System and method for quantitative assessment of bovine mastitis using optical imaging

Abstract

A system and method for in-situ quantitative assessment of Bovine Mastitis (BM) in collected milk samples using optical imaging is disclosed. A bio-functionalized assay tray contains multiple wells, each having a gel with a specially prepared mixture of hemoglobin (Hb) and metal ions. At least one of the wells is filled with a reference milk sample which is characteristic of milk with no BM disease. Other wells are filled with collected milk samples. After waiting an interaction time, luminol and hydrogen peroxide solutions are added to the wells, causing chemiluminescent (CL) light emission. The intensity of CL light emitted by wells containing haptoglobin (Hp) is inhibited due to Hb-Hp binding. After waiting a CL light stabilization time, the tray is placed in a dark box, and a camera and image processor are used to analyze the CL light intensities. A calibration curve yields the Hp concentration in each well.

Description

[0001] System and Method for Quantitative Assessment of Bovine Mastitis Using Optical Imaging

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is related to and claims priority from commonly owned US Provisional Patent Application No. 63 / 734887, entitled “Method and System for Bovine Mastitis Assessment Using Optical Imaging”, filed on December 16, 2024, the disclosure of which is incorporated by reference in its entirety herein.

[0004] TECHNICAL FIELD

[0005] The present invention relates to systems and methods for quantitative assessment of bovine mastitis (BM) in milk and specifically to the determination of BM severity using optical imaging of enhanced chemiluminescence (CL) signals.

[0006] BACKGROUND OF THE INVENTION

[0007] BM is one of the most frequent diseases in dairy cattle, having a large adverse effect on farm economics, including increased treatment costs, decreased milk yield, escalation of somatic cell counts, increased risk of removal from the herd or even death. Early detection of new BM cases and the ability to provide quantitative diagnostic measurements of their severity are essential for timely infection control. A portable in-situ system with these capabilities would enable rapid and cost-effective use of antibiotics and thereby enhance the overall economy of dairy farms.

[0008] Traditionally, BM detection depends on the efficacy and reliability of a conventional laboratory technique, such as Enzyme-Linked Immunosorbent Assay (ELISA), which is designed to measure somatic cell counts (SCC), detect causative pathogenic bacteria, and reveal inflamed status associated with secreted biomarkers, such as Haptoglobin (Hp). The latter are secreted into plasma or milk upon inflammation, infection or trauma and are used as analytical biomarkers for clinical status assessment.

[0009] US patent number 9,927,447 to A. R. Lippert et al., entitled “Composition, device and imaging system for analysis using chemiluminescent probes”, and dated March 27, 2018, provides a method for the rapid monitoring of biological analytes in a point-of-care setting by providing a smart phone; providing a sample chamber; providing a sample; providing a dark box with a smartphone holder attached to the dark box top with the camera opening positioned about the aperture, adding a biological specimen suspected of containing a biological analyte in the sample chamber; adding a bis(2,4,6-trichlorophenyl) oxalate, an imidazole and a fluorophore to the sample chamber to react with the biological analyte; placing the sample chamber into the dark box; generating an emission from the fluorophore in response to the reaction with the biological analyte; and recording a set of time-lapse images of the emission with the smartphone. U.S. Patent No. 10,866,250, to J. Lehmann et al., entitled “Method and Apparatus for Monitoring the State of Health of Dairy Cows”, and dated December 15, 2020, teaches methods and apparatuses for monitoring the state of health of dairy cows based on analyzing the Hp biomarker and part of the polymeric immunoglobulin receptor (PIGR), the secretory component (SC), in a milk sample. This allows diagnosis of mastitis or systemic diseases which occur outside the udder on the basis of the protein biomarker described here.

[0010] Promising research results for BM detection based upon a chemiluminescence (CL) assay have appeared in an article by N. R. Nirala et al., entitled “Milk Haptoglobin Detecting Based on Enhanced Chemiluminescence of Gold Nanoparticles”, published in Taianta vol. 197, pp. 257- 263, 2019. In this technique, the presence of Hp protein in a milk sample is indicated by a decrease in the CL emission of light at a specific wavelength, when the sample is mixed with a liquid-phase biofunctionalized assay containing a luminol-oxidant-Hb system and gold nanoparticles.

[0011] US patent number 11,307,198 to Y. Lin et al., entitled “Compositions and Methods for Antigen Detection Incorporating Inorganic Nanostructures to Amplify Detection Signals”, and dated April 19,2022, discloses antigen detection reagents and related methods, systems, and kits. The reagents comprise an antigen-binding molecule conjugated to an inorganic component. In some embodiments, the inorganic component possesses catalytic functionality to provide a detectable signal. In some embodiments, the catalytic inorganic component is or comprises a bimetallic nanoparticle. In other embodiments, the inorganic component is a nanoflower that provides a physical scaffold onto which the antigen-binding component and a reporter component can be loaded, resulting in augmented antigen-binding and reporting capabilities.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is directed to an in-situ system and method for quantitative assessment of BM predicting biomarkers (haptoglobin, Hp), to serve as a basis for timely, evidence-based therapy. The system includes an integrated CL imaging and image analysis platform, which enables rapid and accurate assessment of the quality and safety of whole milk samples, without specialized laboratory equipment.

[0014] According to the teachings of an embodiment of the present invention, there is provided a system for in-situ quantitative assessment of Bovine Mastitis in collected milk samples using optical imaging. The system includes a bio-functionalized assay tray having a multiplicity of wells, a chemiluminescence (CL) solution, a hydrogen peroxide solution, a reference milk sample, a dark box, a camera and an image processor. Each well of the bio-functionalized assay tray includes an agarose gel with predetermined concentrations of metal ions and hemoglobin (Hb). At least one cell of the tray receives a specified amount of the reference milk sample, and one or more cells of the tray each receive the same amount of the collected milk samples. Each well with a milk sample then receives a specified amount of the CL solution and a specified amount of the hydrogen peroxide solution. The dark box, camera and image processor are used to acquire and analyze images of CL light emission, to calculate normalized CL light intensities, and to determine quantitative estimates of Haptoglobin (Hp) concentration in the collected milk samples. The determination is based upon a calibration curve which relates a value of normalized CL light intensity to a value of Hp concentration.

[0015] According to some aspects, the normalized CL light intensity is calculated by dividing a CL light intensity value for a well containing a collected milk sample by a CL light intensity value for a well containing the reference milk sample.

[0016] According to some aspects, the calibration curve represents a linear relationship between the value of the normalized CL light intensity and a logarithm of the value of Hp concentration.

[0017] According to some aspects, the metal ions are copper ions.

[0018] According to some aspects, the luminol solution includes a colloidal suspension of reflective nanoparticles configured to enhance the CL light emission.

[0019] According to some aspects, the reception of milk samples in the wells of the tray is followed by a predetermined Hb-Hp interaction time duration (Tl).

[0020] According to some aspects, Tl has a value between 5 and 60 minutes

[0021] According to some aspects, reception of the CL and hydrogen peroxide solutions in the wells of the tray is followed by a predetermined CL stabilization time duration (T2).

[0022] According to some aspects, T2 has a value greater than or equal to 0.5 minutes.

[0023] According to some aspects, the camera and image processor are integral components of a smartphone.

[0024] According to some aspects, the camera is fitted with a blue filter having a transmission band which includes a light wavelength of 425 nanometers.

[0025] According to some aspects, the bio-functionalized assay tray is stored in refrigeration before it receives milk samples.

[0026] According to some aspects, the image processor is in communication with a display device in order to present a BM diagnostic report to a user. There is also provided according to the teachings of an embodiment of the present invention, a method for in-situ quantitative assessment of Bovine Mastitis in collected milk samples using optical imaging, consisting of: preparing a bio-functionalized assay tray with multiple wells each containing a gel with hemoglobin (Hb) and metal ions; preparing a chemiluminescence (CL) solution, a hydrogen peroxide solution, a reference milk sample, a dark box, a camera, and an image processor; collecting milk samples on-site and applying specified amounts of the reference and collected milk samples to wells of the assay tray; waiting a predetermined Hb-Hp interaction time duration (Tl); adding specified amounts of CL and hydrogen peroxide solutions to the wells containing milk samples; waiting a predetermined CL stabilization time duration (T2); inserting the tray into the dark box, positioning the camera, acquiring an image, and transferring the image to the image processor; calculating normalized CL light intensity values; and using a predetermined calibration curve to determine values of Hp concentration in each of the collected milk samples.

[0027] According to some aspects, Tl has a value between 5 and 60 minutes and T2 is greater than or equal to 0.5 minutes.

[0028] According to some aspects, the bio-functionalized assay tray is stored in refrigeration prior to the collecting of milk samples in step c).

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings. With regard to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0031] FIG. 1: A schematic of an exemplary in-situ system for quantitative assessment of BM in whole milk samples using optical imaging, according to the invention.

[0032] FIGs. 2A-2D: Exemplary bar graphs for optimizing the metal ion, luminol, peroxide and hemoglobin concentrations, respectively.

[0033] FIGs. 3A and 3B: Exemplary bar graphs for optimizing the Hb-Hp interaction time duration (Tl) and the CL stabilization time duration (T2), respectively.

[0034] FIG. 4: An exemplary bar graph of enhanced CL intensity vs. shelf life (in weeks) of the biofunctionalized assay tray. FIG. 5: An exemplary graph of a calibration curve relating normalized CL intensity to Hp concentration in a milk sample.

[0035] FIG. 6: A block diagram of an exemplary method for quantitative BM assessment using optical imaging, according to the invention.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] FIG. 1 shows a schematic of an exemplary in-situ system 100 for quantitative assessment of BM in whole milk samples using optical imaging, according to the invention. A biofunctionalized assay tray 120 includes multiple wells 125, each of which contains an agarose hydrogel blended with metal ions, e.g. copper ions (Cu+2), and then functionalized with hemoglobin (Hb) for target of Hp. The relative amounts of hydrogel, copper ions and Hb are optimized for producing CL (or enhanced CL) emission of light, when luminol and hydrogen peroxide (H2O2) are added to the wells. The optimization process is described in detail below, in connection with FIGs. 2A-2D.

[0038] Pre-fabrication of the bio-functionalized assay tray 120 proceeds as follows. A 3% agarose solution in ultrapure water is brought to a boil and then cooled to about 40°C. 10 mL of this solution is blended with 0.5 mL of metal-ion solution, e.g. an aqueous solution of Cu+2ions with a concentration of 10 mg / mL. A volume of 200 pL of this hydrogel mixture is poured into each well 125 and given 5 minutes to solidify. Finally, 100 pL of Hb in aqueous solution with a concentration of 1 pg / mL is applied to each well. After incubating for an hour and several rinsings with ultrapure water, the assay tray 120 is ready for use. To maintain its effectiveness, the assay tray is stored at 4 degrees Celsius (°C) and a relative humidity of at least 60% until it is used.

[0039] For BM assessment, each well of the assay tray 120 is filled with a predetermined amount of a whole milk sample 110, for example having a volume of 100 milliliters (mL). After an interaction time duration of T1 minutes, the Hp bio markers (if any) that are present in the milk sample bind with the Hb to form Hp-Hb complexes. The choice of a value for T1 is explained in detail, in connection with FIG. 3A.

[0040] Each well is then treated with a solution 130a of luminol and a solution 130b of hydrogen peroxide (H2O2). Under alkaline conditions, the Hb and metal ions in the well catalyze the luminol-peroxide mixture and produce a distinct emission of CL, light, peaked at a wavelength of approximately 425 nm. This emission is caused by the transition of 3-amino phthalate ions from an excited energy state to their ground state. The intensity of the emitted CL light is strongly affected by the presence of Hp, and depends upon reaction conditions, such as Hb and metal ion concentrations, luminol and oxidant levels, and the CL stabilization time duration T2. The choice of a value for T2, which optimizes the intensity of the CL emission, is explained in detail in connection with FIG. 3B.

[0041] In some embodiments, the luminol solution 130a also contains a colloidal suspension of reflective nanoparticles.

[0042] In FIG. 1, the assay tray 120 is inserted into a dark box 160 through input port 160a and a digital camera 170 is positioned to receive CL emitted photons 165 thereby forming a CL image 175 of the assay tray. The image is then transferred to an image processor for determining the intensity of CL emission in each well

[0043] In some embodiments, the camera is fitted with a blue filter with a transmission band containing a wavelength of 425 nanometers.

[0044] In some embodiments, a smartphone with an image processing application may be used to integrate the camera 170 together with the image processor 180. When using a smartphone camera placed in direct contact with the dark box 160, a setting of maximum ISO (above 1600) with an exposure time of 8 seconds provides adequate sensitivity for forming the CL image 175.

[0045] The CL intensity in each well is related to the concentration of Hp biomarkers in the well. The relation is explained in detail, in connection with FIG. 5. An output BM diagnostic report 190 contains the results of the optical imaging assay together with specific recommendations for BM infection control. In some embodiments, the image processor is configured to prepare a BM diagnostic report 190, which may be displayed on a peripheral display device.

[0046] FIG. 2A shows an exemplary bar graph for optimizing the metal ion concentration of Cu+2in units of milli-moles per liter, also known as milli-Molar (mM), so as to distinguish between different levels of BM infection. On the horizontal axis, P+ is a low SCC reference level, typically below 200 thousand white blood cells per milliliter, corresponding to a healthy reference milk sample. The healthy, low sick, medium (or mid-) sick, and sick levels correspond to SCC levels (in thousands of counts) of <200, 200-400, 400-800, and >800, respectively. The metal ion concentration is varied from 4 to 149 mM, while maintaining fixed conditions of: hemoglobin (Hb) of 1 pg / mL in the well, luminol sodium salt solution of 50 mM, hydrogen peroxide (H2O2) solution of 3.0 mM, and sodium hydroxide (NaOH) solution of 15 mM. The latter is used to maintain a pH of about 12, as higher pH values were observed to significantly reduce the enhanced CL intensity.

[0047] The height of each colored bar in FIG. 2A is the enhanced CL intensity in arbitrary units (a.u.), and the “stem” at the top indicates the ± one-sigma standard deviation. The figure indicates that a choice of 37 mM (the gray bar) is most effective at distinguishing between healthy and sick BM levels.

[0048] FIG. 2B shows an exemplary bar graph for optimizing the luminol concentration, to distinguish between different levels of BM infection. The luminol concentration varies from 13 mM to 100 mM, while maintaining the other conditions fixed at the values in FIG. 2A. A concentration of 50 mM (the dark green bar) provides good differentiation between different levels of BM infection. Higher luminol concentrations tend to lead to self-absorption of emitted radiation and a reduced CL. intensity.

[0049] FIG. 2C shows an exemplary bar graph for optimizing the peroxide concentration, to distinguish between different levels of BM infection. The peroxide concentration varies from 1.5 mM to 8.0 mM, while maintaining the other conditions fixed at the values in FIG. 2A. A concentration of 3.0 mM (the gray bar) provides good differentiation between healthy and sick levels of BM infection.

[0050] FIG. 2D shows an exemplary bar graph for optimizing the hemoglobin (Hb) concentration immobilized in each well of the assay tray, so as to distinguish between different levels of BM infection. The Hb concentration is varied from 0.5 to 5.0 pg / mL, while fixing the other conditions as before. A concentration of 1.0 pg / mL (the orange bar) provides good differentiation between healthy and sick levels of BM infection. Higher concentrations tend to reduce CL intensity and do not improve the differentiation.

[0051] FIG. 3 A is an exemplary bar graph for optimizing the Hb-Hp interaction time duration T1 so as to distinguish between different levels of BM infection. The value of T1 is varied from 5 minutes (min.) to 60 minutes. An interaction time duration of 20 min. (the dark blue bar) provides good differentiation between healthy and sick levels of BM infection.

[0052] FIG. 3B is an exemplary bar graph for optimizing the CL stabilization time duration T1 so as to distinguish between different levels of BM infection. The value of T2 varies from 0.4 min. to 4 min. A CL stabilization time of 1 min. (the reddish-brown bar) provides good differentiation between healthy and sick levels of BM infection.

[0053] FIG. 4 shows an exemplary bar graph of enhanced CL intensity vs. shelf life (in weeks) of the bio-functionalized assay tray. Stems above each bar indicate +1 standard deviation (based on three samples). The data indicates that storing the tray in refrigeration, for up to five weeks, does not cause deterioration of the effectiveness of the bio-functionalized assay tray, as indicated by the normalized intensity of CL emission. Table 1 summarizes exemplary optimal conditions for system 100 of FIG. 1, providing quantitative assessment of BM in whole milk samples using optical imaging. The values shown are based on the extensive experimental results shown in the figures.

[0054] Table 1: Optimal reagant concentrations and time durations Quantity Unit Value

[0055] Quantitative Estimation of Hp Concentration

[0056] FIG. 5 shows an exemplary graph of a calibration curve relating enhanced CL intensity to Hp concentration in a milk sample. Milk samples with Hp concentrations ranging from 0 to 50 micrograms per milliliter (pg / mL) were prepared by diluting with healthy milk (rather than water) in order to simulate correctly any interferences from (healthy) milk constituents.

[0057] The graph is a plot of normalized CL intensity in percent, on the vertical axis, vs. Hp concentration, marked by a base- 10 logarithmic scale on the horizontal axis. The points indicate normalized CL intensity values and the error bars indicate ± one standard deviation. The vertical color bar shows the CL intensity as it typically would appear in a CL image 175.

[0058] The normalized CL intensity values are calculated by dividing the CL intensity measured in a well containing a BM sample by the intensity measured in a reference cell filled with a low SCC reference milk sample.

[0059] The dotted (blue) line is a linear fit to the normalized intensity measurements having the semi-log form:

[0060] Normalized Intensity (in %) = A { logeHp [in mg / mL] } + B (equation 1) where logedenotes the natural logarithm, and a least- squares fit in the exemplary graph of FIG. 5 has the values A= -18.69 and B=92.68. The R-squared coefficient of the fit is 0.99, indicating an excellent goodness of fit between the normalized measurements and the linear fit.

[0061] Using equation 1 for quantitative estimation of BM disease in milk samples provided by system 100 is found to be in excellent quantitative agreement with benchmark ELISA measurements provided by a laboratory facility. Table 2 summarizes the results of extensive experimentation using ensembles of milk samples having Hp concentrations corresponding to healthy, low sick, medium sick, and sick BM levels.

[0062] Table 2: Accuracy of CL imaging results compared with an ELISA benchmark

[0063] In Table 2, the “Recovery Rate” in percent is defined as 100 times the ratio between the Hp concentration in pg / mL measured by CL imaging to that measured by laboratory ELISA. In all four rows, the relative variation (i.e. standard deviation divided by the mean) is about 8-10%.

[0064] For the sake of completeness, the data on which table 2 is based are presented in Tables 2A-2D below.

[0065] Table 2 A: Accuracy comparison for Healthy BM cases Table 2B: Accuracy comparison for Low Sick BM cases Table 2C: Accuracy comparison for Medium Sick BM cases

[0066] Table 2D: Accuracy comparison for Sick BM cases

[0067] Recove

[0068] Sample SCC CL imaging ELISA ry

[0069] # (xlOOO) assay assay (%)

[0070] FIG. 6 shows a block diagram of an exemplary method 600 for quantitative BM assessment using optical imaging, according to the invention. The method consists of the following sequential steps:

[0071] Step 610: Prepare a bio-functionalized Hb-modified assay tray with a multiplicity of wells, a reference sample, a peroxide solution, a dark box, a camera, and an image processor;

[0072] Step 620: Collect milk samples on-site and apply predetermined amounts of the reference sample and the collected milk samples to wells of the assay tray;

[0073] Step 630: Wait a first predetermined Hb-Hp interaction time duration (Tl);

[0074] Step 640: Add CL and peroxide solutions to wells of the assay tray;

[0075] Step 650: Wait a second predetermined CL stabilization time duration (T2);

[0076] Step 660: Insert the tray into the dark box, position the camera, acquire an image, and transfer it to the image processor;

[0077] Step 670: Calculate normalized CL intensity values; Step 680: Use a predetermined calibration curve to determine Hp concentration estimates.

[0078] In some embodiments, the camera and image processor in step 610 are integrated in a smartphone and steps 660-680 involve use of the smartphone.

[0079] In some embodiments, the image processor is configured to provide a BM diagnostic report to a peripheral display device.

[0080] The descriptions of the various embodiments of the present disclosure have been presented for purposes of illustration but are not intended to be exhaustive or limited to the embodiments disclosed.

[0081] Many other modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. CLAIMS1. A system for in-situ quantitative assessment of Bovine Mastitis in collected milk samples using optical imaging, the system comprising: a bipo-functionalized assay tray having a multiplicity of wells; a chemiluminescence (CL) solution, a hydrogen peroxide solution and a reference milk sample; a dark box, a camera and an image processor; wherein, each well of the bio-functionalized assay tray comprises a gel with predetermined concentrations of metal ions and hemoglobin (Hb); at least one cell of the tray receives a specified first amount of the reference milk sample; one or more cells of the tray each receive said first specified amount of the collected milk samples; each well with a milk sample receives a second specified amount of the CL solution and a third specified amount of the hydrogen peroxide solution; and the dark box, camera and image processor are configured to acquire and analyze images of CL light emission, to calculate normalized CL light intensities, and to determine quantitative estimates of Haptoglobin (Hp) concentration in the collected milk samples, based upon a predetermined calibration curve relating a value of normalized CL light intensity to a value of Hp concentration.

2. The system of claim 1 wherein the normalized CL light intensity is calculated by dividing a CL light intensity value for a well containing a collected milk sample by a CL light intensity value for a well containing the reference milk sample.

3. The system of claim 1 wherein the calibration curve represents a linear relationship between the value of the normalized CL light intensity and a logarithm of the value of Hp concentration.

4. The system of claim 1 wherein the metal ions are copper ions.

5. The system of claim 1 wherein the luminol solution comprises a colloidal suspension of reflective nanoparticles configured to enhance the CL light emission.

6. The system of claim 1 wherein reception of milk samples in the wells of the tray is followed by a predetermined Hb-Hp interaction time duration (Tl).

7. The system of claim 6 wherein Tl has a value between 5 and 60 minutes.

8. The system of claim 1 wherein reception of the CL and hydrogen peroxide solutions in the wells of the tray is followed by a predetermined CL stabilization time duration (T2).

9. The system of claim 8 wherein T2 has a value greater than or equal to 0.5 minutes.

10. The system of claim 1 wherein the camera and image processor are integral components of a smartphone.

11. The system of claim 1 wherein the camera is fitted with a blue filter having a transmission band which includes a light wavelength of 425 nanometers.

12. The system of claim 1 wherein the bio-functionalized assay tray is stored in refrigeration before it receives milk samples.

13. The system of claim 1 wherein the image processor is in communication with a display device in order to present a BM diagnostic report to a user.

14. A method for in-situ quantitative assessment of Bovine Mastitis in collected milk samples using optical imaging, the method comprising the steps: a) preparing a bio-functionalized assay tray with a multiplicity of wells each containing a gel with hemoglobin (Hb) and metal ions; b) preparing a chemiluminescence (CL) solution, a hydrogen peroxide solution, a reference milk sample, a dark box, a camera, and an image processor; c) collecting milk samples on-site and applying specified amounts of the reference and collected milk samples to wells of the assay tray; d) waiting a predetermined Hb-Hp interaction time duration (Tl); e) adding specified amounts of CL and hydrogen peroxide solutions to the wells containing milk samples; f) waiting a predetermined CL stabilization time duration (T2); g) inserting the tray into the dark box, positioning the camera, acquiring an image, and transferring the image to the image processor; h) calculating normalized CL light intensity values; and i) using a predetermined calibration curve to determine values of Hp concentration in each of the collected milk samples.

15. The method of claim 14 wherein the normalized CL light intensity values are calculated by dividing CL light intensity values for wells containing collected milk samples by a CL light intensity value for a well containing the reference milk sample.

16. The method of claim 14 wherein the predetermined calibration curve represents a linear relationship between the value of the normalized CL light intensity and a logarithm of the value of Hp concentration.

17. The method of claim 14 wherein T1 has a value between 5 and 60 minutes and T2 has a value greater than or equal to 0.5 minutes.

18. The method of claim 14 wherein the camera and image processor are integral components of a smartphone.

19. The method of claim 14 wherein the camera is fitted with a blue filter having a transmission band which includes a light wavelength of 425 nanometers.

20. The method of claim 14 wherein the bio-functionalized assay tray is stored in refrigeration prior to the collecting of milk samples in step c).

Images