In this work, electrochemical biosensors utilizing tyrosinase (Tyr) for the detection of the nonselective beta-adrenergic agonist isoproterenol (ISO) are presented. Three different configurations for immobilizing Tyr on a graphite electrode (GE) are compared: (1) GE modified with poly(diallyldimethylammonium chloride) (PDADMAC), PDADMAC/Tyr/GE; (2) PDADMAC combined with iridium nanoparticles (IrNPs) in a stepwise preparation, resulting in PDADMAC/IrNPs/Tyr/GE; and (3) a composite of PDADMAC and IrNPs mixed with Tyr at a 1:1 (v:v), forming PDADMAC/(IrNPs-Tyr)/GE. Surface morphology was characterized using scanning electron microscopy (SEM). Cyclic voltammetry (CV) and amperometry were applied to characterize the biosensor's performance. Within the linear range of 5 μM to 211 μM, the biosensor PDADMAC/Tyr/GE exhibited a limit of detection (LOD) of 1.4 μM and a limit of quantification (LOQ) of 4.1 μM. PDADMAC/IrNPs/Tyr/GE displayed improved sensitivity with an LOD of 0.9 μM and an LOQ of 2.8 μM. The configuration PDADMAC/(IrNPs-Tyr)/GE demonstrated the best performance with an LOD of 0.3 μM and an LOQ of 0.8 μM. The slopes (0.0147 μA/M, 0.0096 μA/M, and 0.0031 μA/M for PDADMAC/(IrNPs-Tyr)/GE, PDADMAC/IrNPs/Tyr/GE, and PDADMAC/Tyr/GE, respectively) of the concentration dependencies for the three sensor modifications (which represent the analytical sensitivity) demonstrate the achieved enhancement of analytical performance by IrNPs. Furthermore, the biosensor's ability to detect ISO in the presence of potential interferences, such as ascorbic acid, uric acid, and paracetamol, was assessed. Additionally, we demonstrated the biosensor's potential to detect ISO in diluted spiked human serum samples.

Molecularly imprinted polymers (MIPs) have been established as effective specifiers in clinical diagnostics and environmental analysis for a large spectrum of analytes spanning from low-molecular-weight substances to proteins, including enzymes. The synergism of MIP technology and enzyme technique is mirrored on the one hand by the high affinity, which results in an excellent analytical performance for the determination of enzymes by MIP-sensors, and on the other hand, in the improvement of preparation and readout of MIPs by using enzymes as tools. In addition, the creation of catalytically active MIP has been derived from the transition state concept of enzymatic substrate conversion. The integration of MIPs for enzymes in electrochemical sensors offers distinct benefits, e.g., the direct readout of the catalytic activity or the direct electron transfer with redox-active groups ensures excellent specificity. On the other hand, enzymes simplify the MIPs synthesis and improve the signal processing of electrochemical sensors.

Objective: In this study, it was aimed to develop a voltammetric method using sensors prepared with the molecular imprinting technique for the detection of Captopril, an antihypertensive drug. Material and Method: With the molecular imprinting method, molecularly imprinted polymers were formed on the surfaces of glassy carbon electrodes. The analysis of Captopril was carried out using the differential pulse voltammetry method, and the performance of the sensor was examined. Result and Discussion: A linear analysis was performed up to 50 pM Captopril with a limit of detection value of 2.62 pM. Selectivity studies have shown that Captopril has a higher electrochemical response than other interfering substances, such as paracetamol, ascorbic acid, and L-proline.

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.

Sensitivity in the sub-nanomolar concentration region is required to determine important protein biomarkers, e.g., ferritin. As a prerequisite for high sensitivity, in this paper, the affinity of the functional monomer to the macromolecular target ferritin in solution was compared with the value for the respective molecularly imprinted polymer (MIP)-based electrodes, and the influence of various surface modifications of the electrode was investigated. The analytical performance of ferritin sensing was investigated using three different carbon electrodes (screen-printed carbon electrodes, single-walled-carbon-nanotube-modified screen-printed carbon electrodes, and glassy carbon electrodes) covered with a scopoletin-based MIP layer. Regardless of the electrode type, the template molecule ferritin was mixed with the functional monomer scopoletin, and electropolymerization was conducted using multistep amperometry. All stages of MIP preparation were followed by evaluating the diffusional permeability of the redox marker ferricyanide/ferrocyanide through the polymer layer by differential pulse voltammetry. The best results were obtained with glassy carbon electrodes. The MIP sensor responded up to 0.5 µM linearly with a Kd of 0.30 µM. Similar results were also obtained in solution upon the interaction of scopoletin and ferritin using fluorescence spectroscopy, resulting in the quenching of the scopoletin signal, with a calculated Kd of 0.81 µM. Moreover, the binding of 1 µM ferritin led to 49.6% suppression, whereas human serum albumin caused 8.6% suppression.

Molecularly Imprinted Polymers (MIPs) are potential tools in pharmaceutical analysis and for determining protein biomarkers. This review presents a comprehensive comparison of MIP synthesis concepts, i.e., segment vs. whole template imprinting and polymerization of the template/monomer mixture vs. “hierarchical (oriented) imprinting” and combinations of MIPs with enzymes, antibodies, and aptamers, respectively. For low-molecular-weight substances such as drugs and biomacromolecules, the hierarchical polymer synthesis around the oriented template results in MIPs with higher affinity than MIPs prepared by polymerizing a mixture of the template and functional monomers. Application of the target molecule fragments as the template, so-called epitopes, gives MIPs that possess comparable affinity towards the whole analyte as the “Whole-molecule”-MIPs, but the synthesis costs are considerably lower. The combination of MIPs with enzymes, antibodies, and aptamers allows the expansion of the analyte spectrum, amplifies the signal, and suppresses interfering substances. Catalytically active MIPs may, in the future, substitute enzymes and catalyze unnatural reactions.

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via imprinting of the widely used Strep-tag II affinity peptide (Strep-MIP). Electrochemistry, surface-sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tagged enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology.

Around 30% of the scientific papers published on imprinted polymers describe the recognition of proteins, nucleic acids, viruses, and cells. The straightforward synthesis from only one up to six functional monomers and the simple integration into a sensor are significant advantages as compared with enzymes or antibodies. Furthermore, they can be synthesized against toxic substances and structures of low immunogenicity and allow multi-analyte measurements via multi-template synthesis. The affinity is sufficiently high for protein biomarkers, DNA, viruses, and cells. However, the cross-reactivity of highly abundant proteins is still a challenge.

Surfactants (surface-active agents) are substances that, when used in very small amounts, significantly reduce the surface tension of water. Surfactants are usually organic compounds with hydrophobic groups (water repellent) that play the tail and hydrophilic groups (water absorbent) that play the role of head, so they dissolve in organic solvents and water according to their molecular structure. They can be classified as anionic, cationic, amphoteric, and nonionic. Amino acids are the primary building blocks of proteins, and extensive research has been dedicated to amino acids and biologically important compounds. Many methods have been developed for detecting amino acids and biologically important compounds based on high-performance liquid chromatography, colorimetry, fluorimetry, molecular imprinted polymer methods, capillary electrophoresis, etc. Hence, this chapter will deal with the electrochemical sensors and biosensors for detecting amino acids and biologically important compounds.

Aptamers and imprinted polymers (MIPs) for the recognition of low-molecular substances, proteins, nucleic acids, viruses and cells have been developed with the advantage over antibodies of not having to use animals for their production. They can also be generated against toxic substances and structures of low immunogenicity. Their affinities to the target molecules, especially for protein biomarkers, are mostly comparable to those of antibodies. However, the cross-reactivity of highly abundant interferences is still a challenge for MIPs.

Correction for ‘Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein’ by Zsófia Bognár et al. , Chem. Sci. , 2022, 13 , 1263–1269, https://doi.org/10.1039/D1SC04502D.

The need for fast, selective, accurate, and cost-effective in situ analysis in various fields like medical diagnosis, environmental, food, or pharmaceutical analysis has pushed the development of biosensors. Due to their fragility, lower stability under harsh conditions such as high/low pH, organic solvents, high pressure, or temperature, the biological recognition elements have been substituted by fully synthetic counterparts like aptamers and molecularly imprinted polymers (MIPs). MIPs have recently found applications for the detection of SARS-CoV-2. Herein we describe the potential and challenge of application in this field.

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 (KD) 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.

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.

Since the first reported case of COVID-19 in 2019 in China and the official declaration from the World Health Organization in March 2021 as a pandemic, fast and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has played a major role worldwide. For this reason, various methods have been developed, comprising reverse transcriptase-polymerase chain reaction (RT-PCR), immunoassays, clustered regularly interspaced short palindromic repeats (CRISPR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and bio(mimetic)sensors. Among the developed methods, RT-PCR is so far the gold standard. Herein, we give an overview of the MIP-based sensors utilized since the beginning of the pandemic.

After the pioneering work of Wulff and Mosbach in the development of MIPs by chemical polymerization, in the 1980s synthesis of MIPs by electropolymerization has been successfully introduced. Electrosynthesis of MIPs can be performed in aqueous solutions, where protein molecules preserve their natural conformation. The layer thickness can be precisely tuned by controlling the amount of charge passed. A frequently applied indirect method for the characterization of MIPs exploits voltammetry and impedance spectroscopy of ferricyanide. The changes of the current signals are caused by the removal or binding of the target, but also by “nonspecific” pores. Furthermore, target binding brings about minute decreases of the big current signal. Nevertheless, several papers describing MIPs for both low and high molecular weight substances claim measuring ranges over several orders of magnitude with subnanomolar lower limits of detection. On the other hand, evaluation of the enzymatic activity or of direct electron transfer gives a direct quantification of the target bound to the MIP. MIPs can be synthesized from only one monomer and exhibit measuring ranges from micromolar up to the subnanomolar concentration range. On the other hand, many basic and technological problems have not yet been adequately tackled. We describe in the present talk the electrosynthesis of MIPs and the analytical performance of the electrochemical MIP-sensors for the following proteins: Acetylcholinesterase, Butyrylcholinesterase, Cytochrome P450, Laccase, Tyrosinase, Ferritin, Transferrin, Hemoglobin, and Serum Albumin.