

[0152] In one embodiment, the external reference electrode 604 can be a silver / silver chloride (Ag / AgCl) electrode. In other embodiments, the external reference electrode 604 can include a saturated calomel reference electrode (SCE) or a copper-copper(II) sulfate electrode (CSE). The external reference electrode 604 can also be a pseudo reference electrode including any metal that is not part of the active electrode, such as platinum, silver, gold, or combinations thereof, any metal oxide material or semiconductor oxide material, such as aluminum oxide, iridium oxide, silicon oxide, or any conductive polymer electrode, such as polypyrrole, polyaniline, polyacetylene, or combinations thereof.

[0143]

[0153] The active electrode 602 may include multiple conductive layers (e.g., a stack of metal layers) and a redox active layer 610 or layer, such as a gold layer, a platinum layer, a metal oxide layer, a carbon layer, a combination thereof, on the multiple conductive layers. In some embodiments, the metal oxide layer may include an iridium oxide layer, a ruthenium oxide layer, or a combination thereof. The parameter analyzer 120 may be coupled to the active electrode 602 and the external reference electrode 604.

[0144]

[0154] The parameter analyzer 120, the computing device 116, or a combination thereof can determine the ORP of the sample solution 612 by measuring the potential difference between the external reference electrode 604 and the active electrode 602 instantaneously or over a time range. As shown in FIG. 6A, the parameter analyzer 120 can be a voltmeter or any other type of high impedance amplifier or source meter. The voltmeter can measure the relative change in equilibrium potential at the interface between the redox active layer 610 of the active electrode 602 and the sample solution 612 containing the electroactive redox species. The parameter analyzer 120 can also be used to apply a voltage or current to the active electrode and the external reference electrode 604.

[0145]

[0155] The solution properties of the sample solution 612 can change with changes in the amount of electroactive redox species due to build-up and breakdown of metabolically associated energy of the pathogen or oxygen depletion or release. For example, the amount of electroactive redox species in the sample solution 612 can change as a result of cellular activity performed by an infectious agent in the solution. As a more specific example, the amount of electron donors (e.g., the amount of energy carriers such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2)) in Table 1 in the sample solution 612 can change due to growth of an infectious agent in the solution. As another more specific example, the amount of depleted oxygen in the sample solution 612 can change due to growth of an infectious agent in the solution.

[0146]

[0156] In one embodiment, the active electrode 602 may include a metal layer. The metal layer may include a gold layer, a platinum layer, or a combination thereof. The active electrode 602 may also include multiple layers including a semiconductor layer with a redox active metal oxide layer, such as iridium oxide or ruthenium oxide, on top of the multiple layers. In other embodiments, the active electrode 602 may include one or more metal layers, one or more redox active metal oxide layers, one or more semiconductor layers, or any combination or stacking arrangement thereof.

[0147]

[0157] FIG. 6B illustrates a schematic diagram of another embodiment of an ORP sensor 600 used as part of the methods and systems described herein. The sensor 600 of FIG. 6B can be or refer to any of the sensors 122 shown in FIGS. 1, 2, and 4. The sensor 600 can have an on-chip reference electrode 614 disposed on the substrate layer 606 in place of the external reference electrode 604 of FIG. 6A. In some embodiments of the sensor 600, the only electrodes of the sensor 600 are the active electrode 602 and the on-chip reference electrode 614. The parameter analyzer 120 can also be used to apply a voltage or current to the active electrode and the on-chip reference electrode 614.

[0148]

[0158] In these and other embodiments, the on-chip reference electrode 614 can be coated with a polymeric coating. For example, the on-chip reference electrode 614 can be coated with a polyvinyl chloride (PVC) coating, a perfluorosulfonate coating (e.g., Nafion™), or a combination thereof.

[0149]

[0159] The on-chip reference electrode 614 may serve the same purpose as the external reference electrode 604, except that it is fabricated on or integrated into the backing layer 606. The on-chip reference electrode 614 may be positioned adjacent or near the active electrode 602. The sensor 600 of Figure 6B may serve the same function as the sensor 600 of Figure 6A. Similar to the active electrode 602 of Figure 6B, the on-chip reference electrode 614 may also be in fluid communication or contact with the sample solution 612 held within the wall 608.

[0150]

[0160] The on-chip reference electrode 614 can be made of a metal, a semiconductor material, or a combination thereof. The metal of the on-chip reference electrode 614 can be covered by an oxide layer, a silane layer, a polymer layer, or a combination thereof. In another embodiment, the on-chip reference electrode 614 can be a metal combined with a metal salt, such as an Ag / AgCl on-chip reference electrode. In another embodiment, the on-chip reference electrode can be a small electrode with a well-defined potential. In some embodiments, multiple sensors can share and use the same on-chip reference electrode 614. The on-chip reference electrode 614 can include a saturated calomel reference electrode (SCE) or a copper-copper(II) sulfate electrode (CSE). The on-chip reference electrode 614 may also include a pseudo reference electrode comprising any metal that is not part of the active electrode, such as platinum, silver, gold, or a combination thereof, any metal oxide material or semiconductor oxide material, such as aluminum oxide, iridium oxide, silicon oxide, or any conductive polymer electrode, such as polypyrrole, polyaniline, polyacetylene, or a combination thereof. In other embodiments, the on-chip reference electrode 614 may be covered by a small cell that stabilizes a small internal potential similar to a calomel or Ag / AgCl electrode.

[0151]

[0161] FIG. 7A illustrates a schematic diagram of one embodiment of a sensor 700 used as part of the methods and systems described herein. The sensor 700 of FIG. 7A may be or may refer to any of the sensors 122 shown in FIGS. 1, 2, and 4. The sensor 700 may be or may include an electrochemical cell having a container wall 702, a first active electrode 704 and a second active electrode 706 positioned on a substrate layer 708, and an external reference electrode 710. Although two active electrodes are shown in FIG. 7A, it should be understood by one of ordinary skill in the art that more than two active electrodes or multiple reference electrodes can be positioned on a substrate layer as contemplated by the present disclosure.

[0152]

[0162] The first active electrode 704 may include a redox active layer 712 disposed or otherwise positioned on a conductor layer 714. The second active electrode 706 may include a functionalization layer 716 disposed or otherwise positioned on the conductor layer 714. In some embodiments, the functionalization layer 716 may be a pH sensitive layer. In these and other embodiments, the first active electrode 704 may function as part of an ORP sensor and the second active electrode 706 may function as part of a pH sensor.

[0153]

[0163] The vessel walls 702 of the sensor 700 can be configured to receive and hold the sample solution 718. The vessel walls 702 can be made of an inert or non-conductive material. The vessel walls 702 can include, but are not limited to, polymeric materials such as polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), ceramic, glass, or combinations thereof.

[0154]

[0164] In other embodiments not shown but contemplated by the present disclosure, one or more layers of the sensor 700 may be in fluid contact or communication with the sample solution 718, even if the sample solution 718 is not held within a container wall 702 of the sensor 700 or the sensor 700 does not have a container wall 702. The sample solution 718 may be any of the diluted samples or aliquots thereof described herein.

[0155]

[0165] 7A, one or more parameter analyzers 120 can be coupled to both the external reference electrode 710 and the conductor layer 714 of the first active electrode 704 and the second active electrode 706. The parameter analyzer 120 can be coupled to the external reference electrode 710 and the conductor layer 714 through one or more other layers of the sensor 700. The parameter analyzer 120 can be coupled to the first active electrode 704, the second active electrode 706, the external reference electrode 710, and any other active or reference electrodes, and can multiplex signals from each of the electrodes in parallel or alternating fashion.

[0156]

[0166] At least a portion of the external reference electrode 710 and the two active electrodes may be in fluid communication with or in fluid contact with the sample solution 718. As shown in Figure 7A, at least a portion of the external reference electrode 710 may extend into or be submerged in the sample solution 718. The sample solution 718 may refer to either the diluted sample 112, the reference sample 208, or an aliquot thereof.

[0157]

[0167] The external reference electrode 710 may also have a stable or known internal voltage and may act as a differential noise filter to remove electrical noise from measurements made using the sensor 700. In one embodiment, the external reference electrode 710 may be a stand-alone probe or electrode coupled to the parameter analyzer 120. In other embodiments, the external reference electrode 710 may be integrated with the parameter analyzer 120. As shown in FIG. 7A, the first active electrode 704 and the second active electrode 706 may be coupled to and share the same external reference electrode 710. Although FIG. 7A shows the first active electrode 704 and the second active electrode 706 coupled to separate parameter analyzers 120, it should be understood by one of ordinary skill in the art that the first active electrode 704 and the second active electrode 706 may be coupled to the same parameter analyzer 120 as is contemplated by the present disclosure.

[0158]

[0168] In one embodiment, the external reference electrode 710 can be or include a silver / silver chloride (Ag / AgCl) electrode. In other embodiments, the external reference electrode 710 can be or include a saturated calomel reference electrode (SCE) or a copper-copper (II) sulfate electrode (CSE). The external reference electrode 710 can also be a pseudo reference electrode including any metal that is not part of the active electrode, such as platinum, silver, gold, or combinations thereof, any metal oxide material or semiconductor oxide material, such as aluminum oxide, iridium oxide, silicon oxide, or any conductive polymer electrode, such as polypyrrole, polyaniline, polyacetylene, or combinations thereof.

[0159]

[0169] 7A, each of the first and second active electrodes 704, 706 can include at least one conductor layer 714 disposed or otherwise positioned on a substrate layer 708. The substrate layer 708 can be composed of any non-conductive material, such as, but not limited to, a polymer, an oxide, a ceramic, or a composite thereof.

[0160]

[0170] The conductor layer 714 can be composed of, but is not limited to, a metal, a semiconductor material, a metal / metal salt, or a combination thereof. For example, but not limited to, the conductor layer 714 can be composed of silicon, gold, silver, aluminum, platinum, or a composite thereof. The conductor layer 714 can also be an organic semiconductor, a carbon nanotube, graphene, an organic conductor, such as derived from polyacetylene, polyaniline, quinacridone, poly(3,4-ethylenedioxythiophene) or PEDOT, PEDOT:polystyrenesulfonate (PSS), or a combination thereof. The conductor layer 714 can be composed of any conductive material that allows for the measurement of a change in an electrical property, such as, but not limited to, a voltage change, a capacitance change, a conductance change, and / or a current change measured through the conductor layer 714, the redox active layer 712 or the functionalization layer 716, and the sample solution 718. The conductor layer 714 can also refer to a stack of multiple conductive layers, such as metal layers. For example, the metal layer may include a gold layer, a platinum layer, or a combination thereof.

[0161]

[0171] The first active electrode 704 may include a layer disposed on or otherwise covering the redox active layer 712 or the conductor layer 714. The redox active layer 712 may include a gold layer, a platinum layer, a metal oxide layer, a carbon layer, or a combination thereof on the conductor layer 714 (or multiple conductor layers 714). In some embodiments, the metal oxide layer may include an iridium oxide layer, a ruthenium oxide layer, or a combination thereof. In other embodiments, the conductor layer 714 may be the redox active layer 712 and may include a gold layer, a platinum layer, a metal oxide layer, a carbon layer, or a combination thereof.

[0162]

[0172] A parameter analyzer 120 coupled to the first active electrode 704 and the external reference electrode 710 (or another device coupled to the parameter analyzer 120, such as a computing device 116, not shown) can determine the ORP of the sample solution 718 by measuring the potential difference between the external reference electrode 710 and the first active electrode 704.

[0163]

[0173] In some embodiments, the parameter analyzer 120 can be a voltmeter or any other type of high impedance amplifier or source meter. The parameter analyzer 120 can measure the relative change in equilibrium potential at the interface between the redox active layer 712 and the sample solution 718 containing the electroactive redox species. The parameter analyzer 120 can also measure the relative change in equilibrium potential at the interface between the conductor layer 714 and the sample solution 718 containing the electroactive redox species. The change in equilibrium potential can be measured relative to the external reference electrode 710. The parameter analyzer 120 can also be used to apply a voltage or current to the external reference electrode 710 or to the active electrode.

[0164]

[0174] The solution properties of the sample solution 718 can change with changes in the amount of electroactive redox species due to energy use, oxygen uptake or release, growth, or metabolism of an infectious agent in the solution. For example, the amount of electroactive redox species in the sample solution 718 can change as a result of cellular activity performed by an infectious agent in the solution. As a more specific example, the amount of electron donors (e.g., the amount of energy carriers such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2)) in the sample solution 718 can change due to the growth or lack thereof of an infectious agent in the solution. As another more specific example, the amount of depleted oxygen in the sample solution 718 can change due to the growth or lack thereof of an infectious agent in the solution.

[0165]

[0175] The second active electrode 706 may include a functionalization layer 716 disposed on or otherwise covering the conductor layer 714. The functionalization layer 716 may include an oxide, a silane, DNA, a protein, an antibody, a self-assembled monolayer (SAM), an oxide, a buffered hydrogel, PVC, parylene, polyACE, or any other biochemically active material. The functionalization layer 716 may be a pH-sensitive or pH-active layer configured to interact with ions, analytes, or other molecules or byproducts in the sample solution 718. For example, the functionalization layer 716 may react with hydrogen ions (H + ) may contain hydroxyl groups capable of interacting with the

[0166]

[0176] A parameter analyzer 120 coupled to the second active electrode 706 and the external reference electrode 710 (or another device coupled to the parameter analyzer 120, such as a computing device 116, not shown) can determine the pH of the sample solution 718 by measuring the potential difference between the external reference electrode 710 and the second active electrode 706.

[0167]

[0177] The parameter analyzer 120 can measure the relative change in equilibrium potential at the interface between the functionalized layer 716 and a sample solution 718 containing ions, analytes, or other molecules. The parameter analyzer 120 can also measure the relative change in equilibrium potential at the interface between the conductor layer 714 and a sample solution 718 containing ions, analytes, or other molecules. The solution properties of the sample solution 718 can change with changes in the amount of ions, analytes, or other molecules due to energy use, oxygen uptake or release, growth, or metabolism of infectious agents in the solution. For example, the amount of hydrogen ions (H + ) can change as a result of cellular activity caused by infectious agents in the solution. The change in equilibrium potential can be measured relative to an external reference electrode 710. In such a case, it is the relative change in the electrical property of the sensor 700 that is measured by the parameter analyzer 120 (or a computing device 116 (not shown) coupled to the parameter analyzer 120).

[0168]

[0178] 7B illustrates a schematic diagram of another embodiment of a sensor 700 that may be used as part of the methods and systems described herein. The sensor 700 of FIG. 7B may be or may represent any of the sensors 122 shown in FIGS. 1, 2, and 4.

[0169]

[0179] The sensor 700 may be or may include an electrochemical cell having a container wall 702, a first active electrode 704 and a second active electrode 706 positioned on a substrate layer 708, and an on-chip reference electrode 720 positioned on the same substrate layer 708. Although two active electrodes are shown in Figure 7B, it should be understood by one of ordinary skill in the art that more than two active electrodes or multiple reference electrodes can be positioned on a substrate layer as contemplated by the present disclosure.

[0170]

[0180] The vessel wall 702, first active electrode 704, second active electrode 706, and backing layer 708 of Figure 7B may be identical to the vessel wall 702, first active electrode 704, second active electrode 706, and backing layer 708, respectively, of Figure 7A. A sample solution 718 may be in fluid communication with or otherwise exposed to the on-chip reference electrode 720, the first active electrode 704, and the second active electrode 706 simultaneously.

[0171]

[0181] Although not shown in FIG. 7B, a passivation layer may be disposed on or cover the on-chip reference electrode 720. The passivation layer may be configured to prevent interaction between the on-chip reference electrode 720 and redox active species, analytes, ions, or other molecules in the sample solution 718. For example, the passivation layer may be a pH-insensitive layer. The passivation layer may include a silane, a self-assembled monolayer (SAM), a buffered hydrogel, parylene, polyACE, or any other biochemically inert material.

[0172]

[0182] In this embodiment, the parameter analyzer 120 may have a lead connection wire, such as a copper wire, coupled to the conductor layer 714 of the active electrode and another lead connection wire connected to an on-chip reference electrode 720. The parameter analyzer 120 may be coupled to the first active electrode 704, the second active electrode 706, the on-chip reference electrode 720, and any other active or reference electrodes, and may multiplex signals from each of the electrodes in parallel or alternating fashion. The parameter analyzer 120 may also be used to apply a voltage or current to the on-chip reference electrode 720 or the active electrodes.

[0173]

[0183] In this and other embodiments, the sensor 700 shown in FIG. 7B miniaturizes the sensor configuration shown in FIG. 7A. The on-chip reference electrode 720 avoids the need for an external reference electrode, such as the external reference electrode 710. The on-chip reference electrode 720 can also be a silver / silver chloride (Ag / AgCl) electrode. In other embodiments, the on-chip reference electrode 720 can be, but is not limited to, a saturated calomel reference electrode (SCE) or a copper-copper(II) sulfate electrode (CSE). The on-chip reference electrode 720 provides a similar function to the external reference electrode 710.

[0174]

[0184] In one embodiment, the conductor layer 714 can be used as an on-chip reference electrode 720. The conductor layer 714 functioning as the on-chip reference electrode 720 can be a metal covered with a metal salt, such as a metal chloride. In another embodiment, the conductor layer 714 functioning as the on-chip reference electrode 720 can also be covered with an oxide. For example, the conductor layer 714 can be a silver / silver chloride contact. In some embodiments, the conductor layer 714 can be covered with a passivation layer, such as a KCL electrolyte gel or KCL solution, to prevent interaction of the conductor layer 714 with redox active species, analytes, ions, or other molecules in the sample solution 718 and to act as a reference electrode. In another embodiment, the on-chip reference electrode 720 can be covered with a small cell that stabilizes a small internal potential, such as a calomel electrode.

[0175]

[0185] Each of the individual variations or embodiments described and illustrated herein has discrete components and features that may be readily separated from or combined with any features of the other variations or embodiments. Modifications may be made to adapt a particular situation, material, composition of matter, process, process acts, or steps to the objective, spirit or scope of the present invention.

[0176]

[0186] The methods recited herein may be carried out in any order of the recited events that is logically possible, even in the recited order of events. For example, the flowcharts or process flows depicted in the Figures do not require the particular order depicted to achieve desired results. Moreover, additional steps or operations may be provided or steps or operations may be eliminated in order to achieve desired results.

[0177]

[0187] Those skilled in the art will appreciate that all or part of the methods disclosed herein may be embodied in a non-transitory machine-readable or accessible medium containing instructions readable or executable by a processor or processing unit of a computing device or other type of machine.

[0178]

[0188] Moreover, when a range of values ​​is provided, every intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the scope of the invention. Additionally, any optional feature of the described inventive variations may be defined or claimed independently or in combination with any one or more of the features described herein.

[0179]

[0189] All pre-existing subject matter cited herein (e.g., publications, patents, patent applications, and hardware) is incorporated herein by reference in its entirety, except to the extent that the subject matter conflicts with that of the present invention, in which case the subject matter of the present specification shall take precedence. The referenced items are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such material by virtue of prior invention.

[0180]

[0190] Reference to a singular item includes the possibility that a plural of the same item exists. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," "said," and "the" include plural references unless the context clearly dictates otherwise. It is further noted that the claims may be formulated to exclude any optional element. Thus, this statement is intended to serve as a prior basis for the use of exclusive terms such as "sole," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0181]

[0191] The present disclosure is not intended to be limited in scope to the specific embodiments defined, but is intended to cover alternatives, modifications, and equivalents of the variations or embodiments described herein. Moreover, the scope of the present disclosure fully encompasses other variations or embodiments that may become apparent to those skilled in the art in view of the present disclosure. The scope of the present invention is limited only by the scope of the appended claims.

Claims

1. 1. A method for preparing an output sample of a defined concentration, comprising the steps of: diluting an aliquot of the original sample containing the infectious agent by a dilution factor to produce a diluted sample; exposing one or more sensors to the diluted sample, wherein at least a portion of each of the one or more sensors is in fluid communication with the diluted sample upon exposure to the diluted sample; incubating the diluted sample at an incubation temperature, wherein the diluted sample is incubated while the one or more sensors are exposed to the diluted sample; monitoring the diluted sample for changes in solution properties using a parameter analyzer or computing device coupled to the one or more sensors; and cooling the diluted sample to a cooling temperature when a solution property of the diluted sample changes by a threshold amount to generate an output sample of the specified concentration; A method comprising:

2. retrieving a universal lookup table from a database prior to monitoring the change in solution properties of the diluted sample; and setting the threshold amount based on the specified concentration, concentration data obtained from the universal lookup table, and solution property data obtained from the universal lookup table; 10. The method of claim 1, further comprising: The universal lookup table is created from a plurality of strain-specific lookup tables that represent data measured from a plurality of reference samples monitored over time; and A method, wherein at least one of said plurality of reference samples comprises a reference infectious agent of a different species than the infectious agent in said original sample.

3. Each of the plurality of strain-specific lookup tables comprises: monitoring changes in solution properties of the reference sample over a time range; performing a sample enumeration assay of said reference sample over the same time period; converting the result of the sample enumeration assay to a reference sample concentration using a conversion factor; and correlating the reference sample concentration to a change in a solution property of the reference sample; The method of claim 2 , wherein the composition is produced by:

4. 4. The method of claim 3, wherein the universal lookup table is created by averaging all solution property variations obtained from the plurality of strain-specific lookup tables for each of the reference sample concentrations, and relating each of the reference sample concentrations to the averaged solution property variation.

5. 4. The method of claim 3, wherein the sample enumeration assay comprises an optical density measurement, a plate count assay, a flow cytometry assay, or a combination thereof.

6. retrieving a species-specific lookup table from a database based on the species of infectious agent in the original sample before monitoring the change in solution properties of the diluted sample; and setting the threshold amount based on the specified concentration, concentration data obtained from the species-specific lookup table, and solution property data obtained from the species-specific lookup table; 10. The method of claim 1, further comprising: The species-specific lookup table is generated from a plurality of strain-specific lookup tables that represent data obtained from a plurality of reference samples monitored over time; and A method, wherein each of the plurality of reference samples comprises a reference infectious agent of the same species as an infectious agent in the original sample.

7. Each of the plurality of strain-specific lookup tables comprises: monitoring changes in solution properties of the reference sample over a time range; performing a sample enumeration assay of said reference sample over the same time period; converting the result of the sample enumeration assay to a reference sample concentration using a conversion factor; and correlating the reference sample concentration to a change in a solution property of the reference sample; The method of claim 6 , wherein the composition is produced by:

8. 8. The method of claim 7, wherein the species-specific lookup table is created by averaging all solution property variations obtained from the plurality of strain-specific lookup tables for each of the reference sample concentrations, and relating each of the reference sample concentrations to the averaged solution property variation.

9. 8. The method of claim 7, wherein the sample enumeration assay comprises an optical density measurement, a plate count assay, a flow cytometry assay, or a combination thereof.

10. 2. The method of claim 1, wherein the incubation temperature is about 33°C to 37°C.

11. 2. The method of claim 1, wherein the cooling temperature is between about 4°C and 25°C.

12. 10. The method of claim 1, further comprising diluting the output sample at another dilution factor to generate a further diluted sample, the further diluted sample comprising an infectious agent concentration required for downstream testing.

13. 2. The method of claim 1, wherein the solution property is oxidation-reduction potential (ORP) and the one or more sensors are ORP sensors, each of the one or more ORP sensors comprising a redox active layer, and the ORP is monitored without the presence of any added reporter molecule in the diluted sample.

14. 14. The method of claim 13, wherein each of the one or more ORP sensors includes at least an active electrode and a reference electrode.

15. The method of claim 13 , wherein the redox active layer comprises a gold layer, a platinum layer, a metal oxide layer, a carbon layer, or a combination thereof.

16. 2. The method of claim 1, wherein the solution property is pH and the one or more sensors are pH sensors, each of the one or more pH sensors comprising a pH sensitive layer, and the pH is monitored without the presence of any added reporter molecule in the diluted sample.

17. 17. The method of claim 16, wherein each of the one or more pH sensors includes at least an active electrode and a reference electrode.

18. 17. The method of claim 16, wherein the pH sensitive layer comprises an oxide layer, a silane layer, a self-assembled monolayer (SAM), a hydrogel layer, a protein layer, a polymer layer, or a combination thereof.

19. 10. The method of claim 1, wherein the original sample comprises a body fluid, a wound swab or sample, a rectal swab or sample, other types of biological samples, cultures derived therefrom, or combinations thereof.

20. 20. The method of claim 19, wherein the bodily fluid comprises urine, blood, sputum, saliva, breast milk, spinal fluid, semen, vaginal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, amniotic fluid, a culture of the bodily fluid that tests positive for growth of an infectious agent, or a combination thereof.

21. 10. The method of claim 1, wherein the infectious agent comprises a bacterium, a fungus, a mold, or a combination thereof.

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