Multiplex biosensing for multi-omics analyses

Abstract

A multiplex biosensing device and system are disclosed, including a plurality of multiplex biosensing electrodes configured for simultaneous, real-time detection of multiple biomolecule markers. The multiplex biosensing electrodes may comprise working electrodes modified with diverse biosensing platforms. The biosensing platforms may include molecularly imprinted polymers, immunocomplex reactions, enzymatic reactions, or DNA hybridization, each customized to detect specific biomarkers. The device further includes a microfluidic delivery mechanism designed for the sequential processing and delivery of biofluid samples and reagents, using a separation or filter media to remove impurities. The multiplex biosensor system integrates the multiplex biosensing device and a detection apparatus may be wirelessly connected via Bluetooth or other communication modules, enabling remote, real-time data monitoring and analysis. The system supports various biofluids, including blood, plasma, urine, saliva, sputum, and sweat, facilitating diverse applications in health diagnostics, environmental monitoring, and food safety.

Description

MULTIPLEX BIOSENSING FOR MULTI-OMICS ANALYSESFIELD

[0001] The present disclosure relates to biosensing methods and devices, including but not limited to multiplex biosensor systems and methods.BACKGROUND

[0002] Multi-omics analyses may be useful in gaining a thorough understanding of biological activities. Multi-omics integrated analysis pertains to the statistical examination of biomolecular data across various levels, such as genome, transcriptome, proteome, metabolome, and lipidome. By integrating data from diverse molecular levels, this analysis mutually validates and complements each component, enabling a comprehensive understanding of overall biological changes.

[0003] Monitoring different biomolecules and their presence in bodily fluids that are used as diagnostic or prognostic indicators for the beginning, progression, and remission of diseases is important for early-stage disease diagnosis and effective treatment of disease.

[0004] Clinical evaluation based on a single biomarker may not be adequate for timely and early diagnostics and therapy monitoring. Multiplexed biosensing provides more accurate diagnosis, treatment, and prevention for health conditions and leads to reducing analytical time, detection cost, and the pain and effort of patients that suffer from several diseases simultaneously.

[0005] Improvements in approaches for multiplexed biosensing of biomarkers are desirable.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0007] FIG. 1 illustrates a block diagram of a multiplex biosensing device in accordance with one or more embodiments.

[0008] FIG. 2 illustrates a block diagram of a multiplex biosensing device in accordance withone or more embodiments.

[0009] FIG. 3 illustrates a block diagram of a multiplex biosensor system in accordance with one or more embodiments.

[0010] FIG. 4A and FIG. 4B illustrate a schematic overview of a multiplex biosensing device in 2D and 3D in accordance with one or more embodiments.

[0011] FIG. 5 illustrates a schematic of a multiplex biosensing device including a plurality of multiplex electrodes, with an insulation layer and a temporary or permanent well cell removed and separated in accordance with one or more embodiments.

[0012] FIG. 6 illustrates different parts of a multiplex biosensing device including a plurality of multiplex electrodes in accordance with one or more embodiments.

[0013] FIG. 7 A, FIG. 7B and FIG. 7C illustrate a multiplex biosensing device including two, three and four working electrodes, respectively, in accordance with one or more embodiments.

[0014] FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D illustrate a multiplex biosensing device including a plurality of multiplex electrodes with varied sizes and configurations for four working electrodes, in accordance with one or more embodiments.

[0015] FIG. 9 illustrates a multiplex biosensing device including a plurality of multiplex electrodes for eight working electrodes, in accordance with one or more embodiments.

[0016] FIG. 10 illustrates a multiplex biosensing device including a plurality of multiplex electrodes for a plurality of working electrodes, in accordance with one or more embodiments.

[0017] FIG. 11 illustrates a schematic overview of a multiplex biosensing device including a plurality of multiplex biosensing electrodes, an insulation layer, a temporary or permanent well cell, a delivery mechanism, and a separation or filter media in 2D and 3D, in accordance with one or more embodiments.

[0018] FIG. 12 illustrates a schematic overview of a connector that may be used during the fabrication or testing of a multiplex biosensing device as the interface between the device and a detection apparatus, in accordance with one or more embodiments.

[0019] FIG. 13 illustrates a schematic representation of a surface modification process to convert multiplex bare electrodes to multiplex biosensing electrodes, in accordance with one or more embodiments.

[0020] FIG. 14 illustrates a schematic overview of a multiplex biosensor system in 3D,involving the multiplex biosensing device connected to a detection apparatus via a connector, in accordance with one or more embodiments.

[0021] FIG. 15 shows measured calibration curves obtained from electrochemical response studies of implementations of embodiments of the present disclosure for different analytes analysis (cortisol, agmatine, succinate, and glial fibrillary acidic protein (GFAP)).

[0022] FIG. 16 shows measured calibration curves obtained from electrochemical response studies of implementations of embodiments of the present disclosure for different biofluids (plasma, blood, urine, and sweat).DETAILED DESCRIPTION

[0023] A multiplex biosensing device includes a plurality of multiplex biosensing electrodes configured for simultaneous real-time monitoring of at least two biomarkers. The multiplex biosensing electrodes include at least a first electrode modified with a biosensing platform (molecularly imprinted polymers, immunocomplex reaction, enzymatic reaction or DNA hybridization) customized for a first biomarker, and a second electrode modified with a biosensing platform customized for a second biomarker. A delivery mechanism is provided for delivering at least one biofluid sample and at least one biosensing reagent to the plurality of multiplex biosensing electrodes. A multiplex biosensor system may comprise a multiplex biosensing device as disclosed, and a detection apparatus configured to electrically communicate with the plurality of multiplex biosensing electrodes and to obtain, analyze, and display biomarker data corresponding to the plurality of biomarkers and to provide a readout.

[0024] The plurality of multiplex biosensing electrodes may comprise more than two multiplex biosensing electrodes. Example embodiments will be described later with four, eight and nineteen multiplex biosensing electrodes. In an example implementation, the multiplex biosensing electrodes may comprise a series of electrodes, each functionalized with a distinct biosensing platform — such as molecularly imprinted polymers, immunocomplex reactions, enzymatic reactions, or DNA hybridization — tailored for the detection of specific biomarkers. The first electrode may be modified to detect a first biomarker, while the second electrode is customized for a second biomarker. This configuration may be extended to incorporate additional electrodes, each designed to detect a unique biomarker within the same sensingformat.

[0025] A multiplex biosensing device and system are disclosed, featuring a plurality of multiplex biosensing electrodes configured for simultaneous, real-time detection of multiple biomolecule markers. The plurality of multiplex biosensing electrodes may comprise multiple working electrodes modified with diverse biosensing platforms, an integrated reference electrode shared among the working electrodes, and an integrated counter electrode partially shared with each working electrode. The biosensing platforms may include molecularly imprinted polymers, immunocomplex reactions, enzymatic reactions, or DNA hybridization, each customized to detect specific biomarkers. The device further includes a microfluidic delivery mechanism designed for the sequential processing and delivery of biofluid samples and reagents, using a separation or filter media to remove impurities. The device’s substrate, constructed from flexible polymers, glass, or ceramics, supports varied biosensing environments, while the electrodes use advanced ink materials to enable compatibility with different biosensing platforms. The multiplex biosensor system integrates or comprises the multiplex biosensing device and a detection apparatus that may be wirelessly connected via Bluetooth or other communication modules, enabling remote, real-time data monitoring and analysis. To enhance analytical performance, a ratiometric analysis approach is employed to mitigate matrix effects, and a non-destructive quality control process ensures consistent fabrication and reliability of the device. The system supports various biofluids, including blood, plasma, urine, saliva, sputum, and sweat, facilitating diverse applications in health diagnostics, environmental monitoring, and food safety.

[0026] Certain terms used in this application and their meaning as used in this context are set forth in the description below. To the extent a term used herein is not defined, it should be given the broadest definition persons in the pertinent art have given that term as reflected in at least one printed publication or issued patent. Further, the present processes are not limited by the usage of the terms shown below, as all equivalents, synonyms, new developments and terms or processes that serve the same or a similar purpose are considered to be within the scope of the present disclosure.

[0027] The term “electrode” as used herein, on its own with no modifier, represents an electrode without any biosensing function. A plurality of multiplex electrodes may comprise asubstrate, a plurality of working electrodes, electrode bases, an integrated reference electrode and an integrated counter electrode.

[0028] A “biosensing electrode”, or a “functional electrode”, represents an electrode modified with, or modified to include, a biosensing platform, such that the electrode is functional with respect to biosensing. Modifying an electrode to include a biosensing platform may comprise one or more of a physical modification and a chemical modification of the electrode. Decorating an electrode is an example of a physical modification. A multiplex biosensing device may comprise a plurality of multiplex biosensing electrodes.

[0029] A “biosensing platform” represents a platform or methodology for detection of biomarkers. Some examples of biosensing platforms comprise: molecularly imprinted polymers, immunocomplex reaction, enzymatic reaction or DNA hybridization. An electrode many be modified with a biosensing platform to create a biosensing electrode.

[0030] While the term “multiplex” is used primarily herein, “multiplex” and “multiplexed” may be used interchangeably to refer to the ability to perform simultaneous measurement, such that a multiplex biosensing device is configured to simultaneously perform biosensing, for example for different biomarkers.

[0031] A “multiplex biosensing device” as used herein is a device comprising a plurality of multiplex biosensing electrodes, an insulation layer, a temporary or permanent well cell, a delivery mechanism and a separation or filter media.

[0032] For example, multiplex “bare” electrodes may comprise a substrate, working electrodes without a biosensing platform, an electrode base, a reference electrode, and a counter electrode. These electrodes do not have any biosensing functionality. In contrast, multiplex biosensing electrodes in accordance with one or more embodiments of the present disclosure comprise working electrodes with a biosensing platform, as well as a substrate, an electrode base, a reference electrode, and a counter electrode. The multiplex biosensing electrodes are specifically modified, either physically or chemically, to enable biosensing functionality. The modification of multiplex “bare” electrodes with a biosensing platform createsmultiplex biosensing electrodes in accordance with one or more embodiments. After surface modification of multiplexed (bare) electrodes to make them ready for sensing, they may be referred to as “multiplex biosensing electrodes”. In an example implementation, if all electrodes are not modified for biosensing, the term “multiplex biosensing electrodes” no longer applies, and they would simply be referred to as multiplex (bare) electrodes.

[0033] A “multiplex biosensor system”, or a “multiplex biosensor”, represents a system comprising a multiplex biosensing device and a detection apparatus. A “detection apparatus” may be configured to record, or detect, electrical signals from the multiplex biosensing device and to analyze the signals to enable measurement of a concentration of targets and optionally transmit the results.

[0034] A plurality of “sensing units” may comprise a plurality of multiplex biosensing electrodes and an insulation layer. The insulation layer may be configured to separate the plurality of multiplex electrodes into a plurality of sensing units by providing selective access to a portion of an integrated reference electrode, and to a portion of an integrated counter electrode, for each of the plurality of sensing units.

[0035] A “delivery mechanism” comprises a means for processing a biofluid sample and sequentially directing the processed sample and the relevant biosensing reagents to one or more detection zones. The delivery mechanism may be used to automate one or more biosensing platforms, for example to operate manually or automatically. A delivery mechanism may comprise pipetting the biofluidic samples as a mechanism of delivery.

[0036] A “microfluidic unit” comprises a sample inlet port, a separation or filter media for sample processing, a microfluidic for automating the sensing platform, a plurality of electrochemical detection zones, and one or more vents. A microfluidic unit, or microfluidic section, is an example of a delivery mechanism that may be used with the biosensing electrodes.

[0037] Embodiments of the present disclosure relate generally to the field of multiplexed biosensing technology, biosensor systems, delivery systems, and multi-omics. Embodiments ofthe present disclosure are in particular directed to a self-powered microfluidic-integrated multiplexed biosensing approach for automated biosample preparation and measuring the concentration of target proteins, metabolites, and genomes, in different biofluid samples such as blood, plasma, urine, sweat, saliva, sputum, tears, stool, and general use based on different electrochemical biosensing platforms.

[0038] In an aspect, the present disclosure provides a multiplex biosensing device comprising: a plurality of multiplex biosensing electrodes configured for simultaneous detection of two or more biomarkers in a biosample input, the plurality of multiplex biosensing electrodes comprising: a first biosensing electrode including a first electrode modified with a first biosensing platform for detecting a first biomarker, and a second biosensing electrode including a second electrode modified with a second biosensing platform for detecting a second biomarker; and a delivery mechanism configured for processing at least one biosample and for sequentially delivering the at least one biosample and at least one biosensing reagent to the plurality of multiplex biosensing electrodes as the biosample input.

[0039] In an example embodiment, the plurality of multiplex biosensing electrodes comprises: a substrate; a plurality of working electrodes provided on the substrate, each of the plurality of working electrodes including an electrode base; an integrated reference electrode provided on the substrate and shared between the plurality of multiplex biosensing electrodes, the integrated reference electrode including an electrode base; and an integrated counter electrode provided on the substrate and shared between the plurality of multiplex biosensing electrodes, the integrated counter electrode including an electrode base.

[0040] In an example embodiment, the plurality of working electrodes each define a circular or linear profile, the integrated reference electrode defines a circular or linear profile, and the integrated counter electrode defines a profile of a quarter of a ring or linear.

[0041] In an example embodiment, the device further comprises a patterned insulation layer configured to separate the plurality of multiplex electrodes into a plurality of sensing units by providing selective access to a portion of the integrated reference electrode and to a portion ofthe integrated counter electrode for each of the plurality of sensing units.

[0042] In an example embodiment, the device further comprises a well cell configured to enable the plurality of multiplex electrodes to operate with respect to different biomarkers and / or different biosamples.

[0043] In an example embodiment, the plurality of multiplex biosensing electrodes are configured to simultaneously sense one or more target biomolecules selected from the group consisting of: proteins, metabolites, microbes, genes, or extracellular vesicles.

[0044] In an example embodiment, the biosensing platform used for fabrication of multiplex biosensing device comprises a combination of one of or more of: molecularly imprinted polymers (MIP), immunocomplex reaction, enzymatic reaction and DNA hybridization mechanisms.

[0045] In an example embodiment, the biosensing platform comprises a molecularly imprinted polymer (MIP) biosensing electrode selected from the group consisting of: aniline, pyrrole, thiophene, dopamine, o-Phenylenediamine (OPD) and acetylene electropolymerized on an embedded redox layer.

[0046] In an example embodiment, the delivery mechanism comprises a microfluidic unit with an automated liquid handling configured to deliver at least one biosample, from the group consisting of blood, serum, plasma, urine, sweat, tears, saliva, sputum, and the at least one bioreagent to the plurality of multiplex biosensing electrodes.

[0047] In an example embodiment, the device further comprises a microfluidic unit integrated with a separation or filter media configured to prepare the biofluid sample by removing impurities before exposure to the plurality of multiplex biosensing electrodes.

[0048] In an example embodiment, the delivery mechanism comprises a self-powered delivery and automated system. In an example embodiment, the delivery mechanism is configured to support multiple biofluid types, including blood, plasma, urine, saliva, sputum, andsweat, through optimized microfluidic pathways that facilitate bubble-free and uniform distribution across detection zones.

[0049] In an example embodiment, the plurality of working and counter electrodes are constructed using ink materials selected from the group consisting of poly(3,4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT), polyaniline, MXene, graphene, and graphite, enabling compatibility with various biosensing platforms and electrode functionalization processes.

[0050] In an example embodiment, the plurality of multiplex biosensing electrodes employ a ratiometric analysis method to mitigate matrix effects, enhancing the accuracy and analytical performance of biomarker detection in complex sample matrices. In an example embodiment, the ratiometric analysis method includes simultaneous detection of a target analyte response and a stable internal reference response, with a ratio of such signals being used to correct for variations caused by matrix effects.

[0051] In an example embodiment, the plurality of multiplex biosensing electrodes are screened via a non-destructive quality control process based on an electro-fabrication process, thereby ensuring repeatability and reliability in biosensor performance.

[0052] In an example embodiment, the working electrodes are constructed of ink materials selected to enhance electrode conductivity and compatibility with specific biosensing platforms, including molecularly imprinted polymers, immunocomplex reactions, and DNA hybridization.

[0053] In a further aspect, the present disclosure provides a multiplex biosensor system comprising: a multiplex biosensing device according to one or more embodiments as described and illustrated herein; and a detection apparatus configured to electrically communicate with the plurality of multiplex biosensing electrodes and to obtain, analyze, and display biomarker data corresponding to the plurality of biomarkers and to provide a readout.

[0054] In an example embodiment, the detection apparatus comprises a communicationmodule allowing for remote data transmission and real-time monitoring of biomarker levels.

[0055] In an example embodiment, the multiplex biosensor system comprises a single-time detection, near-real time, or real-time model for detection of target biomarkers.

[0056] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the features illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Any alterations and further modifications, and any further applications of the principles of the disclosure as described herein are contemplated as would normally occur to one skilled in the art to which the disclosure relates. It will be apparent to those skilled in the relevant art that some features that are not relevant to the present disclosure may not be shown in the drawings for the sake of clarity.

[0057] In accordance with one or more embodiments, a multiplex biosensing method and device are disclosed for rapid, accurate, and quantitative detection of different biomolecules. Such detection may be performed and applicable in the varied fields of proteomics, metabolomics, and genomics with limited sample volumes autonomously. The biomolecules may comprise proteins, metabolites, pathogens, DNAs, RNAs, exosomes, small molecules as well as extracellular vesicles, and microorganisms including, pathogenic and non-pathogenic bacteria, viruses, and / or fungi. The body fluids may comprise blood, plasma, urine, saliva, sputum, tears, stool, and / or sweat.

[0058] The development of a highly sensitive, simple, and self-powered multiplex biosensing device for simultaneous detection of a panel of target biomolecules can dramatically improve diagnostic efficiency, for example through presenting a true sample-to-result diagnostic test that can be performed in any setting by any user. Detecting multiple biomarkers may provide more efficient information for early-stage diagnosis of specific diseases such as neurodegenerative disease, traumatic brain injury (TBI), infectious disease, diabetes, cardiovascular disease, and different types of cancer.

[0059] In accordance with one or more embodiments, different biosensing platforms may beapplied to perform simultaneous detection of the levels of more than two biomarkers in a small volume of biofluidic samples. A delivery mechanism may be used for processing the biofluid sample and sequentially directing the processed sample and the relevant biosensing reagents to the detection zones to automate the biosensing platforms. The delivery mechanism may also enable the simultaneous delivery of multiple biosamples. Furthermore, the multiplex biosensing device may facilitate real-time or near real-time monitoring of multiple biomarkers simultaneously, for example for applications in wearable and implantable biosensors.Additionally, a unique quality control (QC) protocol relying on electrochemical signals obtained during the biosensor production may be used to minimize or eliminate crosstalk among sensing electrodes and enhance the reliability and reproducibility of the results.

[0060] A self-powered multianalyte biosensing device in accordance with one or more embodiments may be used in different applications in which it is desirable to detect different biomarkers very quickly via different biosensing platforms based on electrochemical analysis with or without redox chamber. The different applications may comprise health, environment, food packaging quality control, agriculture, and / or veterinary medicine. The different biosensing platforms may comprise molecularly imprinted polymers, immunocomplex reaction, enzymatic reaction, and DNA hybridization.

[0061] Currently, biosensors utilize biological recognition elements (capture probes) such as enzymes, antibodies, nucleic acids, and aptamers which provide selective affinity toward their target biomolecules. However, these approaches have limited long-term stability / durability at room temperatures, and difficulty of reproducing and repeating biological recognition elements on a large scale. Accordingly, the development of synthetic alternatives is desirable, for example by healthcare authorities, for reliable, sensitive, specific, and rapid detection of target biomolecules. Molecularly imprinted polymers (MIPs) are efficiently developed biomimetic receptors with proven chemical, mechanical, and thermal stability, high specific recognition of target molecules, scalability, excellent reusability and storage stability, low cost, and easy preparation.

[0062] In view of recent advances in medicine and biotechnology and increasing human attention to health, it is desirable to measure small amounts of biomarkers such as proteins, genomes, and metabolites simultaneously, and the need for doing so is growing more andmore.

[0063] Embodiments of the present disclosure have been designed and fabricated in response to this global necessity and challenge to conduct quantification detection with low cost, high sensitivity, and in a manner that is user-friendly. An autonomous multiplex biosensing device according to one or more embodiments enables simultaneous detection of two or more specific biomarkers across different biofluid samples for various applications. According to one or more embodiments, different substrates of detection may be provided on a plurality of multiplex electrodes based on different biosensing platforms, including molecular imprinting technique, immunocomplex reaction (with capture antibodies, nanobodies, or aptamers), enzymatic reaction and / or DNA hybridization. Each multiplexed electrode may include two or more distinct sections for preparing the specific target biomolecule sensing platform. According to one or more embodiments, there is no or minimal signal interference during the preparation of each section, ensuring that each section independently supports its designated biosensing platform.

[0064] Biosensors for detecting various target biomolecules, including GFAP, succinate, agmatine, and cortisol, may be configured within each designated section. Additionally, a delivery mechanism, which may operate manually or automatically, may be integrated within the system according to one or more embodiments, to autonomously channel biofluids — such as plasma, urine, sweat, or whole blood — to multiplex biosensing electrodes. A fiber media may be employed to separate plasma from whole blood or to filter impurities from various biofluids, ensuring cleaner sample delivery to the biosensors.

[0065] FIG. 1 illustrates a block diagram of a multiplex biosensing device 100 in accordance with one or more embodiments. The multiplex biosensing device 100 comprises a substrate 110, and a plurality of multiplex biosensing electrodes 120 provided on the substrate 110. The plurality of multiplex biosensing electrodes 120 are configured for simultaneous detection of two or more biomarkers in a biosample input. The plurality of multiplex biosensing electrodes 120 comprise a first biosensing electrode 122 including a first electrode modified with a first biosensing platform for detecting a first biomarker, and a second biosensing electrode 124 including a second electrode modified with a second biosensing platform for detecting a second biomarker. The plurality of multiplex biosensing electrodes 120 may comprise multiple multiplexbiosensing electrodes. The multiple multiplex biosensing electrodes may include multiple modified working electrodes with diverse biosensing platforms.

[0066] The multiplex biosensing device 100 may comprise an insulation layer 130 and a well cell 140. The insulation layer 130 may be configured to separate the plurality of multiplex biosensing electrodes 120 into a plurality of sensing units. This may be achieved, for example and as described in further detail later, by providing selective access to a portion of an integrated reference electrode, and to a portion of an integrated counter electrode, for each of the plurality of sensing units. The well cell 130 may be configured to enable the plurality of multiplex biosensing electrodes 120 to operate with respect to different biomarkers and / or different biosamples.

[0067] The multiplex biosensing device 100 may further comprise a delivery mechanism 150 configured for processing at least one biosample and for sequentially delivering the at least one biosample and at least one biosensing reagent to the plurality of multiplex biosensing electrodes 120 as the biosample input. In an example embodiment, the delivery mechanism 150 comprises a microfluidic unit 152.

[0068] FIG. 2 illustrates a block diagram of a multiplex biosensing device 200 in accordance with one or more embodiments. In the embodiment of FIG. 2, the substrate 110, insulation layer 130, well cell 140 and delivery mechanism 150 are the same as in FIG. 1. In the embodiment of FIG. 2, the plurality of multiplex biosensing electrodes 120 comprises a first biosensing working electrode 222 and a second biosensing working electrode 224. In an example implementation, the first and second biosensing electrodes 122 and 124 of FIG. 1 are implemented as the first and second biosensing working electrodes 222 and 224 in FIG. 2. Each of the plurality of working electrodes, for example including first and second biosensing working electrodes 222, 224 may include an electrode base. An integrated reference electrode 226 may be provided on the substrate 110 and shared between the plurality of multiplex biosensing electrodes 120. The integrated reference electrode 226 may include an electrode base. An integrated counter electrode 228 may be provided on the substrate 110 and shared between the plurality of multiplex biosensing electrodes 120. The integrated counter electrode 228 may include an electrode base.

[0069] FIG. 3 illustrates a block diagram of a multiplex biosensor system 300 in accordance with one or more embodiments. The multiplex biosensor system 300 comprises a multiplex biosensing device 100, such as those described in relation to FIG. 1 or FIG. 2, or as will be described in relation to other figures. The multiplex biosensor system 300 further comprises a detection apparatus 310 configured to electrically communicate with the plurality of multiplex biosensing electrodes 120 and to obtain, analyze, and display biomarker data corresponding to the plurality of biomarkers and to provide a readout.

[0070] FIG. 4A and FIG. 4B illustrate a schematic overview of a multiplex biosensing device 400 in 2D and 3D in accordance with one or more embodiments. The multiplex biosensing device 400 of FIG. 4A and FIG. 4B comprises a plurality of multiplex biosensing electrodes configured for simultaneous real-time or near real-time monitoring of at least two biomarkers. The plurality of multiplex biosensing electrodes comprise a first electrode modified with a biosensing platform (molecularly imprinted polymers, immunocomplex reaction, enzymatic reaction and / or DNA hybridization) customized for a first biomarker and a second electrode modified with a second biosensing platform customized for a second biomarker. A delivery mechanism (not shown) is configured to deliver at least one biosample (after an electrode modification process) and at least one biosensing reagent to the plurality of multiplex biosensing electrodes.

[0071] In an example embodiment, the first biosensing platform and the second biosensing platform comprise customized molecularly imprinted polymers (MIPs) for each target biomarker. The MIPs may be selected from the group consisting of aniline, pyrrole, thiophene, dopamine, o-Phenylenediamine (OPD) and acetylene electropolymerized on an embedded redox layer. Each of the multiplexed biosensing electrodes is configured to sense one of the target biomolecules selected from the group consisting of: proteins, metabolites, nucleic acids, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), exosomes, extracellular vesicles, and pathogens like bacteria and viruses.

[0072] FIG. 5 illustrates a schematic of a multiplex biosensing device 500 including a plurality of multiplex biosensing electrodes 510, with an insulation layer 520 and a temporary or permanent well cell 530 which can be removed and separated in accordance with one or more embodiments. The plurality of multiplex biosensing electrodes 510 may comprise a substrate502, and a plurality of working electrodes 504 provided on the substrate 502. Each of the plurality of working electrodes 504 may comprise an electrode base 506. The plurality of multiplex electrodes 510 may comprise an integrated reference electrode 508 provided on the substrate 502 and shared between the plurality of working electrodes 504. The plurality of multiplex biosensing electrodes 510 may comprise an integrated counter electrode 509 provided on the substrate 502 and shared partially with each of the plurality of working electrodes 504. The insulation layer 520 may be configured to separate the plurality of multiplex biosensing electrodes 510 into a plurality of sensing units by providing selective access to a portion of an integrated reference electrode 508, and to a portion of the integrated counter electrode 509, for each of the plurality of sensing units.

[0073] An insulation layer 220 may be configured to separate the plurality of multiplex electrodes into a plurality of sensing units by providing selective access to a portion of the integrated reference electrode 208 and to a portion of the integrated counter electrode 209 for each of the plurality of sensing units. A well cell 230 may be provided for different biomarkers and configured to enable the plurality of multiplex electrodes 210. The well cell 230 may be a primary well cell or a temporary well cell. In the example embodiment of FIG. 2, the insulation layer 220 and the well cell 230 cooperate to separate the plurality of multiplex electrodes 210 into four sensing units.

[0074] FIG. 6 illustrates different parts of a plurality of multiplex biosensing electrodes 600 in accordance with one or more embodiments. The plurality of multiplex biosensing electrodes 600 may comprise a substrate 602, and a plurality of working electrodes 604 provided on the substrate 602. Each of the plurality of working electrodes 604 may comprise an electrode base 606. The plurality of multiplex electrodes 600 may comprise an integrated reference electrode 608 provided on the substrate 602 and shared between the plurality of working electrodes 604. The plurality of working electrodes may each define a circular profile. The integrated reference electrode 608 may define an annular profile, such as a circular profile. The plurality of multiplex electrodes 600 may comprise an integrated counter electrode 609 provided on the substrate 602 and shared partially with the plurality of working electrodes 604. The integrated counter electrode 609 may define an annular profile, such as a ring-shaped profile, for example a profile of a quarter of a ring.

[0075] FIG. 7A, FIG. 7B and FIG. 70 illustrate a multiplex biosensing device including two, three and four working electrodes, respectively, in accordance with one or more embodiments.

[0076] In FIG. 7A, a multiplex biosensing device 710 is illustrated in an assembled form, with constituent component parts 712, 714 and 716 of the device 710 shown separately beside the full device. In FIG. 7A, a plurality of multiplex biosensing electrodes 712 includes thereon two multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with the electrodes, such that the integrated counter electrode is divided into portions and each portion is only used by one electrode. An insulation layer 714 may be configured to separate the electrodes into two sensing units. A well cell 716 may be configured to enable the electrodes to operate with respect to two different biomarkers, with respect to the two sensing units. The insulation layer 714 and the well cell 716 in FIG. 7A cooperate to separate the electrodes into two sensing units.

[0077] In FIG. 7B, a multiplex biosensing device 720 is illustrated in an assembled form, with constituent component parts 722, 724 and 726 of the device 720 shown separately beside the full device. In FIG. 7B, a plurality of multiplex biosensing electrodes 722 includes thereon three multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with the electrodes. An insulation layer 724 may be configured to separate the electrodes into three sensing units. A well cell 726 may be configured to enable the electrodes to operate with respect to three different biomarkers, with respect to the three sensing units. The insulation layer 724 and the well cell 726 in FIG. 7B cooperate to separate the electrodes into three sensing units.

[0078] In FIG. 7C, a multiplex biosensing device 730 is illustrated in an assembled form, with constituent component parts 732, 734 and 736 of the device 730 shown separately beside the full device. In FIG. 7C, a plurality of multiplex biosensing electrodes 732 includes thereon four multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with the electrodes. An insulation layer 734 may be configured to separate the electrodes into four sensing units. A well cell 736 may be configured to enable the electrodes to operate with respect to four different biomarkers, with respect to the four sensing units. The insulation layer 734 and the well cell 436in FIG. 70 cooperate to separate the electrodes into four sensing units.

[0079] FIG. 8A, FIG. 8B, FIG. 80 and FIG. 8D illustrate a multiplex biosensing device including a plurality of multiplex electrodes with varied sizes and configurations for four working electrodes, in accordance with one or more embodiments.

[0080] In FIG. 8A, a multiplex biosensing device 810 is illustrated in an assembled form, with constituent component parts 812, 814 and 816 of the device 810 shown separately beside the full device. In FIG. 8A, a plurality of multiplex biosensing electrodes 812 includes thereon four multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with each of the electrodes. The four multiplex working electrodes in FIG. 8A have a different configuration than the four-electrode embodiment shown in FIG. 7C, for example based on the electrode bases that are not provided in a straight line and have a non-uniform width along their length, the electrode bases being wider at an end farther from the electrode tips. An insulation layer 814 may be configured to separate the electrodes into four sensing units. The layout of the elements of the insulation layer 814 is different than the insulation layer 734 in FIG. 7C, which also has four sensing units. A well cell 816 may be configured to enable the electrodes to operate with respect to four different biomarkers, with respect to the four sensing units. The insulation layer 814 and the well cell 816 in FIG. 8A cooperate to separate the electrodes into four sensing units.

[0081] In FIG. 8B, a multiplex biosensing device 820 is illustrated in an assembled form, with constituent component parts 822, 824 and 826 of the device 820 shown separately beside the full device. The embodiment of FIG. 8B also includes a plurality of multiplex biosensing electrodes 822 including four multiplex working electrodes, an insulation layer 824 configured to separate the electrodes into four sensing units, and a well cell 826 configured to enable the electrodes to operate with respect to four different biomarkers, with respect to the four sensing units. Similar to FIG. 8A, the embodiment in FIG. 8B includes electrode bases that are not provided in a straight line and have a non-uniform width along their length, being wider at an end farther from the electrode tip.

[0082] In FIG. 8C, a multiplex biosensing device 830 is illustrated in an assembled form, with constituent component parts 832, 834 and 836 of the device 830 shown separately besidethe full device. In FIG. 80, a plurality of multiplex biosensing electrodes 832 includes thereon four multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with each of the working electrodes. The four multiplex working electrodes in FIG. 80 have a different configuration than the four-electrode embodiment shown in FIG. 70, even though both embodiments have electrode bases that are provided in a straight line. The counter electrode and its electrode base have a different configuration in FIG. 80 than in other embodiments. An insulation layer 834 may be configured to separate the electrodes into four sensing units. The layout of the elements of the insulation layer 834 is slightly different than the insulation layer 734 in FIG. 70, which also has four sensing units. A well cell 836 may be configured to enable the electrodes to operate with respect to four different biomarkers, with respect to the four sensing units. The insulation layer 834 and the well cell 836 in FIG. 80 cooperate to separate the electrodes into four sensing units.

[0083] In FIG. 8D, a multiplex biosensing device 840 is illustrated in an assembled form, with constituent component parts 842, 844 and 846 of the device 840 shown separately beside the full device. In FIG. 5D, a plurality of multiplex biosensing electrodes 842 includes thereon four multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with each of the working electrodes. The four multiplex working electrodes, counter electrode and reference electrode in FIG. 8D have a similar configuration to the four-electrode embodiment shown in FIG. 80. The electrode bases in FIG. 8D are longer than the electrode bases in FIG. 80. An insulation layer 844 may be configured to separate the electrodes into four sensing units. The layout of the elements of the insulation layer 844 is similar to insulation layer 834 in FIG. 8D, which also has four sensing units. A well cell 846 may be configured to enable the electrodes to operate with respect to four different biomarkers, with respect to the four sensing units. The insulation layer 844 and the well cell 846 in FIG. 8D cooperate to separate the electrodes into four sensing units.

[0084] FIG. 9 illustrates a multiplex biosensing device 900 including a plurality of multiplex biosensing electrodes for eight working electrodes, or at least eight working electrodes, in accordance with one or more embodiments. In FIG. 9, a multiplex biosensing device 900 is illustrated in an assembled form, with constituent component parts 902, 904 and 906 of thedevice 900 shown separately beside the full device. In FIG. 9, a plurality of multiplex biosensing electrodes 902 includes eight multiplex working electrodes, an integrated reference electrode shared between the electrodes, and an integrated counter electrode shared partially with each of the working electrodes. An insulation layer 904 may be configured to separate the electrodes into eight sensing units. A well cell 906 may be configured to enable the electrodes to operate with respect to eight different biomarkers, with respect to the eight sensing units. The insulation layer 904 and the well cell 906 in FIG. 9 cooperate to separate the electrodes into eight sensing units.

[0085] FIG.10 illustrates a linear multiplex biosensing device 1000 including a plurality of multiplex biosensing electrodes, for example more than eight working electrodes, in accordance with one or more embodiments. While the embodiment of FIG. 10 is shown with more than eight working electrodes, it is also applicable to an embodiment with two or more working electrodes. In FIG. 10, a multiplex biosensing device 1000 is illustrated in an assembled form, with constituent component parts 1002, 1004 and 1006 of the device 1000 shown separately beside the full device. In FIG. 10, a plurality of multiplex electrodes 1002 includes thereon more than eight multiplex working electrodes. In this example embodiment, nineteen multiplex working electrodes are shown, though blank space is shown between a group of 16 electrodes and a group of 3 electrodes, indicating that there is flexibility and space in this type of design to expand the design to any number of multiplex working electrodes. An integrated reference electrode is shared between the electrodes, and an integrated counter electrode is shared partially with the working electrodes. An insulation layer 1004 may be configured to separate the electrodes into a plurality of sensing units, for example 19 sensing units. A well cell 1004 may be configured to enable the electrodes to operate with respect to a plurality of, for example 19, different biomarkers, with respect to the 19 sensing units. The insulation layer 1004 and the well cell 1006 in FIG. 10 cooperate to separate the electrodes into a plurality of, for example 19, sensing units.

[0086] In an example embodiment, a plurality of multiplex biosensing electrodes is configured for simultaneous real-time monitoring of a plurality of biomarkers. The plurality of multiplex electrodes may each modified with similar or different biosensing platforms configured for simultaneous detection of two or more biomarkers (biomolecules). An insulation layer may be configured to separate the plurality of multiplex electrodes into a plurality of sensing units byproviding selective access to a portion of the integrated reference electrode and to a portion of the integrated counter electrode for each of the plurality of sensing units.

[0087] FIG.11 illustrates a schematic overview of a multiplex biosensing device 1100 including a plurality of multiplex biosensing electrodes, an insulation layer, a temporary or permanent well cell, a delivery mechanism, and a separation or filter media in 2D and 3D, in accordance with one or more embodiments. The device 1100 may include a delivery mechanism configured to sequentially process and deliver at least one biosample and at least one biosensing reagent to the plurality of multiplex electrodes. In the embodiment of FIG. 11 , the delivery mechanism comprises a microfluidic unit 1102, which may include a sample inlet port 1104, a separation or filter media 1106 for sample processing, and a microfluidics 1108 for automating the sensing platform. A plurality of electrochemical detection zones 1110 may be provided, for example by a plurality of sensing units as described in relation to the previous figures. One or more vents 1112 may be provided in communication with the plurality of electrochemical detection zones 1110. The delivery mechanism may comprise a self-powered and automated delivery system.

[0088] FIG.12 illustrates a schematic overview of a connector 1200 that may be used during the fabrication or testing process of a multiplex biosensing device 1202, in accordance with one or more embodiments. The connector 1200 may be configured to receive a multiplex biosensing device 1202 comprising a plurality of multiplex biosensing electrodes, such as described in relation to one or more embodiments. The connector 1200 may be configured to be in communication with the plurality of multiplex biosensing electrodes, and with the plurality sensing units, to obtain data associated with the simultaneous real-time or near real-time monitoring of at least two biomarkers.

[0089] FIG. 13 illustrates a schematic representation of a surface modification process during a fabrication process for a plurality of multiplex biosensing electrodes, in accordance with one or more embodiments. Any suitable surface modification process may be employed to modify a multiplex bare electrode to create a multiplex biosensing electrode in accordance with one or more embodiments, for example by modifying the bare electrode with a biosensing platform. The surface modification process of FIG. 13 includes electrodeposition of Prussian blue nanoparticles onto a substrate. Electropolymerization is performed of the substrate with theelectrodeposited Prussian blue particles, followed by washing and rebinding.

[0090] FIG.14 illustrates a schematic overview of a multiplex biosensor system 1400, in accordance with one or more embodiments. The multiplex biosensor system 1400 comprises a multiplex biosensing device 1402, which may utilize one or more biosensing platforms in accordance with any one or more of the embodiments described and illustrated herein and comprising a plurality of multiplex biosensing electrodes. The multiplex biosensing device 1402 may comprise a delivery mechanism for processing at least one biosample and sequential delivery of biosample(s) and at least one biosensing reagent to the plurality of multiplex biosensing electrodes. The multiplex biosensor system 1400 comprises a detection apparatus 1404 configured to electrically communicate with the plurality of biosensing electrodes, to obtain, analyze, and interpret signals, preferably display, biomarker data corresponding to the plurality of biomarkers and to provide a readout. In an example embodiment, detection system comprises a detection apparatus.

[0091] FIG.15 shows measured calibration curves obtained from electrochemical response studies of implementations of embodiments of the present disclosure for different analytes analysis. The response studies are based on one or more embodiments in PBS solution such as (A) Cortisol in the detection range of 1 nM to 100 pM with the limit of detection of 0.1 nM, (B) Agmatine in the detection range of 0.1 pM to 1 mM with the limit of detection of 0.01 pM, (C) Succinate in the detection range of 0.25 pM to 250 pM with the limit of detection of 0.025 pM, and (D) GFAP in the detection range of 0.002 pg / ml to 200 ng / ml with the limit of detection of 0.3 fg / ml.

[0092] FIG.16 shows measured calibration curves obtained from electrochemical response studies of implementations of embodiments of the present disclosure for different biofluids (plasma, blood, sweat, and urine). The response studies are based on one or more embodiments relating to biofluids such as (A) Plasma in the detection range of 2.5 pM to 2500 pM with the limit of detection of 0.025 pM, (B) Blood in the detection range of 0.25 pM to 250 pM with the limit of detection of 0.02 pM, (C) Artificial sweat in the detection range of 0.2 nM to 50 pM with the limit of detection of 0.02 nM, and (D) urine in the detection range of 0.025 pM to 250 pM with the limit of detection of 0.02 pM.

[0093] Molecularly Imprinted Polymers (MIPs)

[0094] As mentioned earlier, molecularly imprinted polymers (MIPs) are efficiently developed biomimetic receptors with proven chemical, mechanical, and thermal stability, high specific recognition of target molecules, scalability, excellent reusability and storage stability, low cost, and easy preparation. These characteristics of MIPs offer a promising approach to developing a new generation of chemical-biological sensors. These superiorities have provided a new standard for M IP-based biosensors for sensitive, selective, near real-time, or continuous detection of a wide range of biomarkers including metabolites, biological molecules, bioparticles, cells, and pathogens in complex biofluids toward identification specifically targets by polymerizing a functional monomer or a series of functional monomers, and cross-linker around target molecules that create noncovalent detection cavities based on shapes, sizes, geometries, and functionality after the removal of the template molecule.

[0095] MIP technology has been successfully applied as an efficient biosensing platform for the detection of small molecules and DNAs in the nanometer (-0.1-5 nm) range to much larger molecules or bioparticles such as large proteins, extracellular vesicles, bacteria, viruses, in the submicro- or micro-meter (~ 0.1-10 pm) range. Also referred to as “artificial antibodies,” MIP biosensors have similar sensing mechanism as antibody-based biosensors, and provide cavities with high affinity binding sites, that only bind to the molecular structure to which it has been imprinted while having a much longer shelf-life in comparison, with antibodies due to its synthetic nature and can be stored at room temperature without degradation. Using MIPs as an antifouling layer of the electrode surface brings functionality along with antifouling properties. MIPs have great applications in membrane separations, drug delivery systems, biosensors, and environmental assessment of target compounds. In addition, MIPs have also been successfully commercialized as a specific sorption medium in solid-phase extraction (SPE) technique and a stationary phase in separation techniques such as liquid chromatography or electrophoretic chromatography.

[0096] Challenges such as inconsistent control over polymer film thickness, uniform deposition of the film, and template leakage have long limited the effectiveness of MIP biosensors. Electropolymerization of monomers like aniline, pyrrole, thiophene, and acetylene on various electrode surfaces, including gold, pencil graphite, graphite, platinum, and indium tin oxide (ITO), have enabled the formation of electrically conductive polymeric films with precisethickness and surface uniformity control, advancing the development of MIP-based biosensors. Additionally, incorporating boronic acid-derived compounds, such as 3-amino phenyl boronic acid, as crosslinkers enhances target affinity, mechanical and chemical stability, and improves the efficiency of the template-entrapping cavities.

[0097] However, the need to add external redox signaling probes such as ferrocyanide in electrochemical sensing platform has limited the application of M IPs biosensors. Prussian blue (PB) may be used as the embedded redox probe because of its superior specifications such as high electrochemical-electrocatalytic reactivity and low redox potentials; such use has eliminated the need for complex labeling procedures or external redox probes and can be successfully applied to develop reagentless electrochemical sensors. However, the hydrolysis of PB in neutral or alkaline solutions and low electron conductivity limit the use of PB as a sensing probe in ultrasensitive electrochemical biosensors. Electrodeposition of PB on the surface of nanostructure-based supports such as carbon-based materials, graphene, MXene, and so on can effectively stabilize PB and eliminate these constraints.

[0098] Recently, MXene has garnered significant attention in the field of biosensors due to its excellent metal conductivity, hydrophilicity, and large specific surface area for analyte sensing. However, the inherent challenges associated with limited interlayer spacing, self-restacking, and aggregation of MXenes hinder the exploitation of their superior surface metal properties. To overcome these limitations, graphene may be incorporated between MXene layers may serve as an ideal "buffer" and spacer, effectively preventing undesired stacking of MXene nanosheets and enhancing the electrochemical properties of MXene.

[0099] Considering the importance of the controllable patterning step of MXene composites on flexible surfaces which has a great impact on achieving favorable conductivity, using poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) as noncytotoxic and good biocompatible conductive polymer can provide a polymer structure with a doped backbone that can be utilized as interfacial materials in development of biosensors.

[0100] MIP biosensors incorporating nanostructure exhibit excellent sensing performance, specific recognition ability, and massive reactive sites that hold great potential for applications in developing flexible biosensors and provide vast possibilities for disease diagnostics. MIPbiosensors incorporating MXene / graphene exhibit excellent sensing performance, specific recognition ability, and massive reactive sites that provide a wide linear range and high sensitivity in M IPs biosensors and hold great potential for applications in developing flexible biosensors for disease diagnostics.

[0101] It is known that the matrix effect significantly impacts the accuracy and sensitivity of MIP biosensors. This effect arises when components within the sample matrix interfere with the target detection, leading to inaccurate readings and reduced sensitivity. Common sources of interference include proteins, salts, and other endogenous substances present in complex biological or environmental samples. These interferences can hinder the binding of the target molecule to the MIP, thereby affecting the sensor's performance. As a result, the reliability of MIP biosensors in real sample detection is often compromised, necessitating strategies to mitigate these matrix effects.

[0102] To mitigate the matrix effect and enhance the analytical performance of MIP biosensors, several strategies may be employed. One approach is the use of ratiometric biosensors. The core concept of these biosensors is the simultaneous detection of two signals: one that is responsive to the target analyte and another that serves as a stable internal reference. This internal reference is designed to provide a consistent baseline that can be used to normalize the analyte-specific signal. By calculating the ratio of the analyte signal to the reference signal, the biosensor can effectively correct for any variations caused by the matrix, leading to more accurate and sensitive measurements.

[0103] The internal reference in a ratiometric biosensor may be implemented in several ways. One common approach is the use of dual electrochemical probes, where one electrochemical probe responds to changes in the presence of the target analyte, while the other remains constant. By taking the ratio of the electrochemical signal from these two electrochemical probes, any fluctuations due to the matrix can be negated, ensuring that the signal accurately reflects the concentration of the target analyte.

[0104] In this regard, Prussian Blue (PB) may be used in conjunction with various electrochemical probes such as ferrocene derivatives, thionine and methylene blue to create effective ratiometric biosensors. Using electrodeposited Prussian Blue (PB) as an internalreference probe and labeled monomers (boronate affinity) with electrochemical probes which create electrochemical signal at different potential of PB, can be an efficient approach to achieve molecularly imprinted polymer (MlP)-based immunosandwich assay for ultrasensitive and specific detection of biomarkers in complex samples.

[0105] Another approach involves the use of dual-electrode systems in electrochemical biosensors. In this configuration, one electrode is modified with an MIP that is selective for the target analyte, while the other electrode serves as the reference. The signal from the analytesensitive electrode is compared to the signal from the reference electrode, and the ratio of these signals is used to determine the analyte concentration. This method helps to eliminate the impact of matrix components on the sensor's response, providing a more reliable measurement.

[0106] Also, another effective strategy to overcome matrix effects is to employ signal amplification mechanisms that increase the signal response upon target binding, rather than relying on signal reduction mechanisms. This approach can enhance the sensitivity of MIP biosensors, making them more resilient to the noise introduced by the matrix. By increasing the signal output when the target molecule is present, these methods help to distinguish true signals from background noise more effectively.

[0107] Incorporating ratiometric biosensors with internal references may significantly enhance the performance of MIP biosensors in complex sample matrices and for application with the needs of real-time, near real-time or continuous sensing. This approach not only improves the accuracy and sensitivity of the detection but also simplifies the sensor design by reducing the need for extensive sample preparation and purification steps. Consequently, ratiometric biosensors are highly suitable for real-world applications, including medical diagnostics, environmental monitoring, and food safety testing, where matrix effects are often a major concern.

[0108] Despite the notable advantages of MIP biosensors, advancing electrochemical MIP biosensors encountered challenges, including selecting appropriate monomers and crosslinkers, controlling the thickness and uniformity of the polymeric layer, template removal, and producing reproducible MIP films. The electro-fabrication procedure, an important step in biosensor development, demands stringent quality control (QC) measures to ensure thereproducibility and reliability. Electrodeposition of embedded redox probes such as Prussian blue (PB) in electrochemical MIP biosensors enables monitoring the electrical signal at each fabrication step, providing an effective QC measure. Additionally, the electropolymerization of conductive monomers facilitates the direct, in situ fabrication of M IPs on electrode surfaces. This technique offers a rapid and straightforward method to produce electrically conductive polymeric films with controllable thickness, uniform morphology, reproducible outcomes, and real-time polymer growth monitoring, enhancing the precision of M IP-based biosensor development. Such strategic tuning is crucial for modulating charge transfer through the electrode, thereby improving the overall performance of the biosensor.

[0109] Variations in ink properties, substrate characteristics, and fabrication methods of biosensors can lead to disparities in electrochemical performance, impacting the accuracy and precision of MIP biosensors. Thus, developing an efficient QC strategy to address these challenges and enhance the reliability and reproducibility of biosensors is crucial. Screening electrodes during the fabrication process is essential to identify and eliminate defects or irregularities that may compromise the overall performance of the biosensors.

[0110] Use Cases

[0111] Multiplexed biosensors, such as those described in relation to embodiments of the present disclosure, may be used in a plurality of different practical applications and use cases. Some examples use cases will be discussed below.

[0112] A multiplexed biosensor may be used for detection of a heart attack. The ability to detect multiple cardiac biomarkers simultaneously is important from a clinician’s perspective, as it allows for a more accurate and comprehensive diagnosis of heart attacks. A complex multiplex biosensor device may enable simultaneous detection of multiple cardiac biomarkers, including troponin I, troponin T, creatine kinase-MB, myoglobin, and B-type natriuretic peptide. The device may include a data processing unit that analyzes and correlates signals to provide a comprehensive assessment of the biomarkers, along with a display unit that presents real-time results to the user. The impact of this complex biosensor may be profound, as it enables rapid, accurate, and early diagnosis of heart attacks, facilitating timely medical intervention. This early detection capability may greatly increase survival rates and reduce heart damage, enhancing diagnostic precision and reliability, and ultimately saving countless lives through better-informedclinical decisions.

[0113] A multiplexed biosensor may be used for detection of drug overuse. The ability to detect multiple drugs simultaneously is important, as it allows for a more accurate and holistic understanding of a patient's condition, particularly in cases of polydrug use, which is common in overdose situation. A complex multiplex biosensor device may enable simultaneous detection of overuse of drugs, including but not limited to morphine, fentanyl, xylazine, oxycodone, heroin, methadone, and benzodiazepines. The complex biosensor may provide a comprehensive assessment of the presence and / or concentration of these drugs, along with a display unit that presents real-time results to the user. The impact of this complex biosensor may be profound, as may enable rapid, accurate, and early detection of drug overuse, facilitating timely medical intervention. This early detection capability may be important in preventing overdoses, which can significantly increase survival rates and reduce health complications. By enhancing diagnostic precision and reliability, this biosensor may ultimately save countless lives through better-informed clinical decisions and immediate action against potential overdose situations.

[0114] A complex biosensor may be integrated with BacT / Alert system for real-time monitoring of multiple microbial biomarkers. Currently, diagnosing bloodstream infections often depends on these conventional techniques, which can be slow and less precise. Colorimetric assays based on pH and CO2 measurements typically involve lengthy procedures and may not offer the level of specificity required for accurate diagnosis since additional culturing steps is required for identification of bacteria. Combining a complex multiplex biosensor with a current BacT / Alert system may enable real-time monitoring and simultaneous detection of multiple microbial biomarkers in blood samples. This complex biosensor system may provide a comprehensive assessment of microbial biomarkers, addressing the limitations of traditional diagnostic methods that rely on colorimetry, pH, and CO2 measurements. By integrating realtime monitoring with the multiplex biosensor, this system may accelerate the diagnostic process, enabling instant feedback on infection status and biomarker concentrations. This rapid and precise detection may facilitate timely medical intervention, significantly reducing patient suffering and improving outcomes by avoiding the delays and limitations associated with traditional diagnostic methods. The system's ability to deliver immediate and detailed diagnostic information may enhance the management of bloodstream infections and helps preventcomplications and mortality.

[0115] A multiplexed biosensor may be used for real-time monitoring of multiple biomarkers in Organ-on-a-Chip (OOC) models or bioreactors. Current cell culture practices often involve frequent and time-consuming media changes to maintain optimal conditions and prevent cellular stress. This process can be labor-intensive, prone to human error, and disruptive to the cells’ growth and function, impacting the reliability and reproducibility of experimental results. The need for precise and timely adjustments to the culture environment can significantly affect the quality and outcome of research. A multiplexed biosensor may enable the real-time monitoring of multiple biomarkers in cell culture systems, specifically within organ-on-a-chip models or bioreactors. This system may enable the simultaneous detection and quantification of various biomarkers relevant to cellular and tissue responses, including but not limited to cytokines, metabolites, and signaling molecules, from a small volume of culture fluid. The multiplexed biosensor may integrate multiple sensing platforms within a compact device, facilitating the continuous monitoring of biomarkers in real-time. This approach may provide comprehensive insights into cellular behavior and biochemical processes, enabling more precise and dynamic analysis of cell cultures. By incorporating this multiplexed biosensor into organ-on-a-chip models or bioreactors, researchers and clinicians may obtain immediate and detailed information on cellular responses, allowing for more effective monitoring of cell health, tissue development, and biochemical interactions. This real-time capability may address the limitations of traditional methods, which often involve delayed or less precise measurements, thereby improving the efficiency of experimental processes and accelerating the development of biomedical applications. Ultimately, this system may enhance the understanding of cellular mechanisms, may support more accurate drug testing and disease modeling, and may contribute to advancements in tissue engineering and regenerative medicine.

[0116] Alzheimer’s disease (AD) is a neurodegenerative disease that causes severe cognitive decline, irreversible memory loss and endangers physical and mental health. The treatment of Alzheimer's disease after the onset of clinical symptoms can only delay the deterioration of symptoms and cannot stop or reverse the disease progression. Early diagnosis at a preclinical stage is important in that it may can reduce the risk of suffering AD by one-third. Blood examination is the most suitable method for early screening in asymptomatic populationswhich can detect changes in the

[0117] AD biomarker concentration. Clinical studies have shown that the concentration of p-amyloid (A ) in the blood changes about 10-15 years before the appearance of AD symptoms. Subsequently, researchers also found that the concentration of tau protein (tau), and neurofilament light chain protein (NFL) in AD patients’ cerebrospinal fluid (CSF) is two to three times higher than that of normal control. Therefore, the detection of multiple biomarkers in blood with high sensitivity, for example using a multiplexed biosensor as described herein, can be a good approach for the accurate diagnosis of AD especially at an early stage.

[0118] Objective diagnosis of traumatic brain injury (TBI) and accurate risk-stratification of patients with TBI is one of the major challenges with important public health implications. The diagnosis of TBI may occur several days after admission to the hospital or clinically, which can lead to missing the critical window for early intervention. Multiplex detection of related bloodbased biomarkers may include glial fibrillary acidic protein (GFAP) breakdown products and the neuronal proteins, ubiquitin c-terminal hydrolase L1 (LICH-L1), neurofilament light (NF-L), calpain-cleaved all-spectrin (SPTAN1) N-terminal fragment and tau, a microtubule-associated protein found predominantly in cortical nonmyelinated axons, can dramatically improve identification and stratifying TBI and for rapid diagnoses and treatment.

[0119] Diagnosing an infectious disease promptly is one of the most challenging clinical diagnose for the clinician. To perform early diagnosis of an infectious disease, a series of inflammatory biomarkers such as procalcitonin (PCT), interleukin-6 (IL-6), C-reactive protein (CRP), and serum amyloid A (SAA) need to be monitored simultaneously. Since the mentioned inflammatory biomarkers are highly correlated with each other, their simultaneous measurement may effectively improve the accuracy and diagnostic efficiency of infectious diseases and reduce diagnostic costs. For example, PCT is a polypeptide consisting of 116 amino acids and is the precursor of calcitonin, and it increases rapidly in human serum after bacterial infection within 1.5-2 h and reaches a maximum after 24 h. Also, previous studies suggest that inflammatory or autoimmune diseases could influence the level of IL-6 which regulates the immune response and inflammation. Furthermore, IL-6 shows kinetics for monitoring the efficacy of antibiotic treatment. So, simultaneous detection of PCT and IL-6 may greatly improve the accuracy of diagnosis of bacterial infections from nonbacterial infections, guide antibiotictherapy and improve the efficacy of clinical therapy.

[0120] In the early detection of diabetes, which is a systemic disease that affects several organs, it may be necessary to examine several biomarkers simultaneously, and therefore multiparameter detection tests may be required. Such tests may be performed using a multiplexed biosensor in accordance with one or more embodiments. The concept of immunculus method is the investigation of the level and dynamic change of natural autoantibodies and anti-idiotypic antibodies in the body. In other words, immunculus is the mapping of the body’s immunochemical codes. In fact, by this method, a doctor may detect diabetes in the early stages and before the onset of symptoms Some existing challenges include the possibility of human error in conventional ELISA immunculus kits, the relatively high cost of the kit for screening tests, unbearable, unavailable, time-consuming, multi-step test, the long response time, sample volume constraints, and the need for skilled labor. Access to a sensitive, stable, and reproducible multiplexed and multianalyte nano-biosensor system is desirable for measuring biomarkers of early detection of diabetes including, for example, Insulin antibody, Insulin receptors antibody, NF-200 antibody, glial fibrillary acidic protein (GFAP) antibody, Collagen II antibody, TrM-03 antibody, ANCA antibody, and Kim-05 antibody simultaneously. Mapping the immunochemical codes of a person may indicate the status of the disease, and a doctor may make the correct detection and intervention by investigating the changes in the progress of these immunochemical codes.

[0121] Most patients with cancer are diagnosed at a late stage. Due to the high metastatic diffusion capacity and fatality rate, cancer is the second leading cause of death globally. Early-stage clinical diagnosis is one of the key issues in disease control which can improve the chance of patient survival. Since most markers are not specific to a particular disease or physiological change, and most cancers have more than one tumor marker associated with the incidence, a single marker immunoassay is usually not sufficient to diagnose or prognosis of specific cancer. For instance, breast cancer is associated with cancer antigen 153 (CA 15-3), cancer antigen 125 (CA 125), and mucinl (MLIC1), and patients with breast cancer typically show the positive expression of these tumor markers. So, the simultaneous determination of these biomarkers which are often applied in early diagnosis can provide more reliable measurements and show the patterns that contribute to the diagnosis of malignancy. Thus, the development of highly sensitive, specific, and rapid techniques for the simultaneousdetermination of multiple biomarkers can play a critical role in the early diagnosis and management of diseases and improve the survival rate of cancer patients, treatment efficiency, and diagnostic specificity.

[0122] In accordance with one or more embodiments, the present disclosure provides the design and fabrication of an electrochemical multiplex microfluidic biosensing device for the simultaneous detection of several biomarkers such as proteins, genomes, and metabolites in different biofluidic samples. The device may comprise: 1- A plurality of multiplex electrodes preferably the substrate made of flexible polymer with 4 working electrodes preferably made of Graphene, conductive polymers, and Graphite nanocomposite, and one general counter electrode preferably made of the same materials of working electrodes, and one general reference electrode preferably made of silver, 2-An insulation layer for covering the connecting part of the plurality of multiplex electrodes and making the separated counter electrode and reference electrode for each working electrode, 3- Modified the working electrodes with Prussian blue nanoparticles, conductive polymer, cross-linker and specific target (such as Cortisol, Agmatine, Succinate, and glial fibrillary acidic protein (GFAP)) for making the cavities in the polymer media, 4- A permanent well cell for directing different biofluids to the related detection zone without any mixing, 5- A microfluidic layer preferably made of Pressure-Sensitive Adhesive (PSA) for directing biofluidic sample to the plurality of electrochemical detection zones including sample inlet port, a microfluidic channel, A plurality of electrochemical detection zones and at least one vent, 6- A separation fiber of impurities from biofluidic samples, preferably made of nitrocellulose, 7- A top hydrophilic layer for covering the microfluidic channel and plurality of electrochemical detection zones including semicircular-shape sample inputs and three rectangular shape for vents, and 8- A customized external electron receiving device (galvanostat or potentiostat) to record electrical signal and analyze the concentration of targets.

[0123] In accordance with one or more embodiments, a method and mechanism are provided for the prepared multiplex microfluidic biosensing device for multi-omics analysis in different biofluids electrochemically. The approach may comprise the following steps: 1-Separation or removal of impurities via embedded fiber in the sample inlet (sample preparation) 2- Movement of the biofluidic sample towards the plurality of multiplex electrodes with the help of capillary-driven flow and the designed vent, 3- Interaction of targets in the biofluidic sample with specific receptors prepared in each working electrode of the plurality of multiplexbiosensing electrodes, 4- Measurement of changes in the electrical characteristics of the surface of working electrodes by using the detection apparatus and recording the results using Amperometry, electrochemical impedance spectroscopy (EIS), chronoamperometry, square wave voltammetry (SWV), and differential pulse voltammetry (DPV) methods and 5-Determination of targets’ concentration in the biosample by using the calibration curves related to each target that saved in the readout system and report simultaneously.

[0124] In accordance with one or more embodiments, a process fabrication of an autonomous multiplex biosensing device (sample-to-result test) may comprise the following steps: 1-Preparation of the multiplexed electrochemical biosensor, 2- Attaching the permanent well cell, PSA layer and adding the fiber media at the biofluidic sample inlet of microfluidic layer, 3- Covering the microfluidic channel and detection chamber zones via hydrophilic top layer and 4- Inserting the multiplex biosensing device to the customized readout and then it will be ready to add the sample and read the signal.

[0125] In accordance with one or more embodiments, a method of modifying and functionalizing each working electrode surface of an autonomous multiplex biosensing device may include the following steps: 1- Formation of Prussian blue nanoparticles on the bare working electrode surface as the redox by electrodeposition with cyclic voltammetry method, 2-Making the molecularly imprinted polymer platform by electropolymerization of conductive polymer, crosslinkers, and specific target with cyclic voltammetry technique, 3- Washing the biosensing platform for removing the specific target from MIP media with cyclic voltammetry technique or adding / removing of the washing solution.

[0126] In accordance with one or more embodiments, a method of mitigating the matrix effect and enhancing the analytical performance of an autonomous multiplex biosensing device is the use of ratiometric biosensors. The ratiometric method can include the following steps: 1-Simultaneous detection of two signals: the target analyte response and the stable internal reference response, 2- Calculating the ratio of the analyte signal to the reference signal, and 3-Correcting any variations caused by matrix.

[0127] In accordance with one or more embodiments, approaches of measuring the internal response of an autonomous multiplex biosensing device may include 1- use of dualelectrochemical probes, where one electrochemical probe responds to changes in the presence of the target analyte, while the other remains constant and 2-use of dual-electrode systems for each analyte, where one electrode is modified with the Ml P that is selective for the target analyte, and 3- employ signal amplification mechanism that increases the signal response upon target binding, rather than relying on signal reduction mechanisms.

[0128] In accordance with one or more embodiments, a method and strategy are provided for quality control in the fabrication of an autonomous multiplex biosensing device, aimed at achieving repeatable and reliable performance for multi-omics analysis. This strategy may include non-destructive QC steps based on the electro-fabrication process. The strategy may comprise the following quality control steps: 1- Visual test and storage conditions of bare electrodes, 2- Electrodeposition of a redox probe, 3- Electropolymerization of the MIP film using functional monomer, and 4-Extraction of the template molecule in two approaches: electrocleaning and solvent extraction.

[0129] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required. In other instances, well-known mechanical structures are shown with limited details in order not to obscure the understanding.

[0130] The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be affected to the particular embodiments by those of skill in the art without departing from the scope, which is defined solely by the claims appended hereto.

[0131] Embodiments of the disclosure can be described with reference to the following clauses:

[0132] Clause 1. A multiplexed biosensor system for multi-omics analyses with specified features of wash-free, antifouling, portable, user-friendly, antibody-free, and high durability; the system comprises: a multiplex biosensing device containing a plurality of multiplex electrodes modified with different biosensing platforms; an insulation layer; temporary or permanent wellcell; a delivery mechanism designed to enable the sequential delivery of multiple biosamples and biosensing reagents to the plurality of multiplex electrodes; a separation or filter media; and detection apparatus.

[0133] Clause 2. The multiplex biosensing device of Clause 1 wherein the plurality of multiplex electrodes comprises: a substrate; multiple working electrodes; an integrated counter electrode; and an integrated reference electrode made of silver Chloride; electrode bases made of silver.

[0134] Clause 3. A multiplex biosensing device of Clause 1 comprising:(a) Plurality of multiplex biosensing electrodes configured for real-time or near real-time monitoring of multiple biomarkers simultaneously;(b) an insulation layer;(c) a temporary or permanent well cell;(d) a delivery mechanism;(e) a separation or filter media.

[0135] Clause 4. The plurality of multiplex electrodes of Clause 2, wherein a substrate made of at least one of a flexible polymer, glass, and ceramic, the plurality of multiplex electrodes screen-printed on the substrate.

[0136] Clause 5. The plurality of multiplex electrodes of Clause 2, wherein the integrated counter electrode made of at least one of the mixtures of poly (3,4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT: PSS), Polyaniline, MXenes, Graphene, and Graphite.

[0137] Clause 6. The plurality of multiplex biosensing electrodes of Clause 3 wherein the multiple working electrodes comprise: a base nanocomposite surface screen-printed on the substrate; Prussian blue (PB) electrodeposited on the surface of bare electrodes; molecular imprinted polymer platform of specific polymers such as aniline, pyrrole, thiophene, dopamine, o-Phenylenediamine (OPD) and acetylene electropolymerized on the embedded redox layer.

[0138] Clause 7. The multiple working electrodes of Clause 2, wherein the basenanocomposite surface of multiple working electrodes made of at least one of the mixtures of poly (3,4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT: PSS), Polyaniline, MXenes, Graphene, and Graphite.

[0139] Clause 8. The plurality of multiplex electrodes of Clause 2, wherein the multiple working electrode surfaces comprise circular surfaces with a diameter in a range of 2 to 6 mm.

[0140] Clause 9. The plurality of multiplex electrodes of Clause 2, wherein the integrated counter electrode surface linked to each of the working electrode comprises a quarter of a ring with an external diameter of 16 to 32 mm and an inner diameter of 14 to 30 mm.

[0141] Clause 10. The plurality of multiplex electrodes of Clause 2, wherein the integrated reference electrode surface comprises a circular surface with a diameter in a range of 6 to 14 mm.

[0142] Clause 11. The plurality of multiplex electrodes of Clause 2, wherein the multiple working electrode surface and the integrated reference electrode surface could be different shapes such as circle, triangle, square, polygon and oval.

[0143] Clause 12. The plurality of multiplex biosensing electrodes of Clause 3, wherein at least one of the biosensing platforms including molecularly imprinted polymers, immunocomplex reaction, enzymatic reaction and DNA hybridization were prepared on the multiple working electrodes.

[0144] Clause 13. The multiplex biosensing device of Clause 1, wherein the insulation layer is configured to prepare the separated counter electrode and reference electrode for each working electrode and cover and isolate the connecting part of the multiplexed electrode.

[0145] Clause 14. The multiplex biosensing device of Clause 1 , wherein temporary well cell configured for fabrication procedure of multiplexed biosensor for different targets and different biosensing platforms such as molecularly imprinted polymers, immunocomplex reaction,enzymatic reaction, and DNA hybridization.

[0146] Clause 15. The multiplex biosensing device of Clause 1 , wherein permanent well cell configured for detection of single or different targets in different biofluid samples such as blood, serum, urine, sweat, and general biofluid samples.

[0147] Clause 16. The multiplex biosensing device of Clause 1 wherein a delivery mechanism connected with the plurality of multiplex biosensing electrodes, the delivery mechanism comprising: a sample inlet port configured to receive a sample comprising at least one of biofluid samples, like a blood sample, a serum sample, a urine sample, a sweat sample, and a general biofluid sample; a microfluidic channel with varied widths is configured to connect the sample inlet port to the plurality of electrochemical detection zones, enabling bubble-free and uniform distribution of the processed sample in the detection zone; a plurality of electrochemical detection zones, each detection zone connected in fluid communication with a respective microfluidic channel, each detection zone of the plurality of electrochemical detection zones configured to encompass respective electrodes of the plurality of multiplexed biosensing electrodes; and at least one vent is configured to draw air out of the detection zones, to assist in sample collection and direct the sample uniformly to the detection zones.

[0148] Clause 17. The delivery mechanism of Clause 16, wherein at least a portion of the sample is exposed to each sensing unit of the plurality of multiplex biosensing electrodes disposed within each respective detection zone of the plurality of electrochemical detection zones.

[0149] Clause 18. The delivery mechanism of Clause 16, wherein the microfluidic channel comprises a channel with a width of 400 to 2000 micrometers, a length of 2 to 12 mm, and a height of 100 to 200 micrometers.

[0150] Clause 19. The delivery mechanism of Clause 16, wherein a fluid sample is introduced in a self-powered way without the need for an external power source such as a pump.

[0151] Clause 20. The multiplex biosensing device of Clause 1, wherein the single and plurality of collection chambers comprise the separation or fiber media disposed between the sample inlet port and the plurality of detection zones for collecting the samples, separation of impurities from the samples, or plasma separation.

[0152] Clause 21. The multiplex biosensing device of Clause 3, wherein the plurality of multiplex biosensing electrodes is configured to sense one or more target biomolecules such as proteins, metabolites, pathogens, DNAs, RNAs, exosomes, bacteria, and viruses.

[0153] Clause 22. The multiplex biosensing device of Clause 1 , wherein the delivery mechanism is configured to direct different biofluids such as blood, serum, urine, sweat, and general biofluid samples through the plurality of electrochemical detection zones.

[0154] Clause 23. The multiplexed biosensor system of Clause 1 , wherein a customized and low-cost potentiostat detection apparatus is in communication with the multiplex microfluidic biosensing device and configured to record electrical signals from the multiplex biosensing device and to analyze the signals to enable measuring the concentration of targets and optionally transmit the results.

[0155] Clause 24. The multiplexed biosensor system of Clause 1 , wherein the detection apparatus is configured to receive each working electrode signal separately and simultaneously, then analyze them all together.

[0156] Clause 25. The multiplexed biosensor system of Clause 1 , wherein a transceiver with the capability of Wireless or Bluetooth communications is configured for communicating data with the multiplex microfluidic biosensing device.

[0157] Clause 26. The multiplexed biosensor system of Clause 1 , wherein the customized potentiostat is adapted for performing an electrochemical measurement technique such as EIS, SWV, and DPV.

[0158] Clause 27. The multiplex biosensing device of Clause 3, wherein the plurality of multiplex biosensing electrodes is mitigating the matrix effect and enhancing the analytical performance via the use of the ratiometric analysis method.

[0159] Clause 28. The multiplex biosensing device of Clause 3, wherein this ratiometric method comprises the following: simultaneous detection of two signals: the target analyte response and the stable internal reference response; calculating the ratio of the analyte signal to the reference signal; and correcting any variations caused by the matrix.

[0160] Clause 29. The multiplex biosensing device of Clause 3, wherein the ratiometric analysis method and the approach of measuring the stable internal reference response comprises: use of dual electrochemical probe; use of dual-electrode systems for each analyte; employ a signal amplification mechanism that increases the signal response upon target binding.

[0161] Clause 30. The multiplex biosensing device of Clause 3, where the plurality of multiplexed biosensing electrodes is screened via a non-destructive quality control strategy based on the electro-fabrication process, thereby ensuring repeatability and reliability in biosensor performance.

[0162] Clause 31. The multiplex biosensing device of Clause 3, wherein the QC strategy comprises one or more of the following steps: visual test and storage conditions of bare electrodes; electrodeposition of a redox probe; electropolymerization of the MIP film using functional monomer; and extraction of the template molecule.

[0163] Clause 32. The multiplex biosensing device of Clause 3 wherein the extraction of template molecules comprises: electro-cleaning and solvent extraction.

[0164] Clause 33. The multiplex biosensing device of Clause 2, wherein the substrate is composed of a material selected from the group consisting of flexible polymers, glass, and ceramics, thereby providing structural adaptability for diverse biosensing applications.

Claims

CLAIMS:

1. A multiplex biosensing device comprising:a plurality of multiplex biosensing electrodes configured for simultaneous detection of two or more biomarkers in a biosample input, the plurality of multiplex biosensing electrodes comprising:a first biosensing electrode including a first electrode modified with a first biosensing platform for detecting a first biomarker, anda second biosensing electrode including a second electrode modified with a second biosensing platform for detecting a second biomarker; and a delivery mechanism configured for processing at least one biosample and for sequentially delivering the at least one biosample and at least one biosensing reagent to the plurality of multiplex biosensing electrodes as the biosample input.

2. The device of claim 1, wherein the plurality of multiplex biosensing electrodes comprises:a substrate;a plurality of working electrodes provided on the substrate, each of the plurality of working electrodes including an electrode base;an integrated reference electrode provided on the substrate and shared between the plurality of multiplex biosensing electrodes, the integrated reference electrode including an electrode base; andan integrated counter electrode provided on the substrate and shared between the plurality of multiplex biosensing electrodes, the integrated counter electrode including an electrode base.

3. The device of claim 2, wherein:the plurality of working electrodes each define a circular or linear profile,the integrated reference electrode defines a circular or linear profile, andthe integrated counter electrode defines a profile of a quarter of a ring or linear.

4. The device of claim 1 , further comprising:a patterned insulation layer configured to separate the plurality of multiplex electrodes into a plurality of sensing units by providing selective access to a portion of the integrated reference electrode and to a portion of the integrated counter electrodefor each of the plurality of sensing units.

5. The device of claim 1, further comprising:a well cell configured to enable the plurality of multiplex electrodes to operate with respect to different biomarkers and / or different biosamples.

6. The device of claim 1, wherein the plurality of multiplex biosensing electrodes are configured to simultaneously sense one or more target biomolecules selected from the group consisting of: proteins, metabolites, microbes, genes, or extracellular vesicles.

7. The device of claim 1, wherein the biosensing platform used for fabrication of multiplex biosensing device comprises a combination of one of or more of: molecularly imprinted polymers (MIP), immunocomplex reaction, enzymatic reaction and DNA hybridization mechanisms.

8. The device of claim 1 , wherein the biosensing platform comprises a molecularly imprinted polymer (MIP) biosensing electrode selected from the group consisting of: aniline, pyrrole, thiophene, dopamine, o-Phenylenediamine (OPD) and acetylene electropolymerized on an embedded redox layer.

9. The device of claim 1, wherein the delivery mechanism comprises a microfluidic unit with an automated liquid handling configured to deliver at least one biosample, from the group consisting of blood, serum, plasma, urine, sweat, tears, saliva, sputum, and the at least one bioreagent to the plurality of multiplex biosensing electrodes.

10. The device of claim 1, further comprising a microfluidic unit integrated with a separation or filter media configured to prepare the biofluid sample by removing impurities before exposure to the plurality of multiplex biosensing electrodes.

11. The device of claim 9 or claim 10, wherein the delivery mechanism comprises a self- powered delivery and automated system.

12. The device of claim 9 or claim 10, wherein the delivery mechanism is configured to support multiple biofluid types, including blood, plasma, urine, saliva, sputum, and sweat, through optimized microfluidic pathways that facilitate bubble-free and uniformdistribution across detection zones.

13. The device of claim 2, wherein the plurality of working and counter electrodes are constructed using ink materials selected from the group consisting of poly(3,4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT), polyaniline, MXene, graphene, and graphite, enabling compatibility with various biosensing platforms and electrode functionalization processes.

14. The device of claim 1, wherein the plurality of multiplex biosensing electrodes employ a ratiometric analysis method to mitigate matrix effects, enhancing the accuracy and analytical performance of biomarker detection in complex sample matrices.

15. The device of claim 13, wherein the ratiometric analysis method includes simultaneous detection of a target analyte response and a stable internal reference response, with a ratio of such signals being used to correct for variations caused by matrix effects.

16. The device of claim 1, wherein the plurality of multiplex biosensing electrodes are screened via a non-destructive quality control process based on an electro-fabrication process, thereby ensuring repeatability and reliability in biosensor performance.

17. The device of claim 2, wherein the working electrodes are constructed of ink materials selected to enhance electrode conductivity and compatibility with specific biosensing platforms, including molecularly imprinted polymers, immunocomplex reactions, and DNA hybridization.

18. A multiplex biosensor system comprising:the multiplex biosensing device of any one of claims 1 to 17; anda detection apparatus configured to electrically communicate with the plurality of multiplex biosensing electrodes and to obtain, analyze, and display biomarker data corresponding to the plurality of biomarkers and to provide a readout.

19. The system of claim 18, wherein the detection apparatus comprises a communication module allowing for remote data transmission and real-time monitoring of biomarkerlevels.

20. The system of claim 18, wherein the multiplex biosensor system comprises a single-time detection, near-real time, or real-time model for detection of target biomarkers.

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