BIOSENSOR ARRAY FOR RAPID AND MULTIPLEX DETECTION OF FOODBORNE PATHOGENS

Abstract

ABSTRACT The present invention discloses a biosensor array for rapid and multiplex detection of foodborne pathogens, addressing limitations of current technologies. The array comprises multiple sensing elements, each functionalized with specific biorecognition elements, enabling simultaneous detection of various pathogens in a single sample. This enhances efficiency and reduces testing time compared to traditional methods. The invention leverages readily available detection techniques and a user-friendly platform, simplifying operation. Its fabrication process is amenable to mass production, potentially reducing costs. The array's enhanced sensitivity, specificity, simplicity, scalability, and cost-effectiveness make it a novel and inventive solution for food safety testing, contributing to improved public health and reduced economic losses associated with foodborne illness.

Specification

Description:BIOSENSOR ARRAY FOR RAPID AND MULTIPLEX DETECTION OF FOODBORNE PATHOGENS

Field of Invention
The present invention relates to the detection of multiple pathogens. More particularly, a biosensor array for rapid and multiplex detection of foodborne pathogens.

Background of the Invention
Common pathogens in food are a major cause of food poisoning, and can cause a variety of diseases. Pathogens can contaminate food or food animals during production, processing and preparation. Similarly, pathogens can contaminate water sources or seafood harvested from such contaminated waters. Human exposure to pathogens can cause illness, most often gastroenteritis, but also potentially more serious diseases such as salmonellosis and hepatitis A. These diseases are a serious threat to people's health. Rapid and accurate detection of foodborne pathogens are an effective tool to combat such diseases. Most regulatory agencies require, and customers demand specific testing for pathogens that are common to specific food types, and agricultural products, that are capable of in vivo multiplication. Rapid and accurate methods for detection of foodborne and waterborne pathogens are essential, particularly in the context of food manufacturing processes, pharmaceutical industry, drinking water and wastewater utilities, management of fisheries resources and bodies of water.
1. Velusamy, V., et al. (2010). An electrochemical biosensor array for the detection of foodborne pathogens. Sensors and Actuators B: Chemical, 145(1), 180-186. • This study presents an electrochemical biosensor array capable of detecting multiple foodborne pathogens. However, the sensitivity and specificity of this array may be limited, potentially leading to false positives or false negatives in food safety testing.
2. Wang, J., et al. (2016). A label-free electrochemical immunosensor array for simultaneous detection of multiple foodborne pathogens. Biosensors and Bioelectronics, 77, 521-527. • This research demonstrates a label-free electrochemical immunosensor array for the simultaneous detection of multiple foodborne pathogens. However, the complexity and fabrication process of this array might hinder its widespread adoption and scalability for routine food safety testing.
3. Zhao, X., et al. (2018). A microfluidic biosensor array for rapid and sensitive detection of foodborne pathogens. Lab on a Chip, 18(14), 2054-2063. • This study describes a microfluidic biosensor array for the rapid and sensitive detection of foodborne pathogens. However, the microfluidic system involved in this array might introduce operational challenges and increase the complexity of the detection process.
Technical Drawbacks of Prior Art:
• Limited Sensitivity and Specificity: Some prior art biosensor arrays may not offer sufficient sensitivity and specificity for the accurate detection of foodborne pathogens, especially at low concentrations.
• Complexity and Fabrication Challenges: The fabrication and operation ofsome prior art biosensor arrays may be complex and time-consuming, limiting their practicality for routine food safety testing.
• Scalability Issues: Certain prior art biosensor arrays might face challenges in terms of scalability and throughput, making it difficult to adapt them for high-volume food safety testing.
The present invention addresses these limitations by offering a biosensor array with enhanced sensitivity, specificity, simplicity, and scalability, enabling rapid and multiplex detection of foodborne pathogens more practically and efficiently.

Objectives of the Invention
The prime objective of the present invention is to provide a biosensor array for rapid and multiplex detection of foodborne pathogens.
Another object of this invention is to provide the biosensor array for rapid and multiplex detection of foodborne pathogens where the array comprises multiple sensing elements, each functionalized with specific biorecognition elements (e.g., antibodies, aptamers) that target different foodborne pathogens.
Another objective of the present invention is to provide the biosensor array for rapid and multiplex detection of foodborne pathogens where the array enables the simultaneous detection of multiple pathogens in a single sample, offering a faster and more efficient alternative to traditional methods that require separate tests for each pathogen.
Another objective of the present invention is to provide the biosensor array for rapid and multiplex detection of foodborne pathogens where the array's fabrication process is amenable to mass production, potentially reducing costs and facilitating widespread adoption.
Another objective of the present invention is to provide the biosensor array for rapid and multiplex detection of foodborne pathogens where the array prioritizes ease of use, employing readily available detection techniques (e.g., electrochemical, optical) and a user-friendly platform, this simplifies operation and reduces the need for specialized expertise or complex laboratory setups, making the technology more accessible for routine testing and potentially on-site applications.
Another objective of the present invention is to provide the biosensor array for rapid and multiplex detection of foodborne pathogens where the array incorporates integrated sample preparation modules within the array, streamlining the testing process and further enhancing its practicality and efficiency.
Yet another object of this invention is to provide the biosensor array for rapid and multiplex detection of foodborne pathogens where the combination of enhanced sensitivity and specificity, broad-range multiplex detection capability, simplified operation, scalability, cost-effectiveness, and potential for integrated sample preparation sets it apart from prior arts.
These and other objects of the present invention will be apparent from the drawings and descriptions herein. Every object of the invention is attained by at least one embodiment of the present invention.

Summary of the Invention
In one aspect of the present invention provides a biosensor array for rapid and multiplex detection of foodborne pathogens where an integrated sample preparation module, the food sample is first processed within the array itself.

In one of the aspects, in the present invention, the prepared sample is then introduced to the biosensor array, typically through microfluidic channels or other delivery mechanisms.

In one of the aspects, in the present invention, the array contains multiple sensing elements, each functionalized with a different biorecognition element, it can simultaneously detect a panel of foodborne pathogens in a single test.

Brief Description of Drawings
The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure. Further objectives and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawing and wherein:
Figure 1 illustrates the flow diagram according to an embodiment of the present invention.

DETAIL DESCRIPTION OF INVENTION
Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to".
In the following description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present invention. It will be apparent to one skilled in the art that embodiments of the present invention may be practiced without some of these specific details.
Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. Reference will now be made in detail to the exemplary embodiments of the present invention.
The present invention discloses a biosensor array for rapid and multiplex detection of foodborne pathogens, the array comprises of multiple sensing elements, each functionalized with specific biorecognition elements, enabling simultaneous detection of various pathogens in a single sample.
In describing the preferred embodiment of the present invention, reference will be made herein to like numerals refer to like features of the invention.
According to an embodiment of the invention, the biosensor array for rapid and multiplex detection of foodborne pathogens comprises of following parts:
A. Biosensor Array Platform:
• Sub-Part 1: Substrate Material such as Material choice (e.g., glass, silicon, polymer) for supporting the sensing elements. And Surface modification to enhance biocompatibility and immobilization efficiency.
• Sub-Part 2: Sensing Elements such as Microelectrodes, Optical Waveguides, Carbon Nanotubes, Generalized Transduction.
• Sub-Part 3: Array Layout such as Patterning of sensing elements into a microarray format. And Spatial arrangement to allow multiplexing.
B. Biorecognition Elements:
• Sub-Part 1: Antibodies/Aptamers/Nanobodies such as selection and functionalization of specific biorecognition elements for target pathogens. And stabilization to ensure long-term shelf-life and reactivity.
• Sub-Part 2: Immobilization Chemistry such as covalent attachment methods (e.g., EDC/NHS coupling) for stable binding to the sensing elements. And surface blocking to prevent non-specific binding.
C. Detection System:
• Sub-Part 1: Signal Transduction includes electrochemical (e.g., impedance, amperometric) or optical (e.g., fluorescence, SPR) transducers.
• Sub-Part 2: Readout and Data Processing includes electronic interfaces (e.g., potentiostat) for signal acquisition. And software algorithms for signal analysis and pathogen identification.
D. Sample Preparation Unit:
• Sub-Part 1: Sample Pre-treatment Filtration, centrifugation, or microfluidic integration for sample concentration and purification.
• Sub-Part 2: Pathogen Lysis and Extraction Chemical or enzymatic lysis methods to release target analytes.
According to preferred embodiment of the invention, referring to Figure 1, the biosensor array for rapid and multiplex detection of foodborne pathogens comprises of mainly five process steps: Fabrication of the Biosensor Array, Functionalization with Biorecognition Elements, Sample Preparation, Detection Process, Signal Readout and Data Analysis.
According to another embodiment of the invention, the detailed process steps are as follows:
Step 1: Fabrication of the Biosensor Array:
• Process 1.1: Substrate Preparation Clean and activate the substrate surface (e.g., plasma treatment) to enhance functionalization.
• Process 1.2: Microarray Patterning Use lithography or printing techniques to pattern the array with microelectrodes or optical sensors.
• Process 1.3: Surface Functionalization Deposit a functional layer (e.g., self-assembled monolayers) to enable immobilization of biorecognition elements.
Step 2: Functionalization with Biorecognition Elements:
• Process 2.1: Selection and Preparation of Biorecognition Molecules Identify antibodies, aptamers, or nanobodies specific to each target pathogen.
• Process 2.2: Immobilization on Sensing Elements Use covalent attachment chemistry (e.g., EDC/NHS coupling) to immobilize biorecognition elements on the sensor surface.
• Process 2.3: Surface Blocking Apply a blocking agent (e.g., BSA) to minimize non-specific interactions.
Step 3: Sample Preparation:
• Process 3.1: Pre-treatment of the Sample Filter or centrifuge the food sample to concentrate pathogens and remove debris.
• Process 3.2: Lysis and Extraction Apply lysis agents to release target analytes from pathogens for detection.
Step 4: Detection Process:
• Process 4.1: Sample Application
o Apply the prepared sample onto the biosensor array.
• Process 4.2: Binding and Signal Generation
o Allow binding between the target pathogens and immobilized biorecognition elements.
o Signal generation through electrochemical (current change) or optical (fluorescence) methods.
• Sub-Process 4.2.1: Real-time Monitoring o Monitor signal changes in real-time for rapid detection.
Step 5: Signal Readout and Data Analysis:
• Process 5.1: Signal Acquisition
o Utilize electronic interfaces (e.g., potentiostat) to capture the generated signals.
• Process 5.2: Data Processing and Interpretation
o Analyze the data using software algorithms to identify and quantify pathogens.
• Sub-Process 5.2.1: Multiplex Analysis
o Deconvolute signals from different sensing elements to provide a comprehensive detection profile.

According to exemplary embodiment of the invention, it may vary depending on the chosen detection method and array configuration, the general working principle of the invention is described as follows:
1. Sample Preparation: it includes an integrated sample preparation module; the food sample is first processed within the array itself. This may involve steps such as homogenization, filtration, or enrichment to prepare the sample for analysis and enhance the detection of target pathogens.
2. Sample Application: The prepared sample is then introduced to the biosensor array, typically through microfluidic channels or other delivery mechanisms. The sample flows over the sensing elements, allowing the target foodborne pathogens to interact with the biorecognition elements immobilized on the surface.
3. Binding and Signal Generation: The biorecognition elements (e.g., antibodies, aptamers) on the sensing elements are highly specific to their target pathogens. When a target pathogen is present in the sample, it binds to its corresponding biorecognition element, triggering a change in the sensing element's properties. This change generates a measurable signal, such as a change in electrical conductivity, optical signal, or other detectable parameter, depending on the chosen detection method.
4. Signal Transduction and Processing: The generated signal is transduced and processed by the array's electronics or optics. The signal is converted into a readable format (e.g., electrical current, light intensity) and analyzed using sophisticated algorithms to identify the presence and potentially the concentration of the target pathogens.
5. Multiplex Detection: Since the array contains multiple sensing elements, each functionalized with a different biorecognition element, it can simultaneously detect a panel of foodborne pathogens in a single test. The results are displayed or outputted, providing rapid and comprehensive information about the presence of various pathogens in the sample.

According to another embodiment of the invention, it will depend on specific application requirements, but an embodiment utilizing electrochemical detection with integrated sample preparation offer several advantages:
• Electrochemical Detection: This method is often sensitive, cost-effective, and amenable to miniaturization, making it suitable for portable and on-site applications.
• Integrated Sample Preparation: Incorporating sample preparation within the array streamlines the process, reduces handling steps, and minimizes contamination risks.
• Multiplex Capability: The array format allows for the simultaneous detection of multiple pathogens, increasing efficiency and throughput.
• User-Friendly Platform: The combination of electrochemical detection and integrated sample preparation can result in a compact and user-friendly device, potentially requiring minimal training and expertise to operate.

According to another embodiment of the invention, the biosensor array significantly enhances food safety testing by offering a rapid, sensitive, specific, simple, scalable, and cost-effective platform for the multiplex detection of foodborne pathogens.

According to another embodiment of the invention, the biosensor array for rapid and multiplex detection of foodborne pathogens have most probable and impactful industrial applications in food processing and manufacturing facilities.

Although a preferred embodiment of the invention has been illustrated and described, it will at once be apparent to those skilled in the art that the invention includes advantages and features over and beyond the specific illustrated construction. Accordingly it is intended that the scope of the invention be limited solely by the scope of the hereinafter appended claims, and not by the foregoing specification, when interpreted in light of the relevant prior art.


, Claims:1. A biosensor array for rapid and multiplex detection of foodborne pathogens comprises of:
E. Biosensor Array Platform further comprising:
• Sub-Part 1: Substrate Material such for supporting the sensing elements, Surface modification to enhance biocompatibility and immobilization efficiency,
• Sub-Part 2: Sensing Elements,
• Sub-Part 3: Array Layout, Spatial arrangement to allow multiplexing;
F. Biorecognition Elements further comprising:
• Sub-Part 1: Antibodies/Aptamers/Nanobodies such as selection and functionalization of specific biorecognition elements for target pathogens and stabilization to ensure long-term shelf-life and reactivity,
• Sub-Part 2: Immobilization Chemistry for stable binding to the sensing elements, surface blocking to prevent non-specific binding;
G. Detection System further comprising:
• Sub-Part 1: Signal Transduction,
• Sub-Part 2: Readout and Data Processing for signal acquisition, algorithms for signal analysis and pathogen identification;
H. Sample Preparation Unit further comprising:
• Sub-Part 1: Sample Pre-treatment Filtration, centrifugation, or microfluidic integration for sample concentration and purification,
• Sub-Part 2: Pathogen Lysis and Extraction Chemical or enzymatic lysis methods to release target analytes.

2. The biosensor array for rapid and multiplex detection of foodborne pathogens as claimed in claim 1, wherein the array comprises of following process steps:
Step 1: Fabrication of the Biosensor Array:
• Process 1.1: substrate preparation clean and activate the substrate surface to enhance functionalization,
• Process 1.2: microarray patterning use lithography or printing techniques to pattern the array with microelectrodes or optical sensors,
• Process 1.3: surface functionalization deposits a functional layer to enable immobilization of biorecognition elements;
Step 2: Functionalization with Biorecognition Elements:
• Process 2.1: selection and preparation of biorecognition molecules identify antibodies, aptamers, nanobodies specific to each target pathogen,
• Process 2.2: immobilization on sensing elements use covalent attachment chemistry to immobilize biorecognition elements on the sensor surface,
• Process 2.3: surface blocking applies a blocking agent to minimize non-specific interactions;
Step 3: Sample Preparation:
• Process 3.1: Pre-treatment of the sample filter or centrifuge the food sample to concentrate pathogens and remove debris,
• Process 3.2: lysis and extraction apply lysis agents to release target analytes from pathogens for detection;
Step 4: Detection Process:
• Process 4.1: Sample Application for applying the prepared sample onto the biosensor array,
• Process 4.2: binding and signal generation: allowing binding between the target pathogens and immobilized biorecognition elements; signal generation through electrochemical or optical methods;
• Sub-Process 4.2.1: real-time monitoring to monitor signal changes in real-time for rapid detection;
Step 5: Signal Readout and Data Analysis:
• Process 5.1: Signal Acquisition to utilize electronic interfaces to capture the generated signals,
• Process 5.2: Data Processing and Interpretation, analyse the data using algorithms to identify and quantify pathogens,
• Sub-Process 5.2.1: Multiplex Analysis to deconvolute signals from different sensing elements to provide a comprehensive detection profile.
3. The biosensor array for rapid and multiplex detection of foodborne pathogens as claimed in claim 1, wherein the working principle of the array is as follows:
I. Sample Preparation includes an integrated sample preparation module; where the food sample is first processed within the array itself;
II. Sample Application: The prepared sample is then introduced to the biosensor array, through microfluidic channels or other delivery mechanisms, the sample flows over the sensing elements, allowing the target foodborne pathogens to interact with the biorecognition elements immobilized on the surface;
III. Binding and Signal Generation: when a target pathogen is present in the sample, it binds to its corresponding biorecognition element, triggering a change in the sensing element's properties, this change generates a measurable signal depending on the chosen detection method;
IV. Signal Transduction and Processing: The generated signal is transduced and processed by the array's electronics or optics, the signal is converted into a readable format, analysed using algorithms to identify the presence and potentially the concentration of the target pathogens;
V. Multiplex Detection: the array contains multiple sensing elements, each functionalized with a different biorecognition element, it can simultaneously detect a panel of foodborne pathogens in a single test, the results are displayed, providing information about the presence of various pathogens in the sample.

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