key: cord-0825562-f16wyudx
authors: EL Sharif, H.F.; Dennison, S.R.; Tully, M.; Crossley, S.; Mwangi, W.; Bailey, D.; Graham, S.P.; Reddy, S.M.
title: Evaluation of electropolymerized molecularly imprinted polymers (E-MIPs) on disposable electrodes for detection of SARS-CoV-2 in saliva
date: 2022-04-01
journal: Anal Chim Acta
DOI: 10.1016/j.aca.2022.339777
sha: 060c36afd5d342d96ac35b88194aee6dfb254ed5
doc_id: 825562
cord_uid: f16wyudx

We investigate electropolymerized molecularly imprinted polymers (E-MIPs) for the selective recognition of SARS-CoV-2 whole virus. E-MIPs imprinted with SARS-CoV-2 pseudoparticles (pps) were electrochemically deposited onto screen printed electrodes by reductive electropolymerization, using the water-soluble N-hydroxmethylacrylamide (NHMA) as functional monomer and crosslinked with N,N′-methylenebisacrylamide (MBAm). E-MIPs for SARS-CoV-2 showed selectivity for template SARS-CoV-2 pps, with an imprinting factor of 3:1, and specificity (significance = 0.06) when cross-reacted with other respiratory viruses. E-MIPs detected the presence of SARS-CoV-2 pps in <10 min with a limit of detection of 4.9 log(10 )pfu/mL, suggesting their suitability for detection of SARS-CoV-2 with minimal sample preparation. Using electrochemical impedance spectroscopy (EIS) and principal component analysis (PCA), the capture of SARS-CoV-2 from real patient saliva samples was also evaluated. Fifteen confirmed COVID-19 positive and nine COVID-19 negative saliva samples were compared against the established loop-mediated isothermal nucleic acid amplification (LAMP) technique used by the UK National Health Service. EIS data demonstrated a PCA discrimination between positive and negative LAMP samples. A threshold real impedance signal (Z(Re)) ≫ 4000 Ω and a corresponding charge transfer resistance (R(CT)) ≫ 6000 Ω was indicative of absence of virus (COVID-19 negative) in agreement with values obtained for our control non-imprinted polymer control. A Z(Re) at or below a threshold value of 600 Ω with a corresponding R(CT) of <1200 Ω was indicative of a COVID-19 positive sample. The presence of virus was confirmed by treatment of E-MIPs with a SARS-CoV-2 specific monoclonal antibody.

The coronavirus disease 2019 (COVID-19) pandemic has infected millions of people around the globe 48 [1] [2] [3] [4] [5] . The initial symptoms of COVID-19 are similar to other respiratory diseases, but some patients 49 can be asymptomatic [6] or presenting with milder symptoms due to the success of the vaccination 50 programme [7] . Therefore, tracking the causative SARS-CoV-2 is a difficult task. SARS-CoV-2 51 continues to pose a great health risk to the public with the evolution of more transmissible variants 52 [8] . Whilst current vaccines have reduced the risk of infection, hospitalisation, and mortality, they do 53 not absolutely prevent infection and transmission [8] [9] [10] . Therefore, rapid and accurate diagnosis of 54 the virus continues to play a crucial role in the management of the disease enabling a return to near 55 normality in society and in saving lives. 56

The current gold standard method for SARS-CoV-2 diagnosis is the quantitative reverse transcription 57 polymerase chain reaction (qRT-PCR) test [11] [12] [13] . The method detects the presence of viral RNA in 58 nasopharyngeal swab samples with high sensitivity and specificity. . However, this assay does have 59 some disadvantages, as the qRT-PCR requires complex equipment, extensive training for operators, 60 and multiple hours to complete the procedure. These limitations were further accentuated by the 61 rapid growth of the pandemic, as the qRT-PCR did not initially have the screening capacity to keep 62 pace especially at the height of the pandemic [14] . 63

New solutions for COVID-19 detection are in high demand, and one method that has received much 64 traction and also implemented by the National Health Service in the UK is the loop-mediated 65 isothermal amplification (LAMP) method [15, 16] . The LAMP method utilises nucleic acid 66 amplification at one temperature, thereby obviating the need for a thermal cycler, essential for 67 conventional PCR. Albeit less quantitative the LAMP method is inherently faster, easier to use, and 68 more cost effective than qRT-PCR assays. While nasopharyngeal and oral swab samples are the 69 accepted mode of sample collection for qRT-PCR [17] , the LAMP method uses saliva and/or sputum 70 samples. Saliva is a more attractive medium due to simplicity and no discomfort in collection [18] [19] [20] , 71 J o u r n a l P r e -p r o o f obviating the need for skilled personnel to extract the sample to improve accuracy. Importantly, high 72 viral loads can be present in saliva during the first days of infection, further making it a useful 73 medium to investigate [21, 22] . While offering these advantages, there are still disadvantages in the 74 both LAMP and RT-PCR methods affecting accuracy and misdiagnosis or for example cases where 75 individuals have recovered from the infection but are still releasing genetic material from inactivated 76 (non-intact) virus [23] . This can lead to false positive cases skewing case statistics and needlessly 77 taking people out of the workforce. 78

The other diagnostic assay widely utilised by the public is the SARS-CoV-2 lateral flow test (LFT), 79 which relies on immobilised antibodies to detect nucleoprotein antigen from oral and nasal swabs 80 associated with the virus infection [24] . These tests are easy to use with rapid results, but such 81 immunoassays lack the necessary accuracy to be a reliable SARS-CoV-2 diagnostic test due to its low 82 sensitivity and high false negative/positive rates. For example, the Innova SARS-CoV-2 antigen rapid 83

qualitative LFT offers convenience of use [25] [26] [27] [28] . However, data on their efficacy showed that in 84 asymptomatic people, the LFT fails to detect SARS-CoV-2 infection in a substantial proportion. and exposed to the working electrode. Cyclic voltammetry (CV) is used to induce polymerization of 106 the functional monomer at the electrode surface to progressively grow the polymer layer, requiring 107 multiple sequential cycles for optimum film growth. The template, when removed, leaves binding 108 sites selective for the rebinding of target. The subsequent rebinding of target can be investigated 109 also using CV. A small inorganic redox marker such as potassium ferricyanide is used to probe the 110 changing permeability of the MIP depending on whether the target template is bound. In the bound 111 state (when target is present), there is a reduced diffusion of redox marker to the electrode and 112 therefore a small current produced. In the eluted state (when target is absent), the MIP is more 113 permeable to the redox marker resulting in an increase in current. The typical redox marker used is 114 the ferro/ferricyanide redox system. This can be used as a ubiquitous marker for all electrode-based 115

MIPs and obviates the need for the target itself to be electrochemically active. In tandem with cyclic 116 voltammetry, electrochemical impedance spectroscopy (EIS) has been increasingly used to 117 characterise adsorbed biological and polymeric layers on electrode surfaces [42, 43] . Supplementary 118 Figure S1 (A) shows the equivalent circuit model for EIS measurement and Figure S1 (B) shows a 119 typical EIS spectrum displaying a characteristic semi-circle and the inflection point in the real 120 impedance signal (Z Re ) axis marks the charge transfer resistance (RCT). The RCT (i.e. diameter of the 121 semi-circle) typically increases in the presence of bound layers. There is also typically a 122 corresponding increase in the height of the semi-circle (increase in imaginary impedance, Z Im ) with 123 adlayers. Therefore, EIS can be potentially used as a tool to discriminate between different bound 124 and unbound states of, for example, the bare electrode and following MIP integration. 125

In response to the COVID-19 pandemic, there have been some limited reviews exploring but not 126

limited to the deployment of MIPs to detect SARS-CoV-2. have reported detection of the full-length spike protein (FL-S) from nasopharyngeal swabs using 133 electropolymerized 3-aminophenylboronic acid (APBA) as a MIP. Analyte measurement was possible 134

within 15 min and limit of detection (LOD) of 64 fM reported using differential pulse voltammetry. 135

While the APBA serves to attach to cis-diols of glycoproteins such as the FL-S, it should be noted that 136

APBA is not selective to any one glycoprotein and therefore will be prone to interference from other 137 glycoproteins. 138

It should be noted that the spike protein is prone to multiple mutations as the virus evolves, which 139 could limit the application of this approach. A holistic virus imprinting approach, taking into 140

consideration the template's full characteristics (size, shape and presentation of spike) offers the 141 potential for MIP-based diagnostic to evolve with the virus. To this end, we have found that 142

imprinting of the SARS-CoV-2 virion particle (whole intact virus) as a template has been largely 143 overlooked with only one publication [49] at the time of writing this paper. In their work, a 144 graphene-based electrode was integrated with an E-MIP based on pyrrole as functional monomer in 145 conjunction with 3-aminophenylboronic acid (APBA) to imprint whole virus, in a sensor production 146 process taking 1 hr. All rebinding studies were performed in control solutions only and did not use 147 real samples. As mentioned earlier an approach using APBA is prone to interference due to the 148 presence of interfering glycoproteins. 149

In our approach, we use an acrylamide-based hydrogel MIP to electrochemically imprint the whole 150 intact virus in a MIP production process taking 2 -5 min and we demonstrate the measurement of 151 SARS-CoV-2 whole virus in actual biological samples (saliva) within 5 min. Specifically, we evaluate 152 and present proof-of-concept findings that electrochemically grown MIPs (E-MIPs) imprinted with 153 SARS-CoV-2 pseudoparticles (replication incompetent lentiviruses embedded with the SARS-CoV-2 154 spike protein [50]) can be used to selectively recognise the template particles. This is achieved by the 155 MIP identifying with the virion shape and/or the presence of the spike protein. Subsequently, E-MIPs 156 are prepared for virus capture from saliva and analysis. The E-MIPs are interrogated 157 electrochemically (using CV and EIS) with a view to be used by untrained and trained personnel alike 158 (see Figure 1 ). We present an evaluation of an evolving (MIP) technology and demonstration of 159 proof of concept. We explain the simplicity of the method to determine SARS-CoV-2 in untreated 160 saliva samples, which in itself is an indication of how it can easily replace the laboratory-based LAMP 161 method, with minimal labour and skill required. MIP is electrodeposited onto a disposable screen-printed electrode (SPE). In the presence of SARS-167

CoV-2 pps the anodic/cathodic peak currents to an external redox marker (ferricyanide) decrease 168

and EIS spectra show a corresponding change, leading to qualitative and quantitative approaches to 169

in-situ SARS-CoV-2 detection. with 0.1% β-propiolactone (BPL; Sigma) at 4°C whilst rolling. Following this, incubated at 37°C for 2 194 hr to hydrolyse BPL. SARS-CoV-2 pps were titrated before and after BPL inactivation by titration on 195

HEK293T cells, previously transfected with 500 ng of a human ACE2 expression plasmid (Addgene, 196 Cambridge, MA, USA Figure S1 ). All electrochemical measurements were performed using a Metrohm Autolab PGSTAT204 232 and NOVA2. Figure S2) . This was 276

found to effectively remove the template without apparently compromising the integrity of the pre-277

formed E-MIP. Ferricyanide was used as a redox label to confirm that E-MIP or E-NIP was deposited on the bare 293 electrode. A reduction in peak current to the redox marker was a firm indicator that a polymer layer 294 was formed on the electrode surface. Figure 2C shows the ferro-ferricyanide cyclic voltammograms 295 comparing the bare electrode and following MIP formation, template elution and template 296 rebinding. Figure 2D shows the corresponding cyclic voltammograms for the E-NIP control polymer. 297

The E-MIP follows the expected pattern in change in peak currents. There is a significant decrease in 298 current upon MIP formation, compared with the bare electrode (SPE). Upon template elution, the 299 redox peak increases to an intermediate stage but still lower than the bare electrode, confirming 300 that template has been removed (allowing more redox marker to diffuse to the electrode). Upon 301 template rebinding, the current decreases due to a decrease in permeability of the redox marker to 302 the electrode. The E-NIP also behaves as expected. Since there is no template to remove, it also 303 confirms that electrochemical elution conditions do not affect the integrity of the polymer adlayer. 304

Indeed, the NIP layer is more uniform and homogenously produced compared with the MIP. The 305 presence of the virus serves to impede the otherwise natural course of monomer polymerization 306 and crosslinker incorporation. The polymerization process therefore needs to navigate around the 307 template and entraps it between the growing polymer chains during the process resulting in a more 308 porous polymer layer structure on the electrode. Polymer growth in the NIP, by contrast, is less 309 tortuous due to the absence of template, allowing it to form a more rigidly coupled, homogeneous 310 and dense layer with inherently low porosity. Therefore, for an equivalent number of polymerization 311 cycles, the NIP will incorporate more monomer and crosslinker than the MIP. 

Cyclic voltammograms for SARS-CoV-2 pps template rebinding at a high viral titre (5.6 log10 pfu/mL) 317 are shown in Figure 2C and D. The decrease in peak current did not correlate with increasing viral 318 titre (data not shown), and so at best, it presents a qualitative (yes/no) approach to determining the 319 presence of virus. We were able to detect the presence of template in the range 4.1 -6.0 log10 320 pfu/mL. This is an acceptable range for the determination of SARS-CoV-2 in saliva levels found using 321 genome copy analysis [58] and the lower limit (calculated based on 3σ/slope) is comparable to the 322 LOD (4 log10 pfu/mL) reported by Hashemi et al in their graphene electrode-based MIP system for 323 measuring SARS-CoV-2 whole virus [49] . Based on the virus particle concentration in their control 324 experiments, they also calculated that this equated to a LOD of 11.3 fg/mL. By extrapolation, this 325

suggests our method has a LOD of 88.0 fg/mL. It is of note that real samples were not tested in their 326 study. 327 PCA and factor analysis was used to discriminate and semi-quantitate between the presence and 328

absence of SARS-CoV-2 pps in MIP and NIP formation. Figure 3 shows a clear discrimination between 329 NIP control (no SARS-CoV-2 pps present) and MIP loaded with SARS-CoV-2 pps, with 99.5% of the of 330 the variance explained by principal components 1 and 2. The factor analysis ( Figure 3A In addition to cyclic voltammetry, EIS was used to further investigate the rebinding of SARS-CoV-2 344 pps to MIP and NIP. Figure 4A compares the Nyquist plots for bare electrode, MIP and NIP. The bare 345 gold SPE (black line) was characterised by a small impedance (≈500 Ω) a resistance to charge transfer 346 (RCT) of 1800±100 Ω, the latter indicating the expected small resistance toward redox conversion and 347 a high electron transfer on the bare electrode surface. It should be noted that the RCT in the EIS 348 spectrum (but not the form of the spectrum) for the bare SPE can vary between electrodes due to 349 batch-to-batch variation in printed electrode material. Subsequent changes to RCT, real (Z Re ) and 350 imaginary (Z Im ) impedances, will be relative to the baseline EIS response of the SPE used. Both MIP 351 and NIP modified electrodes showed an increase in impedance (Z Re ≈ 3000 Ω) and an increase in RCT 352 to 7000 Ω, demonstrating an increased resistance to redox conversion. This would be expected since 353 both MIP and NIP presented a barrier to diffusion of the ferricyanide redox marker. We 354 subsequently treated MIP and NIP layers with a SARS-CoV-2 specific mAb CR3022. MIP samples 355 elicited a significant change in impedance likely due to antibody binding to entrapped SARS-CoV-2 356 pps (Z Re ≈ 5000 Ω) and an associated RCT ≈10000±1000 Ω demonstrating high resistance to redox 357 conversion. Conversely, the NIP showed only a small increase in impedance upon mAb CR3022 358 treatment (from 3000 to 3400 Ω) with no significant change in RCT. This is possibly attributed to some 359 non-specific binding to the NIP. 360 J o u r n a l P r e -p r o o f curve for SARS-CoV-2 pps rebinding (2min, RT 22±2 °C) using RCT data as input, insert shows LDR 367 following axis titles, data represents mean ± SD, n=3. 368

This was an interesting trend in that the presence of SARS-CoV-2 pps on MIP was defined by RCT = 370

6000±2300 Ω whereas zero pseudovirus loading as represented by the NIP was characterised by a 371 significantly higher signal in Z Re and Z Im impedances. We investigated a range of SARS-CoV-2 pps 372 loadings on the MIPs ( Figure 4C ) which demonstrated that the trend continued with all viral loadings 373

(1.0 -6.0 log10 pfu/mL) being grouped together with RCT values at or below 2000 Ω. However, with 374 the eluted MIP (virus removed) or below 2 log10 pfu/mL viral load on the MIP, the corresponding 375

Nyquist plot resembled that of NIP (zero virus) allowing us to determine a threshold of 2 log10 376 pfu/mL below which the E-MIP sensor could not determine SARS-CoV-2. The clear separation in 377 impedance characteristics between presence and absence of virus on MIP suggests that this method 378 could be used qualitatively to discriminate between positive and negative COVID-19 cases, 379 essentially with no requirement for post measurement data processing through principal component 380

analysis. This radically speeds up the time to a result to less than 5 min. 381

By extracting the charge transfer resistance values, we were able to produce a calibration plot for 382 the semi-quantitative determination of SARS-CoV-2 pps in the range 3.0 -7.0 log10 pfu/mL with a 383 limit of detection of 4.9 log10 pfu/mL ( Figure 4D) . Whereas there was a linear correlation between 384 viral load and RCT at medium to high viral loadings of 4 -7 log10 pfu/mL, the RCT responses in the 385 range 2.0 -4.0 log10 pfu/mL did not differ significantly. Nonetheless, we present a wide linear range 386 and whereas it may not cover 8 logs as with the PCR method, it should be noted that we present a 387 method to detect SARS-CoV-2 in untreated saliva with no amplification of virus material. Our E-MIPs 388 also showed selectivity for template SARS-CoV-2 pps, with an imprinting factor of 3:1, and specificity 389 (significance = 0.06) when cross-reacted with similar concentrations of either porcine reproductive 390 and respiratory syndrome virus 1 (PRRSV) or influenza A virus subtype H9N2 (IAV) (Supplementary 391 Figure S3 ). 392

The richness in information from the whole Nyquist plot and the high discrimination in overall signal 395

for the different E-MIP viral loading conditions ( Figure 4A and C) offers a route to a plausible rapid 396

and sensitive system for SARS-CoV-2 determination. We investigated this further by testing 24 397 patient saliva samples, which had been previously tested for SARS-CoV-2 using the LAMP method. 398

Only a small saliva sample (50 µL) was required with no dilution or pre-treatment. PCA biplots for 399 polymer formation using the 'qualitative' approach were again determined ( Figure 5A ), 400 demonstrating two clusters for each of the positive and negative samples, showing 66.6% 401 agreement with LAMP positive results and 88.9% agreement with LAMP negative results. 402

Interestingly, our study revealed that a degree of overlap was present in the discrimination method, 403

with five samples from the LAMP negative results presenting as positive results using our E-MIP 404 approach. Overall, there was 75% agreement between our E-MIP method and the LAMP method. 405

There is a difference in the EIS response when switching from imprinting of control pps in PBS or PBS 406 diluted synthetic saliva ( Figure 4A and C) to imprinting of virus within a real saliva sample ( Figure  407 5B). Nonetheless, Figure 5B shows that the E-MIP device continues to return the small Z Re and Z Im 408 values when the virus is entrapped in the MIP production process indicating a positive test result. 409

The negative test results possess significantly higher Z Re and Z Im values to those of either the NIP (no 410 virus) or the MIP with virus removed from Figure 4A . As a confirmatory measure of entrapped SARS-411

CoV-2 in positive samples, all representative samples were treated with SARS-CoV-2 S-specific mAb 412 CR3022. Cyclic voltammograms and electrochemical impedance spectra of the ferro/ferricyanide 413 redox marker were subsequently produced. Only the COVID-19 positive samples elicited a significant 414 change in the Nyquist plots apparently due to antibody binding to entrapped virus presenting with 415 the spike protein. Interestingly, the COVID-19 negative samples also showed a small change in Z Re 416 and Z Im upon antibody treatment, possibly due to non-specific binding of antibody. Surprisingly 417 though, the direction of changes in impedance observed for the positive cases upon mAb CR3022 418 treatment was unexpected. Whereas we expected an increase in real and complex impedances due 419

to specific adsorption and layering of antibodies to the MIP-surface-entrapped virus, there was 420 instead a decrease in both Z Re and Z Im as well as in the associated RCT suggesting a decreased 421 resistance to (increased diffusion of) the ferro/ferricyanide redox marker electrode process. It is 422 noteworthy that this unexpected decrease occurred consistently only with the positive cases. We 423 speculate that the antibody binding with SARS-CoV-2 antigen is leading to the antibody-antigen 424 complex stripping away from the electrode into the surrounding solution, and hence the decrease in 425 Z Re and Z Im . The binding affinities of acrylamide-based MIPs for proteins are typically in the sub-426 micromolar range [59] . Given that the affinity of CR3022 for the RBD is very high at 0.125 nM [52], 427 such a stripping mechanism by the antibody is plausible. The cyclic voltammograms ( Figure 5C Prof. Munir Iqbal, The Pirbright Institute, for providing inactivated influenza A virus, and Dr James 504

Kelly and Ahmed Mohamed for assistance with SARS-CoV-2 propagation and purification. 505 rebinding (50 µL, 5.6 log10 pfu/mL, 2 min, RT, 22±2 °C); using resulting RCT data from EIS as input, data 523

represents mean ± SD, n=3. 524 525 J o u r n a l P r e -p r o o f

Overview of COVID-19 Pandemic: Transmission, Epidemiology and Diagnosis

COVID-529 19: Understanding the Pandemic Emergence, Impact and Infection Prevalence Worldwide

Wuhan to World: The COVID-19 Pandemic, 533 Frontiers in Cellular and Infection Microbiology

COVID-19: breaking down a global health 536 crisis

Outbreak of COVID-19: An emerging global pandemic threat

Impact of the COVID-19 pandemic on the 541 management of chronic noninfectious respiratory diseases

Considerations and guidance to control the rebound in COVID-544 19 cases

549 Increased mortality in community-tested cases of SARS-CoV-2 lineage B

Transmission 553 event of SARS-CoV-2 delta variant reveals multiple vaccine breakthrough infections

Current Status of Diagnostic Testing for SARS-CoV-2 Infection and Future 556 Developments: A Review

558 Point-of-Care PCR Assays for COVID-19 Detection

The Dynamics of SARS-CoV-2 (RT-PCR) Testing, Case Reports in 560

Advancements in detection of SARS-CoV-2 infection for 562 confronting COVID-19 pandemics

Development and Validation of a Rapid

Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-566

Throughput Screening of COVID-19

Extraction-free RT-LAMP to detect SARS-CoV-2 is less 568 sensitive but highly specific compared to standard RT-PCR in 101 samples

Performances, feasibility and acceptability of nasopharyngeal swab, saliva and oral-self sampling 573 swab for the detection of severe acute respiratory syndrome coronavirus 2

Preliminary optimisation of a simplified sample preparation method to permit direct 579 detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal 580 amplification (RT-LAMP)

Saliva as a Potential Diagnostic Tool

Consistent Detection of 2019 Novel Coronavirus in Saliva, Clinical Infectious 586 Diseases

Saliva as a Potential Diagnostic Specimen for COVID-19 Testing

Temporal 592 profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during 593 infection by SARS-CoV-2: an observational cohort study

Evaluation of 596 droplet digital PCR for quantification of SARS-CoV-2 Virus in discharged COVID-19 patients

Operational characteristics of 30 lateral flow immunoassays used to identify 600 COVID-19 immune response

point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 605 infection

Performance of the Innova SARS-CoV-2 antigen rapid 608 lateral flow test in the Liverpool asymptomatic testing pilot: population based cohort study, Bmj-609

COVID-19: Rapid antigen detection for SARS-CoV-2 by lateral flow assay: A 611 national systematic evaluation of sensitivity and specificity for mass-testing, Eclinicalmedicine

Diagnostic 616 accuracy of rapid antigen tests in asymptomatic and presymptomatic close contacts of individuals 617 with confirmed SARS-CoV-2 infection: cross sectional study

Molecularly imprinted polymers with specific recognition for 619 macromolecules and proteins

Selective extraction of proteins and other 621 macromolecules from biological samples using molecular imprinted polymers

Advances in imprinting strategies for selective virus recognition 624 a review

Molecularly imprinted polymer for human viral 626 pathogen detection

Toward Rational Design of 629 Selective Molecularly Imprinted Polymers (MIPs) for Proteins: Computational and Experimental 630 Studies of Acrylamide Based Polymers for Myoglobin

Evaluation of Molecularly Imprinted Polymers as Synthetic Virus Neutralizing Antibody 634 Mimics

Design and 637 development of plastic antibodies against SARS-CoV-2 RBD based on molecularly imprinted polymers 638 that inhibit in vitro virus infection

Molecularly imprinted polymers for the 640 recognition of proteins: The state of the art

Recent advances and future prospects in molecularly imprinted 642 polymers-based electrochemical biosensors

Molecularly Imprinted Polymers 644 (MIP) in Electroanalysis of Proteins

MIP-based 647 electrochemical protein profiling

Green synthesis as a simple 649 and rapid route to protein modified magnetic nanoparticles for use in the development of a 650 fluorometric molecularly imprinted polymer-based assay for detection of myoglobin

Electrosynthesized molecularly 653 imprinted polymers for protein recognition

Electrochemical biosensor based on 655 biomimetic material for myoglobin detection

Dual-template rectangular 657 nanotube molecularly imprinted polypyrrole for label-free impedimetric sensing of AFP and CEA as 658 lung cancer biomarkers

Development of a portable MIP-660 based electrochemical sensor for detection of SARS-CoV-2 antigen

Current methods and prospects of 663 coronavirus detection

665 Strategies and perspectives to develop SARS-CoV-2 detection methods and diagnostics

An ultrasensitive molecularly 668 imprinted polymer-based electrochemical sensor for the determination of SARS-CoV-2-RBD by using 669 macroporous gold screen-printed electrode

Molecularly imprinted polymer based 671 electrochemical sensor for quantitative detection of SARS-CoV-2 spike protein

Graphene-Based Femtogram-Level

Sensitive Molecularly Imprinted Polymer of SARS-CoV-2

The SARS-CoV-2 679 Spike protein has a broad tropism for mammalian ACE2 proteins

Production of Recombinant Replication-defective

Lentiviruses Bearing the SARS-CoV or SARS-CoV-2 Attachment Spike Glycoprotein and Their 682 Application in Receptor Tropism and Neutralisation Assays

Human monoclonal antibody combination against SARS coronavirus: Synergy and 686 coverage of escape mutants

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike

MIP-based protein profiling: A method for interspecies 693 discrimination

Application of thymine-based nucleobase-695 modified acrylamide as a functional co-monomer in electropolymerised thin-film molecularly 696 imprinted polymer (MIP) for selective protein (haemoglobin) binding

Enhanced selectivity of hydrogel-based molecularly 698 imprinted polymers (HydroMIPs) following buffer conditioning

Investigation of protein imprinting in hydrogel-based 701 molecularly imprinted polymers (HydroMIPs)

SARS-CoV-2 viral 706 load is associated with increased disease severity and mortality

Determination of protein binding 708 affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)