Patent Application
20220339556
It is provided a nanostructured biosensor based on gold nano/micro islands (NMI) for detecting proteins.
The global wave of the coronavirus pandemic has exposed critical gaps in monitoring, and mitigating the spread of respiratory viral infections, highlighting the need for rapid, reliable, and accurate diagnostic tests for the detection of viruses like SARS-CoV-2 and Influeza A at the point-of-need. The existing gold standard detection techniques have failed to satisfy the ongoing need for early-diagnosis and serological testing on demand, being hindered by the need of specialized laboratory equipment and inherent assay limitations, like long turnarounds time in centralized laboratory facilities and lengthy protocols. The effective control of a fast-growing outbreak requires major in-community diagnosis to contain the high transmission potential in early infection stages. An ideal test platform should detect the level of the infection and the body's immunity by targeting both the virus and specific antibodies.
Ceasing the SARS-CoV-2 pandemic requires the application of diagnostic testing on an immense scale. In-community testing could successfully identify cases enabling contact tracing and containment without lockdowns. The speed of testing process could be significantly increased using Point of Care assays that would allow testing to be carried out in remote locations (particularly at homes). Rapid antigen tests have won the competition and are in the hands of individuals, however, the tests using nasal swabs as the targeted sample for detection, are challenged with numerous false results with low reported sensitivities.
Means for an early and accurate diagnosis of SARS-CoV-2 present a great concern due to the substantial transmission potential occurring in the pre-symptomatic phase. Currently, there are many available diagnosis tests including nucleic acid test (NATs), serological tests, antigen, and ancillary (based on personal devices or hospital laboratory tests). NATs are the most used with more than 80 tests approved by the Food and Drugs Agency (FDA). However, the assays requirements hinder the screening and diagnosis of a suspected infected person of less than 24 h or after the acute phase of infection. Despite the overall effectiveness of the diagnostics methods, most are prone to long assay times and high false-negative rates. Moreover, several methods are not suitable for early diagnosis or require access to expensive and highly specialized equipment. Thus, effective diagnosis in resource-limited settings remains a challenge.
The long virus incubation period of up to 14 days can accelerate the spread of the virus, where an average of 5-6 days is expected between incubation and symptom onset, thus prompting the need for early-detection. Further concerns are the new variants that arise with mutations in viral strains over many infections; (e.g. Alpha B.1.1.7, Delta B.1.617.2, Omicron B.1.1.529, amongst others). These variants exhibit increased transmissibility and potential resistance to vaccines and their detection.
To date, the U.S. Food and Drug Administration (FDA) has solely issued approvals for saliva collection kits for at-home self-collection of biofluids that are stabilized in a diluent buffer and shipped to a centralized facility for subsequent sample pre-treatment and testing. When compared to self-administered nasopharyngeal swabs (71%, 4.93 mean log copies. ml−1), the self-collected saliva (81%, 5.58 mean log copies. ml−1) provides higher sensitivity when the diagnosis is performed in the first five to ten days of infection. However, untreated saliva typically requires lengthy pre-treatment protocols negatively affecting its potential as target biofluid.
It is thus still highly desired to be provided with a simple and reliable tool for detecting a viral infection such as COVID-19.
The combination of molecular diagnostic and serological testing on a single platform improves the robustness of the result due to the heterogeneity in disease responses. Testing via molecular diagnostics alone is shown to have a positive predictive agreement from 51.9%-79.2%, likely due to viral load clearance from the upper respiratory tract over time. Meanwhile, a combined approach with tandem serology testing increases the positive detection rate to 98.6%-100%, allowing for more reliable responses during the acute and convalescent phase of infection. In addition, the multiplex detection of IgG and IgM antibodies enables the serosurveillance of current and past infections based on the temporal prevalence of the immunoglobulins in response to infection; sequential seroconversion is confirmed to occur first in IgM followed by IgG in early infection, with waning levels of IgM and high levels of IgG during late infection. Finally, the earliest humoral response after the first week of symptom onset has been shown in antibodies targeting the receptor binding domain (RBD) on the spike protein (37.2% and 38.5% positive samples for IgM-RBD and IgG-RBD, respectively) as opposed to their nucleocapsid (N) antibody counterparts (20.5% and 37.2% for IgM-N and IgG-N, respectively). Nonetheless, the diagnostic community has found it highly challenging to achieve the accuracy for parallel diagnosis and serological testing in one single attempt from the easily accessible body fluids.
It 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, the MIP consisting of a conductive monomer comprising a built-in recognition site of the target protein, wherein the charge transfer resistance and the impedance of the MIPs change upon binding of the target protein.
In an embodiment, the NMIs are electrodeposited on a conductive glass with a reference electrode of Ag/AgCl and a counter electrode of platinum wire.
In another embodiment, the conductive glass is a tin oxide (ITO) substrate.
In a further embodiment, the conductive monomer is polyaniline (PANI) or o-phenylenediamine (o-PD).
In an additional embodiment, the target protein is an antibody, a viral protein or a heart fatty acid binding protein (H-FABP).
In an embodiment, the antibody is a viral antibody.
In a further embodiment, the viral protein is from SARS-CoV-2 or Influenza.
In an added embodiment, the viral protein is from a SARS-CoV-2 variant.
It is also provided a microfluidic read-out apparatus for detecting a target protein in a subject comprising the biosensor defined herein and microfluidic reader.
In an embodiment, the microfluidic read-out apparatus is a multiplex microfluidic apparatus.
In an additional embodiment, the microfluidic reader allows multiplexing different human samples.
In an embodiment, the apparatus recited herein further comprises a WiFi adapter for transferring the read-out signals from the microfluidic reader to a platform.
In an embodiment, the WiFi adapter is a Bluetooth low energy (BLF) connector.
In another embodiment, the platform is a computer or a smartphone.
It is additionally provided a method of detecting a target protein in a subject comprising the steps of providing a sample from the subject, contacting the sample with a biosensor as defined herein, 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 transferring the change in charge transfer resistance or impedimetric signal to a microfluidic reader for transforming the signal into a cyclic voltammetry signal or impedimetric signal, wherein the cyclic voltammetry signal or impedimetric signal indicates the presence of the target protein.
In an embodiment, the subject sample is saliva, plasma, or whole blood.
In a particular embodiment, the subject is a human or an animal.
In another embodiment, the method provided herein further comprises transmitting the cyclic voltammetry signal to a platform.
In an embodiment, the cyclic voltammetry signal or impedimetric signal is transmitted by Wi-Fi to the platform.
In another embodiment, the platform depicts the cyclic voltammetry signal or impedimetric signal in a gauge.
In a further embodiment, the cyclic voltammetry signal or impedimetric signal is transmitted to a computer or a smartphone.
In another embodiment, the cyclic voltammetry signal or impedimetric signal indicates the presence of the target protein in 1 min to 11 min.
Reference will now be made to the accompanying drawings.
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.
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.
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.
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.
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.
However, MIPs' known low sensitivity towards the detection of some proteins and biological compounds have hindered their use for highly sensitive applications.
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−1; Alpha variant: 6.48 pg.ml−1; Delta variant: 8.13 pg.ml−1; Omicron variant: 7.62 pg.ml−1 in human saliva with a linear range of 10 pg ml−1-105 pg. ml−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−1 with a linear range of 10 pg. μl−1-104 pg. All LODs for original variant, SP and Abs are detailed in Table 1.
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.
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.
It is encompassed that the device provided herein allows detection of any proteins, not just viruses such as SARS-CoV2 (see
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.
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.
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−1; Alpha variant: 6.48 pg.ml−1; Delta variant: 8.13 pg.ml−1; Omicron variant: 7.62 pg.ml−1l in human saliva with a linear range of 10 pg. ml−1-105 pg. ml−1. Additionally, the NMIs/MIPs for the spike protein antibody was tested in undiluted human plasma reaching a LOD of 3.13 pg. μl−1 with a linear range of 10 pg. μl−1-104 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.
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.
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
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.
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
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.
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
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 (
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−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 SiO2 via BOE etching (
The electrochemical characterization after each step of the electrode fabrication is shown in
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−1 to μg. ml−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 (
The sensitive signal transduction at a low concentration of SARS-CoV-2 (10 pg. ml−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) (
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 (
87%
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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 (
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−1-104 pg. μl−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 (
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.
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 (
aSignificant p values are denoted by a one (1) and non-significant p values are denoted by a zero (0).
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 (
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 (
aSignificant p values are denoted by a one (1) and non-significant p values are denoted by a zero (0).
aSignificant p values are denoted by a one (1) and non-significant p values are denoted by a zero (0).
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 (
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 (
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 (
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 (
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 (
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−1-105 pg. ml−1 at a low LOD was observed in both buffer and saliva (
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 (
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 (
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.
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−1 H-FABP. The clinical samples without MI symptoms showed very low (i0−i)/i0 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 (i0−i)/i0 (less than 0.4) compared with the Pos. Ctrl while showed higher (i0−i)/i0 in comparison with the Neg. Ctrl. This implies that the level of H-FABP is lower than 8 ng mL−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−1, despite the negligible interference from Tnl. Sample 5 (tested negative for H-FABP and Tnl) showed higher (i0−i)/i0 (approximately 0.5). According to the calibration plot, the level of H-FABP in this sample is lower than 10 pg mL−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.
The other two samples (samples 4, and 7) that tested positive H-FABP on the LFA cassettes, displayed (i0−i)/i0 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−1. Sample 6 tested positive for H-FABP and Tnl by LFA. However, the (i0−i)/i0 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−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.
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.
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−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.
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.
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 H2SO4. 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]3-/4- solution was prepared for electrochemical experiments.
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.
Solutions spiked with heat inactivated SARS-CoV-2 virus (105 pg. ml−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−1-105 pg. ml−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.
Device, MNIs and MIPs Fabrication
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 HAuCl4 (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 H2SO4 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−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.
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]3-/4 PBS solution containing.
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.
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). (
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 (
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 (
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 RCT for the MIP biosensor (virus and antibodies) was recorded after 7 days, and 21 days of storage in a shelf which demonstrated that the RCT 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.
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 (
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.
The present application claims benefit of U.S. Provisional Application No. 63/180,327 filed Apr. 27, 2022, the content of which is herewith incorporated in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63180327 | Apr 2021 | US |
