WO2024250102A1

WO2024250102A1 - Food packaging system for detecting pathogenic bacteria

Info

Publication number: WO2024250102A1
Application number: PCT/CA2024/050753
Authority: WO
Inventors: Tohid DIDAR; Yingfu Li; Carlos Filipe; Akansha PRASAD; Shadman KHAN; Jiuxing LI
Original assignee: Toyota Tsusho Canada Inc
Current assignee: Toyota Tsusho Canada Inc
Priority date: 2023-06-09
Filing date: 2024-06-05
Publication date: 2024-12-12
Anticipated expiration: 2025-12-09

Abstract

Provided herein is a food packaging system, comprising: (i) an inclined packaging compartment; (ii) the inclined packaging compartment comprises (a) a top opening, (b) a bottom opening or a bottom wall comprising a bottom opening, and (c) side walls of the compartment upstanding from the bottom opening or the bottom wall; (iii) a sensing interface (1801) at the bottom opening comprising a reagent-saturated membrane (100) and a detector (102) subjacent to the reagent-saturated membrane (100), the detector (102) comprising a polyolefin substrate and a biosensor for detecting a bacterial pathogen; wherein the side walls are for fluid-interface; wherein the side walls extend downward and inward toward the bottom opening or bottom wall at an interior angle from about 20 degrees to about 90 degrees; and wherein the inclined packaging compartment is configured to localize a test sample from the food product onto the sensing interface (1801).

Description

FOOD PACKAGING SYSTEM FOR DETECTING PATHOGENIC BACTERIA

Cross-reference to Related

[0001] This application claims the benefit of United States Provisional Patent Application No. 63/472,169 filed June 9, 2023, herein incorporated by reference in its entirety.

[0002] A computer readable form of the Sequence Listing “30012- P71959PC00_SequenceListing” (15,767 bytes) was created on June 3, 2024, is filed herewith by electronic submission and is incorporated by reference herein.

Field

[0003] The present disclosure relates to food packaging system for detecting pathogenic bacteria.

[0004] Foodborne illness represents a growing crisis, with an estimated annual caseload surpassing 600 million globally - largely attributed to the consumption of pathogen-contaminated food products.¹¹¹ In the United States alone, there are an average of 37 million annual incidences of foodborne illness, with associated treatment costs exceeding $14 billion.^{12 31} Concerningly, the growing demand for food and climate change-induced shifts in the prevalence of pathogens such as Escherichia coli and Salmonella typhimurium (i.e. Salmonella enterica serovar Typhimurium) are expected to intensify this crisis in the coming years.^14-61 While various procedural and regulatory interventions have been introduced to reduce the incidence of food contamination, testing all foods as they transition through the food production pipeline remains instrumental in illness prevention. That being said, ready-to-eat (RTE) food products are of particular concern, given the lack of a cooking step prior to consumption.¹⁷¹ Unfortunately, culturing protocols remain the industry standard for such testing, despite their costly, laborious, and time-consuming nature. [0005] In an effort to address these limitations, several on-site detection platforms have been developed.^18-131 However, such systems yield significant food waste and operate under the assumption that the food products selected and opened for testing are representative of all products within a given batch. To advance individual product monitoring, in-package incorporation of pathogen sensors is required - a premise that has been explored using various sensing modalities.^114-19’ ³⁷¹ Specifically, functional bacteriophages, oligonucleotides, and antibodies have all been used in situ as highly specific biorecognition probes for the detection of pathogens within complex food matrices. While promising proof-of-concepts have been reported, the means by which these systems have been validated are not commercially feasible.^120-221 Namely, these probes all rely on complementary reagents such as buffers and metal ions for functionality, meaning that food samples must be extensively treated with these agents prior to testing - a measure that severely alters their organoleptic properties. Further, reported tests largely involve contamination and testing of small food samples, wherein the target bacteria is localized to the test site. In reality, contamination can occur at any given site on bulk food products with large surface areas. Given that placing sensors across an entire food surface is commercially unfeasible, testing in a manner that provides results representative of the entire food sample is difficult. Lastly, these sensors largely detect target pathogens within liquid test media, which is not easily isolated within closed packaging systems. Consequently, there exists a critical gap between the myriad of sensing platforms discovered in recent works and their real-world implementation into food packaging for real-time food monitoring, necessitating the innovation of traditional packaging.

[0006] While food packaging has been reengineered significantly in recent years to create intelligent packaging materials that are more sustainable, delay food spoilage, and have innate antibacterial capabilities, redesign aimed at improved in situ sensing has not been proposed. ^123-271

Summary

[0007] The present disclosure describes a food packaging system for detecting a bacterial pathogen. The system's components include an inclined packaging tray (105), a sensing interface (1801 ) comprising a reagent saturated membrane (100) and a detector (102), wherein the detector features a polyolefin substrate and a biosensor.

[0008] Accordingly, herein provided is a food packaging system, comprising:

(i) an inclined packaging compartment for receiving and containing a food product;

(ii) the inclined packaging compartment comprises (a) a top opening, (b) a bottom opening or a bottom wall comprising a bottom opening, and (c) side walls of the compartment upstanding from the bottom opening or the bottom wall;

(iii) a sensing interface (1801 ) at the bottom opening comprising a reagent- saturated membrane (100) and a detector (102), wherein the detector is subjacent to the reagent-saturated membrane (100), and the detector (102) comprises a polyolefin substrate and a biosensor for detecting a bacterial pathogen; and

(iv) optionally, a wrap for covering and sealing the top opening; wherein the side walls are for fluid-interface; wherein the side walls extend downward and inward toward the bottom opening or bottom wall at an interior angle from about 20 degrees to about 90 degrees; and wherein the inclined packaging compartment is configured to localize a test sample from the food product onto the sensing interface (1801).

[0009] In some embodiments, the top opening has a square, a rectangular, a circular, a triangular, an oval, a pentagonal, a hexagonal, an octagonal, a rhomboidal, a parallelogrammatic, a trapezoidal, or an irregular shape. In some embodiments, the interior angle is about 40 degrees to about 90 degrees. In some embodiments, the interior angle is about 45 degrees to about 90 degrees (107). In some embodiments, the interior angle is about 45 degrees, about 60 degrees, or about 90 degrees. In some embodiments, the interior angle is about 45 degrees. In some embodiments, the side walls and bottom wall comprise polylactic acid (PLA), polyethylene terephthalate glycol (PETG), thermoplastic polyurethane (TPU), Nylon, high-density polyethylene (HDPE), and/or polyether ether ketone (PEEK). In some embodiments, the membrane comprises cotton, cotton-cellulose, cellulose, cellulose-polyester, and/or polyester. In some embodiments, the membrane comprises cotton. In some embodiments, the reagent comprises a buffer, a divalent metal ion, and/or a salt.

[0010] In some embodiments, the bacterial pathogen is at least one of Salmonella, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, Campylobacter jejuni, Staphylococcus aureus, Clostridium, Vibrio, Shigella, and Yersinia enterocolitica. In some embodiments, the Salmonella is Salmonella typhimurium. The Escherichia coli is Escherichia coli O157:H7. In some embodiments, the Listeria monocytogenes is Listeria monocytogenes. In some embodiments, the Clostridium is Clostridium perfringens or Clostridium botulinum. In some embodiments, the Vibrio is Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and/or Vibrio alginolyticus. In some embodiments, the Shigella is Shigella dysenteriae, Shigella flexneri, Shigella boydii, and/or Shigella sonnei.

[0011] In some embodiments, the biosensor comprises a nucleic acid probe. In some embodiments, the nucleic acid probe is attached to the polyolefin substrate. In some embodiments, the polyolefin substrate is COOH-activated polyolefin substrate, and the nucleic acid probe is attached to the COOH-activated polyolefin substrate connected through a N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide/N- Hydroxysuccinimide (EDC/NHS) cross-linker. In some embodiments, the nucleic acid probe is capable of detecting a RNase H2. In some embodiments, the RNase H2 is Salmonella typhimurium RNase H2, Listeria monocytogenes RNase H2, or Escherichia coli RNase H2. In some embodiments, the RNase H2 is Salmonella typhimurium RNase H2. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 5, 6, or 7. In some embodiments, the biosensor is configured for generating a fluorescent signal upon detection of the bacterial pathogen. In some embodiments, the biosensor is configured for generating a colorimetric signal upon detection of the bacterial pathogen. In some embodiments, the biosensor has a limit-of-detection of 10³ CFU/mL. In some embodiments, the food product is meat, produce, a dairy product, and/or a ready-to-eat food product. In some embodiments, the meat is beef, veal, pork, lamb, mutton, chicken, turkey, duck, goose, quail, pheasant, rabbit, venison, bison, elk, goat, horse, kangaroo, ostrich, alligator, frog, snail, squab, or guinea fowl. In some embodiments, the produce is a fruit or a vegetable. In some embodiments, the vegetable is a lettuce. In some embodiments, the ready-to-eat food product is a salad, a pre-cooked meal, a deli meat, a cheese, a bakery item, or a dessert. In some embodiments, the ready-to-eat food product is a ready-to-eat chicken product.

[0012] Also provided is a method of detecting bacterial pathogens on a food product, the method comprising:

(i) providing the food packaging system described herein;

(ii) placing the food product into the inclined packaging compartment;

(iii) allowing a test sample from the food product to localize onto the sensing interface (1801 ) facilitated by the angled side walls;

(iv) allowing the test sample to interact with the reagent-saturated membrane (100); and

(v) detecting the presence of a bacterial pathogen in the test sample using the biosensor of the detector (102) in the sensing interface (1801 ).

[0013] Also provided is a kit for detecting a bacterial pathogen on a food product, comprising:

(A) a food container comprising:

(i) an inclined packaging compartment for receiving and containing the food product;

(ii) the inclined packaging compartment comprises (a) a top opening, (b) a bottom opening, and (c) side walls of the compartment upstanding from the bottom opening or the bottom wall; wherein the side walls are for fluid-interface; wherein the side walls extend downward and inward toward the bottom opening or bottom wall at an interior angle from about 20 degrees to about 90 degrees; and wherein the inclined packaging compartment is configured to localize a test sample from the food product onto a sensing interface (1801),

(B) a reagent-saturated membrane (100) or a membrane (101 ) for receiving reagent; and a detector (102) comprising a polyolefin substrate and a biosensor for detecting the bacterial pathogen, wherein the detector (102) and the reagent-saturated membrane (100) are configured to form the sensing interface (1801).

[0014] In some embodiments, the top opening has a square, a rectangular, a circular, a triangular, an oval, a pentagonal, a hexagonal, an octagonal, a rhomboidal, a parallelogrammatic, a trapezoidal, or an irregular shape. In some embodiments, the kit further comprises at least one of a wrap, a buffer, a divalent metal salt, a dropper, a sealer, a pair of gloves, and instruction for use.

[0015] Also provided is a method of detecting a bacterial pathogen on a food product, the method comprising:

(i) providing a kit described herein;

(ii) applying the detector (102) to the bottom opening, optionally covering the bottom opening with a transparent seal;

(iii) applying to the bottom opening overlying the detector (102) (a) the reagent-saturated membrane (100) or (b) the membrane (101) for receiving reagent and adding the reagent onto and saturate the membrane (100), whereby the detector and the reagent-saturated membrane form a sensing interface (1801);

(iv) placing the food product into the inclined packaging compartment;

(v) allowing a test sample from the food product to localize onto the sensing interface (1801) facilitated by the angled side walls;

(vi) allowing the test sample to interact with the reagent-saturated membrane (100); and (vii) detecting the presence of a bacterial pathogen in the test sample using the biosensor of the detector (102).

[0016] In some embodiments, after step (iv), applying a wrap to cover and seal the top opening. In some embodiments, the reagent comprises a buffer, a divalent metal ion, and/or a salt.

[0017] These and other features and advantages of the present disclosure will become apparent from the following detailed description taken together with the accompanying drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred implementations of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from this detailed description.

Brief Description of the Drawings

[0018] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments can be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0019] FIG. 1A shows a schematic illustration of Lab-in-a-Package platform, in an exemplary embodiment of the present disclosure. FIG. 1 A shows a complete Lab- in-a-Package in situ detection platform with inclined packaging tray (105), reagent- saturated membrane (100), fluorescence imager (103) and sensor (102) incorporation shown for ready-to-eat (RTE) chicken products.

[0020] FIG. 1 B shows a schematic illustration of Lab-in-a-Package platform, in an exemplary embodiment of the present disclosure. FIG. 1 B shows inclined food packaging trays (105) with angles ranging from 45° to 90° (107) for test sample localization.

[0021] FIG. 1C shows a schematic illustration of Lab-in-a-Package platform, in an exemplary embodiment of the present disclosure. FIG. 1C shows a depiction of membrane (101) saturation with reagent components (100), diffusion of buffer components and target analyte to sensor (102) surface, and fouling prevention.

[0022] FIG. 1 D shows a schematic illustration of Lab-in-a-Package platform, in an exemplary embodiment of the present disclosure. FIG. 1 D shows a fluorescent nucleic acid probe (FNAP) sensor (102) development with corresponding material surface and biochemical modifications (104, 106, 108).

[0023] FIG. 2A shows the characterization of packaging models and membrane candidates based on application-relevant properties, in an exemplary embodiment of the present disclosure. FIG. 2A shows CAD models for all packaging models with top (202), bottom (204), and orthogonal views shown (206). Reported values represent the mean of all samples with error bars representing sample standard deviation. Asterisks represent significant differences at corresponding significance levels.

[0024] FIG. 2B shows the time required for a water droplet to fall down packaging edge, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0025] FIG. 2C shows the time required for 5 mL of buffer to reach sensing window when dispensed at a rate of 0.5 mL/s, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0026] FIG. 2D shows the percentage of original PBS volume localized on sensing window after 1 minute when dispensed at a rate of 0.2 mL/s, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0027] FIG. 2E shows the percentage of original chicken purge volume localized after 24h at 37°C, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0028] FIG. 2F shows SEM images of candidate membranes at 100X with overlays at 500X, in an exemplary embodiment of the present disclosure.

[0029] FIG. 2G shows the mean background fluorescence of candidate membranes, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0030] FIG. 2H shows absorption capacity of candidate membranes, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0031] FIG. 2I shows the volume of buffer diffused through candidate membranes after 2 minutes, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0032] FIG. 2J shows membrane effects on bacterial growth following a 6h incubation with E. coli, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0033] FIG. 2K shows bacterial diffusion through unsaturated membranes onto underlying substrates following a 6h incubation at 37°C with E. coli, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0034] FIG. 2L shows bacterial diffusion through buffer-saturated membranes onto underlying substrates following a 6h incubation at 37°C with E. coli, in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0035] FIG. 2M shows the membrane effects on bacterial growth following a 6h incubation with S. enterica serovar Typhimurium in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0036] FIG. 2N shows bacterial diffusion through unsaturated membranes onto underlying substrates following a 6h incubation at 37°C with S. enterica serovar Typhimurium in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0037] FIG. 20 shows bacterial diffusion through buffer-saturated membranes onto underlying substrates following a 6h incubation at 37°C with S. enterica serovar Typhimurium in an exemplary embodiment of the present disclosure. All reported values represent the mean of all samples with error bars representing sample standard deviation. All asterisks represent significant differences at corresponding significance levels.

[0038] FIG. 3A shows S. enterica serovar Typhimurium sensor development and testing, in an exemplary embodiment of the present disclosure. FIG. 3A shows a schematic illustration of S. enterica serovar Typhimurium-responsive nucleic acid probe (SEQ ID NO: 7) cleavage activity within food matrices, with associated precleavage, cleavage, and quencher separation states.

[0039] FIG. 3B shows S. enterica serovar Typhimurium sensor development and testing, in an exemplary embodiment of the present disclosure. FIG. 3B shows the sensitivity testing of nucleic acid probe using bacterial dilutions in chicken purge, with associated images with 100 pm scale bars. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0040] FIG. 3C shows S. enterica serovar Typhimurium sensor development and testing, in an exemplary embodiment of the present disclosure. FIG. 3C shows the temperature profile of nucleic acid probe with bacterial species of 10⁷ and 10⁵ CFU/mL at 4°C, 25°C, 37°C, and 45°C. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0041] FIG. 3D shows S. enterica serovar Typhimurium sensor development and testing, in an exemplary embodiment of the present disclosure. FIG. 3D shows the covalent attachment confirmation of nucleic acid probe on substrate surface. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0042] FIG. 3E shows S. enterica serovar Typhimurium sensor development and testing, in an exemplary embodiment of the present disclosure. FIG. 3E shows the stability testing of developed sensor tested with 10⁶ to 10³ CFU/mL of bacteria after storage for three months at 4°C. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0043] FIG. 3F shows S. enterica serovar Typhimurium sensor development and testing, in an exemplary embodiment of the present disclosure. FIG. 3F shows the specificity testing of nucleic acid probe using various bacterial species at 10⁶ CFU/mL, with associated images with 100 pm scale bars. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0044] FIG. 4A shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4A shows a schematic illustration of in situ sensing interface (1801) with FNAP-based S. enterica serovar Typhimurium detection. FIG. 4A was created using BioRender. [0045] FIG. 4B shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4B shows images of a top view (202) of the packaging platform assembly, involving (i) sensor (102) implantation within sensing window, (ii) membrane (100) incorporation, and (iii) food addition into the package. Scale bars represent 3 cm on printed packaging tray.

[0046] FIG. 4C shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4C shows the inherent fluorescence of chicken purge at four fluorescence wavelengths with the mean fluorescent values of overlayed cotton membranes shown with shaded boxes. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0047] FIG. 4D shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4D shows the MgCL concentration effects for membrane absorption and diffusion. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0048] FIG. 4E shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4E shows the sensitivity testing following in situ full platform testing of contaminated whole chicken sample, with associated images with 100 pm scale bars. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0049] FIG. 4F shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4F shows contamination of food products from (i) various avenues of contamination, introduced during (ii) stages of the production process. FIG. 4F was created using BioRender.

[0050] FIG. 4G shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4G shows induced real- world contamination detection in situ with Lab-in-a-Package platform. All reported values represent the mean of all samples with error bars representing standard error of the mean. All asterisks represent significant differences at corresponding significance levels.

[0051] FIG. 4H shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4H shows an optical image of an experimental set-up for handheld fluorescence scanner with associated smartphone readout.

[0052] FIG. 4I shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. S. enterica serovar Typhimurium detection using FNAP sensor (102) as visualized using a handheld scanner, and associated images with 3.33 mm scale bars.

[0053] FIG. 4J shows the Lab-in-a-Package platform development and testing, in an exemplary embodiment of the present disclosure. FIG. 4J shows the handheld fluorescence detection of S. enterica serovar Typhimurium in Lab-in-a-Package, with associated sensor images with 3.33 mm scale bars.

[0054] FIG. 5 shows the top (202) views of 45-, 60-, and 90-degrees 3D-printed packaging models (left to right) with bottom view (204) images overlayed, in an exemplary embodiment of the present disclosure. Scale bars represent 2.5 cm on printed packaging trays. Printed trays were smoothened.

[0055] FIG. 6 shows 2D drawings of packaging trays with key dimensions shown in mm in isometric (600), side (602) and bottom views (604), in an exemplary embodiment of the present disclosure. FIG. 6 shows the sensor window dimensions highlighted in the bottom view (204) along with dimensions for the inner edges used to secure sensors in place.

[0056] FIG. 7 shows the volume localization over time for all packaging models based on an original applied volume of 5 mL across 10 and 20 seconds and 10 mL across 30 to 60 seconds, in an exemplary embodiment of the present disclosure. Reported values represent mean of all samples with error bars representing sample standard deviation. [0057] FIG. 8 shows microscopic images of tested membrane materials at 4X with overlays of 10X images, in an exemplary embodiment of the present disclosure. Scale bars represent 500 pm at 4X and 100 pm at 10X.

[0058] FIG. 9A shows membrane absorption capacity over time based on volume of buffer absorbed, in an exemplary embodiment of the present disclosure. FIG. 9A shows carrying capacity of cotton-cellulose and cellulose membranes over 30 seconds, in an exemplary embodiment of the present disclosure. Reported values represent the mean of all samples with error bars representing sample standard deviation. Graphs with different axes ranges were used based on the higher absorption capacities of cellulose and cotton-cellulose materials compared to cotton, polyester, and cellulose polyester.

[0059] FIG. 9B shows membrane absorption capacity over time based on volume of buffer absorbed, in an exemplary embodiment of the present disclosure. FIG. 9B shows carrying capacity of cellulose-polyester, cotton, and polyester membranes over 10 seconds. Reported values represent the mean of all samples with error bars representing sample standard deviation. Graphs with different axes ranges were used based on the higher absorption capacities of cellulose and cotton-cellulose materials compared to cotton, polyester, and cellulose polyester.

[0060] FIG. 9C shows membrane absorption capacity over time based on volume of buffer absorbed, in an exemplary embodiment of the present disclosure. FIG. 9C shows the carrying capacity of all five membrane materials over 30 minutes. Reported values represent the mean of all samples with error bars representing sample standard deviation. Graphs with different axes ranges were used based on the higher absorption capacities of cellulose and cotton-cellulose materials compared to cotton, polyester, and cellulose polyester.

[0061] FIG. 10 shows membrane buffer retention over 120 hours as a percent of the original volume of buffer applied for all five membrane materials, in an exemplary embodiment of the present disclosure. Membranes were submerged in excess PBS buffer for 1 minute and stored for 120 hours. Reported values represent the mean of all samples with error bars representing sample standard deviation. [0062] FIG. 11 shows membrane porosity characterization based on percent area covered by pores compared to total sample area, in an exemplary embodiment of the present disclosure. Unmodified membrane SEM images are shown in the top row and analyzed SEM images with pores are shown in the bottom row. Scale bars represent 500 pm.

[0063] FIG. 12A shows the characterization of membrane antifouling capabilities, in an exemplary embodiment of the present disclosure. FIG. 12A shows optical density measurement of chicken purge, chicken purge filtered through a cotton membrane, and water (control). Asterisks represent a significant difference between OD of membrane filtered chicken purge and unfiltered chicken purge at the corresponding significance level.

[0064] FIG. 12B shows a SEM image of cotton membrane saturated in chicken juice at 100X with 500X overlay. Scale bars represent 500 pm at 100X and 100 pm at 500X.

[0065] FIG. 13 shows the calibration curve for the determination of probe density on sensing interface (1801), in an exemplary embodiment of the present disclosure. TRITC-labelled single-stranded DNA molecules were used to establish a calibration curve correlating fluorescence per unit area and oligonucleotide content. The resultant linear relationship was used to quantify immobilized sensing probe density based on NaOH-induced maximal fluorescence per unit area. The average of sensing probe values are denoted as a unique data point.

[0066] FIG. 14 shows the effect of chicken purge on bacterial growth, in an exemplary embodiment of the present disclosure. FIG. 14 shows the level of bacterial concentration present in chicken purge contaminated with bacteria, bacteria resuspended in PBS buffer, and uncontaminated chicken purge. Reported values represent the mean of all samples with error bars representing sample standard deviation.

[0067] FIG. 15 shows a linear regression analysis on FNAP sensor (102) sensitivity data (FIG. 3B), in an exemplary embodiment of the present disclosure. Regression coefficient and model equation are shown. Model significance was evaluated based on slope coefficient value (P<0.001).

[0068] FIG. 16 shows a linear regression analysis on Lab-in-a-Package sensitivity (FIG. 4E), in an exemplary embodiment of the present disclosure. Regression coefficient and model equation are shown. Model significance was evaluated based on slope coefficient value (P<0.05).

[0069] FIG. 17 shows optical images of complete Lab-in-a-Package set-up in an exemplary embodiment of the present disclosure. FIG. 17 shows a whole, unprocessed RTE chicken product sample and polyolefin food wrap from the top view (202). FIG. 17 shows a saturated membrane (100), FNAP sensor (102), and inclined tray (105) shown in the bottom view (204).

[0070] FIG. 18 shows an overview of the Lab-in-a-Package sensing window in an exemplary embodiment of the present disclosure. FIG. 18 shows an optical image of the sensing interface (1801) with saturated membrane (100) and FNAP sensor (102) shown within the sensing window of the redesigned packaging tray. Scale bars represent 0.6 cm. FIG. 18 shows a zoomed-in optical image of FNAP sensor arrays (1802) with 2.5 cm scale bars.

[0071] FIG. 19 shows a S. enterica serovar Typhimurium growth study in an exemplary embodiment of the present disclosure. FIG. 19 demonstrates exponential growth of an original 10² CFU/mL sample over a 4-hours timespan.

[0072] FIG. 20 shows a full system specificity testing where samples were contaminated with a mixture of common food contaminants including E. coli O157:H7 (EC), Listeria monocytogenes (LM), and S. enterica serovar Typhimurium (ST), in an exemplary embodiment of the present disclosure. Reported values represent the mean of all samples with error bars representing standard error of the mean. Asterisks represent a significant difference at corresponding significance level.

[0073] FIG. 21 shows a target verification study comparing the concentration of S. enterica serovar Typhimurium recovered from the sensor surface after an 8-hour incubation period compared to the initial contaminated purge sample, in an exemplary embodiment of the present disclosure. Reported values represent the mean of all samples with error bars representing standard deviation. [0074] FIG. 22A shows the full system testing with S. enterica serovar Typhimurium contaminated lettuce samples, in an exemplary embodiment of the present disclosure. FIG. 22A shows an optical image of experimental set-up.

[0075] FIG. 22B shows the full system testing with S. enterica serovar Typhimurium contaminated lettuce samples, in an exemplary embodiment of the present disclosure. FIG. 22B shows the quantification of sensor signals from lettuce samples contaminated with 10⁶ CFU/mL of spiked produced washing water. Reported values represent the mean of all samples with error bars representing standard deviation. Asterisks represent a significant difference at corresponding significance level.

[0076] Further aspects and features of the example embodiments described herein will appear from the following description taken together with the accompanying drawings.

Detailed Description of the Disclosure

[0077] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0078] All features disclosed in the specification, including the claims, abstract, and drawings, and all the steps in any method or process disclosed, can be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise.

I. Definitions

[0079] The term “food packing system” as used herein refers to materials used to contain, protect, preserve, transport, and provide information about food products from the point of production to the point of consumption. It includes packaging that houses the food and associated assembly. The system ensures food safety and quality, extends shelf life, and maintains the sensory and nutritional properties of the food. Additionally, the system can provide essential information to consumers about the product inside, such as its ingredients, nutritional facts, expiration date, cooking or usage instructions. Food packaging systems can also include intelligent packaging that uses sensor technology to provide information about the condition of the food product over time. It can include time-temperature indicators, freshness indicators, or sensors that provide signals to indicate if the food product is fresh, has been properly stored, or is past its shelf life. Intelligent packaging can also use RFID tags or QR codes to provide more information about the food product or its journey through the supply chain. A biosensor can be incorporated in intelligent packaging to determine contamination such as bacterial contamination.

[0080] As used herein, the term “sample” or "test sample" refers to any material in which the presence or amount of an analyte is unknown and can be determined in an assay. The sample can be from any source, for example, any biological (e.g. human or animal samples, including clinical samples), environmental (e.g. water, soil or air) or natural (e.g. plants, food) source, orfrom any processed, manufactured or synthetic source (e.g. food or drinks). The sample can be comprised or is suspected of comprising one or more analytes.

[0081] The term “bacterial pathogen”, as used herein, refers to bacteria capable of causing disease or illness in hosts. Food-borne bacterial pathogens, which include but are not limited to Salmonella, Campylobacter, Listeria, and Escherichia coli, can induce foodborne illnesses, commonly known as food poisoning, when ingested through contaminated food. Contamination can occur at various stages of the food supply chain, from production and processing to preparation and consumption. The symptoms of illnesses caused by these pathogens can vary based on the bacteria type and individual’s health status. Prevention of bacterial contamination is a critical aspect of food safety practices, which involves maintaining hygiene and sanitation during all stages of food production, preparation, and storage, as well as avoiding consumption of contaminated food.

[0082] The term “fluid-interface” as used herein refers to liquid-solid interface, for example, between a packaging tray and fluids released by packaged food product. [0083] The term “nucleic acid” or “nucleic acid molecule” as used herein refers to biopolymer comprising monomers of nucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), hybrid DNA/RNA, and other polynucleotides of modified nucleotides and/or nucleotide derivatives, and can be double stranded (ds), single stranded (ss), or a multiplex molecule. The nucleic acid can contain multiple strands held together by complementarity or partial complementarity. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule”, “DNA molecule”, and “RNA molecule” embrace chemically, enzymatically, or metabolically modified forms. Examples of modified nucleotides which can be used to generate the nucleic acids disclosed herein include xanthine, hypoxanthine, 2-aminoadenine, 6- methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8- aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8- substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5- trifluoro cytosine. Alternatively, the nucleic acid molecules can be produced biologically using an expression vector. In some embodiments, modified nucleotides comprise one or more modified bases (e.g. unusual bases such as inosine, and functional modifications to the bases such as amino modifications), modified backbones (e.g. peptide nucleic acid, PNA) and/or other chemically, enzymatically, or metabolically modified forms. The term “functional fragment” as used herein refers to a fragment of the nucleic acid that retains the functional property of the full-length nucleic acid, for example, the ability of the fragment to act as a substrate for detecting a particular analyte, for example, Salmonella typhimurium RNase H2.

[0084] As used herein, the term "signal" refers to the measurable response or output that a detectable label or moiety produces when it interacts with a specific target or undergoes a particular reaction. This signal is used to indicate the presence, quantity, or condition of the target of interest. Detectable labels can be fluorescent dyes, enzymes, chromogenic substrates, or any other molecules or substances that produce a signal. This signal can be detected using appropriate equipment or methods, including those that allow for direct visualization by the human eye. The detection and analysis of these signals facilitate the interpretation of results. For example, in a fluorescence-based detection system, the signal would be the light emitted by the fluorescent label when it is excited by a specific wavelength of light. The intensity of this emitted light can be measured and used to determine the presence or concentration of the target substance.

[0085] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

[0086] Further, terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0087] More specifically, the term “about” means plus or minus 0.1 to 50%, 5- 50%, or 10-40%, 10-20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made. [0088] As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds.

[0089] In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0090] The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

[0091] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

II. Systems, nucleic acid, kits, and methods

[0092] The food packaging system provided herein offers high detection performance and real-world viability, marking a significant advancement in the field of food safety monitoring. By addressing sensor visualization, sample localization, reagent retention, analyte diffusion, and specific pathogen detection, this food packaging system serves as a potent tool for in situ, real-time monitoring of packaged foods. The inventors herein disclose a food monitoring packaging system that: (A) enables sensor visualization without disrupting the closed package, (B) localizes all sample solution released by the food matrix onto the sensor, (C) retains necessary reagents within the food packaging in a manner that minimizes organoleptic alterations to the adjacent food, and (D) facilitates analyte diffusion from the food matrix onto the sensor surface. Achieving these four objectives then enables (E) the in situ integration of biorecognition elements, to advance complete hands-free, real-time monitoring of packaged foods. The disclosed system employs food safe materials to ensure regulatory and commercial viability. The system’s primary components include an inclined packaging tray (105), a sensing interface (1801 ) comprising a reagent- saturated membrane (100) and a detector (102). The inclined packaging tray (105), with fluid-interface angles between about 20 degrees to about 90 degrees, or between about 45 degrees to about 90 degrees (107), helps localize the test sample from the food matrix onto the sensing interface (1801). This unique design facilitates sensor visualization without disrupting the closed package and ensures the full capture of the sample solution released by the food matrix. The reagent-saturated membrane (100), located at the packaging tray’s base, retains necessary reagents within the food packaging in a way that minimizes organoleptic alterations to the adjacent food. This membrane also facilitates the diffusion of analytes from the food matrix onto the detector (102) surface. The detector (102), subjacent to the reagent-saturated membrane (100), comprises a polyolefin substrate and a biosensor for detecting bacterial pathogens. The polyolefin substrate enables the integration of various pathogen sensing platforms, enhancing the system’s versatility.

[0093] Accordingly, the present disclosure provides a food packaging system, comprising:

(iii) a sensing interface (1801 ) at the bottom opening comprising a reagent- saturated membrane (100) and a detector (102), wherein the detector is subjacent to the reagent-saturated membrane (100), and the detector (102) comprises a polyolefin substrate and a biosensor for detecting a bacterial pathogen; and (iv) optionally, a wrap for covering and sealing the top opening; wherein the side walls are for fluid-interface; wherein the side walls extend downward and inward toward the bottom opening or bottom wall at an interior angle from about 20 degrees to about 90 degrees; and wherein the inclined packaging compartment is configured to localize a test sample from the food product onto the sensing interface (1801).

[0094] In some embodiments, the top opening has a square, a rectangular, a circular, a triangular, an oval, a pentagonal, a hexagonal, an octagonal, a rhomboidal, a parallelogrammatic, a trapezoidal, or an irregular shape. In some embodiments, the top opening has a rectangular shape. In some embodiments, the detector (102) covers and seals the bottom opening. In some embodiments, a transparent seal covers and seal the bottom opening. In some embodiments, the interior angle is about 20 degrees to about 85 degrees. In some embodiments, the interior angle is about 20 degrees to about 80 degrees. In some embodiments, the interior angle is about 20 degrees to about 75 degrees. In some embodiments, the interior angle is about 20 degrees to about 70 degrees. In some embodiments, the interior angle is about 20 degrees to about 65 degrees. In some embodiments, the interior angle is about 20 degrees to about 60 degrees. In some embodiments, the interior angle is about 20 degrees to about 55 degrees. In some embodiments, the interior angle is about 20 degrees to about 50 degrees. In some embodiments, the interior angle is about 20 degrees to about 45 degrees. In some embodiments, the interior angle is about 20 degrees to about 40 degrees. In some embodiments, the interior angle is about 20 degrees to about 35 degrees. In some embodiments, the interior angle is about 20 degrees to about 30 degrees. In some embodiments, the interior angle is about 20 degrees to about 25 degrees. In some embodiments, the interior angle is about 25 degrees to about 85 degrees. In some embodiments, the interior angle is about 25 degrees to about 80 degrees. In some embodiments, the interior angle is about 25 degrees to about 75 degrees. In some embodiments, the interior angle is about 25 degrees to about 70 degrees. In some embodiments, the interior angle is about 25 degrees to about 65 degrees. In some embodiments, the interior angle is about 25 degrees to about 60 degrees. In some embodiments, the interior angle is about 25 degrees to about 55 degrees. In some embodiments, the interior angle is about 25 degrees to about 50 degrees. In some embodiments, the interior angle is about 25 degrees to about 45 degrees. In some embodiments, the interior angle is about 25 degrees to about 40 degrees. In some embodiments, the interior angle is about 25 degrees to about 35 degrees. In some embodiments, the interior angle is about 25 degrees to about 30 degrees. In some embodiments, the interior angle is about 30 degrees to about 85 degrees. In some embodiments, the interior angle is about 30 degrees to about 80 degrees. In some embodiments, the interior angle is about 30 degrees to about 75 degrees. In some embodiments, the interior angle is about 30 degrees to about 70 degrees. In some embodiments, the interior angle is about 30 degrees to about 65 degrees. In some embodiments, the interior angle is about 30 degrees to about 60 degrees. In some embodiments, the interior angle is about 30 degrees to about 55 degrees. In some embodiments, the interior angle is about 30 degrees to about 50 degrees. In some embodiments, the interior angle is about 30 degrees to about 45 degrees. In some embodiments, the interior angle is about 30 degrees to about 40 degrees. In some embodiments, the interior angle is about 30 degrees to about 35 degrees. In some embodiments, the interior angle is about 35 degrees to about 85 degrees. In some embodiments, the interior angle is about 35 degrees to about 80 degrees. In some embodiments, the interior angle is about 35 degrees to about 75 degrees. In some embodiments, the interior angle is about 35 degrees to about 70 degrees. In some embodiments, the interior angle is about 35 degrees to about 65 degrees. In some embodiments, the interior angle is about 35 degrees to about 60 degrees. In some embodiments, the interior angle is about 35 degrees to about 55 degrees. In some embodiments, the interior angle is about 35 degrees to about 50 degrees. In some embodiments, the interior angle is about 35 degrees to about 45 degrees. In some embodiments, the interior angle is about 35 degrees to about 40 degrees. In some embodiments, the interior angle is about 40 degrees to about 85 degrees. In some embodiments, the interior angle is about 40 degrees to about 80 degrees. In some embodiments, the interior angle is about 40 degrees to about 75 degrees. In some embodiments, the interior angle is about 40 degrees to about 70 degrees. In some embodiments, the interior angle is about 40 degrees to about 65 degrees. In some embodiments, the interior angle is about 40 degrees to about 60 degrees. In some embodiments, the interior angle is about 40 degrees to about 55 degrees. In some embodiments, the interior angle is about 40 degrees to about 50 degrees. In some embodiments, the interior angle is about 45 degrees to about 90 degrees (107). In some embodiments, the interior angle is about 45 degrees to about 85 degrees. In some embodiments, the interior angle is about 45 degrees to about 80 degrees. In some embodiments, the interior angle is about 45 degrees to about 75 degrees. In some embodiments, the interior angle is about 45 degrees to about 70 degrees. In some embodiments, the interior angle is about 45 degrees to about 65 degrees. In some embodiments, the interior angle is about 45 degrees to about 60 degrees. In some embodiments, the interior angle is about 45 degrees to about 55 degrees. In some embodiments, the interior angle is about 40 degrees to about 50 degrees. In some embodiments, the interior angle is about 40 degrees to about 45 degrees. In some embodiments, the interior angle is about 45 degrees to about 50 degrees. In some embodiments, the interior angle is about 45 degrees to about 85 degrees. In some embodiments, the interior angle is about 20 degrees, about

25 degrees, about 30 degrees, about 35 degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39 degrees, about 40 degrees, about 41 degrees, about 42 degrees, about 43 degrees, about 44 degrees, about 45 degrees, about 46 degrees, about 47 degrees, about 48 degrees, about 49 degrees, about 50 degrees, about 55 degrees, about 60 degrees, about 65 degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85 degrees, or about 90 degrees. In some embodiments, the interior angle is about 40 degrees, about 41 degrees, about 42 degrees, about 43 degrees, about 44 degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85 degrees, or about 90 degrees. In some embodiments, the interior angle is about 20 degrees, about 30 degrees, about 45 degrees, about 60 degrees, or about 90 degrees. In some embodiments, the interior angle is about 45 degrees.

[0095] The materials used in the food packaging system, including the membrane and the substrate, are all food-safe to ensure the system’s regulatory and commercial viability. In some embodiments, the food packaging system comprises a wrap. In some embodiments, the wrap comprises polyethylene, polyvinylidene chloride, polypropylene, cellophane, parchment paper, beeswax, silicone, cellulose, and/or compostable plastic material. In some embodiments, the inclined packaging compartment comprises polylactic acid (PLA), polyethylene terephthalate glycol (PETG), thermoplastic polyurethane (TPU), Nylon, high-density polyethylene (HDPE), and/or polyether ether ketone (PEEK). In some embodiments, the side walls comprise PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the bottom wall comprises PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises a top wall covering the opening. In some embodiments, the top wall comprises PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the Nylon is Nylon 6 or Nylon 12. In some embodiments, the inclined packaging compartment is smoothened by sanding, heat, or a chemical. In some embodiments, the chemical is acetone or XTC-3D. In some embodiments, the inclined packaging compartment comprises sanded-PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises heat-treated PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises chemically treated PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises heat-treated PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises acetone- treated PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises XTC-3D-coated PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. In some embodiments, the inclined packaging compartment comprises heat-treated PLA, PETG, TPU, Nylon, HDPE, and/or PEEK. The present inventors have brought to light certain materials which demonstrate significant utility as membrane material, based on background fluorescence, reagent absorption, reagent diffusion, bacterial diffusion capabilities, and anti-fouling abilities. In some embodiments, the membrane comprises any food-safe material. In some embodiments, the membrane comprises cotton, cotton-cellulose, cellulose, cellulosepolyester, and/or polyester. In some embodiments, the membrane comprises cotton. In some embodiments, the membrane comprises an anti-fouling barrier for the detector (102). In some embodiments, the polyolefin substrate comprises polyethylene, polypropylene, ethylene vinyl alcohol, polybutene-1 , and/or metallocene polyolefin. In some embodiments, the polyethylene comprises low-density polyethylene (LDPE). In some embodiments, the reagent comprises a buffer, a metal ion, and/or a salt. In some embodiments, the buffer is at a concentration of about 20 mM to about 100 mM. In some embodiments, the buffer is any suitable food-safe buffer. In some embodiments, the buffer comprises citric acid, acetic acid, lactic acid, malic acid, phosphoric acid, tartaric acid, potassium citrate, calcium lactate, potassium phosphate, sodium acetate, sodium citrate, sodium lactate, and/or sodium phosphate. In some embodiments, the metal ion is a divalent metal ion. In some embodiments, the divalent metal ion is Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, and/or Mn²⁺. In some embodiments, the divalent metal ion is Mg²⁺. In some embodiments, the Mg²⁺ is at a concentration of about 20 mM to 50 about mM in the buffer. In some embodiments, the Mg²⁺ is at a concentration of about 25 mM to about 35 mM in the buffer. In some embodiments, the Mg²⁺ is at a concentration of about 25 mM, about 30 mM, or about 35 mM in the buffer. In some embodiments, the Mg²⁺ is at a concentration of about 30 mM in the buffer. In some embodiments, the salt is CaCl2, CeH CaOe, CaCOs, MgSO4, MgC , ZnSO4, FeSO4, Ci2H22FeOi4, MnSO4, and/or CuSCk Salt is MgCh In some embodiments, the MgCl2 is at a concentration of about 20 mM to 50 about mM in the buffer. In some embodiments, the MgCl2 is at a concentration of about 25 mM to about 35 mM in the buffer. In some embodiments, the MgCl2 is at a concentration of about 25 mM, about 30 mM, or about 35 mM in the buffer. In some embodiments, the MgC is at a concentration of about 30 mM in the buffer.

[0096] The food packaging system described herein can be configured to detect any food-borne pathogenic bacteria, provided that an appropriate biosensor for identifying the bacteria is available. For example, the bacterial pathogens that the food packaging system can detect include, but are not limited to, Salmonella, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, Campylobacter jejuni, Staphylococcus aureus, Clostridium, Vibrio, Shigella, and Yersinia enterocolitica. In some embodiments, the bacterial pathogen is at least one of Salmonella, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, Campylobacter jejuni, Staphylococcus aureus, Clostridium, Vibrio, Shigella, and Yersinia enterocolitica. In some embodiments, the Salmonella is Salmonella typhimurium. In some embodiments, the Salmonella is Salmonella enterica serovar Typhimurium. In some embodiments, the Escherichia coli is Escherichia coli O157:H7. In some embodiments, the Listeria monocytogenes is Listeria monocytogenes V₂a. In some embodiments, the Clostridium is Clostridium perfringens or Clostridium botulinum. In some embodiments, the Vibrio is Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and/or Vibrio alginolyticus. In some embodiments, the Shigella is Shigella dysenteriae, Shigella flexneri, Shigella boydll, and/or Shigella sonnei.

[0097] The biosensor can be a nucleic acid probe. In some embodiments, the biosensor comprises a nucleic acid probe. The polyolefin substrate can be activated to facilitate the attachment of the nucleic acid probe, for example, with the activation achieved through a COOH-activation process. The nucleic acid probe is then attached to the COOH-activated polyolefin substrate using a chemical such as a N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide/N-Hydroxysuccinimide (EDC/NHS) crosslinker. In some embodiments, the nucleic acid probe is attached to the COOH- activated polyolefin substrate using an epoxy, amine, carboxyl, aldehyde or EDC/NHS cross-linker. In some embodiments, the nucleic acid probe is attached to the COOH- activated polyolefin substrate using an epoxy cross-linker. In some embodiments, the nucleic acid probe is attached to the COOH-activated polyolefin substrate using an amine cross-linker. In some embodiments, the nucleic acid probe is attached to the COOH-activated polyolefin substrate using a carboxyl cross-linker. In some embodiments, the nucleic acid probe is attached to the COOH-activated polyolefin substrate using an aldehyde cross-linker. In some embodiments, the nucleic acid probe is attached to the COOH-activated polyolefin substrate using a N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide/N-Hydroxysuccinimide (EDC/NHS) crosslinker. In some embodiments, the nucleic acid probe is attached to the polyolefin substrate. In some embodiments, the polyolefin substrate is COOH-activated polyolefin substrate and the nucleic acid probe is attached to the COOH-activated polyolefin substrate connected through an EDC/NHS cross-linker.

[0098] The system described herein comprises a biosensor that is configured to generate a signal upon detecting the bacterial pathogen. This signal can either be, for example, fluorescent or colorimetric, depending on the design of the specific biosensor used. The nucleic acid probe used in the system can be designed, for example, as a synthetic fluorescent acid probe (FNAP) to detect, for example, RNase H2, a protein found in Salmonella typhimurium (i.e. Salmonella enterica serovar Typhimurium), Listeria monocytogenes, and Escherichia coli. This nucleic acid probe, when integrated into the system, acts as a highly specific substrate for cleavage by the RNase H2 of the bacteria. This reaction is trackable through the integration of, for example, a fluorophore-quencher pairing into the FNAP construct, providing real-time read out on the presence of pathogens within the packaged food.

[0099] In some embodiments, the nucleic acid probe is capable of detecting a RNase H2. In some embodiments, the RNase H2 is Salmonella typhimurium RNase H2, Listeria monocytogenes RNase H2, or Escherichia coli RNase H2. In some embodiments, the RNase H2 is Salmonella typhimurium RNase H2. In some embodiments, the sensing surface has a nucleic acid probe density of about 1.3 x 10' ⁵ nmol per array spot. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 5, 6, or 7. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the biosensor is configured for generating a fluorescent signal upon detection of the bacterial pathogen. In some embodiments, the fluorescent signal is detected by a fluorescent scanner. In some embodiments, the fluorescent scanner is a handheld fluorescent scanner. In some embodiments, the handheld fluorescent scanner is a smartphone. In some embodiments, the biosensor is configured for generating a colorimetric signal upon detection of the bacterial pathogen. The biosensor described herein is also stable and sensitive in detecting bacterial pathogens. In some embodiments, the biosensor is stable at 25°C for 5 days in the presence of the bacteria. In some embodiments, the biosensor is stable at 4°C for three months. In some embodiments, the biosensor is stable at 4°C for three months in the absence of the bacteria. In some embodiments, the biosensor has a limit-of-detection of 10³ CFU/mL for detecting pathogenic bacteria. In some embodiments, the biosensor has a limit-of-detection of 10³ CFU/mL for detecting Salmonella typhimurium. In some embodiments, the biosensor has a limit-of-detection of 10³ CFU/g for detecting bacteria.

[00100] The food packaging system described herein is versatile and can be used with various types of food matrices, including meat, produce, a dairy product, and a ready-to-eat food product. In some embodiments, the food product is meat, produce, a dairy product, and/or a ready-to-eat food product. In some embodiments, the meat is beef, veal, pork, lamb, mutton, chicken, turkey, duck, goose, quail, pheasant, rabbit, venison, bison, elk, goat, horse, kangaroo, ostrich, alligator, frog, snail, squab, or guinea fowl. In some embodiments, the produce is a fruit or a vegetable. In some embodiments, the fruit is an apple, an orange, a banana, a strawberry, a pineapple, a mango, a pomegranate, a kiwi, a blueberry, a raspberry, a blackberry, a melon, a watermelon, an apricot, a pear, a cherry, a plum, a grapefruit, a lemon, a lime, a fig, a guava, a papaya, a passionfruit, a lychee, a starfruit, a tangerine, a coconut, a date, a dragon fruit, a gooseberry, a jackfruit, a nectarine, a peach, and a persimmon. In some embodiments, the vegetable is a carrot, a potato, a tomato, a cucumber, a lettuce, a spinach, a bell pepper, an onion, a garlic, a broccoli, a cauliflower, a Brussel sprout, a zucchini, a squash, a pumpkin, a beetroot, a radish, a celery, a cabbage, a kale, a Swiss chard, a leek, a green bean, a pea, an asparagus, a corn, an eggplant, a turnip, a sweet potato, a parsnip, a yam, an artichoke, a mushroom, an okra, a jalapeno, a fennel, a chive, a ginger, a shallot, a rhubarb, an arugula, and a bok choy. In some embodiments, the vegetable is a lettuce. In some embodiments, the ready-to-eat food product is a salad, a pre-cooked meal, a deli meat, a cheese, a bakery item, or a dessert. In some embodiments, the ready-to-eat food product is a ready-to-eat chicken product. In some embodiments, the read-to-eat chicken product is a ready-to-eat rotisserie chicken.

[00101] In another aspect, the present disclosure provides a method of detecting a bacterial pathogen in a food product using the food packaging system described herein. Accordingly, also provided is a method of detecting bacterial pathogens in a food product, the method comprising:

(i) providing the food packaging system described herein;

(ii) placing the food product into the inclined packaging compartment;

[00102] In some embodiments, the test sample from the food product localizes onto the sensing interface (1801) facilitated by the angled side walls via gravity.

[00103] Also provided is kit for detecting a bacterial pathogen on a food product, comprising:

(A) a food container comprising:

(ii) the inclined packaging compartment comprises (a) a top opening, (b) a bottom opening or a bottom wall comprising a bottom opening, and (c) side walls of the compartment upstanding from the bottom opening or the bottom wall; wherein the side walls are for fluid-interface; wherein the side walls extend downward and inward toward the bottom opening or bottom wall at an interior angle from about 20 degrees to about 90 degrees; and wherein the inclined packaging compartment is configured to localize a test sample from the food product onto a sensing interface (1801 ).

(B) a reagent-saturated membrane (100) or a membrane (101) for receiving reagent; and

(C) a detector (102) comprising a polyolefin substrate and a biosensor for detecting the bacterial pathogen, whereby the reagent-saturated membrane (100) and the detector (102) are configured to form the sensing interface (1801).

[00104] In some embodiments, the top opening has a square, a rectangular, a circular, a triangular, an oval, a pentagonal, a hexagonal, an octagonal, a rhomboidal, a parallelogrammatic, a trapezoidal, or an irregular shape. In some embodiments, the kit further comprises at least one of a wrap, a buffer, a divalent metal salt, a dropper, a sealer, a pair of gloves, and instruction for use.

[00105] The kit described herein is useful for detecting pathogenic bacteria. The steps for packaging a food product using the kit involves, for example, the steps of sensor placement, membrane placement, buffer infusion, addition of food product such as ready-to-eat chicken, and product packaging. The step of sensor placement involves placing, for example, a polyolefin-wrapped glass sensor substrate with immobilized FNAP microarrays within the sensor window of the packaging tray. The step of membrane placement involves placing, for example, sensor-sized cotton membrane on top of FNAP sensor. The step of buffer infusion involves, for example, saturating the membrane with, for instance, 1 mL of MgCl2 buffer. The step of food addition to the food packaging system involves, for example, placing, for instance, a ready-to-eat chicken what has been sliced and pre-weighed into the packaging tray. The step of product packaging involves, for example, wrapping the entire food packaging system with, for instance, polyolefin food wrap to seal all components. The finished packaged food product is now ready for in situ detection of pathogenic bacteria. [00106] Accordingly, also provided is a method of detecting a bacterial pathogen on a food product, the method comprising:

(i) providing the kit described herein;

(iii) applying to the bottom opening overlying the detector (102) (a) the reagent-saturated membrane (100) or (b) the membrane (101 ) for receiving reagent and adding the reagent onto and saturate the membrane (100), whereby the detector (102) and the reagent-saturated membrane (100) form a sensing interface (1801 );

(iv) placing the food product into the inclined packaging compartment;

(v) allowing a test sample from the food product to localize onto the sensing interface (1801 ) facilitated by the angled side walls;

(vi) allowing the test sample to interact with the reagent-saturated membrane (100); and

(vii) detecting the presence of a bacterial pathogen in the test sample using the biosensor of the detector (102).

[00107] In some embodiments, after step (iv), applying a wrap to cover and seal the top opening. In some embodiments, the reagent-saturated membrane (100) or the membrane (101 ) for receiving reagent is applied to the bottom opening from above the bottom opening. In some embodiments, the reagent-saturated membrane (100) or the membrane (101 ) for receiving reagent is applied to bottom opening from below the bottom opening. In some embodiments, the reagent comprises a buffer, a metal ion, and/or a salt. In some embodiments, the buffer comprises citric acid, acetic acid, lactic acid, malic acid, phosphoric acid, tartaric acid, potassium citrate, calcium lactate, potassium phosphate, sodium acetate, sodium citrate, sodium lactate, and/or sodium phosphate. In some embodiments, the metal ion is a divalent metal ion. In some embodiments, the divalent metal ion is Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, and/or Mn²⁺. In some embodiments, the divalent metal ion is Mg²⁺. In some embodiments, the Mg²⁺ is at a concentration of about 20 m M to 50 about m M in the buffer. I n some embodiments, the Mg²⁺ is at a concentration of about 25 mM to about 35 mM in the buffer. In some embodiments, the Mg²⁺ is at a concentration of about 25 mM, about 30 mM, or about 35 mM in the buffer. In some embodiments, the Mg²⁺ is at a concentration of about 30 mM in the buffer. In some embodiments, the salt is CaCl2, CeH CaOe, CaCOs, MgSO4, MgCh, ZnSO4, FeSO4, Ci2H22FeOi4, MnSO4, and/or CuSCU. Salt is MgCh In some embodiments, the MgCl2 is at a concentration of about 20 mM to 50 about mM in the buffer. In some embodiments, the MgCl2 is at a concentration of about 25 mM to about 35 mM in the buffer. In some embodiments, the MgCl2 is at a concentration of about 25 mM, about 30 mM, or about 35 mM in the buffer. In some embodiments, the MgC is at a concentration of about 30 mM in the buffer.

[00108] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[00109] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.

Example

[00110] The following non-limiting Example is illustrative of the present disclosure:

Example 1 - Lab-in-a-Packaqe

[00111] Herein described is a food packaging system useful for detecting pathogenic bacteria. Methods and Materials

[00112] Materials. 3D printing filament was obtained from Creality 3D Technology (Shenzhen, China). Membrane materials were acquired from TNG Worldwide (Michigan, United States), Superscandi (London, United Kingdom), EcoJeannie (New Jersey, United States), Shoppers Drug Mart (Ontario, Canada), and Walmart Canada (Ontario, Canada). Polyethylene wraps were sourced from Thomas Scientific (New Jersey, United States). N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS), 2-(N-morpholino) ethanesulfonic acid (MES) buffer, and MgCL were purchased from Millipore Sigma (Ontario, Canada). Oligonucleotide sequences were ordered from Integrated DNA Technologies (Iowa, United States). Adenosine triphosphate (ATP), polynucleotide kinase buffer A, polynucleotide kinase, T4 DNA ligase buffer, and T4 DNA ligase were purchased from Thermofisher Scientific (Ontario, Canada). Fluorophore-quencher substrate sequences were acquired from the Keck Oligonucleotide Synthesis Facility at Yale University (Connecticut, United States). Ready-to-eat chicken products were sourced from local grocery stores.

[00113] Packaging Tray Fabrication. All three packaging trays and their associated 2D drawings were developed using 3D computer assisted design (CAD) software (Autodesk Fusion) and then 3D printed using PLA filament (Ender 3 V2, Shenzhen Creality 3D Technology Co., Ltd., China). These packages were then smoothened using acetone to lower the coefficient of friction on the fluid-interface. ^[331 All packages were printed at a 50% scaled down rendering to improve characterization efficiency.

[00114] Fluid Transfer and Localization Efficacy. The fluid transfer efficiency involved recording the time it took for a droplet to transport down the edge of each packaging model, when dispensed at a rate of 16.54 pL/s using an automated syringe (DSA30, Kriiss Scientific, Hamburg, Germany). This study was videotaped using the Kriiss Advance software and then viewed in slow motion to accurately quantify time measurements. The time required to localize 5 mL of deionized water into the sensing window was quantified through the time required for 5 g of water to collect within a weigh boat positioned directly below the sensing window, as PBS was dispensed onto the trays’ edges from above. Here, PBS was dispensed at a rate of 0.5 mL/s. All studies had at least triplicate measurements. Fluid localization based on volume was characterized as the percent of solution that reached a weigh boat collection basin that was attached to the bottom of each packaging model, relative to the total applied volume. PBS studies involved dispensing the buffer over 1 minute at a rate of 0.2 mL/s. Chicken purge studies involved applying 4 mL of chicken purge onto 62 g samples of RTE chicken and assessing the percent volume collected within an attached collection basin after 24 hours storage at 37°C. Timepoint readings were also done to develop trends for volume localization across a 60 second timespan. Here, 5 mL of solution were applied for 10 seconds and 20 seconds on each packaging model and 10 mL of solution were applied for timepoints from 30 to 60 seconds and the total volume of fluid collected in collection basins after each time period was recorded. All values were considered relative to the initial volume applied.

[00115] Preliminary Membrane Characterization. All membrane candidates were first cut to the same size of 4 cm x 2.5 cm. Their thicknesses were then matched to the same general thickness of approximately 1.1 mm. The samples were then microscopically imaged using an inverted microscope (Nikon Eclipse Ti2, Nikon Instruments Inc.) at 4X and 10X magnifications. Fluorescence analysis was done on the same samples using the same imaging system, with at least three samples imaged for each membrane. Samples were imaged across DAPI, FITC, TRITC, and Cy5 fluorescence wavelengths. Membrane samples were also cut to approximately 1 x 1 cm sizes and then mounted using carbon tape and nickel paste. A sputter coater (Polaron model E1500, Polaron Equipment Ltd., Watford, Hertfordshire) was then used to coat the samples with 10 nm of gold, which were then imaged using the TESCAN VEGA-II LSU SEM.

[00116] Membrane Absorption Quantification. The absorption capacity of each candidate membrane was assessed using 3.5 cm x 2.0 cm x 1 .1 mm samples, wherein samples were weighed when dry, submerged in PBS for 1 minute, and then reweighed. The density of PBS was then used to convert the weight readings into volumetric measurements, which yielded the total absorption capacity of each membrane. To prevent measurement error, the hydrated samples were briefly shaken to remove residual, unabsorbed PBS. Additionally, a timepoint study was also created to determine that the membranes were saturated in 1 minute. This involved repeating the above protocol for different submersion times spanning 5 seconds to 30 minutes.

[00117] Membrane Retention Analysis. Membranes were submerged in PBS for 1 minute, shaken to remove unabsorbed solution, and then weighed as the initial starting weight. Membrane weight was measured and then converted to volumetric values using the density of PBS. These samples were then stored within packaging. Membranes were reweighed at 24h and 120h to quantify the volume of buffer retained within the membranes over time.

[00118] Membrane Diffusion Analysis. Buffer diffusion was assessed by quantifying the volume of PBS that diffused through 5 cm x 2.5 cm pre-saturated membrane samples over 2 minutes of continuous flow at a rate of 0.1 mL/s. Samples were supported by a plastic scaffold and placed on top of a collection basin. PBS was then pipetted onto the top surface of the membrane and the amount of buffer which diffused through each membrane into the basin below was collected and quantified. Triplicate measurements were obtained to reduce experimental error.

[00119] Membrane Bacterial Studies. To assess membrane effects on bacterial proliferation and survival, 1 cm x 1 cm membrane samples were incubated with 10⁸ CFU/mL of bacteria for6 hours. The contaminated membranes were then vortexed for 1 minute to extract bacteria from the membrane into solution. This solution was then serially diluted and plated onto Gram-negative selective MacConkey agar (MilliporeSigma) plates. MacConkey agar was used based on its selectivity for gramnegative bacteria. ^[34'³⁶¹ A control (no membrane) condition consisting of 10⁸ CFU/mL of bacteria was maintained for the same incubation period and concurrently plated. The bacterial plates were incubated overnight at 37°C. Following incubation, the total number of colony-forming units were counted for each membrane and compared to that of the control. Bacterial diffusion through the membranes was assessed across both unsaturated and saturated membranes (100), where the latter had 1 mL of PBS buffer added on the surface. Both groups of membranes were then placed on top of glass substrates and 10⁶ CFU/mL of bacteria was distributed on top of each membrane candidate. After 6 hours, any solution which diffused through the membrane onto the glass substrate below was collected, serially diluted, and plated onto the same selective plates. Once again, the total number of colony-forming units formed after the overnight incubation was used to determine the overall bacterial diffusion through each membrane.

[00120] Bacteria Preparation. Salmonella enterica serovar Typhimurium, E. coli K12, E. coli O157:H7, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes 1 a, and Bacillus subtilis were cultured in appropriate media for 18 hours at 37 °C under constant agitation at 180 RPM from glycerol stock solutions. The bacteria from these overnight incubations were then centrifuged at 7000 RPM for 15 minutes to form a bacterial pellet. This pellet was then resuspended in PBS buffer solution for use in all bacterial studies.

[00121] Membrane Antifouling Assessment. Chicken purge was extracted from chicken samples and heated in a water bath at 60°C to melt any solidified lipid molecules. The filtered chicken purge was then pipetted through a cotton membrane and then collected. Both the filtered and unfiltered membrane samples were pipetted into a well plate and had their optical density measured using a Synergy Neo2 plate reader (Aligent Technologies). Samples were measured across an absorbance spectrum ranging from 400 nm to 700 nm in increments of 10 nm. Deionized (DI) water was also assessed for baseline readings. Quadruplicate readings were obtained. SEM images of cotton membranes saturated in chicken purge were obtained to visualize antifouling properties. 1 x 1 cm² cotton membrane samples were saturated with chicken purge and then dried in ambient conditions for 24 hours. These samples were then mounted, coated, and imaged in a method identical to the SEM procedures outlined in Preliminary Membrane Characterization.

[00122] FNAP Synthesis. All relevant sequences are listed in Table 1. 3’ aminomodified probe fragments were phosphorylated using ATP, T4 polynucleotide kinase buffer A, and T4 polynucleotide kinase in-solution, over 30 minutes at 37°C. Substrate fragments (FQ30, TB30) and ligation template fragments were then added, heated for 1 minute at 90 °C, and cooled at ambient temperature, to mediate the annealing of the three fragments. T4 DNA ligase buffer, T4 DNA ligase, and water were then added and incubated at ambient temperature for 1 hour to mediate ligation of the probe and substrate fragments. The sample was then ethanol precipitated and centrifuged for 20 minutes at 4°C, 20000 x g. A polyacrylamide gel was used to purify the ligated product. The final nucleic acid probe product was resuspended in water. Table 1. Oligonucleotide sequences used for S. enterica serovarTyphimurium sensor.

[00123] Sensor Development and Preliminary Characterization. Nucleic acid probe was first mixed with EDC-NHS crosslinker in MES buffer to facilitate covalent attachment to polyethylene substrates. A GeSiM Nano-Plotter piezoelectric printer was used to deposit nucleic acid probe onto the sensor surface. The sensors were then incubated in a 75% humidity environment for 2 hours and then washed in a water bath at 220 RPM for 30 minutes on a platform shaker (VWR International) to remove any unbound probe molecules. They were then dried and imaged using an inverted fluorescent microscope. Covalent attachment was shown by comparing the fluorescence of nucleic acid probes both with and without the EDC-NHS covalent crosslinker before and after the aforementioned water washing step. Next, a calibration curve was developed as described in Yousefi etal 2021 ^[15] to determine the density of nucleic acid probe added to the sensor surface. This curve was created using intensity measurements of arrays composed of known concentrations of fluorescent, single-stranded TRITC DNA molecules. Maximal fluorescence intensity of the nucleic acid probe was obtained using 1 M NaOH, at which point probe density was quantified using the curve. The resultant linear relationship was used to quantify immobilized sensing probe density based on NaOH-induced maximal fluorescence per unit area as described in Li et al 2023 and Yousefi et al 2018.^[13’ ²²¹ The average of sensing probe values are denoted as a unique data point. The calibration curve was developed as described in Yousefi et al 2021. ^[15]

[00124] Sensor Sensitivity and Specificity Testing. The effects of chicken purge on bacterial proliferation and survival were assessed through the resuspension of 10⁶ CFU/mL E. coli in chicken purge and in PBS. These solutions were then plated on selective MacConkey agar plates, alongside chicken purge alone. These plates were incubated overnight at 37°C and then the colony-forming units were quantified. Sensor sensitivity was tested by incubating printed, pre-imaged sensors with S. enterica serovar Typhimurium concentrations ranging from 10⁷ to 10³ CFU/mL, where all dilutions were performed using chicken purge. 100 mM MgCh was also added into the incubation solution. Control samples were composed of chicken purge and MgCL alone. After an 8-hour incubation at 37°C, all test solution was removed from the sensor surface. The sensors were then briefly washed in DI water and re-imaged to assess their fluorescence fold change. Sensor selectivity was tested in a similar manner except this time a constant bacterial concentration of 10⁶ CFU/mL was tested using S. enterica serovar Typhimurium, Escherichia coli O157:H7, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes 1 a, and Bacillus subtilis, while the control solution remained the same.

[00125] Sensor Stability and Temperature Profile Development. Printed sensors were washed, imaged, and then stored at 4°C. After three months of storage, the sensors were incubated with S. enterica serovar Typhimurium in concentrations ranging from 10⁶ to 10³ CFU/mL and were then re-imaged to show sensor viability. A temperature profile of sensor performance was also developed to show their functionality in a variety of food storage conditions. To this end, sensors were incubated with 10⁷ to 10⁵ CFU/mL contaminated chicken purge and incubated for 24 hours at 4°C, 25°C, 37°C, and 45°C and then reimaged.

[00126] Proof-of-Concept Testing. Full-scale models of the 45° packaging tray were 3D printed for the proof-of-concept testing. A printed sensor was placed in the window at the base of the packaging and an MgCh buffer-saturated membrane of the same size as the sensor was placed directly on top. Cooked RTE rotisserie chicken purchased from local grocery stores was cut into 250 g samples and placed on the packaging trays. These chicken samples were contaminated with 10 mL volumes of S. enterica serovar Typhimurium chicken purge suspensions to yield the desired CFU/g concentrations. Control samples were treated with 10 mL of uncontaminated chicken purge. The samples were incubated at 37°C for 8 hours. After 8 hours, the packaging was opened, the membrane was removed, and the extracted sensor was fluorescently imaged. Application-based testing followed the exact same protocol, except introduction of the bacteria onto food samples was done via a contaminated surface, glove, and knife, rather than through contaminated chicken purge.

[00127] S. enterica serovar Typhimurium Growth Study. An original concentration of 10² CFU/mL of S. enterica serovar Typhimurium suspended in chicken purge was selectively plated at a timepoint of 0 hours. It was then incubated at 37°C, with selective plating repeated at 2 hour and 4 hours timepoints. The total number of colony-forming units formed after an overnight incubation at 37°C was used to quantify the growth of the original 10² CFU/mL of S. enterica serovar Typhimurium.

[00128] Full System Specificity Testing. Full system specificity was tested in a similar manner to sensor selectivity testing. Equal amounts of 10⁶ CFU/mL of Escherichia coli O157:H7, Listeria monocytogenes 1 a, and S. enterica serovar Typhimurium where resuspended in chicken purge and applied onto chicken samples within sensor and membrane containing packaging trays for an 8 hour incubation at 37°C. After this incubation, sensors were fluorescently images as previously described.

[00129] Target Verification Study. Chicken samples were contaminated with 10⁶ CFU/g S. enterica serovar Typhimurium. The final target that reached the sensor interface was collected after the 8 hour incubation period and selectively plated along with some of the original contaminated chicken purge that was applied. After the plates were stored for a standard overnight incubation at 37°C, the total number of colonyforming units formed for both the initial and post-incubation samples were compared to both assess that the collected target contained S. enterica serovar Typhimurium and that there was no significant change in the overall bacterial concentration.

[00130] S. enterica serovar Typhimurium Detection in Lettuce Samples. Bagged lettuce was obtained from a local grocery store and washed thoroughly. Water used to wash the lettuce was collected as the wash fluid, which was then spiked with 10⁶ CFU/mL of S. enterica serovar Typhimurium to simulate contaminated lettuce. Lettuce leaves were placed within the Lab-in-a-Package system and 10 mL of the contaminated wash fluid was readministered onto the samples. The sensors were reimaged after a 24 hour incubation at 25°C to assess contamination detection.

[00131] Handheld Fluorescence Detection. A handheld fluorescence scanner (Dino-Lite Edge, Dino-Lite US, Dunwell Tech., Inc.) was used to image S. enterica serovar Typhimurium contamination in sensor samples and Lab-in-a-Package. Initial characterization was performed with 10⁸ CFU/mL contaminated FNAP sensor samples. In situ detection was performed with chicken samples that were contaminated with 10⁶ CFU/g chicken purge. In this case, the handheld microscope was used to image the sensor window on the base of the packaging, without opening the package or extracting the sensor. The scanner can be connected to either an associated computer software or smartphone application for sensor visualization and final signal readout, through which all images were obtained.

Results and Discussion

Lab-in-a-Package platform design

[00132] Overarchingly, the inventors sought to develop Lab-in-a-Package to facilitate test sample localization, in situ reagent incorporation, target diffusion, and prevention of fouling on the sensing interface (1801). Collectively, these efforts all contribute to pathogen detection - all while eliminating the need to open food packaging or manipulate food products. The integration of the aforementioned universally applicable novel food packaging tray design, membrane interface, and pathogen sensor are shown in FIG. 1A to FIG. 1 D. Importantly, all materials considered in this Example were selected from the Indirect Food Substances provisions, ensuring Generally Recognized As Safe (GRAS) designation from the Food and Drug Administration (FDA), to ensure regulatory viability.¹²⁸¹

Test sample localization determination

[00133] A dramatic shift in packaging design compared to traditional packaging was performed to facilitate sensor visualization and localization of sample fluids. A sensing window was first introduced to enable the integration of fluorescent sensing interfaces that can be monitored without opening packaged foods. Determination of fluid localization from target food matrices was then explored through three packaging trays with varying levels of incline - 45°, 60°, and the traditional 90°, which were fabricated using 3D printing (FIG. 2A, FIG. 5, and FIG. 6). Here, the angle refers to the incline at the fluid-package interface. The 60° model was developed as an intermediary model to substantiate any trends observed with changes in incline angle.

[00134] Fluid transfer efficiency of the models was first assessed, where the 90° model significantly outperformed the other models (P < 0.01). This was attributed to its steeper angle inducing the strongest forces of downward acceleration (FIG. 2B). Accordingly, the 60° model also outperformed the 45° model. Yet, while fluid transfer efficiency offers an important preliminary understanding of the models’ fluid transport capabilities on a droplet scale, macroscale fluid accumulation at a central collection site better defines suitability for the desired application. As such, subsequent tests focused on characterizing the localization efficiency of each model. This was first accomplished by measuring fluid localization over time (FIG. 2c). Here, the 45° model significantly outperformed the 60° (P < 0.001) and 90° models (P < 0.0001). This improved performance was attributed to the 45° model offering a consistent decline directly into the sensor window for the milliliter-scale test volumes. Fluid localization was then evaluated based on the total volume localized onto the sensing window within a fixed period of time with both phosphate buffered saline (PBS) (FIG. 2D) and chicken purge (FIG. 2E). Specifically, this was represented as the percentage of volume localized and was calculated according to Equation (1) below.

% volume localized = —

x 100% Equation (1 )

[00135] Here, Vf represents the final volume collected from the base of the packaging tray after a constant timepoint, and V, is the initial volume of fluid applied to the trays at the start of the study. In both studies, the 45° model exhibited significantly higher fluid localization compared to both the 60° (P < 0.001 , 0.01 ) and 90° models (P < 0.0001 , 0.001 ). However, the overall localization with chicken purge was lower due to a combination of viscosity-induced slow fluid transfer and a longer incubation time. Together, these factors led to purge drying along the fluid-package interface prior to reaching the sensor window, decreasing the fluid volume available for localization. A timepoint analysis of this experiment was also conducted (FIG. 7). Ultimately, the 45° model was selected for use within the final Lab-in-a-Package platform given its superior localization when compared to the collective properties exhibited by the other models as quantitatively summarized in Table 2.

Table 2. Summary of packaging tray model characterization.

Absorption and diffusion-focused materials characterization

[00136] With regards to the membrane interface, the buffer absorption, macromolecular filtration, and target diffusion properties of five candidate materials were evaluated (FIG. 1 C). Namely, cotton, cotton-cellulose, cellulose, cellulosepolyester, and polyester materials were considered, owing to their inherent biocompatibility, filtering potential, and permeability. The candidate membranes were imaged via optical microscopy (FIG. 8) and scanning electron microscopy (FIG. 2F) to visualize their fibrous structures. Cotton exhibited the most unique structure due to its convoluted fibril arrangement, with comparatively larger pores.¹²⁹¹ All materials exhibited low fluorescence across the visible light spectrum, substantiating further characterization given their applicability to fluorescent systems (FIG. 2G).

[00137] After optical characterization, the absorption capacity of candidate membranes was assessed by volume (FIG. 2H) and over time (FIG. 9A to FIG. 9C). Here, cotton-cellulose significantly outperformed all other candidate membranes (P < 0.0001 ). This was attributed to the abundance of cellulose hydroxyl groups present within the matrix, giving the material high hydrophilicity and affinity for moisture.¹³⁰¹ While pure cellulose offers a larger number of hydroxyl groups, cotton-cellulose has comparatively higher crystallinity, resulting in the formation of complex hydrogen bonds that further elevate hydrophilicity and in turn, buffer absorption.¹³¹¹ As polyester fibres are densely packed and lack polar groups, they are intrinsically hydrophobic and thus had the lowest absorption.¹³²¹ Buffer retention was subsequently evaluated within a closed package environment, wherein all of the candidate materials exhibited limited changes in buffer volume after 120 hours (FIG. 10).

[00138] Next, diffusion testing was performed to ensure that agents of interest can permeate through the membrane matrix onto the detector (102). Polyester, cellulose-polyester, and cotton membranes performed best in preliminary buffer diffusion studies (FIG. 2I). Bacterial diffusion was subsequently evaluated using E. coli. A control study showed that the candidate materials did not influence bacterial proliferation or survival (FIG. 2J). Two bacterial diffusion studies were then performed with both unsaturated and buffer-saturated membranes (100) to evaluate the effects of hydration on bacterial diffusion (FIG. 2K and FIG. 2L). In both studies, cotton significantly outperformed all other candidate materials, facilitating the diffusion of 10⁹ CFU/mL of E. coli in both unsaturated (P < 0.001 ) and saturated states (P < 0.01) given its unique fibrous arrangement. Membrane performance was also tested with S. enterica serovar Typhimurium (FIG. 2M, FIG. 2N, and FIG. 20). Again, no effect on bacterial proliferation or survival was observed. Similar trends were observed with the five membrane candidates with regards to bacterial diffusion, except that the magnitude of diffusion was much lower in the unsaturated state with S. enterica serovar Typhimurium. This is attributed to its larger size, requiring saturation-mediated pore expansion for substantial diffusion opportunities.^{138 391}

[00139] Finally, porosity was also directly quantified as the percentage of the total top surface covered by pores within previously collected SEM images (FIG. 11). Here, the cotton membrane had the best combination of surface porosity and low fiber density, making its pores better suited for the facilitation of buffer and bacterial diffusion. Consequently, cotton membranes were selected given their high buffer retention, diffusion, and porosity when compared to the other materials, as summarized in Table 3. The material offered comparatively lower absorption capacity, and this could be offset through the use of a more concentrated reagent mix.

Table 3. Summary of membrane materials characterization.

[00140] Lastly, the benefits of a cotton membrane as a physical barrier between a food sample and the sensing substrate were explored. Specifically, recognizing that food represents a very complex matrix, fouling of the sensing interface (1801) is a major concern. The optical density of crude chicken purge and chicken purge processed through cotton membranes was measured for each sample (FIG. 12A). The collected values showed that the cotton membrane significantly decreased the presence of macroscale entities present within the chicken purge test samples (P < 0.0001). This effect can be expected to be amplified with the saturation of the cotton membrane, as wet cotton fibres offer a larger physical footprint and higher mechanical strength.¹²⁹¹ To visualize anti-diffusive effects towards macroscale entities, buffer- saturated cotton membranes were visualized via SEM after chicken purge processing (FIG. 12B). These images indicated lipid deposition onto the cotton fibres at a macroscale, while retaining sufficient porosity to permit analyte and buffer diffusion at the microscale, further substantiating the use of this membrane within the proposed platform.

S. typhimurium sensor development

[00141] With packaging and membrane determination complete, the inventors next incorporated a compatible sensor into the developed platform. Growing concerns surrounding S. enterica serovar Typhimurium contamination led the inventors to develop a new sensor for S. typhimurium. Using systematic evolution of ligands by exponential enrichment (SELEX), a highly sensitive nucleic acid probe that cleaves in the presence of RNase H2 from S. enterica serovar Typhimurium was identified. This probe was hybridized with a substrate strand embedded with a fluorophore-quencher pairing. S. enterica serovar Typhimurium-induced cleavage induced quencher separation, yielding an increase in fluorescence (FIG. 3A). The complete sequence for this construct is provided in Table 1 .

[00142] The surface-immobilized sensor was developed through the covalent attachment of an aminated, FITC-labelled version of the S. enterica serovar Typhimurium-responsive probe to polyethylene food packaging substrates. To ensure an adequate sensor signal, probe surface density was adjusted and subsequently quantified to be 1.3 x 10'⁵ nmol per array spot (FIG. 13). The sensitivity, stability, and specificity of this surface sensor was then evaluated. Given that chicken food matrices are most commonly contaminated by S. typhimurium, and RTE foods offer the highest risk of illness, RTE chicken products were selected as the target matrix for the present studies. Sensitivity testing was thus performed using contaminated chicken purge samples. The effects of chicken purge on bacterial proliferation and survival were first assessed, to ensure consistency between reported and experimental bacterial concentrations. Chicken purge spiked with bacteria, bacteria resuspended in buffer, and chicken purge alone were all selectively plated (FIG. 14). No significant changes in bacterial count were observed within any of the tested conditions.

[00143] Subsequent sensitivity testing was performed using spiked chicken purge, wherein sensors were incubated with the bacterial test solutions for eight hours at 37°C, which are environmental conditions in line with RTE chicken storage within grocery stores (FIG. 3B). The limit-of-detection of the developed sensor was determined to be 10³ CFU/mL of S. enterica serovar Typhimurium, where a distinct 2.34-mean fold change was observed following incubation - significantly higher than the non-specific 1 ,50-mean fold change of the control condition (P < 0.05). Chicken purge samples contaminated with S. enterica serovar Typhimurium concentrations ranging from 10⁸ to 10⁴ CFU/mL were also tested, showing a linear relationship between bacterial concentration and mean fluorescence fold change, with the 10⁸ CFU/mL showing the highest significant mean fold change at 4.36 (P < 0.0001). The developed sensor exhibited a significant linear operating range based on linear regression analysis (R² = 0.98, P < 0.001 ), further validating its detection efficacy (FIG. 15). The detection range of this sensor is summarized by Equation (2) below, where y represents the fluorescence signal fold change and x is the logarithmic S. enterica serovar Typhimurium concentration in CFU/mL. y = 0.3952% + 1.113 Equation (2)

[00144] Importantly, higher variations in fluorescence signals were observed at high bacterial concentrations. This is of limited real-world concern however, as industry guidelines for S. enterica serovar Typhimurium monitoring seek positive versus negative detection results. Variations at these high concentrations do not affect the accuracy of such Yes/No test results. The functionality of the sensors was then further evaluated at 4°C, 25°C, and 45°C, wherein maintained performance was observed (FIG. 3C). Sensing performance was maintained at 45°C, whereas reduced activity was observed at 25°C. Sensing activity was not observed at 4°C. These results are in line with the temperature-dependent properties of the nucleic acid probe, wherein its activity is limited at low temperatures and peaks around 37°C.

[00145] Next, the stability of these sensors was further established to ensure long-term storage viability. First, covalent attachment of the nucleic acid probe onto the polyethylene substrate was carried out to ensure the sensors could withstand induced shear stresses (FIG. 3D). Here, probes that were covalently attached with crosslinker retained 85% of their original fluorescence signal, significantly higher (P < 0.0001) than the 14% retention of the probes that were not covalently attached. To evaluate sensing performance after prolonged storage, sensors were tested three months after fabrication (FIG. 3E). Sensors tested with 10⁶ CFU/mL S. enterica serovar Typhimurium exhibited a slight increase in signal, which can be attributed to the variation that is observed at higher target concentrations. With all other concentrations, sensing performance deteriorated to a degree, but a significant limit of detection of 10⁴ CFU/mL was obtained (P < 0.0001), showing sensor resilience.

[00146] Specificity testing then involved incubating the sensors with S. enterica serovar Typhimurium and other common foodborne pathogens - namely, Klebsiella pneumoniae, Escherichia coli O157:H7, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus subtilis (FIG. 3F). The developed sensors exhibited a significantly higher 2.59-mean fold (P < 0.0001 ) change in fluorescence when incubated with S. enterica serovar Typhimurium, compared to the less than 1.10-mean fold change observed with other bacterial species. Accordingly, after sensitivity, stability, and specificity were determined, the sensors were then applied for proof-of- concept testing of the developed packaging platform using commercial-scale, solid food products.

Proof-of-concept testing of sensor-embedded packaging platform

[00147] The complete Lab-in-a-Package platform was then tested by integrating the newly developed FNAP sensor (102) into the aforementioned packaging and membrane system (FIG. 4A). The final in situ detection platform comprised of a concave packing tray with a 45° incline and a sensing window, a S. enterica serovar Typhimurium-responsive real-time fluorescence sensor embedded within this window, and an adjacent buffer-infused cotton membrane (FIG. 4B). Recognizing that chicken exhibits significant fluorescence noise at the FITC emission/excitation wavelengths of 490nm/525m, the inventors transitioned to a TAMRA fluorophore-labelled version of the probe with excitation and emission wavelengths of 557nm/576 nm (FIG. 4C). Importantly, the activity of enzymatic oligonucleotide probes largely relies on the presence of divalent metal ions, substantiating the need for a buffer immobilizing membrane within this in situ monitoring platform. The developed S. enterica serovar Typhimurium-responsive probe in particular, offers excellent performance with Mg²⁺ ions - a metal safe for consumption, which made the leaching of buffer from the membrane onto adjacent foods of limited concern. The concentration of the MgC ions at 30 mM within the Lab-in-a-Package platform was found to be useful for surfacebased sensing (FIG. 4D).

[00148] RTE rotisserie chicken products were weighed and placed within inclined packaging trays, which already contained FNAP sensors and MgCl2 saturated membranes (FIG. 17 and FIG. 18). Collected chicken purge was spiked with S. enterica serovar Typhimurium, and this contaminated purge was then applied to the test products to perform a sensitivity analysis of the complete system, with concentrations ranging from 10⁶ to 10² CFU/g. Concurrently, uncontaminated chicken purge was applied onto control chicken samples. The samples were then incubated at 37°C for eight hours to simulate grocery store RTE chicken storage environments conditions. After eight hours, sufficient localization was visually shown through the accumulation of significant chicken purge on the cotton membrane and subjacent sensor (/.e. detector (102)). Specifically, the top surface of the membrane was coated with macroscale fouling agents such as lipids, showing the membrane’s anti-fouling capabilities within an in situ environment. With regards to sensitivity, sensors incubated with contaminated chicken samples exhibited a significantly higher (P < 0.0001 ) mean fold change of up to 2.83 at 10⁶ CFU/g compared to the 1 .09 mean fold change of the control samples (FIG. 4E). The limit of detection of Lab-in-a-Package was determined to be 10³ CFU/g, which exhibited a significant mean fold change of 1.54 (P < 0.0001) compared to the control. The operating range of the complete system was significant (R² = 0.98, P < 0.05) based on linear regression analysis (FIG. 16). The linear operating range of Lab-in-a-Package is summarized by Equation (3). In this model, y represents the fluorescence signal fold change and x is the logarithmic S. enterica serovar Typhimurium concentration in CFU/g. y = 0.4135% + 0.2820 Equation (3)

[00149] Further, a bacterial growth study was conducted to assess opportunities for improved sensitivity using this platform. It was shown that 10² CFU/mL of S. enterica serovar Typhimurium suspended in chicken purge grows to 10⁴ CFU/mL within 4 hours. This shows that extended incubation periods offer improved detection limits (FIG. 19).

[00150] The specificity of Lab-in-a-Package was also shown through sensor incubation with a mixture of common food contaminants. Specifically, FNAP sensors were placed at the base of the packaging and then incubated with control chicken samples contaminated with a 10⁶ CFU/g mixture of E. coli O157:H7 and Listeria monocytogenes. Test samples also contained S. enterica serovar Typhimurium (FIG. 20). Here, the test samples which contained S. enterica serovar Typhimurium exhibited a significantly higher mean fold change of 2.70 compared to the 1 .25 mean fold change of the control samples (P < 0.0001 ). To further show the specificity of the system, a target verification study was also performed (FIG. 21). Here, spiked purge and purge localized on the sensing window after 8 hours of incubation were selectively plated. Insignificant differences were observed between the two samples. These results showed that the developed packaging platform successfully mediated the localization of representative test solution onto the sensing interface (1801 ) for detection, alongside buffers required for biomolecular functionality with a good degree of sensitivity and specificity.

[00151] Next, to further simulate real-world food contamination, the platform was used to detect contamination within samples that were spiked via means of handling and processing. Specifically, the test chicken samples were contaminated through contact with a contaminated knife, glove, and surface (FIG. 4F) spiked with a solution corresponding to 10⁷ CFU/g of the corresponding chicken sample. The contaminated chicken samples exhibited high mean fold changes of 4.03 (P < 0.0001), 3.73 (P < 0.0001), and 3.00 (P < 0.001 ) following contamination induced from the knife, glove, and surface, respectively, further showing the presented platform as a means of in situ contamination detection in real-world settings (FIG. 4G).

[00152] To further evaluate the generalizability of the presented platform, contamination detection was performed in spiked lettuce samples at 25°C (FIG. 22A and FIG. 22B). Test samples that were spiked with 10⁶ contaminated wash water exhibited significant mean fold changes of 1 .51 (P < 0.001 ) compared to the 1.15-fold change of the control samples. These results show the use of this system for other food products including produce and consumer goods.

[00153] Finally, the present disclosure shows simulation of real-world use of the developed platform to evaluate its full in situ sensing capabilities using a portable handheld fluorescence scanner that visualizes images onto a smartphone (FIG. 4H). The capabilities of the handheld scanner were first evaluated with the sensor alone, wherein a significant mean fold change of 2.76 (P < 0.0001 ) was observed following incubation with a 10⁸ CFU/mL solution of S. enterica serovar Typhimurium (FIG. 4I). When used to monitor a sealed Lab-in-a-Package set-up containing 10⁶ CFU/g contaminated RTE rotisserie chicken, a significant fold change of 3.27 (P < 0.01 ) was observed without any disruption to the closed food package (FIG. 4J). Using such a portable system over a laboratory-scale microscope makes sensor monitoring possible across the entire food production pipeline on an individual product level, emulating real-time, hands-free, in situ detection.

Conclusion

[00154] The inventors have developed Lab-in-a-Package, a revolutionary solution to actualize in situ, real-time food contamination detection - bridging the gap between the myriad of developed food sensors and their adoption into food products at the retail and consumer levels. The platform combined a newly designed food packaging tray and a buffer-infused membrane to address the complete lack of an in situ monitoring-compatible packaging platform. This combinatory approach met the key objectives required to facilitate real-time, hands-free detection as it: (A) enabled sensor imaging within a closed package format, (B) localized sample solution onto the sensing interface (1801), (C) retained all necessary buffers inside the food packaging, (D) facilitated sample diffusion from the food matrix to the sensor, and (E) enabled the in situ incorporation of novel biorecognition elements. The designed 45° inclined packaging model displayed the highest levels of fluid localization and fluid transfer when compared to a traditional food packaging tray and an intermediately inclined one. Moreover, the selected cotton membrane was rigorously tested through a variety of experiments to insure adequate diffusivity and buffer retention. Complete proof-of- concept testing with a newly-develop S. enterica serovar Typhimurium sensor demonstrated the successful in situ detection of this target pathogen within food products, with high sensitivity and specificity. The efficacy of the developed solution was also shown via application-specific testing that involved contaminating food samples with a S. enterica serovar Typhimurium-contaminated glove, surface, and knife, to better simulate real-world conditions. Finally, real-world use was simulated using a handheld fluorescence scanner attached to a smartphone for sensor visualization. This solution has immense application for a variety of food samples beyond RTE chicken, including the packaging trays used for other meats and seafood products, as well as for the plastic containers used for produce. Its generalized design, cost-effectiveness, and food-safe nature positions it well to forward commercial implementation of in situ food sensors. Overarchingly, Lab-in-a-Package represents a paradigm shift, as the first packaging technology designed for in situ monitoring. Whereas existing traditional food testing protocols only test select products at central laboratories via time-consuming and laborious procedures, this system offers: (A) continuous monitoring of packaged foods, (B) with results available on an hour-scale, (C) without the need for any product processing, and (D) in a manner that does not destroy tested foods. Collectively, these traits define the system’s delivery of (E) individual product monitoring in situ.

[00155] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. [00156] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present disclosure is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

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Claims

Claims:

1 . A food packaging system, comprising:

2. The food packaging system of claim 1 , wherein the top opening has a square, a rectangular, a circular, a triangular, an oval, a pentagonal, a hexagonal, an octagonal, a rhomboidal, a parallelogrammatic, a trapezoidal, or an irregular shape.

3. The food packaging system of claim 1 or 2, wherein the interior angle is about 45 degrees to about 90 degrees (107).

4. The food packaging system of any one of claims 1 to 3, wherein the interior angle is about 45 degrees, about 60 degrees, or about 90 degrees.

5. The food packaging system of claim 4, wherein the interior angle is about 45 degrees.

6. The food packaging system of any one of claims 1 to 5, wherein the side walls and bottom wall comprise polylactic acid (PLA), polyethylene terephthalate glycol (PETG), thermoplastic polyurethane (TPU), Nylon, high-density polyethylene (HDPE), and/or polyether ether ketone (PEEK).

7. The food packaging system of any one of claims 1 to 6, wherein the membrane comprises cotton, cotton-cellulose, cellulose, cellulose-polyester, and/or polyester.

8. The food packaging system of claim 7, wherein the membrane comprises cotton.

9. The food packaging system of any one of claims 1 to 8, wherein the reagent comprises a buffer, a divalent metal ion, and/or a salt.

10. The food packaging system of any one of claims 1 to 9, wherein the bacterial pathogen is at least one of Salmonella, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Bacillus subtilis, Campylobacter jejuni, Staphylococcus aureus, Clostridium, Vibrio, Shigella, and Yersinia enterocolitica.

11 . The food packaging system of claim 10, wherein the Salmonella is Salmonella typhimurium.

12. The food packaging system of claim 10 or 11 , wherein the Listeria monocytogenes is Listeria monocytogenes 1 a.

13. The food packaging system of any one of claims 10 to 12, wherein the Clostridium is Clostridium perfringens or Clostridium botulinum.

14. The food packaging system of any one of claims 10 to 13, wherein the Vibrio is Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and/or Vibrio alginolyticus.

15. The food packaging system of any one of claims 10 to 14, wherein the Shigella is Shigella dysenteriae, Shigella flexneri, Shigella boydii, and/or Shigella sonnei.

16. The food packaging system of any one of claims 1 to 15, wherein the biosensor comprises a nucleic acid probe.

17. The food packaging system of claim 16, wherein the nucleic acid probe is attached to the polyolefin substrate.

18. The food packaging system of claim 17, wherein the polyolefin substrate is COOH-activated polyolefin substrate and the nucleic acid probe is attached to the COOH-activated polyolefin substrate connected through a N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide/N-Hydroxysuccinimide (EDC/NHS) crosslinker.

19. The food packaging system of any one of claims 16 to 18, wherein the nucleic acid probe is capable of detecting a RNase H2.

20. The food packaging system of claim 19, wherein the RNase H2 is Salmonella typhimurium RNase H2, Listeria monocytogenes RNase H2, or Escherichia coli RNase H2.

21. The food packaging system of claim 20, wherein the RNase H2 is Salmonella typhimurium RNase H2.

22. The food packaging system of claim 21 , wherein the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

23. The food packaging system of any one of claims 1 to 22, wherein the biosensor is configured for generating a fluorescent signal upon detection of the bacterial pathogen.

24. The food packaging system of any one of claims 1 to 21 , wherein the biosensor is configured for generating a colorimetric signal upon detection of the bacterial pathogen.

25. The food packaging system of any one of claims 1 to 24, wherein the biosensor has a limit-of-detection of 10³ CFU/mL.

26. The food packaging system of any one of claims 1 to 25, wherein the food product is meat, produce, a dairy product, and/or a ready-to-eat food product.

27. The food packaging system of any one of claims 1 to 26, wherein the meat is beef, veal, pork, lamb, mutton, chicken, turkey, duck, goose, quail, pheasant, rabbit, venison, bison, elk, goat, horse, kangaroo, ostrich, alligator, frog, snail, squab, or guinea fowl.

28. The food packaging system of any one of claims 1 to 26, wherein the produce is a fruit or a vegetable.

29. The food packaging system of claim 28, wherein the vegetable is lettuce.

30. The food packaging system of claim 26, wherein the ready-to-eat food product is a salad, a pre-cooked meal, a deli meat, a cheese, a bakery item, or a dessert.

31 . The food packaging system of claim 26, wherein the ready-to-eat food product is a ready-to-eat chicken product.

32. A method of detecting bacterial pathogens on a food product, the method comprising: (i) providing the food packaging system of any one of claims 1 to 31 ;

(ii) placing the food product into the inclined packaging compartment;

(iii) allowing a test sample from the food product to localize onto the sensing interface (1801) facilitated by the angled side walls;

(v) detecting the presence of a bacterial pathogen in the test sample using the biosensor of the detector (102) in the sensing interface (1801).

33. A kit for detecting a bacterial pathogen on a food product, comprising:

(A) a food container comprising:

34. The kit of claim 33, wherein the top opening has a square, a rectangular, a circular, a triangular, an oval, a pentagonal, a hexagonal, an octagonal, a rhomboidal, a parallelogrammatic, a trapezoidal, or an irregular shape.

35. The kit of claim 33 or 34, further comprising at least one of a wrap, a buffer, a divalent metal salt, a dropper, a sealer, a pair of gloves, and instruction for use.

36. A method of detecting a bacterial pathogen on a food product, the method comprising: (i) providing the kit of any one of claims 33 to 35;

(iii) applying to the bottom opening overlying the detector (102) (a) the reagent- saturated membrane (100) or (b) the membrane (101 ) for receiving reagent and adding the reagent onto and saturate the membrane (100), whereby the detector and the reagent-saturated membrane form a sensing interface (1801);

(iv) placing the food product into the inclined packaging compartment;

37. The method of claim 36, after step (iv), applying a wrap to cover and seal the top opening.

38. The method of claim 36 or 37, wherein the reagent comprises a buffer, a divalent metal ion, and/or a salt.