WO2019028162A1 - Determination of bacteria viability by measuring transient biogenic amine production - Google Patents

Abstract

Disclosed embodiments provide a method and product for rapid determination of microorganism viability. The method involves measuring transient biogenic amine production of a microorganism after adding an exogenous amino acid cocktail. The product comprises a biosensor having a biosensor surface that is adsorbed with a glycoenzyme and a capture probe, wherein the capture probe comprises a binding material that is capable of binding to a microorganism.

Description

DETERMINATION OF BACTERIA VIABILITY BY MEASURING TRANSIENT BIOGENIC AMINE PRODUCTION

GOVERNMENT INTEREST STATEMENT

[0001] The disclosed subject matter was made with the United States government support under Grant No. 1511953-Nanobioensing awarded by the National Science Foundation Nanobiosensors program. The government has certain rights in the disclosed subject matter.

BACKGROUND

Technical Field

[0002] The presently disclosed subject matter relates to generally to a method, system, and products for determining bacteria viability.

Background

[0003] Contamination of food with pathogens not only sickens, but can lead to hospitalization and even death in people with compromised immune systems. Public demand for organic, non-pasteurized food products is inducing pressure on the food industry to provide high quality/safe products, which requires rapid sensors to test food products in situ. While many detection strategies exist, there are few approaches for determining pathogen viability.

SUMMARY

[0004] According to a first broad aspect, the presently disclosed subject matter provides a product that comprises a biosensor having a sensor surface, a glycoenzyme, and a capture probe. The glycoenzyme and the capture probe are adsorbed to the sensor surface. The capture probe comprises a binding material that is capable of binding to a microorganism.

[0005] According to a second broad aspect, the presently disclosed subject matter provides a kit that comprises a glycoenzyme, a capture probe, and an exogenous amino acid (EAA) cocktail comprising an amino acid. The glycoenzyme and the capture probe are in a formula to be adsorbed to a sensor surface of a biosensor. The capture probe comprises a binding material that is capable of binding to a microorganism. The EAA cocktail is placed separately from the glycoenzyme and the capture probe.

[0006] According to a third broad aspect, the presently disclosed subject matter provides a method for determining the viability of a microorganism. The method comprises: capturing a microorganism in a sample with a capture probe adsorbed to a sensor surface of a biosensor, wherein a glycoenzyme is adsorbed to the sensor surface; adding an EAA cocktail to the sensor surface to form a mixture comprising the glycoenzyme, the EAA cocktail, and the microorganism; and measuring the change in pH or hydrogen peroxide of the mixture, thereby determining the microorganism viability. A quantifiable change in pH or hydrogen peroxide of the mixture is associated with the number of viable microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate exemplary embodiments of the disclosed subject matter, and, together with the general description given above and the detailed description given below, serve to explain the features of the disclosed subject matter.

[0008] FIG. 1 is a schematic diagram illustrating a general scheme for viability detection combined with a generic capture step according to one embodiment of the presently disclosed subject matter.

[0009] FIG. 2 is a schematic illustration showing an example of reaction cascade for a two-step analysis of bacterial capture/detection (step 1) and viability testing (step 2) according to one embodiment of the presently disclosed subject matter.

[0010] FIG. 3 is a schematic illustration showing stimulus-response LBL protein nanobrush on reduced graphene oxide-nanoplatinum (rGO-nPt) electrodes according to one embodiment of the presently disclosed subject matter.

[0011] FIG. 4 is a graph showing representative Nyquist plots for LBL assembly of lectin- glycoenzyme nanobrush according to one embodiment of the presently disclosed subject matter.

[0012] FIG. 5 illustrates graphs showing chronoamperometric detection results of biogenic amine production using the LBL protein nanobrush (5 protein layers) according to one embodiment of the presently disclosed subject matter.

[0013] FIG. 6 illustrates graphs showing average impedance (cutoff frequency = 1 Hz) for rGO-nPt nanobrush electrodes after capture of 10³ CFU/mL E. coli 0157:H7 according to one embodiment of the presently disclosed subject matter.

[0014] FIG. 7 illustrates graphs showing proof of concept demonstration of capture and viability cascade according to one embodiment of the presently disclosed subject matter.

DETAILED DESCRIPTION

Definitions

[0015] Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated. [0016] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0017] For purposes of the presently disclosed subject matter, the term "comprising", the term "having", the term "including," and variations of these words are intended to be open- ended and mean that there can be additional elements other than the listed elements.

[0018] For purposes of the presently disclosed subject matter, directional terms such as "top," "bottom," "upper," "lower," "above," "below," "left," "right," "horizontal," "vertical," "up," "down," etc., are used merely for convenience in describing the various embodiments of the presently disclosed subject matter. The embodiments of the presently disclosed subject matter can be oriented in various ways. For example, the diagrams, apparatuses, etc., shown in the drawing figures can be flipped over, rotated by 90° in any direction, reversed, etc.

[0019] For purposes of the presently disclosed subject matter, a value or property is "based" on a particular value, property, the satisfaction of a condition, or other factor, if that value is derived by performing a mathematical calculation or logical decision using that value, property or other factor.

[0020] For purposes of the presently disclosed subject matter, it should be noted that to provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about." It is understood that whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including approximations due to the experimental and/or measurement conditions for such given value.

[0021] For purposes of the presently disclosed subject matter, the term "biosensor" refers to a device that is used for detecting or monitoring organisms, such as microorganisms, cells, bacteria, pathogens, etc., based on the specific molecular (or macromolecular) interaction between a target organism and a sensor coating. The interaction results in an intermediate complex that leads to transduction of signal to an acquisition system. A biosensor can be an analytical device which converts a biological response into an electrical signal, optical signal, or other transduction scheme.

[0022] For purposes of the presently disclosed subject matter, the term "exogenous amino acid (EAA) cocktail" refers to a solution including one or more exogenous amino acids. "Exogenous" refers to amino acids that are not endogenous to the specific sample (for example a food product, water sample, surface, etc. that contains natural amino acids). The term "cocktail" refers to a mixture of amino acids and micronutrients that is suitable for a particular type of bacteria and a particular sample.

[0023] For purposes of the presently disclosed subject matter, the term "enhance" refers to increasing or prolonging either in potency or duration of a desired effect.

[0024] For purposes of the presently disclosed subject matter, the term "free amino acid" refers to amino acids that are available to a microorganism for metabolic activity within a 24 hour time frame, and excludes amino acids that may be available to bacteria over long periods of time (such as amino acids that result from the decay of natural material).

[0025] For purposes of the presently disclosed subject matter, the term "micronutrients" refers to nutrients that an organism needs for healthy growth and development. A non- limiting list of examples of micronutrients includes carbon, hydrogen, nitrogen, oxygen, phosphorus, potassium, sodium, calcium and magnesium, as well as trace elements such as iron, sulfur, chlorine, manganese, zinc, nickel, molybdenum, copper, iodine, selenium and cobalt.

[0026] For purposes of the presently disclosed subject matter, the term "target" refers to an organism or a biological molecule to which some other entity, like a molecule, is directed and/or binds.

[0027] For purposes of the presently disclosed subject matter, the term "room temperature" refers to a temperature of from about 20 °C to about 25 °C.

[0028] For the purposes of the presently disclosed subject matter, the term "real time" refers to a transaction that is processed fast enough for the result to come back and be acted on as transaction events are generated. For example, a real time measurement refers to the measurement of reactants or end-products during a chemical or other dynamic process.

Description

[0029] While the disclosed subject matter is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the disclosed subject matter to the particular forms disclosed, but on the contrary, the disclosed subject matter is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the disclosed subject matter.

[0030] Embodiments disclosed herein provide an approach for rapid determination of organism (e.g. bacteria) viability by measuring transient biogenic amine production (TBAP) after adding an EAA cocktail.

[0031] The sustainable production of food and water are intricately intertwined, and each of these is susceptible to contamination by pathogenic bacteria.¹ Contamination of food and/or water with pathogens not only sickens, but can lead to hospitalization and even death in people with compromised immune systems. Among global pathogen outbreaks approximately 10% are zoonotic, 15% are a result of person-to-person transmission, 50% are foodborne, and 25% are waterborne (primarily from drinking water).² In 2015 alone, diarrheal and intestinal infectious diseases caused 38 million global cases of disease with 1.5 million deaths, leading to a loss of 84 million Disability Adjusted Life Years. In addition to direct effects, these diseases are also major contributors to malnutrition, as asymptomatic infections with enteric pathogens can lead to stunting through chronic inflammations of the intestines (environmental enteric dysfunction).

[0032] This problem is not constrained to developing regions. In developed regions, growing populations exacerbate the need for securing the cleanest water sources to be used for production of potable water. This leaves the food industry with either surface waters and/or reused wastewater for all processes and operations, markedly increasing the risk of potential pathogen exposure from farm to fork. In addition to this, the public demand for organic, non-pasteurized food products is inducing additional pressure to provide high quality/safe products.

[0033] To meet the safety standards for potable water and fresh produce, a wide range of rapid point of use biosensors are being developed to test drinking water and food products.³ In fact, for drinking water, the world health organization (WHO) mandates that independent surveillance be carried out under the WHO Guidelines for Drinking Water Quality (GDWQ). This is often neither cost effective nor feasible for socioeconomic challenged regions using traditional analytical techniques, which is why many groups around the world are developing low cost biosensors using acquisition systems such as smartphones.⁴

[0034] Biosensors are devices that are used for monitoring pathogens based on the specific molecular (or macromolecular) interaction between the target organism and a sensor coating. The interaction results in an intermediate complex that leads to transduction of signal to an acquisition system. Using this approach, many rapid biosensors have been developed for measuring pathogens based on interactions of the target cell with coatings composed of nucleotides, viruses, proteins, polymers, or peptides.⁵'⁶'⁷'⁸'⁹'¹⁰ To date, nearly all of these sensors lack the ability of discerning pathogen viability after capture, which persists as one of the most difficult challenges in sensing.

Overview

[0035] While traditional microbiological culture-based methods discern viability, only a small percentage of microbes can be cultured in a petri dish,¹¹ and the method thus underestimates microbial diversity. On the other hand, molecular methods such as PCR,¹² or metagenomic methods can capture viability of diverse microorganisms but require repetitive analysis over a long period of time from sub sample populations, producing limited information on the viability of a specific sample (i.e., cross sectional viability determination).¹³ Further, differentiating between viable cells and free DNA fragments with traditional PCR or metagenomics analysis is extremely challenging. Among the technologies that avoid some of the inherent problems with false positives (e.g., PCR) and false negatives (e.g., culture based methods), most techniques focus on cell membrane permeability as the marker of cell viability, assuming that lysis is the most dominant outcome following cell death. Examples of cell permeability analytical techniques include cell live/dead labeling assays,¹⁴ vPCR,^{15 16} and molecular viability testing, or MVT.¹³'¹⁷'¹⁸

[0036] Cell labeling uses a combination of membrane-impermeable stain (e.g., propidium iodide) and membrane-permeable stain (e.g., SYT09) for analysis by fluorescence microscopy and/or flow cytometry. However, live/dead stains are known to have major issues with false positives.¹⁹ The vPCR also uses the live/dead tagging concept, but the detection mechanism is based on inhibition of PCR amplification by a cell impermeant, photoactivated reagent (e.g., propidium monoazide). MVT is also a PCR based method but uses RT-qPCR to detect production of a species specific macromolecule in response to exogenous nutrients.

[0037] Other strategies for determining viability are based on the ability to maintain metabolism and homeostasis. As noted by Cangelosi et al,¹³ these efforts have been marginally successful due to the stochastic variations that occur in the local microenvironment, particularly for single cells or small populations of cells. 20 ' 21 The challenge of current technology for monitoring bacteria viability is the capability to detect a small number of viable cells, or as defined by Cangelosi the "...detectable number of proliferation-competent cells". Indeed, vPCR and MVT are based on this premise and use qPCR-based detection for measuring the molecular markers of proliferation-competent cells. The method herein uses a similar principle, but instead focuses on monitoring homeostasis and metabolic byproducts based on a multiplexing biosensor.

[0038] There is a need for simple, rapid viability detection methods and products that can be directly incorporated into the plethora of biosensor platforms on the market and under development (e.g., smartphone based devices or handheld sensors).

EAA metabolism as a viability marker

[0039] The concept for using amino acid metabolism as a rapid biomarker of viability is derived from the wealth of literature in the food safety industry that targets endogenous biogenic amines (BA) as a biomarker of microbial activity. When food is contaminated by either specific spoilage organisms (SSO) or foodborne pathogens (FP), endogenous amino

22 23 24 25 26

acids present in the food sample are decarboxylated to produce BA. ' ' ' '

[0040] There are a wide range of analytical techniques for detecting BA, including standard analytical tools such as spectrometry, chromatography,²⁷ PCR,²⁸ and biosensors.²⁹'³⁰'³¹'³² While high levels of BA are indeed a qualitative indicator of microbial spoilage, measurement of BA alone does not correlate with cell concentration or viability. Rather, the measurement of BA alone is used as a rapid qualitative screening tool for food samples as BA (such as histamine) can be directly toxic to humans upon ingestion. Amino acid content of various foods has been studied in detail.³³'³⁴ In such work, BA are used as a marker of amino acid metabolism by bacteria that are present in a sample. While BA and AA have been studied in food as qualitative indicators, to date there are no analytical techniques that measure the transient production of BA after addition of exogenous amino acids. This subtle difference is important in rapid biosensing, as to date there are few, if any, methods that can be widely adopted by electrochemical biosensors without adding significant cost or requiring additional equipment.

[0041] Disclosed embodiments of the presently disclosed subject matters provide a novel, simple, low cost method for measuring bacteria viability without the need for expensive equipment or reagents. In the approach disclosed herein, a single enzyme system is used to monitor or test the transient production of BA using a reaction that is similar to a glucometer. The testing period for monitoring the transient production can be within about 60 minutes at room temperature.

[0042] According to disclosed embodiments of the presently disclosed subject matter, a system is developed for measuring microorganism viability without the need for expensive equipment or reagents. The system comprises a biosensor having a sensor surface, a diamine oxidase adsorbed to the sensor surface, and a capture probe adsorbed to the biosensor surface. The biosensor can be any suitable sensor, such as a colorimetric paper test strip, a commercial metal electrode, a flexible electrochemical biosensor, etc. The capture probe comprises a binding material that can bind to a microorganism. The capture probe can be a mannose binding lectin, a DNA aptamer, or any other kind of binding material that can bind to a microorganism such as a bacterium or a pathogen. The system can be used to determine microorganism viability, e.g., bacteria viability or pathogen viability, by measuring transient biogenic amine production produced by the microorganism after the addition of an exogenous amino acid.

[0043] The system disclosed herein further comprises an EAA cocktail comprising an exogenous amino acid. An exogenous amino acid can be tyramine, histamine, agmatine, etc. The EAA cocktail can be a specific cocktail that is prepared to represent the free amino acid and micronutrient content of a particular sample. In one embodiment, the EAA cocktail is a solution comprising one or more amino acids that can be adsorbed to a sensor surface of a biosensor. The EAA is to be added to a sensor assay to rapidly assess microorganism viability based on the BA production produced by a microorganism after the addition of an exogenous amino acid. The EAA cocktail is used for measuring transient biogenic amine production of a sample based on the chemical reaction between BA and a glycoenzyme used in the test solution. The EAA cocktail is placed separately from the glycoenzyme, the capture probe, and the biosensor.

[0044] According to certain embodiments, a lectin-based capture of target pathogens can be combined with a viability assay using a glycoenzyme such as diamine oxidase (DAOx). Particularly, a system for microorganism viability, such as bacterial viability, can comprise a biosensor fabricated by developing a layer by layer (LBL) protein nanobrush on graphene- nanoplatinum electrodes. The protein nanobrush comprises two distinct features: 1) the ability to selectively capture microorganism, e.g., a pathogen, a bacterium, based on stimulus- response capture (measured with impedance spectroscopy), and 2) determination of viability of the microorganism based on the decarboxylation of exogenous amino acids to biogenic amines based on enzymatic activity of DAOx (measured with chronoamperometry). If a viable organism is present, decarboxylation of the amino acids by the viable organism produces biogenic amines that can be measured in real time due to enzymatic production of peroxide by diamine oxidase.

[0045] According to certain embodiments, the protein nanobrush can be a multilayer nanobrush comprising stimulus-response materials, such as, but not limited to, a capture probe (e.g. mannose binding lectin such as concanavalin A,Con A) and a glycoenzyme diamine oxidase (DAOx), assembled layer-by-layer on a surface of a g-nanoplatinum electrode. The nanobrush can be created by drop-casting a solution of Con A and a solution of DAOx on an electrode surface and rinsing the electrode surface in distilled water. The concentration of the solution of Con A can be about 0.1 mg/ml. The concentration of the solution of DAOx can be about 0.1 mg/ml. The lectin and enzyme concentration for creating the nanobrush layers can be about 0.8 mg/mL and about 1.0 mg/mL, respectively; with a time of about 20 min at room temperature using PBS as a binding buffer. A multilayer nanobrush can comprise 3, 5, or 7 layers of the stimulus-response materials. A multilayer nanobrush can also contain layers of the stimulus-response materials with other numbers.

[0046] The outermost layer of the stimulus-response material can be terminated with a 64mer DNA aptamer specific to a microorganism (e.g. Listeria monocytogenes ) for targeted capture based on impedance. Alternatively, the outermost layer of the stimulus-response material can be terminated with a generic capture probe such as Con A for detecting gram negative bacteria, for example. Lectin-mediated organism capture is interrogated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. The protein nanobrush further contains a glycoenzyme (e.g., diamine oxidase) that is used to monitor the production of biogenic amine by viable cells of microorganism in real time using DC potential amperometry.

[0047] In some disclosed embodiments, a kit is provided for rapid determination of microorganism viability. The kit comprises a glycoenzyme, a capture probe, and an EAA cocktail comprising an amino acid and, optionally, one or more micronutrients. A non- limiting list of examples of micronutrients includes carbon, hydrogen, nitrogen, oxygen, phosphorus, potassium, sodium, calcium and magnesium, as well as trace elements such as iron, sulfur, chlorine, manganese, zinc, nickel, molybdenum, copper, iodine, selenium and cobalt. In a specific embodiment, the micronutrients included in the EAA cocktail and amounts thereof will be adjusted depending on the nature of the sample tested. In the kit, the EAA cocktail is placed separately from the glycoenzyme and the capture probe before use. The capture probe and the glycoenzyme are prepared in a formula, respectively, for being adsorbed to a sensor surface of a biosensor. In one embodiment, the glycoenzyme comprises diamine oxidase. The capture probe can be, for example, a mannose binding lectin, a DNA aptamer, or a binding material that is suitable or capable of binding to a microorganism such as a microorganism (e.g. bacterium or a pathogen). The capture probe and the glycoenzyme can be prepared in solutions respectively and be drop-cast to a sensor surface of a biosensor to form a layer of the capture probe and the glycoenzyme on the sensor surface. Alternatively, a glycoenzyme and a capture probe can be assembled in a layer-by-layer (LBL) approach on a sensor surface. For example, a mannose binding lectin and a diamine oxidase can be assembled in a layer-by-layer approach on a sensor surface of an electrode to form a LBL protein nanobrush.

[0048] Disclosed embodiments of the presently disclosed subject matter further provide a method for rapid determination or detection of microorganism viability. This method includes capturing microorganisms such as bacteria from a sample and measuring in real time the transient biogenic amine production (TBAP) produced by viable microorganisms after adding an EAA cocktail. More specifically, in one embodiment, a microorganism in a sample is first captured with a capture probe adsorbed to a sensor surface of a biosensor. Then an EAA cocktail is added to the sensor surface to form a mixture comprising the EAA cocktail, the microorganism, and the glycoenzyme. After the addition of the EAA cocktail, the change in pH of the mixture or the change in hydrogen peroxide contained in the mixture is measured. A quantifiable change in pH or hydrogen peroxide is associated with the number of viable microorganisms. Thereby, the microorganism viability is detected or determined.

[0049] Method embodiments described herein can be applied with a system described herein. For example, in an alternative embodiment, the glycoenzyme is diamine oxidase (DAOx). The capture probe used to capture a microorganism in a sample can be a mannose binding lectin, a DNA aptamer, or any other kind of binding material that can bind to a microorganism such as a bacterium or a pathogen. The EAA cocktail can comprises be tyramine, histamine, agmatine, and other amino acids. The EAA cocktail can be a specific cocktail that is prepared to represent the free amino acid and micronutrient content of a particular sample. This method can be used for rapid determination of bacteria viability in food, water, or other sample.

[0050] This method can be applied to any pathogen biosensor, with a specific EAA solution tailored to match the endogenous amino acid and micronutrient concentration of a particular sample (e.g., a specific food product such as lettuce). The method can be incorporated into any electrochemical sensor directly, and can also be modified for use as a colorimetric assay in optical biosensors.

[0051] FIG. 1 is a scheme illustrating the general scheme for viability detection according to one embodiment of the presently disclosed subject matter. Panel a) of FIG. 1 is a scheme for analysis of bacteria viability based on a cascade reaction involving metabolism of an EAA cocktail according to one embodiment of the presently disclosed subject matter. As shown in Panel a) of FIG. 1, a glycoenzyme (diamine oxidase, DAOx) is adsorbed to a sensor surface 110 of a biosensor 106. A secondary capture probe 120, such as a mannose binding lectin or a DNA aptamer, is also adsorbed to the biosensor surface 110 of the biosensor 106. Step 1 is a capture step to capture a microorganism such as a bacterium using the biosensor 110. After capturing of a target microorganism 130 (as shown in capture step) and detecting the microorganism using a biosensor (of any type), a viability test is conducted. The viability test includes adding an EAA cocktail to the sensor surface 110 and monitoring the transient byproduct of EAA metabolism (biogenic amines). In a specific example, the microorganism can be a bacterium or pathogen. The term "bacterium" is in one sense broader than pathogen as it may include non-pathogenic bacteria. Pathogens may be bacteria or other microorganisms that cause disease.

[0052] Panel b) of FIG. 1 shows a detailed schematic of the reaction cascade in data analysis. The YES state represents a presence of a viable population of microorganism such as a bacterium or pathogen. In the YES (viable) state, the EAA is metabolized by a viable microorganis in the sample within the testing period, leading to a quantifiable change in pH and a concomitant increase in hydrogen peroxide due to the oxidation of B A by diamine oxidase (DAOx), where the pH is measured in real time during the viability test with a pH electrode. Simultaneously, or alternatively, oxidation of BA on the sensor surface is monitored using chronoamperometry, which produces a second signal in the YES (viable) state. Changes in pH or hydrogen peroxide can also be measured using a variety of other transduction schemes such as optical techniques. Thus, a quantifiable change in pH and hydrogen peroxide that is measured is linked to the number of cells detected in the capture step. The NO state represents to a state that a non-viable microorganism such as a bacterium or a pathogen is present. In the NO state (non-viable), no significant metabolism of EAA occurs, the cascade is not activated and no significant change in pH and/or BA occurs within the testing period; no significant change in pH and/or hydrogen peroxide is measured. Thus, the quantitative cell numbers detected in the capture step can be attributed to dead cells and/or cell fragments. In either case, the concentration of cells is established in the first step.

[0053] Embodiments of the presently disclosed subject matter further provide a stimulus- response biosensor for determining bacteria viability using lectin-glycoenzyme nanobrushes. FIG. 2 illustrated a detailed exemplary scheme of the cascade using a nanobrush biosensor. The scheme includes two step analysis of bacteria capture/detection (step 1) and viability testing (step 2). In the nanobrush biosensor shown in FIG. 2, a mannose binding lectin Concavilin A (Con A) 210 is used as the capture probe. A layer-by-layer protein nanobrush is prepared with alternating layers of Con A 210 and the glycoprotein diamine oxidase (DAOx) 212. For the nanobrush biosensor, lectin-mediate capture facilitates binding of gram negative cells from a food sample. Lectin-mediated cell capture can be measured using an electrochemical impedance spectroscopy (EIS) or cyclic voltammetry. After lectin-mediated capture, an EAA cocktail comprising exogenous amino acids is added. Viable cells captured on the sensor surface decarboxylate the amino acids to produce the biomarker of viability (BA). In the final step of the cascade, transient BA production is monitored in real time by measuring the enzymatic production of hydrogen peroxide by DAOx. A DC potential amperometry can be used to detect microbe-produced BA. The total assay time for capture/viability can be about 40 minutes: about 10 min for cell capture and about 30 min for viability.

[0054] This new sensor approach can be expanded to target specific foodborne pathogens by altering the outermost capture probe of the nanobrush assembly (e.g., use of aptamer- decorated polymer nanobrushes), enabling rapid determination of food pathogen presence and viability without addition of exogenous reagents.

[0055] The method disclosed herein can be applied with colorimetric paper test strips, commercial metal electrodes, and flexible electrochemical biosensors. The method can be validated using a variety of standard approaches, including: a secondary antibody for validating detection (fluorescein-labeled IgG),⁴⁷ an ATP assay for determining live cell metabolic state based on our stratified bienzyme sensor,³⁵ a commercial live/dead stain (BacLight), the paddle tester kit by Hach©, the dead cell stain (efluor 660), etc.

[0056] The product, system, and method disclosed herein can be used to ensure food safety and monitor water quality.

Food Safety

[0057] Traditionally, techniques for measuring pathogen viability in food include use of classic culture based methods, fluorescent viability labels,¹⁴'¹⁹ PCR¹²'¹³'¹⁵'¹⁶'¹⁷'¹⁸ flow cytometry,³⁶ physiological responsiveness or substrate turnover.³⁷ Although culture based methods are the gold standard for viability, the technique has inherent problems due to

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underestimation of the number of sublethally damaged organisms, fastidious uncultivable bacteria,³⁹ and viable but non-culturable cells.¹¹ Other than culture based methods, the other techniques have merit, but are limited due to the long analysis time and use of expensive equipment/reagents. Further, these stand-alone methods cannot be easily incorporated into existing biosensors/biochips, and require an independent subset of equipment, reagents, and trained personnel. Thus, there is a need for rapid techniques that can be integrated into handheld biosensor schemes for on site analysis.

[0058] Disclosed embodiments provide a product or system, and a method for measuring pathogen viability in food or other sample. For example, a multilayer pH-sensitive nanobrush described herein can be used for measuring pathogen viability in food. The multilayer pH- sensitive nanobrush comprises a mannose binding lectin (concanavalin A, Con A) and a glycoenzyme diamine oxidase (DAOx) assembled in a layer-by-layer approach. The outermost layer of the brush was terminated with Con A or a 64mer aptamer to facilitate capture of Escherichia coli 0157:H7 (E.coli). Lectin-mediated cell capture is interrogated using electrochemical impedance spectroscopy and cyclic voltammetry. After cell capture, exogenous amino acids are added and metabolism by viable E. coli produced biogenic amines (BA). DC potential amperometry is used to detect microbe-produced BA at +400mV. For creating the biosensor, the optimal lectin and enzyme concentrations for creating the nanobrush layers are determined to be about 0.8 mg/mL and about 1.0 mg/mL, respectively; with an optimum time of lectin/enzyme adsorption of about 20 min at room temperature using PBS as a binding buffer. Once the biosensor is assembled and the test is conducted, the total assay time for capture/viability is about 40 minutes: about 10 min for cell capture and about 30 min for viability.

[0059] This new sensor approach can be expanded to target specific foodborne pathogens by altering the outermost capture probe of the nanobrush assembly (e.g., use of aptamer- decorated polymer nanobrushes), enabling rapid determination of food pathogen presence and viability without addition of expensive reagents. In addition, the amino acid concentration/type can be further optimized for a particular food.

Water Quality

[0060] According to the World Health Organization,⁹² 3.4 million people, mostly children and those with compromised immune systems, die each year from waterborne disease. Even in the US, 7.1 million people suffer from mild to moderate waterborne bacteria infections, and 560,000 people suffer from severe associated diseases, resulting in approximately 12,000 deaths per year.⁹³

[0061] Since the NRC report in 2008 on the biological threat risk assessment (BTRA) tool established after 9/11 (NRC, 2008), there have been a number of improvements to enhance risk assessment.⁹⁶ Although probability risk assessment tools are highly valuable, increased risk and subsequent economic impacts require a new dynamic real time early warning system that builds community resilience (NRC, 2012). Given the growing list of biological threat agents and evolving number of non-zero risk scenarios based on the BTRA event tree (representing millions of numerated scenarios), there is a pressing need for new rapid quantitative threat monitoring technologies. Current threat monitoring kits for the food/water chain do not support such an early warning system, as they are expensive, laborious, and time consuming. These kits produce data that restricts the accuracy of predictive risk models (most test kits for chemical/biological threats require at least 24 hours to merge with a data analytics platform). Warning systems that use statistical risk models have high levels of uncertainty which stems from: i) low resolution/incomplete sensor data sets, and ii) lack of understanding of chemical/biological threat dynamics under "real world" situations. To manage threat risk and response, new decision analytic tools must be developed that are based on accurate/precise real time sensor data linked to risk models, and these tools should be interfaced with Integrated Public Alert and Warning Systems (IPAWS) established by the Federal Emergency Management Agency (FEMA).

[0062] Clean drinking water is a mission-critical asset for deployed defense personnel. The presence of pathogenic bacteria in water systems can cause widespread illness and even death, and thereby significantly hinder warfighter readiness and effectiveness. Army, Air Force and Navy preventive medicine personnel all share similar potable water monitoring requirements, which includes monitoring indicator organisms that are linked to fecal contamination such as generic Escherichia coli (E. coli.) and coliform bacteria.⁵¹ The total concentration of bacterial coliforms (i.e., non spore-forming Gram negative bacteria) is used as an indication of overall sanitary quality, and generic E. coli presence is a hallmark indicator for fecal contamination and potential waterborne pathogens. However, the current approved methods require extensive time and personnel training and thus there is a critical technology gap for rapid, on-site detection of microbial contamination, which is especially important for testing drinking water sources such as surface water, ground water, or host- nation municipal water systems. Further, current field-ready methodologies for detecting fecal organism(s) have challenges related to false positives from non- targets (e.g., microorganisms, native flora, particulate matter), and lack the ability to discriminate between viable and non-viable bacteria.

[0063] Existing methods for bacteriological water analysis (assessment of total coliform bacteria and E. coli) suffer from one or more critical limitations prohibiting their applicability for portable, rapid, low-cost field-testing. Currently, such analysis is governed by the U.S. Environmental Protection Agency (USEPA), as described in the Revised Total Coliform Rule. The USEPA-approved Standard Methods, generally take a minimum of 16 hours to complete, causing significant delays in the evaluation of field drinking water supplies.

[0064] Existing mechanisms for bacteria detection include cell culture and colony counting, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR), each with key limitations for portable, rapid field-testing. Overviews/surveys of bacteria detection approaches, tradeoffs, and limitations are well reported in the scientific literature.^{4*3*71*72*73*74}

[0065] Culture methods (multiple tube method, plate count, membrane filtration, pour plate) are extremely accurate and can discriminate viable from non- viable cells, but the test requires long incubation times and highly skilled personnel. Biochemical methods, such as testing cytosolic biomarkers, are faster than culture methods (<10 min) but cannot trace the results back to specific detection of indicator organisms and also produce false negatives due to incomplete cell lysis.⁵⁴ Enzyme-linked immunosorbent assay (ELISA) is a highly specific quantitative method that permits species or serotype level confirmation, but is expensive, low throughput, demands highly skilled personnel, and also requires long-time frame to obtain results (usually at least 24 hours). Labeling techniques using flow cytometry, laser scanning, luminometry, or epifluorescence are rapid (typically < 1 hour), but fluorescent labels are expensive, susceptible to photobleaching, and some are cytotoxic. In addition, these acquisition systems are cost-prohibitive for large scale or high throughput field monitoring campaigns.

[0066] Molecular methods for monitoring nucleic acids, including polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and nucleic acid sequence -based amplification (NASBA) have been used extensively, but the techniques are time consuming (e.g. 8-24 hr for sample filtration, DNA amplification, and PCR detection).⁵⁵ Although a few recent techniques have used PCR to distinguish viable from non- viable pathogens (known as molecular viability analyses), the variable persistence of nucleic acids in cells post-death leads to low accuracy in "real" environmental samples, which adds to the analysis time and induces error.⁵⁶ In addition, molecular methods require that viability markers be measured over long periods of time, extending the detection time considerably and deterring development of "one pot" rapid testing schemes.

Assessment of Bacteria Viability [0067] In addition to detecting the presence of pathogenic organisms in drinking water, it is of upmost importance to determine cell viability to infer whether the microbes pose a threat to health.

[0068] Methods that detect the viability of a microorganism must provide rapid throughput under a range of operating conditions (temperature, pH, turbidity, total organic carbon, etc). Use of fluorescent labels,¹⁹ or nucleotide markers,¹² are the most common methods, although the use of labels is generally more robust under varying source water characteristics. Commercial methods such as the live/dead assay (Bac Light) use DNA- selective labels such as propidium iodide (PI) and SYT09, the former is not cell permeable and will only stain dead (permeabilized) cells while the latter stains cytosolic DNA. While the technique has become standard for laboratory screens, the labels are cost prohibitive for large scale/high throughput monitoring and subject to photobleaching at room temperature. Further, as discussed in,¹⁹ PI can be toxic at concentrations that are conducive to quantitative imaging.

[0069] Monitoring bacteria viability is a vital and perhaps the most challenging aspect of pathogen detection. The standard methods involve plate based assays, and these techniques are accurate (although they require 24-48 hours for processing). Further complicating the problem, under stress conditions, such as exposure to low levels of disinfectants, many species of bacteria enter a metabolic state that leads to a physiological condition known as viable but non-culturable (VBNC). Several human pathogenic bacteria have been reported to enter into the VBNC state under stress conditions, and culture based techniques are not capable of measuring the presence/viability of these bacteria.¹¹

[0070] Due to the clear limitations of culture based techniques for rapidly assessing viability, a number of other methods have been developed over the last few decades for differentiating viable cells, including atomic force microscopy,⁷⁵'⁷⁶ nucleic acid amplification,⁷⁷ surface-enhanced Raman scattering,⁷⁸ Fourier transform infrared spectroscopy,⁷⁹ and fluorescence techniques combined with flow cytometry or the

80 81 82

polymerase chain reaction. ' ' However, these techniques are time consuming, require training of personnel, and are laborious and costly. Fluorescent stains are traditionally used for detecting viability, but the fluorescent molecules have a poor shelf life due to photobleaching or thermal decomposition above 4C. Additionally, some stains (such as propidium iodide) can be cytotoxic at concentrations that are conducive to quantitative imaging.¹⁹ There are shortcomings of viability assays. [0071] Different from convention technologies, the disclosed embodiments described herein provide a water analysis system that is field-portable, low-cost, and easy-to-use. This water analysis system can detect presence and viability of fecal indicator organisms with a sensitivity of about 1 CFU/100 mL in total test time less than about 4 nr. The technology disclosed herein combines: a rapid concentration/purification of target bacteria from raw water samples, viability discrimination using fluorescent labels, and subsequent analysis using simple imaging via a portable microscope or smartphone. The disclosed technology improves water-testing protocols by reducing test time, cost, and logistical burden. The technology further provides advancements that not only fill a critical technology gap unmet by any other competing technology for defense environments, but also potentially improve sanitation and food safety in other public and private domains (farms, municipal water systems, hospitals/clinics, etc.).

[0072] In one embodiment, the disclosed water analysis system is used to rapidly test for presence and viability of coliform bacteria and E. coli in field water samples. The disclosed water monitoring system is portable, battery-powered, reusable, easy to use, and selective to the specific indicator organism. The disclosed water monitoring system leverages two emergent technologies that have independently developed for biosensing applications. The first emergent technology involves a magnetic pre-concentration step using micrometer- sized magnetic microdiscs coated with capture probes (e.g., aptamers, proteins, etc.) that selectively bind to target bacteria and enable rapid concentration of those targets from about 100 mL samples in a matter of just seconds. The second emergent technology involves the use of carbon quantum dots to enable live/dead viability assay on the magnetically concentrated discs via optical inspection using a smartphone based microscope. The combination of highly selective capture-probe-functionalized microdiscs, magnetic pre- concentration, viability assay, and a portable microscopy system improves measurement time, sensitivity, and viability discrimination compared to other competing technologies.

[0073] Having described the many embodiments of the presently disclosed subject matter in detail, it will be apparent that modifications and variations are possible without departing from the scope of the disclosed subject matter defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure, while illustrating many embodiments of the disclosed subject matter, are provided as non-limiting examples and are, therefore, not to be taken as limiting the various aspects so illustrated.

[0074] Embodiments of the presently disclosed subject matter are further defined in the following examples. It should be understood that these examples are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of embodiments of the presently disclosed subject matter. Without departing from the spirit and scope thereof, one skilled in the art can make various changes and modifications of the disclosed subject matter to adapt it to various usages and conditions. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference herein.

EXAMPLES

Example 1: Food Safety

[0075] In this example, the disclosed technique described herein is applied by using a layer by layer protein nanobrush impedimetric sensor for detection of E. coli 0157:H7. The nanobrush is terminated with a generic capture probe (Concavilin A, a mannose-binding lectin) for detecting gram negative bacteria. The nanobrush also contains the glycoenzyme diamine oxidase, which is used to monitor the production of BA by viable cells in real time using DC potential amperometry. The total assay time for capture and viability test is about 42 minutes: about 10 min for cell capture, about 2 min for adding EAA, and about 30 min for detection of viability. The technique is incorporated with three different sensors, namely a commercial Pt/Ir electrode, a laser inscribed graphene flexible electrode, and a nanomaterial colorimetric test strip.

Methodology

[0076] FIG. 3 is a schematic illustrating a stimulus-response LBL protein nanobrush on reduced grapheme oxide-nanoplatnum (rGO-nPt) electrodes according to one embodiment of the presently disclosed subject matter. In FIG. 3, a mannose binding lectin (Con A) and diamine oxidase (DAOx) are assembled in a LBL approach for developing a multilayer nanobrush. Yao et al,⁴⁰ first developed a LBL stimulus response nanobrush for peroxide sensing using Con A and the glycoenzyme glucose oxidase (GOx), and later extended this to include glucose sensing.⁴¹ Using the methods developed by Yao,⁴⁰'⁴¹ the LBL protein nanobrush as shown in FIG. 3 is prepared on an electrode surface of a reduced graphene oxide-nanoplatinum (rGO-nPt) electrode.⁴²'⁴³ Stock solutions (0.1 mg/mL) of Con A (104 kDa) or DAOx (172 kDa) are drop-cast on the electrode surface 316 to form drop 312, the electrode surface 316 is rinsed in DI, and then the charge transfer resistance (Ret) is measured [0077] To test whether the protein nanobrush is capable of measuring biogenic amines (first in the absence of bacteria), DC potential amperometry (DCPA) is conducted at +350 mV in PBS buffer at room temperature. Stock solutions of tyramine, histamine, or agmatine are added stepwise to the electrochemical cell following established protocol.⁴²'⁴³'⁴⁵'⁴⁶

[0078] For organism capture, the outermost layer of the nanobrush is terminated with a capture probe (e.g. Con A to facilitate capture of Escherichia coli 0157:H7, or other bacteria known to bind to Con A). The sample is collected in a sample tube, and drop cast onto the sensor surface, or in other applications with high levels of bacteria the sensor may be immersed into a liquid sample that is agitated with a magnetic stir bar or other similar mixing equipment. After a contact time of 20 min, the sensor surface is washed with distilled water or buffer at least three times using a spray bottle prior to testing. Lectin-mediated cell capture is interrogated using EIS. After cell capture, exogenous amino acids are added and metabolism by viable microbes (e.g. E. coli) produce biogenic amines (BA). DC potential amperometry is used to detect microbe-produced BA at +350mV.

[0079] All measurements are performed in at least triplicate. Analysis of variance (ANOVA model I) or one-tailed student' s t-test are used to test for significance as noted. All error bars represent standard deviation of the arithmetic mean, and values of n are reported for each data set.

Results and discussion

[0080] FIG. 4 shows representative Nyquist plots obtained during LBL assembly on rGO- nPt electrodes according to one embodiment of the presently disclosed subject matter. The charge transfer resistance increases significantly with each addition of protein, indicating that the alternating charge interactions lead to formation of a chained nanobrush. In particular, after adding Con A, electrostaitc interaction with the metal surface caused a significant increase in the charge transfer resistance (Ret); the mannose binding site on Con A is highly negative and adsorbs well to platinum as shown by many others.⁴⁰'⁴¹'⁴⁸ Subsequent addition of the glycoenzyme (DAOx) further increases Ret, and this trend continues for up to 7 layers of protein (alternating between Con A 210 and DAOx 212 as shown in FIG 2). Assembly of the nanobrush is based on repeating adsorption of proteins in solution with alternating charges on the surface of the rGO-nPt electrode. As reviewed by Sato et al,⁴⁸ lectin-glycoenzyme nanobrushes have stimulus responsive properties, switching between on/off states with changes in temperature, pH, or different chemical environments.

[0081] The optimal lectin and enzyme concentrations for creating the nanobrush layers are determined to be about 0.8 mg/mL and about 1.0 mg/mL, respectively; with an optimum time of about 20 min at room temperature using PBS as a binding buffer. Using these optimum parameters, a LBL nanobrush with 5 layers is prepared and the sensitivity toward biogenic amines (no cell capture) is measured using DCPA at +350 mV. FIG. 5 illustrates graphs showing the results of chronoamperometric detection of biogenic amines using the LBL protein nanobrush (5 protein layers). Panel a) of FIG. 5 illustrates the calibration response of a biosensor toward tyramine, histamine, or agmatine during initial testing (no bacteria). Panel b) of FIG. 5 illustrates shows the linear behavior of the biosensor for detecting either tyramine, histamine, or agmatine based on the average sensor output from panel a). As shown in FIG. 5, the nanobrush system is responsive to tyramine (LOD = 640 + 30 μΜ), histamine (LOD = 510 + 20 μΜ), and agmatine (LOD = 320 + 25 μΜ) with an average response time of 5 s for the range tested. After this proof of concept, the nanobrush is used to capture E. coli 0157:H7.

[0082] In an alternative embodiment, LBL nanobrush sensors are prepared using 5 layers or 7 layers, and EIS is used to test capture efficiency of E. coli 0157:H7 at a cell concentration of 10³ CFU/mL based on R_ct. FIG. 6 shows the average impedance (cutoff frequency - 1 Hz) for rGO-nPt nanobrush electrodes after capture of 10³ CFU/mL E. coli 0157:H7. Panel A of FIG. 6 is a plot illustrating the charge transfer resistance measured after addition of layers in the synthesis of LBL. Panel B of FIG. 6 are graphs illustrating the testing of 10³ CFU/mL bacteria capture using a 5 layer system and a 7 layer system. As shown in Panel B of FIG. 6, for nanobrush structures with 5 layers, the net impedance (cutoff frequency of lHz from Bode plot) increased significantly after cell capture, but with 7 layers the nanobrush is unstable and the impedance (-Z") decreases to levels that are similar to a bare rGO-nPt electrode. This result demonstrates that the LBL capture system cannot be too long or the bacteria will cause the structure to collapse after capture. In preliminary studies, cell capture is highest for a 3 or 5 protein LBL nanobrush. In a specific embodiment, the LBL is not less than 2 layers or more than 6 layers.

[0083] FIG. 7 illustrates the proof of concept demonstration of capture and viability cascade. In FIG. 7, oxidative current is a marker of viable cells captured by the nanobrush. The inset plot shows the differential current versus a control electrode with no cells. E. coli 0157:H7 is captured with a 5 layer nanobrush (EIS plots not shown for brevity). After cell capture, exogenous histidine (0.5mM) is added every 45 min. The noted increase in oxidative current (at +350mV) in FIG. 7 is due to the production of biogenic amine (histamine) by viable cells. In the cascade reaction, this histamine is then oxidized by DAOx, producing hydrogen peroxide. The deprotonation of peroxide at +350mV increases oxidative current due to the liberation of free electrons. In a control study (with no cell capture), the increase in signal due to addition of histidine is not significant. The inset plot shows the differential current versus a control electrode with no cells, which is a more accurate measurement regarding signal to noise ratio. This plot is evidence that histamine produced by cell metabolism can be easily measured with oxidative amperometry using this cascade reaction, without the need for any cofactors or mediators. The average time for a step change response is about 30 min, indicating that this method is a rapid tool that can be used for both bacteria detection and viability measurement.

[0084] While FIG. 7 is strong evidence that the concept for viability has merit, lectins contain one or more carbohydrate recognition domain, and the structure of the CRD determines the overall specificity. Con A is a mannose binding lectin that has affinity for a wide range of carbohydrate patterns on bacteria, viruses, protozoa and fungi.⁴⁹'⁵⁰ Thus, Con A is fairly non-specific and is used as a proof of concept in this work. Alternatively, the lectin can be replaced with a strain-specific aptamer to enhance capture efficiency to specific organisms or foodborn pathogens. As a result, the kinetics of biogenic amine production by the specific spoilage organisms or foodborn pathogens can be examined.

Example 2: Water Quality

[0085] This example includes side-by-side comparisons of the described methods versus established EPA methods using a variety of water samples based on established quality assurance and quality control (QA/QC) protocol. The objective of this example is to detect about 1 CFU/100 mL with test time less than about 8 hr using a laboratory bench-top demonstration. Prior to sample analysis, all source water is characterized for general water quality parameters, including: temperature, pH, turbidity, total organic carbon, free and total disinfection residual, and heterotrophic plate count.

[0086] In this example, the Alternate Test Procedure undergoes a side-by-side comparison to the USEPA approved reference method for total coliforms (number SM9221B) and E. coli (number SM9221F). The pathogen analysis follows comprehensive QA/QC guidelines form EPA/USDA/CDC. The method development can include appropriate QA/QC according to USEPA approved standards, including replicate spiked reagent water, positive/negative spike controls, duplicate samples, method blanks, and media sterility checks. Characterization of method performance includes data on: precision/bias, specificity, detection limit, recovery, precision, and false positive/negative rates. Viability methodology

[0087] In this example, a test system is demonstrated to meet the USEPA-Alternate Test Procedure (ATP).⁸⁹ Using a disposable biosensor on cellulose paper,⁴⁴'⁴⁵ an electrochemical (amperometric) viability assay is implemented based on direct measurement of metabolism using a cascade reaction with the bacteria captured on microdiscs. The technique is based on electrochemical detection of amino acid metabolism as described herein. As described above, the sensor may utilize enzymatic activity of diamine oxidase (DAOx), which oxidizes biogenic amines produced by viable cells, resulting in oxidative current. The working electrode for the disposable sensor is composed of graphene coated cellulose paper,⁴⁴ or laser inscribed graphene on plastic films.⁹⁰ After capture of the bacteria on discs and primary confirmation of viability, bacterial cells are positioned on a conductive surface for the secondary confirmation step, which requires about 20 min and produces quantitative output regarding cell metabolism within about 30 min.

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[0089] All documents, patents, journal articles and other materials cited in the present application are incorporated herein by reference.

[0090] While the presently disclosed subject matter has been disclosed with references to certain embodiments, numerous modification, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the presently disclosed subject matter, as defined in the appended claims. Accordingly, it is intended that the presently disclosed subject matter not be limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.

Claims

WHAT IS CLAIMED IS:

1. A product comprising:

a biosensor having a sensor surface;

a glycoenzyme; and

a capture probe,

wherein the glycoenzyme and the capture probe are adsorbed to the sensor surface, and wherein the capture probe comprises a binding material that is capable of binding to a microorganism.

2. The product of claim 1, wherein the microorganism is a pathogen.

3. The product of any of claims 1 or 2, wherein the microorganism is a bacterium.

4. The product of any of claims 1-3, wherein the glycoenzyme comprises diamine oxidase.

5. The product of any of claims 1-4, wherein the capture probe comprises a mannose binding lectin.

6. The product of any of claims 1-5, wherein the capture probe comprises a DNA aptamer.

7. The product of any of claims 1-6, wherein the biosensor comprises a colorimetric paper test strip.

8. The product of any of claims 1-6, wherein the biosensor comprises a metal electrode.

9. The product of any of claims 1-6 or 8, wherein the biosensor is a flexible electrochemical biosensor.

10. The product of any of claims 1-9, wherein the biosensor is comprises graphene oxide and a metal constituent.

11. The product of any of claims 1-6, 8 or 10, wherein the metal constituent is nanoplatinum.

12. A kit comprising:

a glycoenzyme,

a capture probe, and

an EAA cocktail comprising an amino acid, wherein the capture probe and the glycoenzyme are in a formula to be adsorbed to a sensor surface of a biosensor,

wherein the capture probe comprises a binding material that is capable of binding to a microorganism, and

wherein the EAA cocktail is placed separately from the glycoenzyme and the capture probe.

13. The kit of claim 12, wherein an EAA cocktail is prepared to represent a free amino acid and micronutrient content of a particular sample.

14. The kit of any of claims 12 or 13, wherein the glycoenzyme comprises diamine oxidase.

15. The kit of any of claims 12-14, wherein the capture probe comprises a mannose binding lectin.

16. The kit of any of claim 12-15, wherein the exogenous amino acid comprises tyramine.

17. The kit of any of claims 12-16, wherein the exogenous amino acid comprises histamine.

18. The kit of any of claims 12-17, wherein the exogenous amino acid comprises agmatine.

19. The kit of any of claims 12-18, further comprising a biosensor.

20. A method for determining a microorganism viability comprising:

capturing a microorganism in a sample with a capture probe adsorbed to a sensor surface of a biosensor, wherein a glycoenzyme is adsorbed to the sensor surface; adding an EAA cocktail to the sensor surface to form a mixture comprising the glycoenzyme, the EAA cocktail, and the microorganism; and

measuring a change in pH or hydrogen peroxide of the mixture, thereby determining the microorganism viability,

wherein a quantifiable change in pH or hydrogen peroxide of the mixture is associated with the number of viable microorganisms.

21. The method of claim 20, wherein the EAA cocktail comprises at least one of histamine, tyramine or agmatine.

22. The method of any of claims 20 or 21, wherein the glycoenzyme comprises diamine oxidase.

23. The method of any of claims 20-22, wherein the capture probe comprises a mannose binding lectin.

24. The method of any of claims 20-23, wherein the capture probe comprises a DNA aptamer.

25. The method of any of claims 20-24, wherein the biosensor comprises a colorimetric paper test strip.

26. The method of any of claims 20-25, wherein the biosensor comprises a metal electrode.

27. The method of any of claims 20-26, wherein the biosensor is a flexible electrochemical biosensor.

28. The method of any of claims 20-27, wherein the biosensor is comprises graphene oxide and a metal constituent.

29. The method of any of claims 20-28, wherein the metal constituent is nanoplatinum.