Specific features of epitope-MIPs and whole-protein MIPs as illustrated for AFP and RBD of SARS-CoV-2.

Molecularly imprinted polymer (MIP) nanofilms for alpha-fetoprotein (AFP) and the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 using either a peptide (epitope-MIP) or the whole protein (protein-MIP) as the template were prepared by electropolymerization of scopoletin. Conducting atomic force microscopy revealed after template removal and electrochemical deposition of gold a larger surface density of imprinted cavities for the epitope-imprinted polymers than when using the whole protein as template. However, comparable affinities towards the respective target protein (AFP and RBD) were obtained for both types of MIPs as expressed by the KD values in the lower nanomolar range. On the other hand, while the cross reactivity of both protein-MIPs towards human serum albumin (HSA) amounts to around 50% in the saturation region, the nonspecific binding to the respective epitope-MIPs is as low as that for the non-imprinted polymer (NIP). This effect might be caused by the different sizes of the imprinted cavities. Thus, in addition to the lower costs the reduced nonspecific binding is an advantage of epitope-imprinted polymers for the recognition of proteins.

Multiparameter Sensing of Oxygen and pH at Biological Interfaces via Hyperspectral Imaging of Luminescent Sensor Nanoparticles.

Chemical dynamics in biological samples are seldom stand-alone processes but represent the outcome of complicated cascades of interlinked reaction chains. In order to understand these processes and how they correlate, it is important to monitor several parameters simultaneously at high spatial and temporal resolution. Hyperspectral imaging is a promising tool for this, as it provides broad-range spectral information in each pixel, enabling the use of multiple luminescent indicator dyes, while simultaneously providing information on sample structures and optical properties. In this study, we first characterized pH- and O2-sensitive indicator dyes incorporated in different polymer matrices as optical sensor nanoparticles to provide a library for (hyperspectral) chemical imaging. We then demonstrate the successful combination of a pH-sensitive indicator dye (HPTS(DHA)3), an O2-sensitive indicator dye (PtTPTBPF), and two reference dyes (perylene and TFPP), incorporated in polymer nanoparticles for multiparameter chemical imaging of complex natural samples such as green algal biofilms (Chlorella sorokiniana) and seagrass leaves (Zostera marina) with high background fluorescence. We discuss the system-specific challenges and limitations of our approach and further optimization possibilities. Our study illustrates how multiparameter chemical imaging with hyperspectral read-out can now be applied on natural samples, enabling the alignment of several chemical parameters to sample structures.

A generic approach based on long-lifetime fluorophores for the assessment of protein binding to polymer nanoparticles by fluorescence anisotropy.

Quantitation of protein-nanoparticle interactions is essential for the investigation of the protein corona around NPs in vivo and when using synthetic polymer nanoparticles as affinity reagents for selective protein recognition in vitro. Here, a method based on steady-state fluorescence anisotropy measurement is presented as a novel, separation-free tool for the assessment of protein-nanoparticle interactions. For this purpose, a long-lifetime luminescent Ru-complex is used for protein labelling, which exhibits low anisotropy when conjugated to the protein but displays high anisotropy when the proteins are bound to the much larger polymer nanoparticles. As a proof of concept, the interaction of lysozyme with poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid) nanoparticles is studied, and fluorescence anisotropy measurements are used to establish the binding kinetics, binding isotherm and a competitive binding assay.

Correction: Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein.

[This corrects the article DOI: 10.1039/D1SC04502D.].

Highly hydrophobic TEMPO-functionalized conducting copolymers for solid-contact ion-selective electrodes.

Solid-contact ion-selective electrodes (SCISEs) emerged as the best electrode embodiment for miniaturized, wearable and disposable sensors for ion/electrolyte measurements in body fluids. The commercialization of inexpensive single-use "calibration-free" electrodes requires large scale manufacturing of electrodes with reproducible calibration parameters, e.g. E0. This is perhaps the most important shortcoming of SCISEs, beside the many advantages over their conventional liquid-contact counterparts. However, adjusting the E0 value for optimal potential stability is challenging for all state-of-the-art solid-contact materials, which may combine several types of transducing mechanism (e.g. capacitive and redox materials or their combination) for enhanced potential stability and analytical performance. Therefore, here we introduce for the first time the galvanostatic intermittent titration technique (GITT) to determine the best preadjusment potential. The proof of concept is shown for a novel type of solid-contact based on the copolymerization of 3,4-ethylenedioxythiophene with perfluorinated alkyl side chain (EDOTF) and (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl modified 3,4-ethylenedioxythiophene (EDOT-TEMPO). Such materials that are compliant with local electrodeposition and provide multiple functionalities, i.e. high hydrophobicity by the perfluorinated alkyl side chain, electron-to-ion transduction by the conducting polymer (EDOT) backbone and the confinement of well-defined redox couple (TEMPO), are expected to prevail as solid-contacts.

How an ACE2 mimicking epitope-MIP nanofilm recognizes template-related peptides and the receptor binding domain of SARS-CoV-2.

Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, i.e. the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a ∼5 nm thick polyscopoletin film around the surface confined templates. The interaction of peptides and the parent RBD and spike protein with the imprinted polyscopoletin nanofilm was followed by electrochemical redox marker gating, surface enhanced infrared absorption spectroscopy and conductive AFM. Because the use of non-terminal epitopes is especially intricate, here we characterize the binding pockets through their interaction with 5 peptides rationally derived from the template sequence, i.e. implementing central single amino acid mismatch as well as elongations and truncations at its C- and N- termini. Already a single amino acid mismatch, i.e. the central Cys488 substituted by a serine, results in ca. 15-fold lower affinity. Further truncation of the peptides to tetrapeptide (EGFN) and hexapeptide (YFPLQS) results also in a significantly lower affinity. We concluded that the affinity towards the different peptides is mainly determined by the four amino acid motif CYFP present in the sequence of the template peptide. A higher affinity than that for the peptides is found for the parent proteins RBD and spike protein, which seems to be due to out of cavity effects caused by their larger footprint on the nanofilm surface.

TEMPO-Functionalized Carbon Nanotubes for Solid-Contact Ion-Selective Electrodes with Largely Improved Potential Reproducibility and Stability.

Solid-contact ion-selective electrodes (SCISEs) can overcome essential limitations of their counterparts based on liquid contacts. However, attaining a highly reproducible and predictable E0, especially between different fabrication batches, turned out to be difficult even with the most established solid-contact materials, i.e., conducting polymers and large-surface-area conducting materials (e.g., carbon nanotubes), that otherwise possess excellent potential stability. An appropriate batch-to-batch E0 reproducibility of SCISEs besides aiding the rapid quality control of the electrode manufacturing process is at the core of their "calibration-free" application, which is perhaps the last major challenge for their routine use as single-use "disposable" or wearable potentiometric sensors. Therefore, here, we propose a new class of solid-contact material based on the covalent functionalization of multiwalled carbon nanotubes (MWCNTs) with a chemically stable redox molecule, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO). This material combines the advantages of (i) the large double-layer capacitance of MWCNT layers, (ii) the adjustable redox couple ratio provided by the TEMPO moiety, (iii) the covalent confinement of the redox couple, and (iv) the hydrophobicity of the components to achieve the potential reproducibility and stability for demanding applications. The TEMPO-MWCNT-based SC potassium ion-selective electrodes (K+-SCISEs) showed excellent analytical performance and potential stability with no sign of an aqueous layer formation beneath the ion-selective membrane nor sensitivity toward O2, CO2, and light. A major convenience of the fabrication procedure is the E0 adjustment of the K+-SCISEs by the polarization of the TEMPO-MWCNT suspension prior to its use as solid contact. While most E0 reproducibility studies are limited to a single fabrication batch of SCISEs, the use of prepolarized TEMPO-MWCNT resulted also in an outstanding batch-to-batch potential reproducibility. We were also able to overcome the hydration-related potential drifts for the use of SCISEs without prior conditioning and to feature application for accurate K+ measurements in undiluted blood serum.

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein.

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

In situ silver nanoparticle coating of virions for quantification at single virus level.

In situ labelling and encapsulation of biological entities, such as of single viruses, may provide a versatile approach to modulate their functionality and facilitate their detection at single particle level. Here, we introduce a novel virus metallization approach based on in situ coating of viruses in solution with silver nanoparticles (AgNP) in a two-step synthetic process, i.e. surface activation with a tannic acid - Sn(II) coordination complex, which subsequently induces silver ion (I) reduction. The metalic coating on the virus surface opens the opportunity for electrochemical quantification of the AgNP-tagged viruses by nano-impact electrochemistry on a microelectrode with single particle sensitivity, i.e. enable the detection of particles oherwise undetectable. We show that the silver coating of the virus particles impacting the electrode can be oxidized to produce distinct current peaks the frequency of which show a linear correlation with the virus count. The proof of the concept was done with inactivated Influenza A (H3N2) viruses resulting in their quantitation down to the femtomolar concentrations (ca. 5 × 107 particles per mL) using 50 s counting sequences.

3D-printed manifold integrating solid contact ion-selective electrodes for multiplexed ion concentration measurements in urine.

Urinalysis is a simple and non-invasive approach for the diagnosis and monitoring of various health disorders. While urinalysis is predominantly confined to clinical laboratories the non-invasive sample collection makes it applicable in wide range of settings outside of central laboratory confinements. In this respect, 3D printed devices integrating sensors for measuring multiple parameters may be one of the most viable approaches to ensure cost-effectiveness for widespread use. Here we evaluated such a system for the multiplexed determination of sodium, potassium and calcium ions in urine samples with ion-selective electrodes based on state of the art octadecylamine-functionalized multi-walled carbon nanotube (OD-MWCNT) solid contacts. The electrodes were tested in the clinically relevant concentration range, i.e. ca. 10-4 - 10-1 mol L-1 and were proven to have Nernstian responses under flow injection conditions. The applicability of the 3D printed flow manifold was investigated through the analysis of synthetic samples and two certified reference materials. The obtained results confirm the suitability of the proposed system for multiplexed ion analysis in urine.

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications.

Nucleic acid aptamers show clear promise as diagnostic reagents, as highly specific strands were reported against a large variety of biomarkers. They have appealing benefits in terms of reproducible generation by chemical synthesis, controlled modification with labels and functionalities providing versatile means for detection and oriented immobilization, as along with high biochemical and temperature resistance. Aptamers against immunoglobulin targets-IgA, IgM, IgG and IgE-have a clear niche for diagnostic applications, therefore numerous aptamers have been selected and used in combination with a variety of detection techniques. The aim of this review is to overview and evaluate aptamers selected for the recognition of antibodies, in terms of their design, analytical properties and diagnostic applications. Aptamer candidates showed convincing performance among others to identify stress and upper respiratory tract infection through SIgA detection, for cancer cell recognition using membrane bound IgM, to detect and treat hemolytic transfusion reactions, autoimmune diseases with IgG and detection of IgE for allergy diseases. However, in general, their use still lags significantly behind what their claimed benefits and the plethora of application opportunities would forecast.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Potential Reproducibility of Potassium-Selective Electrodes Having Perfluorinated Alkanoate Side Chain Functionalized Poly(3,4-ethylenedioxytiophene) as a Hydrophobic Solid Contact.

The irreproducibility of the standard potential (E°) is probably the last major challenge for the commercialization of solid-contact ion-selective electrodes (SCISEs) as single-use or wearable sensors. To overcome this issue, we are introducing for the first time a perfluorinated alkanoate side chain functionalized poly(3,4-ethylenedioxythiophene) (PEDOTF) as a hydrophobic SC in potassium-selective electrodes (K-SCISEs) based on plasticized poly(vinyl chloride). The SC incorporates the tetrakis(pentafluorophenyl)borate (TFAB-) anion, which is also present as a lipophilic additive in the ion-selective membrane (ISM), thus ensuring thermodynamic reversibility at the SC/ISM interface and improving the potential reproducibility of the electrodes. We show here that the PEDOTF-TFAB solid contact, which was prepolarized prior to the ISM deposition to either its half or fully conducting form (i.e. different oxidation states) in acetonitrile containing 0.01 M KTFAB, had a very stable open-circuit potential and an outstanding potential reproducibility of only ±0.5 mV (n = 6) for 1 h in the same solution after the prepolarization. This shows that the oxidation state of the highly hydrophobic PEDOTF-TFAB film (water contact angle 133°) is stable over time and can be precisely controlled with prepolarization. The SC was also not light sensitive, which is normally a disadvantage of conducting polymer SCs. After the ISM deposition, the standard deviation of the E° of the K-SCISEs prepared on glassy carbon was ±3.0 mV (n = 5), which is the same as that for conventional liquid contact K-ISEs. This indicates that the ISM deposition is the main source for the potential irreproducibility of the K-SCISEs, which has been overlooked previously.

Multiplexed assessment of the surface density of DNA probes on DNA microarrays by surface plasmon resonance imaging.

In terms of hybridization assays surface plasmon resonance imaging (SPRi) offers high throughput, label-free and real-time monitoring of the binding kinetics. This requires DNA microarrays on bare or modified gold SPRi chips, which are generally premade by an off-line microspotting procedure. Therefore, the surface density of the immobilized probes is not known although it is an essential quality control parameter, especially, when it can vary in a broad range as in case of self-assembled thiol-labeled DNAs on gold surface. Here we show that the small molecular weight ruthenium(III) hexamine complex (RuHex) introduced earlier for electrochemical quantitation of DNA coverage on gold electrodes can be used also in SPRi to assess the surface density of DNA probes in DNA microarrays. A single injection of RuHex solution allows the simultaneous visualization and quantification of the surface density of DNA probes (ranging in this study from 4 × 1011 to 1.7 × 1013 molecules cm-2) on all spots of a microarray made by microspotting thiol labeled short DNA probes both in prehybridized and single-stranded form on a gold SPRi chip. The methodology was applied to determine the effect of the surface density of DNA probes on the hybridization efficiency and kinetics of complementary microRNAs, using hsa-miR-208a-3p as model. Single mismatch duplexes were found to be more effectively destabilized than fully complementary duplexes by steric hindrance at large surface densities of the DNA probes, which offers an effective mean to increase single mismatch selectivity.

Selective counting and sizing of single virus particles using fluorescent aptamer-based nanoparticle tracking analysis.

Detection and counting of single virus particles in liquid samples are largely limited to narrow size distribution of viruses and purified formulations. To address these limitations, here we propose a calibration-free method that enables concurrently the selective recognition, counting and sizing of virus particles as demonstrated through the detection of human respiratory syncytial virus (RSV), an enveloped virus with a broad size distribution, in throat swab samples. RSV viruses were selectively labeled through their attachment glycoproteins (G) with fluorescent aptamers, which further enabled their identification, sizing and counting at the single particle level by fluorescent nanoparticle tracking analysis. The proposed approach seems to be generally applicable to virus detection and quantification. Moreover, it could be successfully applied to detect single RSV particles in swab samples of diagnostic relevance. Since the selective recognition is associated with the sizing of each detected particle, this method enables to discriminate viral elements linked to the virus as well as various virus forms and associations.

Ion-Selective Electrodes Based on Hydrophilic Ionophore-Modified Nanopores.

We report the synthesis and analytical application of the first Cu2+ -selective synthetic ion channel based on peptide-modified gold nanopores. A Cu2+ -binding peptide motif (Gly-Gly-His) along with two additional functional thiol derivatives inferring cation-permselectivity and hydrophobicity was self-assembled on the surface of gold nanoporous membranes comprising of about 5 nm diameter pores. These membranes were used to construct ion-selective electrodes (ISEs) with extraordinary Cu2+ selectivities, approaching six orders of magnitude over certain ions. Since all constituents are immobilized to a supporting nanoporous membrane, their leaching, that is a ubiquitous problem of conventional ionophore-based ISEs was effectively suppressed.

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered.

Electrosynthesized molecularly imprinted polyscopoletin nanofilms for human serum albumin detection.

Molecularly imprinted polymers (MIPs) rendered selective solely by the imprinting with protein templates lacking of distinctive properties to facilitate strong target-MIP interaction are likely to exhibit medium to low template binding affinities. While this prohibits the use of such MIPs for applications requiring the assessment of very low template concentrations, their implementation for the quantification of high-abundance proteins seems to have a clear niche in the analytical practice. We investigated this opportunity by developing a polyscopoletin-based MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. As reference for a low abundance protein ferritin-MIPs were also prepared by the same procedure. Under optimal conditions, the imprinted sensors gave a linear response to HSA in the concentration range of 20-100 mg/dm3, and to ferritin in the range of 120-360 mg/dm3. While as expected the obtained limit of detection was not sufficient to determine endogenous ferritin in plasma, the HSA-sensor was successfully employed to analyse urine samples of patients with albuminuria. The results suggest that MIP-based sensors may be applicable for quantifying high abundance proteins in a clinical setting.

Aptamers for respiratory syncytial virus detection.

The identification of the infectious agents is pivotal for appropriate care of patients with viral diseases. Current viral diagnostics rely on selective detection of viral nucleic acid or protein components. In general, detection of proteins rather than nucleic acids is technically more suitable for rapid tests. However, protein-based virus identification methods depend on antibodies limiting the practical applicability of these approaches. Aptamers rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Although aptamers have been generated for virus identification in laboratory settings, their introduction into routine virus diagnostics has not been realized, yet. Here, we demonstrate that the rationally designed SELEX protocol can be applied on whole virus to select aptamers, which can potentially be applied for viral diagnostics. This approach does not require purified virus protein or complicated virus purification. The presented data also illustrate that corroborating the functionality of aptamers with various approaches is essential to pinpoint the most appropriate aptamer amongst the panel of candidates obtained by the selection. Our protocol yielded aptamers capable of detecting respiratory syncytial virus (RSV), an important pathogen causing severe disease especially in young infants, at clinically relevant concentrations in complex matrices.

Multivalent foldamer-based affinity assay for selective recognition of Aß oligomers.

Mimicking the molecular recognition functionality of antibodies is a great challenge. Foldamers are attractive candidates because of their relatively small size and designable interaction surface. This paper describes a sandwich type enzyme-linked immunoassay with a tetravalent ß-peptide foldamer helix array as capture element and enzyme labeled tracer antibodies. The assay was found to be selective to ß-amyloid oligomeric species with surface features transiently present in ongoing aggregation. In optimized conditions, with special emphasis on the foldamer immobilization, a detection limit of 5 pM was achieved with a linear range of 10-500 pM. These results suggest that protein mimetic foldamers can be useful tools in biosensors and affinity assays.