System and method for direct in-situ detection and identification of contaminants using optical catalysis

Abstract

A system and method provide direct in-situ detection of contaminants using optical catalysis. The system and method utilize a functionalized assay plate with microwells, a Luminol substrate (LS) solution, a camera for acquiring images of the assay plate produced by chemiluminescence (CL) emission, an image processor for analyzing the images, and a dark box with an entry port for the assay plate, in which the plate is positioned within an optical field-of-view of the camera. The presence of a contaminant in an analyzed sample is detected and identified by a decrease in image intensity of CL emission in one or more microwell. The detection is fast, portable, and sensitive to low levels of contamination.

Description

[0001] System and method for direct in-situ detection and identification of contaminants using optical catalysis

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is related to and claims priority from a commonly owned US Provisional Patent Application No. 63 / 681,913, entitled “System and method for direct, in-situ detection and identification of bacterial contamination using optical catalysis”, filed on August 12, 2024.

[0004] TECHNICAL FIELD

[0005] The present invention relates to systems and methods for direct in-situ detection and identification of contaminants in food or water-based samples using optical catalysis, or photocatalysis.

[0006] BACKGROUND OF THE INVENTION

[0007] According to the World Health Organization, each year about 600 million individuals worldwide fall ill, and about 420,000 die, from contaminated food. Moreover, waterborne pathogens contribute an additional 485,000 deaths annually due to diarrheal diseases caused by unsafe drinking water and inadequate sanitation practices. These figures are based on reported cases; the actual numbers may be considerably higher.

[0008] Traditional methods for detecting bacterial pollutants use culture plate and molecular techniques such as Enzyme-Linked Immuno- Sorbent Assay (ELISA) and Polymerase Chain Reaction (PCR). These highly accurate techniques are time-consuming and typically require laboratory facilities. Thus, there is a significant need for a portable, real-time, in-situ system for rapidly detecting and identifying bacterial contamination.

[0009] A technical paper by W. Wu et al., entitled “Single Escherichia coli bacteria detection using a chemiluminescence digital microwell chip array”, which appeared in Biosensors and Bioelectronics, vol. 215, 2022, pp. 1-9, describes a CL digital microwell array chip based on the hydrolysis of 6- Chloro-4-methylumbelliferyl-P-D-glucuronide by the P-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking advantage of picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. Another method for pathogen detection is described in a technical paper by X. Chen et al. entitled “Long-Lasting Chemiluminescence-Based POCT for Portable and Visual Pathogenic Detection and In Situ Inactivation”, which appeared in Analytical Chemistry, vol. 94 issue 23, June, 2022, pp. 8382-8391. The paper describes a chemiluminescence (CL) system with long, persistent, and intensive intensity, based on the peroxidase-like property of 4-mercaptophenylboronic acid (MPBA)-functionalized CuSe nanoprobes (CUSCNPS@MPBA) for improving detection accuracy and sensitivity. By further integrating a smartphone as an analyzer, quantitative point-of-care testing (POCT) of bacteria was realized with high sensitivity.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention is directed to a system and a method for direct in-situ detection of contaminants using optical catalysis.

[0012] According to the teachings of the present invention, the system includes: a functionalized assay plate with a multiplicity of microwells, a Luminol substrate (LS) solution, a camera for acquiring images of the assay plate produced by chemiluminescence (CL) emission, an image processor for analyzing those images, and a dark box with an entry port for the assay plate, in which the plate is positioned within an optical field-of-view of the camera. The presence of a contaminant in the target analyte is detected and identified by a decrease in image intensity of CL emission in one or more microwell.

[0013] According to a feature, the functionalized assay plate includes a material selected from a group consisting of an avidin, an aptamer, an antibody, an enzyme, a nucleic acid, a whole cell, a molecularly imprinted polymer, an affimer, a bacteriophage, and a peptide.

[0014] According to a feature, the functionalized assay plate includes a solidified hydrogel grid formed by a first concentration of agar and Cobalt ions or peroxidase-like active species in aqueous solution, and the Luminol substrate solution includes a second concentration of Luminol and hydrogen peroxide in aqueous solution.

[0015] According to a feature, the first and second concentrations are predetermined to maximize an intensity level of CL emission.

[0016] According to a feature, at least one of the microwells is a control well for determining an in- situ baseline intensity level in the absence of the target analyte.

[0017] According to a feature, the image processor calculates relative intensity levels by normalizing image intensities by the in-situ baseline intensity level. According to a feature, the decrease in image intensity of CL emission occurs over an interaction time (Tl) which is in a range of five minutes to one hour, depending on a level of contaminant concentration that must be detected with high sensitivity.

[0018] According to a feature, the image processor is configured to output an analyte analysis report, which identifies the contaminants that have been detected.

[0019] According to a feature, the camera and image processor are integrated into a cellphone.

[0020] According to the teachings of the present invention, the method includes the steps of: fabricating a functionalized assay plate with a multiplicity of microwells, fabricating a Luminol substrate (LS) solution, applying a target analyte to one or more of the microwells and waiting an interaction time (Tl), applying an LS solution to one or more microwells and waiting a time (T2) for chemiluminescence (CL) emission to reach a steady-state, transferring the assay plate to a dark box, using a camera linked with an image processor to acquire and analyze assay plate images; and generating, storing, and / or transmitting an analyte analysis report which identifies the contaminants that have been detected.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Some embodiments of the present invention are described herein, 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. The description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0023] FIG. 1 : A schematic of an exemplary optical catalysis system, according to the invention.

[0024] FIGs. 2A-2B: Microwell drawings explaining the principle of direct in-situ sensing of contaminants by their CL emissions.

[0025] FIG. 3A: Exemplary images for different bacterial concentrations of Staphylococcus aureus (S.A). FIG. 3B: An exemplary calibration curve for relative intensity vs. bacterial concentration of S.A. FIGs. 4A-4B: Exemplary bar charts showing enhanced CL image intensity vs. concentration for different solutions used in fabricating the assay plate.

[0026] FIGs. 5A-5B: Exemplary bar charts showing enhanced CL image intensity vs. concentration for different solutions used in fabricating the Luminol substrate solution.

[0027] FIGs. 6A-6B: Exemplary bar charts showing enhanced CL image intensity for different combinations of aptamer content, interaction time, and S.A. log-concentration. FIG. 7: An exemplary block diagram of the method used for direct in-situ detection of bacterial contamination using optical catalysis, according to the invention.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] FIG. 1 shows a schematic of an exemplary optical catalysis system 100 for direct in-situ detection and identification of bacterial contaminants, according to the invention. A functionalized assay plate 120 A with, for example, 15 micro wells arranged in three sample columns 124 and five pathogen rows 126 is fabricated in advance. The pathogens in this example are labelled E.C, S.A, B.C, S.T, and L.T corresponding to Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella typhi, and Listeria. An aqueous target analyte 130 is applied to each of the microwells, except for certain control microwells, which receive distilled water instead, as explained below.

[0030] After an exemplary interaction time (Tl) which is between 5 minutes and 1 hour, during which the analyte interacts with avidin-functionalized selective biotinylated aptamers in each of the microwells, a quantity of typically 100 microliters (|1L) of optimized luminol substrate solution 140 is added to each micro well. Emission of CL blue light, as shown in assay plate 120B, begins immediately and reaches a steady-state intensity after a time (T2) of about 15 seconds. The blue light is intense from all wells that do not contain matching bacterial contaminants. In the example shown in 120B, the lack of strong emission by the E.C wells indicates the presence of E.C contamination.

[0031] The assay plate is transferred manually or automatically through an entry port 174 into a dark box 170. The plate, now labelled 120C, is positioned within the optical field-of-view of a camera 184, which forms images of the assay plate, typically at maximum sensitivity (e.g. ISO above 1600) and with a typical exposure time of two seconds. Image processor 186 acquires and analyzes the camera images and generates an analyte report 190, which may be stored or sent to a user by wireless transmission. In FIG. 1, the camera 184 and image processor 186 are shown as being integrated inside a cellphone 180, which may also facilitate wireless transmission of the analyte report.

[0032] Image processor 186 analyzes the assay plate images by first identifying the regions of the plate corresponding to each of the microwells and then determining an average image intensity (IAVG) for each microwell. One or more of the microwells is not treated with target analyte, but only with distilled water. This is designated as a control microwell, and its image intensity is used to determine an in-situ baseline intensity level (IBIL). A relative intensity value (IREL) is calculated for each of the microwells containing target analyte by normalizing, i.e. dividing, the value of its average image intensity (IAVG) by the value of IBIL. Thresholding is then applied to determine which microwells are likely to contain bacterial contamination. FIGs. 2A and 2B are microwell drawings that explain the principle of direct bacteria sensing. In both figures, there is a red grid of solidified hydrogel, in which there are centers of avidin- functionalized selective biotinylated aptamers, optimized for detecting a particular bacterial species. In FIG. 2A, the particular bacterial species is absent, and the emission of blue CL photons, having wavelengths of approximately 425 nanometers (nm.), is intense. In FIG. 2B, the presence of the bacterial species is signalled by a sharp decrease in the intensity of CL emission caused by binding of the selective aptamer with the particular bacterial species.

[0033] FIG. 3A shows a test assay plate in which all fifteen microwells have been prepared to detect the presence of S.A. Each of the three columns is treated with a different log-concentration of S.A. in a phosphate-buffered saline (PBS) solution, in units of log CFU / mL where CFU is a Colony Forming Unit, used to count the number of viable microbes in a sample. The threshold for reliable detection appears to be roughly at a value of log-concentration equal to 1 , marked with an arrow.

[0034] FIG. 3B shows an exemplary calibration curve 250 of the relative image intensity on the vertical axis (y-axis) in arbitrary units (a.u.) versus the S.A. log-concentration on the horizontal axis (x-axis). The linear fit for y(x) has a slope of about -8.8, indicating a drop of 8.8 a.u. for each factor of ten increase in bacterial concentration. The coefficient of fit (R2value) of 0.9888 indicates that the linear fit is highly accurate.

[0035] A series of experiments were carried out to optimize the composition and method of preparing the functionalized assay plate 120A and the Luminol substrate solution 140. The results of these experiments are presented in the bar charts of FIGs. 4A-4B and 5A-5B.

[0036] A. Method for Fabricating the Assay Plate

[0037] The method of fabricating an Agarose Gel / Cobalt Ion- modified assay plate is as follows. Mix 10 mL of ultrapure water with a measured amount of agar to obtain an agarose (hydrogel) within a 96-well plate. Bring to a boil, then cool to 40 °C and blend with 0.125 mL of Cobalt ions. Pour 100 pL into each microwell of the assay plate and wait five minutes for the hydrogel to solidify.

[0038] As shown in FIGs. 4A-4B, the highest enhanced CL (or ECL) intensity is obtained with a hydrogel-Cobalt assay plate using 3% agarose hydrogel and a Cobalt ion solution of 3 mg / mL. The optimal bar is marked with an arrow in each figure. In some embodiments, the Cobalt ion solution may be replaced by a peroxidase-like active species.

[0039] B. Method for Fabricating the Aptamer-Modified Assay Plate

[0040] For each microwell, 30 |1L of avidin is mixed with ultrapure water to reach a concentration of 100 |lg / mL and is applied to the assay microwell. Incubate for two hours and do three consecutive washes with PBS solution. Apply 30 |1L of biotin-aptamer against bacteria, 25 micromole (|1M) to the avidin-functionalized assay plate, followed by a 30-minute incubation and three consecutive washes with PBS solution. Conjugation of the avidin and biotin produces the desired functionalized selective biotinylated aptamer-modified assay plate.

[0041] C. Method for Fabricating the Luminol Substrate (LS) Solution

[0042] Prepare 100 pL of LS solution for each micro well by mixing Luminol (10 mg / mL), hydrogen peroxide (H2O2 , 3 mmol / L) and sodium hydroxide (NaOH, 7.5 mmol / L) with 25 mL of distilled deuterium-depleted water (DDW). As shown in FIGs. 5A-5B, the above concentrations of Luminol and H2O2 were found to yield the highest intensity of enhanced CL emission.

[0043] FIGs. 6A and 6B show bar charts of CL intensity for different combinations of aptamer content, interaction time, and S.A. log-concentration. Black, blue, and red bars correspond to aptamer content of 12, 25, and 50 pM, respectively. In FIG. 6A, the CL intensity is seen to diminish with increasing interaction time between aptamer and bacterial suspension (for a fixed S.A. logconcentration of 3), presumably due to an increase in binding efficiency. In FIG. 6B, the CL intensity is seen to diminish with increasing S.A. log-concentration, with an interaction time (Tl) of one hour, also because of an increase in binding efficiency.

[0044] FIG. 7 shows an exemplary block diagram of a method 300 for direct in-situ detection of bacterial contamination, according to the invention. The steps of the method are as follows:

[0045] Step 310: Fabricate and functionalize an assay plate with multiple microwells. This step includes the above fabrication steps A. and B.

[0046] Step 320: Fabricate Luminol Substrate (LS) solution. This step includes the above fabrication step C.

[0047] Step 330: Apply target analyte to microwells and wait an interaction time (Tl).

[0048] Step 340: Apply LS solution to micro wells and wait a time (T2) for CL emission to reach a steady-state.

[0049] Step 350: Transfer assay plate to a dark box.

[0050] Step 360: Use a camera and a signal processor to acquire and analyze assay plate images.

[0051] Step 370: Generate, store and / or transmit an analyte analysis report, which identifies the contaminants that have been detected. 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. 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. For example, the principles of the invention, which have been illustrated in detail herein for detecting and identifying bacterial contaminants, may be extended to detecting and identifying viral and fungal contaminants as well. In this case the material used to functionalize the assay plate may include, for example, an avidin, an aptamer, an antibody, an enzyme, a nucleic acid, a whole cell, a molecularly imprinted polymer, an affimer, a bacteiophage, or a peptide.

[0052] 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

CLAIMS1. A system for direct in-situ detection of contaminants in a target analyte using optical catalysis, the system comprising: a functionalized assay plate with a multiplicity of microwells, a Luminol substrate (LS) solution, a camera in communication with an image processor configured to acquire and analyze images of the assay plate produced by chemiluminescence (CL) emission, and a dark box comprising an entry port for the assay plate, said box configured to position the plate within an optical field-of-view of the camera; wherein the presence of a contaminant in the target analyte is detected and identified by a decrease in image intensity of CL emission in one or more microwell.

2. The system of claim 1 wherein the functionalized assay plate comprises a material selected from a group consisting of an avidin, a biotin-aptamer, an adsorbing antibody, a synthetic polymer, a phage, and a peptide.

3. The system of claim 1 wherein the functionalized assay plate comprises a solidified hydrogel grid formed by a first concentration of agar and Cobalt ions or peroxidase-like active species in aqueous solution, and the Luminol substrate solution comprises a second concentration of Luminol and hydrogen peroxide in aqueous solution.

4. The system of claim 3 wherein the first and second concentrations are predetermined to maximize an intensity level of CL emission.

5. The system of claim 1 wherein at least one of the micro wells is a control well for determining an in- situ baseline intensity level in the absence of the target analyte.

6. The system of claim 5 wherein the image processor calculates relative intensity levels by normalizing image intensities by the in-situ baseline intensity level.

7. The system of claim 1 wherein the decrease in image intensity of CL emission occurs over an interaction time (Tl) which is in a range of five minutes to one hour, depending on a level of contaminant concentration that must be detected with high sensitivity.

8. The system of claim 1 wherein the image processor is configured to output an analyte analysis report which identifies the contaminants that have been detected.

9. The system of claim 1 wherein the camera and image processor are integrated into a cellphone.

10. A method for direct in-situ detection of contaminants in a target analyte using optical catalysis comprising the steps: a) Fabricating a functionalized assay plate with a multiplicity of microwells; b) Fabricating a Luminol substrate (LS) solution; c) Applying a target analyte to one or more of the micro wells and waiting an interaction time (Tl); d) Applying an LS solution to one or more microwells and waiting a time (T2) for chemiluminescence (CL) emission to reach a steady-state; e) Transferring the assay plate to a dark box; f) Using a camera in communication with an image processor to acquire and analyze assay plate images; and g) Generating, storing, and / or transmitting an analyte analysis report which identifies the contaminants that have been detected.

11. The method of claim 10 wherein the assay plate comprises a material selected from a group consisting of an avidin, an aptamer, an antibody, an enzyme, a nucleic acid, a whole cell, a molecularly imprinted polymer, an affimer, a bacteriophage, and a peptide.

12. The method of claim 10 wherein the assay plate comprises a solidified hydrogel grid formed by a first concentration of agar and Cobalt ions or peroxidase-like active species in aqueous solution, and the Luminol substrate solution comprises a second concentration of Luminol and hydrogen peroxide in aqueous solution.

13. The method of claim 12 wherein the first and second concentrations are predetermined to maximize an intensity level of CL emission.

14. The method of claim 10 wherein at least one of the micro wells is a control well for determining an in- situ baseline intensity level in the absence of the target analyte.

15. The method of claim 14 wherein the image processor calculates relative intensity levels by normalizing image intensities by the in-situ baseline intensity level.

16. The method of claim 10 wherein the interaction time (Tl) is in a range of five minutes to one hour, depending on a level of contaminant concentration that must be detected with high sensitivity.

17. The method of claim 10 wherein the camera and signal processor are integrated into a cellphone.

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