Toward nano-sized imprinted norepinephrine-derived biopolymer as artificial receptors for detecting IgG1 by surface plasmon resonance.

Bio-based nanostructured molecularly imprinted polymers (nano-MIPs), also known as 'plastibodies', have a real potential to be used as alternatives to natural antibodies. These nanostructures have recently gained significant attention for diagnostic and therapeutic purposes. In this context, we have developed polynorepinephrine (PNE)-based nano-MIPs using an eco-friendly one-pot process for the sensitive and selective detection of a model biomolecule, immunoglobulin IgG1. We first investigated non-imprinted nanostructures (nano-NIPs) based on polydopamine as reference material, using DLS, SEM, and UV-Vis spectroscopy. Subsequently, PNE scaffolds were characterized, both in the form of nano-NIPs and nano-MIPs. Concerning nano-MIPs, we used the epitope-directed imprinting technology to create binding cavities using a small peptide from the constant region of IgG1 as a template. Nano-MIPs were initially immobilized on a sensing surface to assess their binding capacity via surface plasmon resonance (SPR) spectroscopy. This strategy showed very good sensitivity, outperforming planar PNE-based imprinted films while keeping a high selectivity even in complex biological matrices such as human serum. Furthermore, we confirmed the presence of selective binding sites on nano-MIPs by flowing them, along with nano-NIPs, through a microfluidic SPR system, where they interact with the covalently immobilized analyte. This approach resulted in a good imprinting factor of 4.5. Overall, this study underscores the broad potential of these nanostructures as a viable and reusable alternative to antibodies across a variety of bioanalytical, biochemical, and immunohistochemistry analysis techniques.

Serotonin: A new super effective functional monomer for molecular imprinting. The case of TNF-α detection in real matrix by Surface Plasmon Resonance.

Molecular imprinting and related technologies are becoming increasingly appreciated in bioanalysis and diagnostic applications. Among the imprinted polymers, we have already demonstrated that the endogenous neurotransmitters (NTs) dopamine (DA) and norepinephrine (NE) can be efficiently used as natural and sustainable monomers to straightforwardly design and synthesize a new generation of green and "soft" Molecularly Imprinted BioPolymers (MIBPs). Here, we demonstrated for the first time the ability of a further NT, i.e., serotonin (SE), in forming adhesive imprinted nanofilms coupled to label-free optical biosensing. Its imprinting efficiency is compared with those obtained with PDA and PNE. As a model study, tumor necrosis factor-alpha (TNF-α) was selected as a biomolecular target of interest in clinical diagnostics. The biomimetic receptor was coupled to Surface Plasmon Resonance (SPR), and TNF-α detection was performed in label-free and real-time manner both in buffer and biological matrices, i.e. synovial fluid and human serum. The results indicate that, under the same imprinting and binding conditions, the analytical performances of PSE are impressively superior to those of PDA and PNE. The PSE-based MIBP was able to detect TNF-α in human matrices with a good sensitivity, selectivity, and repeatability.

Advances and perspectives in the analytical technology for small peptide hormones analysis: A glimpse to gonadorelin.

In the last twenty years, we have witnessed an important evolution of bioanalytical approaches moving from conventional lab bench instrumentation to simpler, easy-to-use techniques to deliver analytical responses on-site, with reduced analysis times and costs. In this frame, affinity reagents production has also jointly advanced from natural receptors to biomimetic, abiotic receptors, animal-free produced. Among biomimetic ones, aptamers, and molecular imprinted polymers (MIPs) play a leading role. Herein, our motivation is to provide insights into the evolution of conventional and innovative analytical approaches based on chromatography, immunochemistry, and affinity sensing referred to as peptide hormones. Indeed, the analysis of peptide hormones represents a current challenge for biomedical, pharmaceutical, and anti-doping analysis. Specifically, as a paradigmatic example, we report the case of gonadorelin, a neuropeptide that in recent years has drawn a lot of attention as a therapeutic drug misused in doping practices during sports competitions.

A LysLysLys-tag as trigger in polynorepinephrine epitope imprinting: The case study of soluble PD-L1 detection in serum by optical-based sensing.

Polycatecholamines (pCAs)-based molecularly imprinted polymers (MIPs) represent the new performing generation of biocompatible ligand/receptor mimetics. In this context, dealing with MIPs synthesis for bio-macromolecules detection/extraction, one of the critical steps in ensuring effective binding affinity for the parent molecule is the selection of suitable epitopes for pCAs imprinting. To address this challenge, here we investigated the ability of lysine (K) residues to trigger the epitope imprinting process into a polynorepinephrine (PNE) matrix. To this aim, we first designed a set of model epitopes composed of three K and six alanine (A) residues to investigate the influence of each 'KA' combination on the imprinting process and the resulting binding performance by Surface Plasmon Resonance (SPR). Only the case of three flanking K residues in N-terminus arose as an excellent trigger for epitope imprinting. The efficacy of the 3K-tag strategy was then evaluated on two peptide templates belonging to soluble programmed cell death protein 1 ligand (PD-L1), which is of great interest as a cancer biomarker in liquid biopsies. These templates were selected due to their negligible natural ability to be imprinted into the PNE matrix and were modified with 3K-tags, in N-, C-, and N/C- positions, respectively. The SPR sensor developed by exploiting the N-3K tag strategy allowed us to achieve excellent sensitivity (0.31 ± 0.04 ng mL-1) and repeatability (avCV% = 4.5) in human serum samples. This strategy opens new insights both for epitopes' design for pCAs-based mimetics and as triggering tags when native epitopes display negligible imprinting capabilities.

Imprinted biopolymers as green abiotic route in immunoglobulin affinity plasmonic sensing.

The relentless research in material science is pushing towards sustainable building blocks, which may be exploited in the molecularly imprinting technology, a potentially ground-breaking tool for producing affinity mimetic receptors. In this scenario, we report and characterize a novel polynorepinephrine (PNE)-based mimetic for IgG detection, biomolecules of utmost clinical interest, coupled to a label-free and real-time sensing based on Surface Plasmon Resonance (SPR). A "molecular walk" around the Y-shaped IgG structure is performed to select small peptide portions to be used as templates during the epitope imprinting process. For real-time diagnosis, the mimetic receptor is integrated into SPR sensing platform, to directly target the IgG both in standard solutions and human serum specimens using the standard addition method. The designed platform is characterized in terms of binding kinetic/affinity parameters and analytical figures of merit, (selectivity, repeatability, limit of detection and quantification, namely 0.90 ± 0.02 µg mL-1 and 3.01 ± 0.07 µg mL-1, respectively), displaying excellent promising outcomes also when the material is subjected to thermal stress. Comprehensively, the excellent analytical performances of the MIP-based SPR sensing and the well-known versatility of such biopolymer encourage the further development of serological point-of-care testing for IgG antibodies detection.

A biomimetic enzyme-linked immunosorbent assay (BELISA) for the analysis of gonadorelin by using molecularly imprinted polymer-coated microplates.

An original biomimetic enzyme-linked immunoassay (BELISA) to target the small peptide hormone gonadorelin is presented. This peptide has been recently listed among the substances banned in sports by the World Antidoping Agency (WADA) since its misuse by male athletes triggers testosterone increase. Hence, in response to this emerging issue in anti-doping controls, we proposed BELISA which involves the growth of a polynorepinephrine (PNE)-based molecularly imprinted polymer (MIP) directly on microwells. PNE, a polydopamine (PDA) analog, has recently displayed impressive performances when it was exploited for MIP preparation, giving even better results than PDA. Gonadorelin quantification was accomplished via a colorimetric indirect competitive bioassay involving the competition between biotinylated gonadorelin linked to the signal reporter and the unlabeled analyte. These compete for the same MIP binding sites resulting in an inverse correlation between gonadorelin concentration and the output color signal (λ = 450 nm). A detection limit of 277 pmol L-1 was achieved with very good reproducibility in standard solutions (avCV% = 4.07%) and in urine samples (avCV% = 5.24%). The selectivity of the assay resulted adequate for biological specimens and non-specific control peptides. In addition, the analytical figures of merit were successfully validated by mass spectrometry, the reference anti-doping benchtop platform for the analyte. BELISA was aimed to open real perspectives for PNE-based MIPs as alternatives to antibodies, especially when the target analyte is a poorly or non-immunogenic small molecule, such as gonadorelin. Biomimetic enzyme-linked immunosorbent assay (BELISA).

Colorimetric selective quantification of anthocyanins with catechol/pyrogallol moiety in edible plants upon zinc complexation.

Here is examined the colour development from common anthocyanins (i.e., cyanidin, delphinidin, malvidin, and pelargonidin glycosides) and from anthocyanins-rich extracts (i.e., bilberries, strawberries, and raspberries), using zinc-anthocyanin complexes as molecular probe. We have observed the absorbance increase in the blue region in presence of large excess of zinc ion at acidic pH for cyanidin and delphinidin derivatives, likely due to quinoidal base stabilization from catechol and pyrogallol moiety. The assay condition were studied and applied to natural extracts containing these compounds. The same behaviour was observed for bilberry and, to a minor extent, for raspberry extracts, due to the larger cyanidin/delphinidin contents in the former than in the latter. Anthocyanin standard UV-Vis analysis in buffer has shown a very good linear correlation for cyanidin and delphinidin (R2 = 0.995 and 0.997, respectively), good precision (CV% = 7.4% and 5.3% respectively), high sensitivity (Cyε600nm = 8300 M-1 cm-1, LOD = 0.264 ± 0.005 mg L-1, LOQ = 0.478 ± 0.007 mg L-1, and Dpε600nm = 15,900 M-1 cm-1, LOD = 0.143 ± 0.002 mg L-1, LOQ = 0.478 ± 0.007 mg L-1). The effectiveness of this colorimetric method for the selective quantification of catechol/pyrogallol-based anthocyanins has been demonstrated in the aforementioned complex real matrices and compared to LC-MS/MS analysis and pH-differential method, offering a valuable tool to characterize plant and food extracts particularly rich in zinc-coordinating anthocyanins.

Sensitive 'two-steps' competitive assay for gonadotropin-releasing hormone detection via SPR biosensing and polynorepinephrine-based molecularly imprinted polymer.

The work reports an innovative bioassay for the detection of gonadorelin in urine, a gonadotropin-releasing hormone agonist widely used in fertility medicine and to treat hormonal dysfunctions. Gonadorelin is also a synthetic hormone listed by the World Anti-Doping Agency (WADA) and of interest in anti-doping controls. The main novelty relies on the development of a biocompatible, stable, and low-cost biomimetic receptor alternative to classic antibodies. Starting from norepinephrine monomer, a highly selective and sensitive molecularly imprinted polymer (MIP) was developed and optimized for optical real-time and label-free SPR biosensing. The selectivity has been addressed by testing a series of peptides, from high to low similarity, both in terms of molecular weight and primary sequence. Due to the very low molecular weight of gonadorelin (1182 Da), a 'two-steps' competitive assay was developed. Particular attention has been paid to the design of the competitor and its binding affinity constant towards the MIP, being a key step for the success of the competitive strategy. The SPR assay was first optimized in standard conditions and finally applied to untreated urine samples, achieving the sensitivity required by WADA guidelines. The MIP, tested in parallel with a monoclonal antibody, gave comparable results in terms of affinity constants and selectivity towards possible interfering analytes. However, the biomimetic receptor appears clearly superior in terms of sensitivity and reproducibility. This, together with its preparation simplicity, the extremely low-cost of the monomer and its reusability for hundreds of measurements, make polynorepinephrine-based MIPs powerful rivals to immune-based approaches in the near future for similar applications.

Polydopamine-based quantitation of albuminuria for the assessment of kidney damage.

Proteinuria is considered indicative of kidney damage that can be related to various adverse outcomes. Nowadays, there is a huge demand for routine urine screening methods to assess health risks in clinical setting without expensive procedures and long pretreatment of the sample. To address this issue, a polydopamine-based colorimetric assay to determine urinary albumin concentration in real samples is proposed here. The core of this approach relies on the established competitive adsorption of polydopamine film and human serum albumin onto the polystyrene surface of ELISA plates. Herein, we investigated the influence of temperature and the Tris-HCl buffer concentration on the polydopamine film growth. The absorbance of polydopamine film, after 24 h at 25 °C, decreases with the increase of HSA concentration, allowing the selective detection of HSA down to 0.036 ± 0.001 g L-1 in untreated urine. This simple and low-cost bioanalytical assay exhibited very good reproducibility, %CVmean = 2 in human urine, and was superior in terms of analytical performances to some standard methods available on the market, especially in comparison to the Bradford assay, for early screening and assessment of kidney damage.

3,3',5,5'-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values.

Sanitizing solutions against bacterial and viral pathogens are of utmost importance in general and, in particular, in these times of pandemic due to Sars-Cov2. They frequently consist of chlorine-based solutions, or in the direct input of a certain amount of chlorine in water supply systems and swimming pools. Colorimetry is one of the techniques used to measure the crucial persistence of chlorine in water, including household chlorine test kits commonly based on colorimetric indicators. Here, we show a simple and cheap colorimetric method based on 3,3',5,5'-tetramethylbenzidine (TMB), commonly used as chromogenic reagent for enzyme-linked immunosorbent assays. TMB is converted by chlorine to a colored molecule through a pH-dependent multi-step oxidation process where the chromaticity of TMB is directly proportional to chlorine content. This molecule offers several advantages over other commonly used reagents in terms of safety, sensitivity, and, peculiarly, hue modulation, giving rise to the detection of chlorine in water with a multi-color change of the indicator solution (transparent/blue/green/yellow). Moreover, through the appropriate setting of reaction conditions, such coloration is finely tunable to cover the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools and to test homemade solutions prepared by dilution of household bleach during health emergency events such as during the current pandemic. Graphical abstract.

Colorimetric analysis of the early oxidation of dopamine by hypochlorous acid as preliminary screening tool for chemical determinants of neuronal oxidative stress.

The hypochlorous acid produced by the innate immune system during inflammation characteristic of several neurodegenerative disorders is responsible for the generation of chlorinated byproducts of dopamine in neurons where this neurotransmitter reaches the highest concentration. Therefore, this physiological acid could play a key role in neuronal oxidative stress associated to aberrant dopamine-quinones (DQ) production. Here we report a model study simulating simplified conditions of HOCl reaction with dopamine (DA) in neurons, showing for the first time that DA is immediately converted by HOCl to the yellow colored DQ molecule. The DQ originated from dopamine oxidation results directly proportional to the total amount of the oxidant with excellent reproducibility. Furthermore, following the several evidences of the interplay between cytosolic dopamine and calcium in neurodegenerative disorders, we have verified that the presence of calcium cation influences the dopamine oxidation pathway likely due to the metal chelation by semiquinone formed in the early stage of dopamine oxidation. This experimental approach, based on the isolation of the highly reactive DQ molecule, could be useful for prelaminar investigation of the (putative) determinants of dopamine-poisoning derivatives formation.

Characterization of troponin T binding aptamers for an innovative enzyme-linked oligonucleotide assay (ELONA).

Early diagnosis of acute myocardial infarction (AMI) is of outmost importance to reduce the mortality rate, and cardiac troponins are considered the gold standard biomarkers of myocardial necrosis. In this scenario, the characterization of two troponin T (TnT)-binding aptamers as viable alternative to antibodies employed on clinical immunoassays is here reported for the first time. Their recognition ability was first investigated through surface plasmon resonance (SPR). Subsequently, an enzyme-linked oligonucleotide assay (ELONA) was developed on common 96-well polystyrene plates, both by direct and sandwich detection strategies for comparison. In both cases, the assay exhibits a detection ability of TnT in the range of low nanomolar but a great advantage on serum interference was obtained by using both aptamers in a sandwich format, with excellent reproducibility and recovery values. Despite the sensitivity needing to be enhanced to the low picomolar range, these results are encouraging for the development of new, low-cost, and rapid antibody-free colorimetric assays for AMI studies based on aptamer-Troponin T recognition.