AUTOMATED FLUIDIC ASSAY BASED ON MOLECULARLY IMPRINTED POLYMER FOR COVID-19 DIAGNOSTICS AND SEROSURVEILLANCE
20220339556 · 2022-10-27
Inventors
- Sara MAHSHID (Mont-Royal, CA)
- Roozbeh SIAVASH MOAKHAR (Montréal, CA)
- Carolina DEL REAL MATA (Montréal, CA)
Cpc classification
B01D15/3852
PERFORMING OPERATIONS; TRANSPORTING
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
B01J20/268
PERFORMING OPERATIONS; TRANSPORTING
International classification
B01D15/38
PERFORMING OPERATIONS; TRANSPORTING
Abstract
It is provided a biosensor, a device containing same and method for detecting a target protein, the biosensor comprising a nano/micro islands (NMIs) core of gold spatially oriented with nanorough protrusions, and a layer of electropolymerized molecularly imprinted polymers (MIP) polymerized on the NMIs core, said MIP consisting of a conductive monomer comprising a built-in recognition site of the target protein, wherein the MIPs generate an electrical signal upon binding of the target protein.
Claims
1. A biosensor for detecting a target protein comprising: a nano/micro islands (NMIs) core of gold spatially oriented with nanorough protrusions, and a layer of electropolymerized molecularly imprinted polymers (MIP) polymerized on the NMIs core, said MIP consisting of a conductive monomer comprising a built-in recognition site of the target protein, wherein the charge transfer resistance and/or impedance magnitude of the MIPs change upon binding of the target protein.
2. The biosensor of claim 1, wherein the NMIs are electrodeposited on a conductive glass with a reference electrode of Ag/AgCl and a counter electrode of platinum wire.
3. The biosensor of claim 2, wherein the conductive glass is a tin oxide (ITO) substrate.
4. The biosensor of claim 1, wherein conductive monomer is polyaniline (PANI) or o-phenylenediamine (o-PD).
5. The biosensor of claim 1, wherein the target protein is an antibody, a viral protein or a heart fatty acid binding protein (H-FABP).
6. The biosensor of claim 6, wherein the antibody is a viral antibody.
7. The biosensor of claim 5, wherein the viral protein is from SARS-CoV-2, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), the human coronavirus 229E (HCoV-229E), the Middle East respiratory syndrome coronavirus (MERS-CoV), or Influenza.
8. The biosensor of claim 5, wherein the viral protein is from a SARS-CoV-2 variant.
9. A microfluidic read-out apparatus for detecting a target protein in a subject comprising: i) the biosensor of claim 1; and ii) microfluidic reader.
10. The apparatus of claim 9, wherein the microfluidic read-out apparatus is a multiplex microfluidic apparatus.
11. The apparatus of claim 9, further comprising a WiFi adapter for transferring the read-out signals from the microfluidic reader to a platform.
12. The apparatus of claim 11, wherein the WiFi adapter is a Bluetooth low energy (BLF) connector.13. The device of claim 9, wherein the platform is a computer or a smartphone.
14. A method of detecting a target protein in a subject comprising the steps of: a) providing a sample from the subject; b) contacting said sample with the biosensor of claim 1, wherein the presence of the target protein changes the charge transfer resistance and/or impedimetric of the MIPs upon binding of the target protein; and c) transferring the change in charge transfer resistance signal and/or impedimetric signal to a microfluidic reader for transforming said signal into a cyclic voltammetry signal, wherein the cyclic voltammetry signal and/or impedimetric signal indicates the presence of the target protein.
15. The method of claim 14, wherein the subject sample is a body fluid such as saliva, plasma, or whole blood.
16. The method of claim 14, wherein the subject is a human or an animal.
17. The method of claim 14, further comprising transmitting the cyclic voltammetry signal or impedimetric signal to a platform.
18. The method of claim 17, wherein the cyclic voltammetry signal or impedimetric signal is transmitted by Wi-Fi to the platform.
19. The method of claim 18, wherein the cyclic voltammetry signal or impedimetric signal is transmitted to a computer or a smartphone.
20. The method of claim 14, wherein the cyclic voltammetry signal indicates the presence of the target protein in 1 min to 11 min.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Reference will now be made to the accompanying drawings.
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DETAILED DESCRIPTION
[0056] In accordance with the present invention, there is provided a biosensor for detecting a target protein comprising a nano/micro islands (NMIs) core of gold spatially oriented with nanorough protrusions, and a layer of electropolymerized molecularly imprinted polymers (MIP) polymerized on the NMIs core, said MIP consisting of a conductive monomer comprising a built-in recognition site of the target protein, wherein the MIPs generate an electrical signal upon binding of the target protein.
[0057] It is provided a portable, rapid, quantitative, and inexpensive diagnostic and serological home-test kit analogous to a glucometer for sensitive detection of SARS-CoV-2 viral particles and SARS-CoV-2 nucleocapsid and spike antibody at the point-of- need, in particular at home and in remote locations.
[0058] The tool provided herein address three challenges: (1) detecting the presence of SARS-CoV-2 and emerging variants at the early-stages of the infection from easily accessible body fluids such as saliva, (2) detecting the antibodies in response to the infection from whole blood, and (3) monitoring the efficacy of therapy once the patient is under treatment by quantifying both SARS-CoV-2 spike proteins (SP) and specific antibodies.
[0059] The kit integrates a new biomimetic receptor based on molecularly imprinted polymers (MIP), a new nanostructured sensor based on gold nano/micro islands (NMI) and a portable microfluidic-impedimetric read-out. As encompassed herein, the microfluidic read-out apparatus can be a multiplex microfluidic apparatus. When the virus or the antibody binds to the NMIs/MIPs morphological-detection site, a signal is generated. The NMIs/MIPs microfluidic device achieves a rapid detection in 10 min for the SARS-CoV-2 whole virus in human saliva and SARS-CoV-2 antibodies in undiluted plasma and 1 min in whole blood. Also, the device presents a high specificity towards SARS-CoV-2 over Influenza A virus. In an embodiment, it is encompassed that the present device has a high specificity towards the severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), the human coronavirus 229E (HCoV-229E), or the Middle East respiratory syndrome coronavirus (MERS-CoV). The detection system is integrated with a WiFi adapter to send the readout signals to a smartphone. The WiFi adapter can be for e.g. but not limited to a Bluetooth low energy (BLF) connector The signals are analyzed in a user-friendly smartphone software, and the level of SARS-CoV-2 virus or SARS-CoV-2 antibody is visualized for nonprofessional users.
[0060] MIPs consists of a synthetic polymer with recognitions sites build-up from specific templates through a co-polymerization of functional monomers and cross-linkers evolving a template of the target. Templates species are needed for the generation of recognition sites during the polymerization process. After the template removal, the imprinted polymer can be used for a rebinding step, where the target, present in a solution can be recognized by the blank binding site built-in synthetic polymer matrix. From low molecular weight molecules to micro-organisms MIPs have proven their value as sensors for many different biological applications. MIPs offer the possibility of a sensitive and highly selective biorecognition sensor based solely on synthetic materials, avoiding the need for fragile biomolecules. The main advantages of MIPs are their cost-effectiveness, easy to scale production, improved shelf-life, stability, and versatility. Recently, the development of MIPs for virus detection has attracted attention due to its versatility and capability to be designed to detect the whole virus or specific proteins. The binding of the target on the MIPs changes the electrical properties (charge transfer resistance) of the polymer, which is measured with a cyclic voltammetry signal and/or impedimetric signal.
[0061] However, MIPs' known low sensitivity towards the detection of some proteins and biological compounds have hindered their use for highly sensitive applications.
[0062] It is provided in an embodiment a method for rapid detection of SARS-CoV2 (whole virus) and spike protein antibodies in biological fluids. The innovative part of the assay is a new biomimetic receptor based on highly selective molecularly imprinted polymer (MIP) assay combined with nano/micro islands (NMIs) of gold with spatial orientation and nanorough protrusions NMIs to form a core-shell structure. The gold NMIs, microfluidics, the biomimetic receptor based on MIP and a portable electrical (impedimetric) read-out are provided for sensitive and quantitative detection of SARS-CoV2 and antibodies in human saliva and human whole blood, respectively. In an embodiment, the bio receptor is based on a conductive monomer, which is electropolymerized in the presence of a target (here heat-inactivated SARS-CoV2 and spike protein antibodies). The target will be removed to leave a template, which can rebind to the target and generate an electrical signal. The NMIs enhances the electropolymerizing of the MIPs, overcoming MIPs challenges and offering a highly sensitive and selective technique for an electrochemical readout system. The important part of the MIP assay is optimizing the electropolymerization and determining the type of polymer. Thus, different polymers were evaluated to optimize the sensitivity and selectivity of the assay. The size of the heat-inactivated SARS-CoV-2 and SARS-CoV-2 Abs are about 200-500 nm and 10 nm, respectively. Therefore, polyaniline (PANI) uses whole virus as template and both spike protein and Abs are done with a thin polymer, o-phenylenediamine (o-PD). Further, the electropolymerization process was optimized in terms of polymer concentration, number of deposition cycles and acidity of the solution and by comparing the electrocatalytic activity and charge transfer resistance of formed polymer layers. The NMIs/MIPs sensor were characterized via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) at every step of the fabrication to complete the NMIs/MIPs optimization. Afterwards, the NMIs/MIPs sensor was tested with real samples achieving a rapid detection of the target in 10-15 minutes. The NMIs/MIPs for SARS-CoV-2 presents a low limit of detection (LOD) for SARS-CoV-2 spike protein (Original strain: 5.89 pg.ml.sup.−1; Alpha variant: 6.48 pg.ml.sup.−1; Delta variant: 8.13 pg.ml.sup.−1; Omicron variant: 7.62 pg.ml.sup.−1 in human saliva with a linear range of 10 pg ml.sup.−1-10.sup.5 pg. ml.sup.−1. Moreover, it shows a high specificity towards detection of SARS-CoV-2, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), the human coronavirus 229E (HCoV-229E), the Middle East respiratory syndrome coronavirus (MERS-CoV) vs. Influenza A H1N1. The NMIs/MIPs serological approach for detection of the spike protein antibody was tested in whole blood reaching a LOD of 3.13 pg. μl.sup.−1 with a linear range of 10 pg. μl.sup.−1-10.sup.4 pg. All LODs for original variant, SP and Abs are detailed in Table 1.
TABLE-US-00001 TABLE 1 Limit of detection and linear range of NFluidEX for various targets in saliva, plasma, blood, and buffer. Target Biofluid Limit of detection Linear range Original strain SP Saliva 5.89 pg .Math. ml.sup.−1 1e1-1e5 pg .Math. ml.sup.−1 Buffer 3.79 pg .Math. ml.sup.−1 Alpha variant SP Saliva 6.48 pg .Math. ml.sup.−1 Buffer 4.51 pg .Math. ml.sup.−1 Delta variant SP Saliva 8.13 pg .Math. ml.sup.−1 Buffer 6.28 pg .Math. ml.sup.−1 Omicron variant SP Saliva 7.62 pg .Math. ml.sup.−1 Buffer 4.72 pg .Math. ml.sup.−1 Heat-inactivated Saliva 948.4 number of viral 9.60e3-3.84e8 number of SARS-CoV-2 viral particles .Math. ml.sup.−1 viral particles .Math. ml.sup.−1 particles Buffer 2091.6 number of viral particles .Math. ml.sup.−1 IgG-RBD Plasma 4.06 pg .Math. ml.sup.−1 1e1-1e4 pg .Math. ml.sup.−1 Blood 5.74 pg .Math. ml.sup.−1 Buffer 3.63 pg .Math. ml.sup.−1 IgM-RBD Plasma 2.97 pg .Math. ml.sup.−1 Blood 3.13 pg .Math. ml.sup.−1 Buffer 2.79 pg .Math. ml.sup.−1 IgG-N Plasma 6.94 pg .Math. ml.sup.−1 Blood 7.76 pg .Math. ml.sup.−1 Buffer 5.18 pg .Math. ml.sup.−1 IgM-N Plasma 3.25 pg .Math. ml.sup.−1 Blood 3.58 pg .Math. ml.sup.−1 Buffer 2.99 g .Math. ml.sup.−1 Influenza A SP Saliva 8.63 pg .Math. ml.sup.−1 1e1-1e5 pg .Math. ml.sup.−1 Buffer 3.99 pg .Math. ml.sup.−1 Heat-inactivated Saliva 2,576,415 number of 6.44e6-2.58e9 number of Influenza A viral viral particles .Math. ml.sup.−1 viral particles .Math. ml.sup.−1 particles Buffer 1,105,422 number of viral particles .Math. ml.sup.−1
[0063] The adaptability nature of viruses presents a challenge to the gold standard diagnosis. The proposed assay offers a versatile approach for the fabrication of the biomimetic receptor that can be tuned towards different targets based on their physical and morphological characteristics. This unique aspects of the proposed MIP assay will bypass the need for in-depth knowledge of the virus mutations and chemistry and can be easily adapted for future applications, such as quasi-simultaneus detection of Abs in blood and SP in saliva samples.
[0064] The proposed microfluidic device integrating the proposed biomimetic electrochemical sensor based on a NMIs/MIPs system with facilitated electropolymerization and enhanced signal read-out, provided a fast, portable, stable, achieved a simple system ultrasensitive and selectivity as an alternative diagnostic test for detection of SARS-CoV2.
[0065] It is encompassed that the device provided herein allows detection of any proteins, not just viruses such as SARS-CoV2 (see
[0066] The integration of a new biomimetic receptor based on MIP with a hierarchical gold nanostructure in the form of nano/micro islands (NMIs) with spatial orientation and nanorough protrusions creates a core-shell structure composed of the NMIs (the core) and a thin layer of electropolymerized MIP (the shell) with enhanced sensitivity and selectivity for the detection of SARS-CoV2 whole virus and antibodies. The bio receptor is based on a conductive monomer, which is electropolymerized in the presence of a target (e.g. heat-inactivated SARS-CoV2 and spike protein antibodies). The target will be removed to leave a template, which can rebind to the target and generate an electrical signal. The gold NMIs will provide a large surface area for immobilization of the MIP and enhancement of the sensitivity.
[0067] The MIPs/NMI sensor demonstrates a rapid, sensitive, and selective electrochemical response in a broad range of dilution of SARS-CoV2 and antibodies in saliva and whole blood respectively, confirming its presence in biological fluids.
[0068] The sensor provided herein allows for a rapid response since the NMIs/MIPs time for an optimal incubation was determined to be 10-15 min, for human saliva. This time is highly reduced in comparison to the golden standard qRT-PCR test which takes approximately 4-6 hours to provide results. The exemplified NMIs/MIPs are demonstrated to be highly sensitive for SARS-CoV-2 microfluidic device, with a low limit of detection (LOD) for SARS-CoV-2 spike protein of: original strain: 5.89 pg.ml.sup.−1; Alpha variant: 6.48 pg.ml.sup.−1; Delta variant: 8.13 pg.ml.sup.−1; Omicron variant: 7.62 pg.ml.sup.−1l in human saliva with a linear range of 10 pg. ml.sup.−1-10.sup.5 pg. ml.sup.−1. Additionally, the NMIs/MIPs for the spike protein antibody was tested in undiluted human plasma reaching a LOD of 3.13 pg. μl.sup.−1 with a linear range of 10 pg. μl.sup.−1-10.sup.4 pg. By having both assays in one device for example, e.g. a multiplex microfluidic device, the disease stage limitations presented by a qRT-PCR and serological test can be overcome. The golden standard qRT-PCR is limited to the acute phase of infection, while the serological test is limited to later stages of the disease. The device described herein combines both approaches to overcome these limitations. The device/process is showed to be highly selectivity and specific, as demonstrated by effectively detecting SARS-CoV-2 over highly similar viral load as is Influenza A virus, and also including severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), the human coronavirus 229E (HCoV-229E), the Middle East respiratory syndrome coronavirus (MERS-CoV), thus avoiding possible cross-reactivity with other coronaviruses.
[0069] The electrochemical NMIs/MIPs sensor do not require highly trained technicians nor expensive equipment. It only require a PC/cellphone reader and potentiostat. In that sense is superior to qRT-PCR which requires expensive instrumentation and highly trained laboratory personnel, proficient in performing the test. Moreover, NMIs/MIPs sensor has a simple and straightforward operation since is integrated into a sample delivery microfluidic system to improve the control of conditions and throughput, allowing for multiplexing. Contrary to qRT-PCR, which requires extended sample preparation in addition to the test protocol, the microfluidic device sample preparation is simple. The sensor effectively detects the target via CV and EIS test. A digitalized system for the analysis of the cyclic voltammetry readout is provided to couple with a PC/phone friendly platform.
[0070] A stepwise schematic of the NMIs/MIPs sensor and the detection of SARS CoV-2 through the whole virus and/or a specific antibody is presented in
[0071] The device electrochemical performance is studied, via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), in the presence of two different targets, the heat-inactivated SARS-CoV-2 whole virus 30 or its nucleocapsid or spike protein antibody 32. NMIs provide an ideal extended and surface area and robust core for the NMIs/MIPs fabrication. Specifically, the NMIs/MIPs sensor was tested in a controlled environment with Heat inactivated SARS CoV-2 virus spiked in 1×PBS (7.2 pH) and in real media conditions, for which the heat-inactivated virus was spiked in saliva from healthy donors. Moreover, the NMIs/MIPs sensor for nucleocapsid and spike protein antibody detection was effectively tested in a 1×PBS (7.2 pH) buffer and in two human body fluids, undiluted plasma, and whole blood from healthy donors. Unlike other technique which are functional solely in diluted plasma media, the assay can successfully detect SARS-CoV-2 antibody in undiluted human plasma and in addition to this can detect antibodies in whole blood.
[0072] The tool provided synergistically combine nanostructured gold sensors with microfluidic sample delivery systems and biomolecular assay capabilities. The proposed device is based on a new hierarchical gold nanostructured platform in the form of nano/micro islands (NMIs) with spatial orientation and nanorough protrusions. It is known that gold NMIs enabled sensitive and quantifiable detection of bacteria (E. Coli, MRSA etc.) and small molecules (e.g. H-FABP protein). The gold NMIs, microfluidics, the new biomimetic receptor based on molecularly imprinted polymers (MIP), and the portable electrical (impedimetric) read-out were harnessed for sensitive and quantitative detection of SARS-CoV-2 viral particles and SARS-CoV-2 nucleocapsid and spike antibodies, analogous to a glucometer. MIPs report physical stability, structure predictability, specificity, versatility, simple fabrication, and cost-effectiveness
[0073] The MIP recognition element is based on a thin layer of a conductive monomer, which is electropolymerized in the presence of a target template (whole virus, viral protein, spike antibody, or nucleocapsid antibody). The template will be removed to leave a built-in recognition site, which can rebind to the target and generate an electrical signal. The tens of nanometer thickness of the film ensure a partial coverage of the template molecule, in turn, easing the diffusion of the target analyte. The highly sensitive and selective MIP biorecognition approach is a suitable synthetic alternative for effective detection of SARS-CoV-2. Recently development of custom-made MIPs for whole virus or specific viral proteins detection has grown, expanding MIP's capabilities for accurate binding of viral sub-types.
[0074] Briefly, the MIP biosensor are fabricated through electrodeposition of gold MNIs 22 on a indium tin oxide (ITO) substrate 20 where the analysis wells are patterned through standard lithography (see
[0075] The selectivity of the biomimic NMI/MIP recognition assay relies on the formation of binding sites in the polymeric thin films imprinted by SP, IgG-RBD, or IgM-RBD that is central to selective recognition in saliva and blood, respectively. We used a thin layer (5-10 nm) of o-PD polymer to interact with SP and antibody entities of the virus to record their spatial molecular configuration. We benchmarked the proficiency of templating the polymer layer with the virus entities by investigating the affinity of the antigen-binding fragment of the IgG-RBD and IgM-RBD (7BWJ) and the SP (6VXX) from Protein Data Bank (PDB), with the o-PD polymer layer using molecular docking (MD) simulations to determine the most favourable binding sites (
[0076] The ideal properties in the materials use for the construction of electrodes depend on their application. In this work, a highly sensitive detection of SARS-CoV-2 virus and SARS-CoV-2 antibodies is desired on a stable linear range. Thus, aiming for a material with increased surface area, high electrical conductivity and of facile fabrication. In this work the conductive glass “ITO” is selected for the base of the electrode. An ITO glass-coated wafer while a single-step lithography was utilized to pattern the fluidic channels. Initially the ITO-coated glass was deposited with a 5 mm silicon dioxide insulating layer using plasma-enhanced chemical vapor deposition (PECVD) at a deposition rate of 10 nm. s.sup.−1. Then the electrochemical reference electrode (RE) and counter electrode (CE) were patterned in an AZ9245 photoresist (10 μm) followed by etching the patterned electrodes in the SiO.sub.2 via BOE etching (
[0077] The electrochemical characterization after each step of the electrode fabrication is shown in
[0078] The NMI/MIP enables a multiplex parallel analysis of saliva samples for whole virus detection via its SP, and blood samples for serology testing of IgG-RBD and IgM-RBD antibodies. The analytical assessment of the NMIs/MIPs is evaluated based on the impedance magnitude within the biological concentration ranges (ng. ml.sup.−1 to μg. ml.sup.−1) in buffer solution and in body fluids including saliva, plasma, and blood. All signal recordings were obtained by a potentiostat/galvanostat module with a potential amplitude of 10 mV. The working principle of the NMI/MIP signal transduction is based on the detection of an increase in impedance magnitude of the spectroscopic signal upon interaction of the electrodes with the specific domains of viral SP or antibodies in the designated chambers. An incubation time of 10 min for viral SPs in saliva and 1 min for IgG-RBD and IgM-RBD in whole blood is required for the optimal performance of the assays (
[0079] The sensitive signal transduction at a low concentration of SARS-CoV-2 (10 pg. ml.sup.−1) is achieved due to the NMI/MIP electrodes harbouring the binding sites. This is evident by comparing the impedimetric signal of SARS-CoV-2 viral SP on the biomimic NMI/MIP test assay with imprinted SP binding sites, with that of NMI/non-imprinted polymer (NIP) electrode (without imprinted binding sites) (
[0080] In both buffer and saliva media, and for all tested concentrations, the NMI/MIP electrodes bearing the binding sites generate a sensitive differentiable signal. For the SP, the increase in the impedance signal was positively correlated to the increase in the concentration of the virus (
TABLE-US-00002 TABLE 2 Comparative table for detection of SARS-CoV-2 virus and antibodies Electrodes Media Time Limit of Detection Linear Range Magnetic bead-based Untreated saliva 30 min 19 ng/mL (Spike — immunosensor combined with protein) carbon black-modified screen- 8 ng/mL (Nucleocapsid printed electrode protein) GO-Modified with SP RBD Clinical sera 30 min 1 ng/mL — inmobilized and SKI bloqued p-sulfocalix[8]arene (SCX8) Various clinical specimens Not stated 200 copies/mL — functionalized graphene (SCX8-RGO) GO-8H-EDC-NHS-Au NS Blood, saliva and 1 min 1.68 × 10.sup.−22 μg/mL — oropharyngeal/nasopharyngeal swab Au-thin film electrode (TFE) - Lysis buffer 15 min 15 fM 2.22-111 fM interfaced with a MIP from (poly-m-phenylenediamine (PmPD)) Capture probe-conjugated Processed nasopharyngeal <2 hours 1 copy/μL 1 to 1 × 109 copies/μL magnetic bead particle swab sample (CP-MNB) Tethered Au nanostructurated Unprocessed saliva 5 min 4 × 10.sup.3 particles/mL — bearing an analyte-binding antibody Gold NMIs/PANI Unprocessed saliva 10 min 5.89 pg .Math. ml.sup.−1 1e1-1e5 pg .Math. ml.sup.−1 Gold NMIs/o-PD Whole blood 1 min 3.13 pg .Math. μl.sup.−1 1e1-1e4 pg .Math. μl.sup.−1
TABLE-US-00003 TABLE 3 Limit of detection and linear range of NFluidEX for various targets in saliva, plasma, blood and buffer Target Biofluid Limit of detection Linear range Original strain SP Saliva 5.89 pg .Math. ml.sup.−1 1e1-1e5 pg .Math. ml.sup.−1 Buffer 3.79 pg .Math. ml.sup.−1 Alpha variant SP Saliva 6.48 pg .Math. ml.sup.−1 Buffer 4.51 pg .Math. ml.sup.−1 Delta variant SP Saliva 8.13 pg .Math. ml.sup.−1 Buffer 6.28 pg .Math. ml.sup.−1 Omicron variant SP Saliva 7.62 pg .Math. ml.sup.−1 Buffer 4.72 pg .Math. ml.sup.−1 Heat-inactivated Saliva 948.4 number of 9.60e3-3.84e8 number of SARS-CoV-2 viral viral particles .Math. ml.sup.−1 viral particles .Math. ml.sup.−1 particles Buffer 2091.6 number of viral particles .Math. ml.sup.−1 IgG-RBD Plasma 4.06 pg .Math. μl.sup.−1 1e1-1e4 pg .Math. μl.sup.−1 Blood 5.74 pg .Math. μl.sup.−1 Buffer 3.63 pg .Math. μl.sup.−1 IgM-RBD Plasma 2.97 pg .Math. μl.sup.−1 Blood 3.13 pg .Math. μl.sup.−1 Buffer 2.79 pg .Math. μl.sup.−1 IgG-N Plasma 6.94 pg .Math. μl.sup.−1 Blood 7.76 pg .Math. μl.sup.−1 Buffer 5.18 pg .Math. μl.sup.−1 IgM-N Plasma 3.25 pg .Math. μl.sup.−1 Blood 3.58 pg .Math. μl.sup.−1 Buffer 2.99 g .Math. μl.sup.−1 Influenza A SP Saliva 8.63 pg .Math. ml.sup.−1 1e1-1e5 pg .Math. ml.sup.−1 Buffer 3.99 pg .Math. ml.sup.−1
TABLE-US-00004 TABLE 4 Comparative table for detection of SARS-CoV-2 antigen and antibodies. Portable Without signal Reference SARS-CoV-2 Tests Media Time Limit of Detection Linear Range transduction Measure Magnetic bead-based Untreated 30 min SP: 1.9e4 pg .Math. ml.sup.−1 SP: 1.9e4-1e7 pg .Math. ml.sup.−1 Yes Yes immunosensor saliva, buffer combined with carbon black-modified screen- printed electrode ePAD paper-based Clinical sera 30 min SP: 110 pg .Math. ml.sup.−1 SP: 1000-1000e3 pg .Math. ml.sup.−1 No No sensor: GO-Modified IgG: 0.96 pg .Math. ml.sup.−1 IgG and IgM: 1-1000 pg .Math. ml.sup.−1 with SP RBD IgM: 0.14 pg .Math. ml.sup.−1 immobilized and SKI bloqued NanoSystem: GO-8H- Blood, saliva 1 min SP: 1.68e−16 pg .Math. ml.sup.−1 SP: 1-10e−11 pg .Math. ml.sup.−1 No Yes EDC-NHS-Au NS and nasal swab Tethered Au Unprocessed 5 min SP: 1 pg .Math. ml.sup.−1 SP: 1-100 pg .Math. ml.sup.−1 No Yes nanostructurated saliva bearing an analyte- binding antibody SPEEDS: Patient 13 min IgG-S: 10.1 pg .Math. ml.sup.−1 IgG-S: 10.1-6e4 pg .Math. ml.sup.−1 Yes Yes electrochemical serum IgM-S: 1.64 pg .Math. ml.sup.−1 IgM-S: 1.64-5e4 pg .Math. ml.sup.−1 immunosensor SARS-CoV-2 Serum and 1 min IgG-S: 250 pg .Math. ml.sup.−1 IgG-S: 2e4-4e4 pg .Math. ml.sup.−1 (serum), Yes No RapidPlex: Laser saliva IgM-S: 250 pg .Math. ml.sup.−1 200-500 pg .Math. ml.sup.−1 (saliva) engraved graphene IgM-S: 2e4-5e4 pg .Math. ml.sup.−1 (serum), electrodes 600-500 pg .Math. ml.sup.−1 (saliva) Electrochemical Serum and Not SP: 760 pg .Math. ml.sup.−1 SP: 760-760e3 pg .Math. ml.sup.−1 No No aptamer-based sensor artificial stated saliva Low-cost Saliva 6.5 min SP and Alpha variant: SP and Alpha variant: No No Electrochemical 0.229 pg .Math. ml.sup.−1 0.1-1e3 pg .Math. ml.sup.−1 Advanced Diagnostic (LEAD): modified graphite leads Carbon nanotube field- Saliva and 2-3 min SP: 4.12e−3 pg .Math. ml.sup.−1 SP: 0.1e−3-5.0 pg .Math. ml.sup.−1 No No effect transistor buffer DSA1N5-Cov-eChip: 1:1 diluted 10 min Original strain: 0.438 pg .Math. ml.sup.−1 Original, Alpha and Delta variants: Yes No aptamer functionalized saliva Alpha variant: 1.227 pg .Math. ml.sup.−1 1.752-1927.2 pg .Math. ml.sup.−1 to gold electrodes Delta variant: 1.578 pg .Math. ml.sup.−1 KAUSTat AuNPs-LSG Nasal swab 1 min SP, Alpha, Beta and Delta variants: SP, Alpha, Beta and Delta variants: Yes No sensor 5140 pg .Math. ml.sup.−1 1e3-500e3 pg .Math. ml.sup.−1 Flexible organic Buffer, 5 min IgG: 1.5e−4 pg .Math. ml.sup.−1 (buffer), IgG: 1.5e−3-1.5e3 pg .Math. ml.sup.−1 Yes No electrochemical serum, saliva 1.5e−3 pg .Math. ml.sup.−1 (saliva, serum) transistors NFluidEX: NMI/MIP Untreated 11 min See Table 3 See Table 3 Yes Yes assay saliva Whole blood
TABLE-US-00005 TABLE 5 Comparative table of SARS-CoV-2 FDA EUA Approved Antigen Diagnostic Tests Sampling Response Company Test method PPA NPA LOD time Abbott Panbio COVID-19 Nasal swab 98.1% 99.8% 2.5 × 10.sup.1.8 15-20 Ag Rapid Test TCID.sub.50 .Math. ml.sup.−1 minutes Device Access Bio CareStart COVID- Nasal swab .sup. 87% 98% 2.8 × 10.sup.3 10 Inc. 19 Antigen Home TCID.sub.50 .Math. ml.sup.−1 minutes Test OraSure InteliSwab COVID- Nasal swab .sup. 84% 98% 2.5 × 10.sup.2 30 Technologies 19 Rapid Test Rx TCID.sub.50 .Math. ml.sup.−1 minutes Inc. Lumira LumiraDx SARS- Nasal swab 97.6% 96.6% 32 12 CoV-2 Ag Test TCID.sub.50 .Math. ml.sup.−1 minutes BTNX Inc. Rapid Response Nasal swab 94.55% 100% 2 × 10.sup.2.4 15 COVID-19 TCID.sub.50 .Math. ml.sup.−1 minutes NFluidEX NMI/MIP assay Saliva 100% 100% 14 11 TCID.sub.50 .Math. ml.sup.−1 minutes
TABLE-US-00006 TABLE 6 Comparative table of SARS-CoV-2 FDA EUA Approved Serology Tests Detected Response Company Test Antibodies PPA NPA LOD time Access Bio, Access Bio IgG-S, IgM-S, 98.4% 98.9% Not stated 10 minutes Inc. CareStart IgG-N and (combined) (combined) COVID-19 IgM-N IgM/IgG Abbott AdviseDx IgG-S 98.1% 99.6% ~8.67 pg .Math. ml.sup.−1 Not stated SARS- CoV-2 IgG II (Alinity) Siemens Atellica IM IgG-S 100% 99.9% ~0.84 pg .Math. ml.sup.−1 2 h, 1 min SARS- batch CoV-2 IgG testing (COV2G) Kantaro COVID- IgG-S 99.15% 99.6% ~3.14 pg .Math. ml.sup.−1 30 min Biosciences SeroKlir NFluidEX NMI/MIP IgG-RBD, IgM-RBD, 100% 100% 2.79-7.76 pg .Math. ml.sup.−1 11 min assay IgG-N, IgM-N for IgG-RBD, IgG-N, IgM-N and IgM-RBD
[0081] Translating the performance of the test assay based on viral load compared to the concentration of other viral entities is advantageous by enabling detection without lysis, isolation or concentration of the entities. To assess the biomimic NMI/MIP test assay for the detection of whole viral particles, we calibrated the impedimetric signal based on the tested heat-inactivated SARS-CoV-2 particles in physiological concentrations in both buffer and saliva (
[0082] In parallel, the NFluidEX was tested for the detection of both IgG-RBD and IgM-RBD antibodies to determine the ability of the device in serology testing. In the designated chambers, the biomimic NMIs/MIPs were specifically fabricated for the serological detection of SARS-CoV-2 specific antibodies.The impedimetric signal of antibodies in the concentration range of 10 pg.μl.sup.−1-10.sup.4 pg. μl.sup.−1 was assessed in spiked buffer, undiluted human plasma, and whole blood, demonstrating an increasing trend with respect to the concentration of IgG-RBD and IgM-RBD (
[0083] To explore the efficacy of NFluidEX for selective detection of SARS-CoV-2 SP in saliva, and both IgG-RBD and IgM-RBD in blood, we obtained the impedimetric signal of SARS-CoV-2 in buffer and saliva compared to the signals of other viral infections that can interfere with SARS-CoV-2 detection due to similarities in shape, size and molecular composition. These include the severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), the human coronavirus 229E (HCoV-229E), the Middle East respiratory syndrome coronavirus (MERS-CoV), and Influenza A H1N1.
[0084] The impedimetric signal from biomimic NMIs/MIPs assay in the NFluidEX device imprinted with SARS-CoV-2 SP demonstrated a higher value towards its matching protein (SARS-CoV-2 SP) compared to the SPs from other tested viruses (
TABLE-US-00007 TABLE 7 A summary of statistical significance evaluation using one-way ANOVA with post hoc Holm-Sidak mean comparison test for the diagnostic selectivity of SARS-CoV-2 SP Saliva p value Buffer p value Mean 10 1000 10000 10 1000 10000 Comparisons pg .Math. ml.sup.−1 pg .Math. ml.sup.−1 pg .Math. ml.sup.−1 pg .Math. ml.sup.−1 pg .Math. ml.sup.−1 pg .Math. ml.sup.−1 Sig..sup.a Influenza A H1N1 3.55E−11 4.02E−12 3.79E−14 1.79E−14 2.26E−14 1.75E−13 1 SARS-CoV-2 HCoV-229E 4.58E−11 4.44E−12 4.69E−14 2.23E−14 2.96E−14 2.10E−13 1 SARS-CoV-2 MERS-CoV 4.69E−11 5.33E−12 4.93E−14 2.33E−14 3.01E−14 2.22E−13 1 SARS-CoV-2 Influenza A H1N1 2.91E−10 5.70E−11 3.57E−13 1.70E−13 2.60E−13 1.56E−12 1 SARS-CoV-1 HCoV-229E 3.98E−10 6.50E−11 4.65E−13 2.24E−13 3.68E−13 1.95E−12 1 SARS-CoV-1 MERS-CoV 4.10E−10 8.25E−11 4.96E−13 2.36E−13 3.76E−13 2.09E−12 1 SARS-CoV-1 SARS-CoV-1 1.69E−04 4.71E−06 2.59E−07 1.23E−07 7.65E−08 1.33E−06 1 SARS-CoV-2 Influenza A H1N1 0.42109 0.31383 0.14863 0.12069 0.10126 0.25589 0 MERS-CoV Influenza A H1N1 0.46224 0.51144 0.23302 0.18526 0.11884 0.38013 0 HCoV-229E MERS-CoV 0.94208 0.71207 0.77357 0.78995 0.92318 0.78 0 HCoV-229E F.sub.4, 10 values 442.444 668.2656 1744.077 2025.4021 1896.4195 1293.194 .sup.aSignificant p values are denoted by a one (1) and non-significant p values are denoted by a zero (0).
[0085] To investigate the versatility of the test response towards SARS-CoV-2, SPs from a series of its variants, Alpha B.1.1.7, Delta B.1.617.2, and Omicron B.1.1.529 were tested similarly on the original strain SP imprinted NMI/MIP assay, demonstrating an adaptable impedimetric response to identify the viral SP from different variants (
[0086] The selectivity of the NFluidEX towards SARS-CoV-2 IgG-RBD and IgM-RBD antibodies over those of other similar viruses was investigated. Similarly, the NMI/MIP test chambers imprinted SARS-CoV-2 IgG-RBD and IgM-RBD demonstrated higher impedimetric signals towards their targets with minimal cross-reactivity (
TABLE-US-00008 TABLE 8 A summary of statistical significance evaluation using a one-way ANOVA with post hoc Holm-Sidak mean comparison test for serological selectivity of SARS-CoV-2 IgG-RBD Blood p value Plasma p value Buffer p value 100 50 100 50 100 50 Mean Comparisons pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 Sig..sup.a HCoV-229E (IgG-N) 6.58E−15 6.25E−15 2.55E−14 3.30E−15 6.81E−14 3.43E−13 1 SARS-CoV-2 (IgG- RBD) MERS-CoV (IgG-N) 6.90E−15 6.33E−15 2.85E−14 3.63E−15 7.06E−14 3.67E−13 1 SARS-CoV-2 (IgG- RBD) Influenza A H1N1 7.38E−15 6.51E−15 3.47E−14 3.85E−15 7.51E−14 3.99E−13 1 (IgG-N) SARS-CoV-2 (IgG- RBD) MERS-CoV (IgG-RBD) 7.51E−15 6.88E−15 3.94E−14 4.04E−15 8.30E−14 5.06E−13 1 SARS-CoV-2 (IgG- RBD) Influenza A H1N1 9.09E−15 6.98E−15 3.95E−14 4.39E−15 9.89E−14 5.90E−13 1 (IgG-RBD) SARS-CoV-2 (IgG- RBD) SARS-CoV-2 (IgM- 1.06E−14 1.94E−14 5.32E−14 6.75E−15 1.11E−13 6.92E−13 1 RBD) SARS-CoV-2 (IgG- RBD) HCoV-229E (IgG-N) 0.26238 0.01724 0.12903 0.09116 0.32972 0.21885 0 SARS-CoV-2 (IgM- RBD) MERS-CoV (IgG-N) 0.31235 0.01826 0.1937 0.13861 0.36607 0.26549 0 SARS-CoV-2 (IgM- RBD) Influenza A H1N1 0.39124 0.02095 0.34805 0.17898 0.43301 0.33389 0 (IgG-N) SARS-CoV-2 (IgM- RBD) MERS-CoV (IgG-RBD) 0.41432 0.02725 0.352 0.21783 0.45325 0.33436 0 SARS-CoV-2 (IgM- RBD) HCoV-229E (IgG-N) 0.44137 0.02922 0.36776 0.2977 0.49796 0.39771 0 Influenza A H1N1 (IgG-RBD) MERS-CoV (IgG-N) 0.51213 0.78972 0.4815 0.47602 0.55969 0.48163 0 Influenza A H1N1 (IgG-RBD) Influenza A H1N1 0.6185 0.8119 0.48639 0.60871 0.57842 0.48737 0 (IgG-N) Influenza A H1N1 (IgG-RBD) MERS-CoV (IgG-RBD) 0.64851 0.81726 0.50615 0.63247 0.68638 0.56102 0 Influenza A H1N1 (IgG-RBD) Influenza A H1N1 0.71294 0.83964 0.52601 0.69575 0.72481 0.58141 0 (IgG-RBD) SARS-CoV-2 (IgM- RBD) MERS-CoV (IgG-RBD) 0.74885 0.86583 0.53116 0.74395 0.74143 0.66919 0 HCoV-229E (IgG-N) HCoV-229E (IgG-N) 0.78125 0.89397 0.67052 0.78343 0.81506 0.77794 0 Influenza A H1N1 (IgG-N) MERS-CoV (IgG-N) 0.83913 0.92197 0.77711 0.81117 0.8369 0.77869 0 MERS-CoV (IgG-RBD) MERS-CoV (IgG-N) 0.87256 0.94486 0.78326 0.83766 0.8424 0.7859 0 Influenza A H1N1 (IgG-N) MERS-CoV (IgG-N) 0.9064 0.97151 0.80788 0.87845 0.90086 0.87589 0 HCoV-229E (IgG-N) F.sub.6, 14 values 327.5460 329.3973 263.5958 359.1365 232.9898 181.5124 .sup.aSignificant p values are denoted by a one (1) and non-significant p values are denoted by a zero (0).
TABLE-US-00009 TABLE 9 A summary of statistical significance evaluation using a one-way ANOVA with post hoc Holm-Sidak mean comparison test for serological selectivity of SARS-CoV-2 IgM-RBD Blood p value Plasma p value Buffer p value 100 50 100 50 100 50 Mean Comparisons pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 pg .Math. μl.sup.−1 Sig..sup.a HCoV-229E (IgG-N) 2.27E−12 1.68E−12 3.60E−13 4.12E−12 4.04E−11 1.09E−13 1 SARS-CoV-2 (IgM- RBD) SARS-CoV-2 (IgM- 2.70E−12 2.23E−12 3.61E−13 5.59E−12 4.06E−11 1.12E−13 1 RBD) SARS-CoV-2 (IgG-RBD) Influenza A H1N1 3.09E−12 2.72E−12 3.65E−13 5.70E−12 4.57E−11 1.20E−13 1 (IgM-N) SARS-CoV-2 (IgM- RBD) Influenza A H1N1 3.74E−12 3.01E−12 4.03E−13 5.89E−12 4.72E−11 1.21E−13 1 (IgM-RBD) SARS-CoV-2 (IgM- RBD) MERS-CoV (IgM-RBD) 3.82E−12 3.42E−12 4.57E−13 8.06E−12 5.77E−11 1.21E−13 1 SARS-CoV-2 (IgM- RBD) MERS-CoV (IgM-RBD) 0.2437 0.11387 0.52198 0.16223 0.51845 0.74855 0 HCoV-229E (IgG-N) HCoV-229E (IgG-N) 0.26263 0.18509 0.52751 0.43708 0.52297 0.75508 0 Influenza A H1N1 (IgM-RBD) MERS-CoV (IgM-RBD) 0.43195 0.26597 0.54373 0.43877 0.67162 0.77592 0 SARS-CoV-2 (IgG- RBD) Influenza A H1N1 0.46046 0.33243 0.73458 0.46137 0.7158 0.81735 0 (IgM-RBD) SARS-CoV-2 (IgG- RBD) HCoV-229E (IgG-N) 0.47905 0.49084 0.75998 0.48041 0.77491 0.82406 0 Influenza A H1N1 (IgM-N) MERS-CoV (IgM-RBD) 0.62924 0.50013 0.76659 0.50431 0.78036 0.84544 0 Influenza A H1N1 (IgM-N) Influenza A H1N1 0.66438 0.60008 0.78583 0.50615 0.82139 0.92827 0 (IgM-N) Influenza A H1N1 (IgM-RBD) HCoV-229E (IgG-N) 0.687 0.64575 0.97291 0.90993 0.82694 0.97118 0 SARS-CoV-2 (IgG- RBD) Influenza A H1N1 0.75614 0.76985 0.97986 0.94332 0.9518 0.97808 0 (IgM-N) SARS-CoV-2 (IgG- RBD) MERS-CoV (IgM-RBD) 0.96045 0.81497 0.99304 0.96646 0.99429 0.9931 0 Influenza A H1N1 (IgM-RBD) F.sub.5, 12 values 255.7395 264.3684 361.9643 230.0317 161.3457 442.9206 .sup.aSignificant p values are denoted by a one (1) and non-significant p values are denoted by a zero (0).
[0087] The selectivity of the assay towards the imprinted target was further tested with a determined concentration of the target and analogous viral particles within the linear range response of the assay, demonstrating a high selectivity at even lower concentrations (
[0088] To demonstrate the applicability of the NFluidEX for clinical decision making, 34 COVID-19-positive saliva samples were analysed in contrast to 17 COVID-19-negative saliva samples, while simultaneously testing 10 COVID-19-positive patient blood samples in contrast to 8 COVID-19-negative blood samples for multiplexed serosurveillance of IgG-RBD and IgM-RBD antibodies (
[0089] A set of randomized samples belonging to the SARS-CoV-2 original strain and the Delta B.1.617.2 variant was analysed. In order to quantifiably assess the impedimetric signal of NFluidEX towards SARS-CoV-2 viral concentration, the signals from a cohort of healthy samples (n=17) were compared to the signal from a cohort of patient samples (n=34) clinically diagnosed with SARS-CoV-2 original strain and Delta variant (
TABLE-US-00010 TABLE 10 Summary of NFluidEX performance against current gold standard testing methods RT-qPCR + − Total NFluidEX + 34 0 34 − 0 17 17 Overall result 34 17 51 of NFluidEX ELISA IgG IgM IgG IgM + + − − Total NFluidEX IgG + 10 0 1 0 11 IgM + 0 10 0 1 11 IgG − 0 0 7 0 7 IgM − 0 0 0 7 7 Overall result + 10 0 of NFluidEX − 0 8 +: positive test result, −: negative test result; Note: Although a single false positive was recorded for IgG and IgM, the combined parallel sensitivity and specificity evaluated at two unique test sites yielded 100% accordance with gold standard methods.
[0090] To assess the quantitative nature of the NFluidEX compared to RT-qPCR, the viral load distribution in the patient samples was calculated based on both methods. For the gold standard RT-qPCR method, cycle threshold (Ct) values were obtained for all patient samples regardless of their viral strain, according to the established inversely proportional scaling between Ct values and viral loads. When the estimated viral load distribution of the patient samples was compared with the NFluidEX impedimetric calibration curve (
[0091] As a proof-of-concept to demonstrate the potential of the NFluidEX as provided herewith in the synchronous usage of saliva-based diagnosis and blood-based serology testing, a field study was conducted for a cohort of 10 patients (n=5 patients clinically diagnosed with the SARS-CoV-2 original strain and n=5 patients clinically diagnosed with the SARS-CoV-2 Delta B.1.617.2 variant). The dual detection device allowed for an enhanced combined sensitivity over diverse disease manifestations due to higher positive rates of diagnostic tests during the acute phase of infection and high positive rates of serology biomarkers during the convalescent phase of infection. All the patients in this cohort were evaluated 1 week after symptom onset. Regardless of their viral strain, all patients demonstrated positive results for both the NFluidEX saliva-diagnostic and blood-serosurveillance tests, which were in accordance with their reported RT-qPCR and ELISA results (
[0092] To demonstrate the broad applicability of the NFluidEX test assay, it is demonstrated that the biomimic NMI/MIP assay is amenable to other contagious respiratory infections, like the Influenza A H1N1 virus. The assay was imprinted following the same protocol with the viral haemagglutinin surface protein of Influenza. Electrochemical sensing demonstrated a linearly increasing impedance magnitude over the varied concentration of viral protein. A similar linear trend over a wide linear range from 10 pg. ml.sup.−1-10.sup.5 pg. ml.sup.−1 at a low LOD was observed in both buffer and saliva (
[0093] Finally, an automatic readout system for nonprofessional users was developed. The assembly of the MIP assay with the devolved readout system provides a platform for rapid test, analysis, and monitoring of SARS-CoV-2 in real samples in 10 min or 1 min depending on the media. A smartphone gadget is developed to obtain an Arduino kit's sent or portable impedimetric signal transduction module signals, analyze, and monitor the risk level based on the received signals (
[0094] The signal transduction panel, potentionstat, is a portable cost-effective battery-operated electrochemical workstation that consists of a printed electrical circuit board and a Bluetooth wireless connection to convert the impedimetric signal of the test assay to a quantifiable readout on a smartphone via an Android application within 1 min (
[0095] It is also encompass that the proposed biosensor allows detection of a protein of interest, not only viruses such as SARS-CoV2. As provided, the developed MIP biosensor offers sensitivity, stability, repeatability, and reproducibility towards protein detection.
[0096] The performance of the MIP biosensor was tested for detection of heart fatty acid binding protein (H-FABP) in clinical samples. Eight clinical blood serums samples were tested using commercial lateral flow assay LFA cassettes and the MIP biosensor (Table 2). Positive control (Pos. Ctrl) sample was HS-10D spiked with 100 ng mL.sup.−1 H-FABP. The clinical samples without MI symptoms showed very low (i.sub.0−i)/i.sub.0 and tested negative H-FABP and Tnl on LFA cassettes (samples 1, and 2) which made them as negative control (Neg. Ctrl). The two samples (samples 3 and 8) tested negative H-FABP and positive Tnl on LFA cassettes, demonstrated low (i.sub.0−i)/i.sub.0 (less than 0.4) compared with the Pos. Ctrl while showed higher (i.sub.0−i)/i.sub.0 in comparison with the Neg. Ctrl. This implies that the level of H-FABP is lower than 8 ng mL.sup.−1 (the LOD of the LFA for H-FABP, provided by the company) in these serums. Further, comparing these results with standard calibration plots of H-FABP in HS-10D, revealed that the levels of H-FABP in these samples are lower than 10 pg mL.sup.−1, despite the negligible interference from Tnl. Sample 5 (tested negative for H-FABP and Tnl) showed higher (i.sub.0−i)/i.sub.0 (approximately 0.5). According to the calibration plot, the level of H-FABP in this sample is lower than 10 pg mL.sup.−1, thus the LFA cassette cannot detect it. This clearly highlights the benefits of the developed biosensor with a lower LOD than the commercial device.
[0097] The other two samples (samples 4, and 7) that tested positive H-FABP on the LFA cassettes, displayed (i.sub.0−i)/i.sub.0 close to the Positive Control, confirming the ability of the device to reliably detect H-FABP in clinical samples with levels higher than 1 ng mL.sup.−1. Sample 6 tested positive for H-FABP and Tnl by LFA. However, the (i.sub.0−i)/i.sub.0 recorded by the biosensor was less than 0.4, mainly related to the rather weak performance of the biosensor in high concentrated serum (the Tnl value for this sample was recorded more than 50000 ng mL.sup.−1 according to the Table 2). Consequently, while the LFA needs expensive bioreceptors (antibodies), has a longer analysis time (˜10 minutes) and a high detection limit, the cost-effective MIP biosensor can successfully and specifically detect significantly lower levels of H-FABP in clinical samples, in less than 1 minute.
TABLE-US-00011 TABLE 11 The summary of the clinical samples investigation and comparison of the results of the developed MIP biosensor with the LFA cassettes as a reference method Sample Description of the H-FABP code clinical sample* LFA (TnI) LFA (H-FABP) (i.sub.0 − i)/i.sub.0 value** Sample 1 Patient without MI Negative Negative 0.15 ± 0.02 <<10 pg mL.sup.−1 symptoms Sample 2 Patient without MI Negative Negative 0.12 ± 0.05 <<10 pg mL.sup.−1 symptoms Sample 3 TnI~36.1 Positive Negative 0.29 ± 0.04 <10 pg mL.sup.−1 Sample 4 Negative troponin Negative Positive 0.68 ± 0.05 ~10 ng mL.sup.−1 but MI occurred Sample 5 Negative troponin Negative Negative 0.50 ± 0.05 <10 pg mL.sup.−1 but MI occurred Sample 6 TnI > 50000 Positive Positive 0.36 ± 0.06 <10 pg mL.sup.−1 Sample 7 Negative troponin Negative Positive 0.70 ± 0.05 ~30 ng ml.sup.−1 but MI occurred Sample 8 TnI~27.7 Positive Negative 0.26 ± 0.05 <10 pg mL.sup.−1 *Troponin levels were the approximate values determined by ELISA at the hospital. MI occurrence was detected by chest pain and ST-segment elevation. **The approximate values of H-FABP measured by the developed MIP biosensor. The values were determined through comparison of the recorded (i.sub.0 − i)/i.sub.0 for 10 times diluted clinical serum samples with the calibration plot of HS-10D spiked with various concentrations of H-FABP
[0098] In summary, the electrochemical biosensor provided based on a core-shell structure of NMIs and MIP allows for detection of protein such as e.g., but not limited to, H-FABP in PBS, human plasma, human serum, and bovine serum via a hybrid electrodeposition/electropolymerization fabrication protocol.
[0099] The proposed biosensor was used to detect H-FABP, as one of the early biomarkers of myocardial infarction with concentrations lower than 10 fg mL.sup.−1 in PBS and in a short period of time (30 s). The biosensor showed lower LOD and wider linear range of detection than the commercial H-FABP LFA cassette. The selectivity evaluations also indicated distinguishable detection of H-FABP among other cardiac biomarkers, and in clinical samples owing to the biomimetic MIP layer. Moreover, the polymeric nature of the MIP layer provided high stability of the proposed biosensor at ambient temperature up to 3 weeks, depicting its excellent storage ability than the previously reported antibody-based biosensors that are usually stored at 4° C. As MIP biosensors are more stable, more efficient, and more scalable, than antibody-based biosensors at ambient temperature and in clinical environments, the disclosed biosensor combination provides for fast, sensitive, and affordable detection of proteins of interest.
EXAMPLE I
Device Fabrication and Testing
[0100] Polyaniline and ortho-phenylenediamine (o-PD) were bought from Thermo-fischer. Gold (III) chloride trihydrate was bought in SigmaAldrich. Heat inactivated SARS-CoV-2 (ATCC® VR1986HK™), SARS-CoV-2 Spike Antibody (CR3022) (NBP2-90980), SARS Nucleocapsid Protein Antibody (NB100-56683), and SARS Membrane Protein Antibody (NB100-56569) and Influenza A H1N1pdm (NY/01/09) Culture Fluid (Heat Inactivated) (0810248CFH1) purchased from Cedarlane. SARS-CoV-2 Nucleocapsid Antibody (N009) (NBP3-05721), Influenza A Haemagglutinin H1N1 Antibody (NBP3-06578) was bought from Novusbio. Pooled Saliva (IRHUSL50ML), Single Donor Human Plasma (Blood Derived) (IPLASK2E50ML) and Single Donor Human Whole blood (IWB1K2E10ML). Samples were bought from Innovative Research. Heat inactivated SARSCoV2 (ATCC® VR1986HK™) purchased from Cedarlane. Aniline 99.5%, Sodium Acetate ASC, Acetic Acid, and Phosphate buffer saline (PBS) 10× were bought in the chemical store of the Université du Quebec à Montreal. The chemicals purchased were analytical grade and were used without further purification. All the solutions were prepared using ultrapure water (>18 MΩ cm) from a Millipore Milli-Q water purification system.
Solution Preparations
[0101] The gold solution for NMIs electrodeposition were prepared from Gold (III) chloride trihydrate (AuHCl) in 0.5 M HCl. The 10 mM electro-monomer ortho-phenylenediamine (o-PD) solution was prepared in acetate Buffer. Similarly, 10 mM, 20 mM, 50 mM, and 100 mM aniline electro-monomer solution was prepared in 0.5 M H.sub.2SO.sub.4. Washing solution, 0.1 M NaOH was prepared with ethanol and water in a 5:1 ratio. A 6.7 mM PBS (pH 7.2) containing 5 mM [Fe(CN)6].sup.3-/4- solution was prepared for electrochemical experiments.
Saliva Collection Protocol
[0102] The human saliva samples were collected from healthy donors (2 female and 2 male) with an age range of 25-35 years old. The collection and processing protocols were adapted from Henson's and Alenus publications. Briefly, the donors were restrained from food and oral hygiene one-hour prior. A rinse and a pause step were followed prior the sample collection. The sample was processed through centrifuged at 10,000 rpm for 10 min at 4° C. Followed by separation of the fractions. Through the text, samples 1,2 and samples 3,4 resemble individual female and male human sample, respectively.
Preparation of Viral Samples
[0103] Solutions spiked with heat inactivated SARS-CoV-2 virus (10.sup.5 pg. ml.sup.−1) were prepared in PBS (pH 7.2) and human saliva. Followed by a series of 10-fold dilutions to cover a range of concentrations from (10 pg. ml.sup.−1-10.sup.5 pg. ml.sup.−1) in both media. Heat inactivated Influenza A H1N1 virus solutions were prepared equally. The solution spiked with antibodies against SARS-CoV-2 spike protein (1:100) were prepared in PBS (pH 7.2) and plasma (1:2). A serial of dilutions followed to cover different range of dilutions.
[0104] Device, MNIs and MIPs Fabrication
[0105] The device is based on a SU8 coated ITO-glass surface, where the analysis wells are patterned through standard lithography. Followed by a three-electrode electrodeposition method of gold to generate 3D hierarchical NMIs at the analysis wells base. The electrodeposition was preformed through an Autolab potentiostat/galvanostat (model: PGSTAT204), with a reference electrode of Ag/AgCl and a counter electrode of platinum wire. The supporting electrolyte solution, for the 3D gold NMIs were synthetization, was HAuCl.sub.4 (1 mM) in a HCl (0.5 M). Synthesis was carried out at the applied fix potential of 600 mV vs Ag/AgCl. Electrosynthesis of polymer was carried out following a electropolymerization method. Briefly, a solution containing different concentrations of polymer (aniline, o-PD) was prepared using sodium acetate and H.sub.2SO.sub.4 solutions. SARS-CoV-2 heat-inactivated virus and antibody were added to the solution with a stock concentration and the volume of the monomer solution to the virus/antibody solution was maintained in the ratio of 95:5. Further, electropolymerization of polymer on NMIs electrode was carried out using cyclic voltammetry (CV) technique at a scan rate of 50 mV s.sup.−1. Eventually, the samples were washed with ethanol and water solution (5:1 VN) containing 0.1 M NaOH to remove the template. Similarly, a control assay modified by non-imprinted polymer (NIP) was fabricated without using SARS-CoV-2 heat-inactivated virus or antibody as the template.
Characterization
[0106] The morphology of the proposed sensor was study via scanning electron microscopy (SEM) images were captured with a Quanta FEG 450 ESEM (FE-SEM) and EIS characterization to assess the electrochemical performance of NMIs, NIP and MIP electrodes by using a 10 mM [Fe(CN)6].sup.3-/4 PBS solution containing.
COVID-19 Patient Sample Study
[0107] Human saliva, blood, and plasma samples were collected from the patients who were admitted at “Erythron Laboratory”, a cooperator laboratory of Isfahan University of Medical Sciences (IR.MUI.MED.REC.1400.066 and McGill IRB Internal Study Number: A03-M24-21B). Free authorization and consent forms were signed by patients, and their clinical samples were collected according to the laboratory regulation. 15 saliva samples were collected from adult patients with COVID-19 symptoms such as fever, fatigue, and dry cough were collected and tested with RT-qPCR (LightCycler 480, Roche) using primers (nCoV_IP2-12669Fw, nCoV_IP2-12759Rv, nCoV_IP2-12696bProbe(+)) targeting the RdRp gene/nCoV_IP2 in the ORF1ab prior to electrochemical studies. 5 samples were determined to belong to the original strain of SARS-CoV-2 and 10 samples belonged to the Delta B.1.617.2 variant. In addition, 19 patient saliva samples were collected from the University Health Network's PRESERVE-Pandemic Response Biobank for testing on the assay (REB #20-5364). All samples tested positively using RT-qPCR (QuantStudio 12K Flex, ThermoFisher) and were determined to be from the original strain of SARS-CoV-2 prior to electrochemical sensing. The samples were assessed at a Level 2+ facility situated in the Montreal Jewish General Hospital. In addition, 10 patient blood and plasma samples were collected for antibody evaluations of which 5 samples belonged to the original strain of SARS-CoV-2 and 5 samples belonged to the Delta B.1.617.2 variant. The blood samples were tested with ELISA reader (EUROIMMUN Analyzer I-2P). The ELISA results were presented as the cut-off index (COI) value for IgG-N, targeting nucleocapsid protein of SARS-CoV-2, and the sum of IgM-N and IgM-S, targeting nucleocapsid and spike proteins of SARS-CoV-2, respectively. Accordingly, the samples with a COI of higher than 1.1 and lower than 0.9 were considered as positive and negative samples, respectively.
EXAMPLE II
Electropolymerization of MIP-SARS-CoV-2 Heat-Inactivated Virus and Antibodies
[0108] Chronoamperometry was performed to fabricate gold NMIs and cyclic voltammetry to electropolymerize nonconductive o-PD polymer to fabricate the MIP assay with various template proteins including the SARS-CoV-2 spike protein (SP) and anti-receptor binding domain (RBD) antibodies (IgG-RBD and IgM-RBD). (
[0109] The electrocatalytic performance of the NMI/MIPs electrode in real biological media was further studied. The SARS-CoV-2 heat-inactivated whole virus was spiked in human saliva. All set of experiments were conduncted as follow. First the incubation time needed to accurately detect the viral particle was determined by studying the charge transfer in pooled human saliva (
[0110] The serological electrocatalytic performance of the NMI/MIPs electrode in real biological was assessed by the study of spike and nucleocapsid SARS-CoV-2 antibodies spiked in undiluted plasma and whole blood. The incubation time needed to accurately detect each antibody was determined in undiluted plasma for both IgG-RBD and IgM-RBD (
EXAMPLE III
Stability, Repeatability, and Reproducibility of the MIP Biosensor
[0111] One of the major merits of MIP biosensors is their high stability at ambient temperature. The stability of the MIP biosensor was examined during 21 days storing at ambient temperature. The R.sub.CT for the MIP biosensor (virus and antibodies) was recorded after 7 days, and 21 days of storage in a shelf which demonstrated that the R.sub.CT decrease by 2.5% and 5% after 7 and 21 days, respectively. These results signify that the proposed biosensor offers high stability at the ambient temperature. To ensure the absence of carryover effects upon successive measurements on a single sensor the repeatability of the biosensor was evaluated by the impedimetric repetitive measurements (five times, n=5). Each experiment was repeated for three individual electrodes and the relative standard deviation (RSD) was recorded 5.2%, which is in an acceptable range. Another important feature for practical applications of the biosensor is the reproducibility which was verified by recording the Rct for 5 as-prepared MIP electrodes, each one three times with a total RSD of 4.2% (n=5). A slight high value of this parameter can be related to the effective the distribution of the MIP layer and its thickness.
EXAMPLE IV
Digitalization
[0112] To digitize the detection process and use the built-in platform as a point-of-need device, a cyclic voltammetry technique (Electrochemical impedance spectroscopy) was used (
[0113] While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.